Abstracts of the 8th international geminivirus symposium and the 6th international ssDNA comparative virology workshop, 7–10th November 2016, New Delhi

Factors driving emergence and spread of whitefly-transmitted geminivirusesin India: introduction of genes for susceptibility and evolution of virus--vector--host interactions

Anupam Varma

Email ID for Correspondence: av.acpv@gmail.com

Advanced Centre for Plant Virology, Indian Agricultural Research Institute, Pusa, New Delhi 110012, India

Whitefly-transmitted geminiviruses (WTGs) have emerged as the most destructive pathogens of crop plants, assuming epidemic proportions even in the regions that were earlier free from these viruses. These epidemics are a serious concern to sustainable crop production. The major contributory factors driving emergence and spread of WTGs, include (a) evolution of virulent strains/isolates by recombinant events and mutations under selection pressure created by the deployment of resistant genotypes of the hosts, (b) acquisition of satellite-like DNA molecules with the ability to enhance virulence of the associated WTGs, (c) introduction of crops/genotypes susceptible to the indigenous WTGs, (d) intensive cropping, (e) influence of climate change on population dynamics of Bemisia tabaci, and (f) inadvertent use of infected planting material. In India, whitefly transmitted diseases (WTDs), have been known for a long time, but since the Green Revolution period, severe disease epidemics, driven by a combination of factors listed above, have assumed threatening proportion in a variety of crops. Whiteflies in Indiaare efficient transmitters of WTGs and have a very wide host range. Influenced by climate change, now whiteflies are active and transmit viruses throughout the year in northern India. This may explain the occurrence of a large number of WTGs in the region. Emergence of cowpea golden mosaic, cotton leaf curl and okra (bhindi) leaf curl diseases in epidemic proportion in India are classic examples of epidemics driven by the introduction of genes for susceptibility and evolution of virus-vector-host interactions. In recent years valuable information has been developed on virus-host interactions and diversity of WTGs and whiteflies. Deployment of resistant plant varieties is the most preferred approach to contain WTG epidemics. Transgenic lines have also shown promise, and in the coming years CRISPR-Cas9 system is expected to emerge as a powerful tool for integrated management of WTGs.

Begomoviruses occurring in southern India: Translation of research for practical field application

V. Muniyappa

Email ID for Correspondence: vmuniyappa@gmail.com

Department of Plant Pathology, University of Agricultural Sciences, GKVK, Bengaluru-560 065, Karnataka State, India

Horsegram yellow mosaic virus is one of the earliest geminiviruses isolated and characterised in India. Subsequently, seven whitefly transmitted geminiviruses were differentiated and recognised. Research was carried out on these begomoviruses of crops with respect to vector transmission, host range, genome sequence, phylogenetic relationships, spread and sources of infection, vector population, host resistance and management. Two begomoviruses of weeds were also studied in relation to crop infecting begomoviruses. In tomato several species of begomoviruses were found across India. The most predominant begomoviruses are tomato leaf curl Bengaluru virus (ToLCBV) and tomato leaf curl New Delhi virus (ToLCNDV). Extensive work has been carried out on ToLCBV occurring in southern India. An unusually severe tomato leaf curl epidemic occurred in Kolar and Bangalore districts of Karnataka state. Thousands of adult Bemisia tabaci colonized tomato plants resulting in complete crop failure. The whitefly population associated with the epidemic was identified as the B-biotype of Bemisia tabaci. B-biotype has spread to neighbouring districts in Andhra Pradesh and Tamil Nadu states and also found in Gujarat. Indian B. tabaci segregated into six genetic groups, called Asia I, Asia II-5, Asia II-7, Asia II-8, Asia I India and Middle East Asia Minor 1 (biotype-B). Resistance is the cornerstone of an IPM strategy. Our most significant achievement is the development of three open pollinated tomato varieties Sankranthi, Nandi and Vybhav resistant to ToLCBV which contains Ty2 gene. Recently, AVRDC tomato lines containing different combination of Ty genes were evaluated against ToLCBV. Tomato lines with two genes (Ty2 + Ty3/Ty5 + Ty6) showed high tolerance to the disease than those with only one Ty gene. Our work on epidemiology and molecular biology of the begomoviruses and B. tabaci contributed to the know-how and practical options for managing leaf curl disease in tomato.

Genetic variability and population structure of the begomovirus: Euphorbia yellow mosaic virus

Talita B. Mar^1,2, José C.F. Silva², Angélica M. Nogueira^1,2, César A.D. Xavier^1,2, Roberto Ramos-Sobrinho³, Douglas Lau⁴, F. Murilo Zerbini^1,2*

Email ID for Correspondence: zerbini@ufv.br

¹Departamento de Fitopatologia/BIOAGRO; ²National Research Institute for Plant-Pest Interactions, Universidade Federal de Viçosa, Viçosa, MG 36570-900, Brazil; ³Centro de Ciências Agrárias/Fitossanidade, Universidade Federal de Alagoas, Rio Largo, AL, 57100-000, Brazil; ⁴Embrapa Trigo, Rodovia BR-285, 3081, Passo Fundo, RS, 99001-970, Brazil

The study of viral populations in non-cultivated hosts can provide valuable clues in terms of the likelihood of these populations to evolve and infect crops. The center of diversity of Euphorbia heterophylla (Euphorbiaceae) is located in Brazil and Paraguay, where it is an important invasive species in soybean and other crops. Reports of begomovirus infection of E. heterophylla in Brazil date back to the 1950s. In 2011, Euphorbia yellow mosaic virus (EuYMV) was described in E. heterophylla plants collected in the state of Goiás. Here we assess the genetic variability and population structure of EuYMV. E. heterophyllasamples (n = 165) were collected in nine Brazilian states from 2009 to 2014. EuYMV was the only virus detected. A total of 158 and 57 haplotypes were compared in DNA-A and DNA-B datasets respectively. EuYMV displayed a lower degree of variability compared with other begomovirus populations infecting non-cultivated hosts. Three recombination events were detected in the DNA-B dataset, which was more variable than the DNA-A. The population displayed geographical subdivision, with six subpopulations: sp1, comprised by isolates from southern states; sp 2, comprised by 17 admixed isolates from Rio Grande do Sul; and sp3–6, comprised mainly by isolates from Goiás, Mato Grosso do Sul, Minas Gerais (plus one isolate from Amazonas) and Paraíba, respectively. A wide variation of d_N/d_S values for each gene/population indicated different selective constraints, with a tendency of purifying selection acting on each subpopulation. Interestingly, we were able to reconstruct the phylogeny using only the most contributing sites, demonstrating that these polymorphisms hold supporting information to discriminate between subpopulations. Together, our results support a long co-evolution time between EuYMV and E. heterophylla, and highlight the role of genetic drift and selection in the maintenance of variability in a geographically structured viral population.

The selection-driven emergence of an unusual Tomato yellow leaf curl virus recombinant that displaced its parental viruses

Z. Belabess^1,2,3, C. Urbino¹, M. Granier¹, A. Tahiri², A. Blenzar³ and M. Peterschmitt^1*

Email ID for Correspondence: michel.peterschmitt@cirad.fr

¹CIRAD-INRA-SupAgro, UMR BGPI, CIRAD, TA A-54K, Campus International de Baillarguet, F-34398 Montpellier, France; ²Ecole Nationaled’Agriculture de Meknès, BPS 40, Meknès, Morocco; ³Faculté des Sciences de Meknès BP 11201, Avenue Zitoune, Meknès, Morocco

TYLCV-IS76 (IS76) is a recombinant from Morocco generated between representatives of the Israel strain of Tomato yellow leaf curl virus (TYLCV-IL) and the Spanish strain of Tomato yellow leaf curl Sardinia virus (TYLCSV-ES). Unlike the previously reported TYLCV/TYLCSV recombinants, IS76 has a non-canonical recombination profile and has replaced its parental viruses in Southern Morocco (Belabess et al. 2015, Virology 486, 291–306). As its emergence coincided with the deployment of Ty-1-tolerant tomato cultivars, it was thought that IS76 may have a selective advantage in tolerant plants. This prediction was tested by comparing the fitness of agro-infectious clones of IS76 and representatives of its parental viruses in tomato plants harboring or not the Ty-1 gene. IS76 DNA accumulation was significantly higher than that of TYLCV-IL and TYLCSV-ES in the plants of the tolerant cultivar. The fitness advantage did not incur any accumulation cost in the susceptible plants. Interestingly, the IS76 selective advantage was associated with a dramatic negative impact of IS76 on TYLCV-IL accumulation which was shown to be determined by the recombinant nature of IS76. As the fitness results are consistent with the selection of IS76 by tolerant tomato plants, it was intriguing that IS76 has emerged only in Morocco and not in other countries where Ty-1-tolerant tomato cultivars were similarly deployed. It was hypothesized that the emergence of IS76 requires an extremely rare combination of circumstances which occurred by chance in Morocco. The monitoring of the population of TYLCV/TYLCSV recombinants generated in 10 Ty-1-tolerant and 10 nearly isogenic susceptible plants co-infected with parental viruses, confirmed this hypothesis. Indeed, although 100% of the co-infected plants were positive for TYLCV-IL/TYLCSV-ES recombinants from 60 days post inoculation (dpi), IS76-type recombinants were generated in only 5 susceptible and 1 tolerant plants and they remained among the rarest recombinants even at 365 dpi.

Diversity of alphasatellites in Pakistan: selective preference for a single alphasatellite by a disease complex

Ishtiaq Hassan¹, Komal Siddiqui^1,2, Qamar Abbas^1,2, Mariyam Masood^1,2, Imran Amin¹, Shahid Mansoor¹ and Rob W. Briddon^1*

Email ID for Correspondence: rob.briddon@gmail.com

¹National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan; ²Pakistan Institute of Engineering and Applied Sciences, Islamabad, Pakistan

Begomoviruses (family Geminiviridae) are single-stranded (ss)DNA viruses that are transmitted by the whitefly Bemisia tabaci. These viruses cause significant losses to dicotyledonous crops and are frequently associated with additional ssDNA molecules known as betasatellites and alphasatellites. The betasatellites provide a selective advantage to their helper viruses by virtue of encoding a protein which overcomes gene silencing. Alphasatellites are autonomously-replicating molecules that require a geminivirus for maintenance in plants and transmission between plants. The precise selective advantage to begomoviruses of associating with alphasatellites remains unclear. Recent evidence suggests that they play a part in overcoming host defenses. The study here was designed to investigate the diversity of alphasatellites in Pakistan. Leaf samples were collected between 2009 and 2014 from various crops and weeds across Pakistan but mainly from cotton. The sequences of 227 full-length alphasatellite molecules were obtained. Analysis of the sequences identified 10 alphasatellite “species” occurring in Pakistan, including some “species” which were not previously known and from unexpected hosts. However, in cotton affected by cotton leaf curl disease (CLCuD) the analysis showed Cotton leaf curl Multan alphasatellite (CLCuMA) to be the most prevalent alphasatellite, despite the fact that whitefly vectors where shown to harbour more than one alphasatellite. The findings indicate that the virus complex causing CLCuD has a preference for CLCuMA and that alphasatellites play a more important part in begomovirus disease complexes than previously thought.

Molecular diversity and phylogeography of begomoviruses and associated DNA satellites of okra in India

M. Krishna Reddy^*, V. Venkataravanappa, G. Swaranalatha, Jalali S, and Samuel D.K

Email ID for Correspondence: mkreddy60@gmail.com; mkreddy@iihr.res.in

Division of Plant Pathology, ICAR-Indian Institute of Horticultural Research, Hessaraghatta Lake PO, Bengaluru-560089, Karnataka State, India

Okra (Abelmoschus esculentus; family Malvaceae) is an important vegetable grown extensively in tropical, subtropical and warm temperate regions of the world. Whitefly-transmitted viruses belonging to the genus Begomovirus (family Geminiviridae) represent a substantial threat to Okra in the Indian subcontinent. The rapid evolutionary potential of these single-stranded DNA viruses combined with the polyphagous feeding behaviour of their whitefly vector (Bemisia tabaci) can lead to the emergence of damaging viral strains. Therefore, it is crucial to characterize begomoviruses circulating in different regions in wild and cultivated species of okra. A survey of begomoviruses infecting okra was carried out in different states of India. A total of 126 full-length begomovirus components (119 DNA-A and 21 DNA-B, with three pairs of cognate A and B components) were amplified using rolling-circle amplification, then cloned and sequenced. Sequence analysis indicated the presence of six species, two of them novel. There was evidence for a high degree of genetic variability and subpopulation structure within the okra infecting begomoviruses At least nine betasatellites and 12 alphasatellite molecules were associated with okra infecting begomoviruses. The diversity of beta satellites in okra across the sub-continent is higher than previously realized and is higher than for any other malvaceous plant species so far analysed. The beta satellites identified in okra show geographic segregation, which has implications for the development and introduction of resistant okra varieties. In addition, alphasatellite and betasatellite molecules may recombine, resulting in the emergence of new satellite DNAs. The recent advances made in understanding the molecular biology of the components of the disease complex and their interactions with host plants will aid in development of suitable management strategies.

A pluricellular way of life for nanoviruses

Sicard¹, Y. Michalakis⁴, J-L Zeddam^1,3, M. Yvon¹, M. Peterschmitt², C. Urbino², S. Gutierrez¹ and S. Blanc^1*

Email ID for Correspondence: blanc@supagro.inra.fr

¹INRA, UMR BGPI, Montpellier, France; ²CIRAD, UMR BGPI, Montpellier, France; ³IRD,UMR RPB,Montpellier, France;⁴CNRS, UMR MIVEGEC 5290, Montpellier, France

Nanoviruses are characterized by a genome composed of 6–8 circular ssDNA segments, each encapsidated individually in a distinct virus particle. A classical view in virology assumes that the viral replication cycle occurs within individual cells, where the whole viral genome information is replicated, and is then reiterated in successively infected cells during host invasion. In the context of multipartite viruses and of nanoviruses in particular, this view implies that at least one copy of each of the genome segments must repeatedly enter together in individual cells for successful infection and maintenance of the integral genome. Because one or more genome segments may be missing in numerous susceptible cells, thus aborting infection, the multipartite viral systems are believed to bear an enormous cost, which drastically increases with the number of segments constituting the viral genome. It has even been concluded that multipartite viruses with an elevated number of segments (typically member species of the family Nanoviridae) appear so costly that they should not have evolved and should thus not exist! To address this paradox, we have experimentally tested the thus far undisputed assumption that the segments of a multipartite virus must be together within individual cells for the system to be functional. For this, we used the nanovirus Faba bean necrotic stunt virus, whose genome is composed of 8 segments. Our results indicate that the various segments are not always together within individual cells and yet, that the system scattered over several distinct cells appears functional. This observation has important implications. First, it questions the cost that has always been attributed to multipartite viral systems, where gathering a copy of each segments in single cells was though to be mandatory. Second, it demonstrates that the replication cycle of a virus is not necessarily “cell-autonomous” and that the spatial unit of a virus replication cycle can be, in some cases, an ensemble of interconnected cells within which the various part of viral genetic information are obviously communicating.

Deltasatellites from the New World: maintenance by New World-but not Old World-begomoviruses and transmission by the whitefly Bemisia tabaci

Elvira Fiallo-Olivé^*, Remedios Tovar and Jesús Navas-Castillo

Email ID for Correspondence: efiallo@eelm.csic.es

Instituto de Hortofruticultura Subtropical y Mediterranea “La Mayora”, Universidad de Málaga—Consejo Superior de Investigaciones Cientificas (IHSM-UMA-CSIC), Estación Experimental “La Mayora”, 29750 Algarrobo-Costa, Málaga, Spain

Small non-coding DNA satellites associated with begomoviruses have been recently named deltasatellites. Here, we investigate the biology of deltasatellites found in wild malvaceous plants in the New World (NW). For that, infectious clones of two NW deltasatellites (from Malvastrum coromandelianum and Sidastrum micranthum) and associated begomoviruses were constructed. Infectivity in Nicotiana benthamiana and their natural malvaceous hosts was assessed. The NW deltasatellites were not able to spread in planta by themselves, but they were maintained by the associated bipartite begomovirus. Also, NW deltasatellites were transreplicated by the monopartite NW begomovirus tomato leaf deformation virus. However, they were not maintained by begomoviruses from the Old World (Tomato yellow leaf curl virus, Tomato yellow leaf curl Sardinia virus and African cassava mosaic virus) or a curtovirus (Beet curly top virus). NW deltasatellites did not modify the symptoms induced by the helper viruses but in some cases reduced their accumulation. Also, one NW deltasatellite was shown to be transmitted by the whitefly Bemisia tabaci, the vector of their helper begomoviruses. The availability of infectious clones and the observation that NW deltasatellites reduced virus accumulation paves the way for further studies of the effect on their helper begomoviruses.

A Tomato leaf curl New Delhi virus strain with low pathogenicity in tomato is spreading in the western Mediterranean basin: risk is rising for vegetable crops

I.M. Fortes, S. Sánchez-Campos, E. Fiallo-Olivé, J.A. Díaz-Pendón, J. Navas-Castillo and E. Moriones^*

Email ID for Correspondence: moriones@eelm.csic.es

Instituto de Hortofruticultura Subtropicaly Mediterránea “La Mayora” (IHSM-UMA-CSIC), Algarrobo-Costa, Málaga, Spain

Tomato leaf curl New Delhi virus (ToLCNDV) (genus Begomovirus, family Geminiviridae) comprises a complex group of bipartite begomoviruses widespread in the Indian subcontinent andother Asian countries. Recently, outbreaks of ToLCNDV have been reported inthe western Mediterranean basin (Spain, Tunisia and Italy) causing severe damage tocucurbit crops. Here, epidemics in Spain are studied showing that outbreaks are caused by a new strain of ToLCNDV with evidence of recombination in the genome. Construction of infectious clones and analysis of natural isolates showed that although highly pathogenic in zucchini squash and other cucurbit species, low pathogenicity was observed in tomato. Nevertheless ToLCNDV infections in tomato occur and could be maintained through time. Therefore, coinfections with begomoviruses already present in this crop such as those associated with tomato yellow leaf curl disease (TYLCD) (e.g. tomato yellow leaf curl virus, TYLCV) might happen with unknown pathogenic consequences. Complementation and synergism between ToLCNDV and TYLCV and consequences for infection of tomatoes resistant to TYLCD werestudied. Risks for tomato cultivation and alternative begomovirus control measures are analyzed.

Monitoring Leaf Curl Disease incidence and begomoviral sequence diversity in cultivated and feral Papaya growing in New Delhi and Haryana

Pratibha Singh-Pant¹, Prashant Pant¹, S. Hamsa¹, Tabasum Akhter¹, Parul Bhardwaj¹, Aashima Mehra¹, Sunil Mukherjee² and Sudeshna Mazumdar-Leighton^1*

Email ID for Correspondence: smazumdar@botany.du.ac.in

¹Plant Biotic Interactions Lab, Department of Botany, University of Delhi, Delhi-7, ²DBT-NERPMC, Government of India

Results from epidemiological screenings using DAS-ELISA, PCR-based coat protein gene, and DNA A genome sequences from plants exhibiting leaf curl disease symptoms will be presented. Data from field-based surveys and molecular analyses of begomoviruses detected in papaya samples collected from Delhi NCR from 2005 to 2015 will be used to discuss trends in prevalence of leaf curl disease in the region. Comparisons with sequence-based reports of TYLCV and ToLCNDV from the literature will be presented to emphasize the need for long-term regional surveillance of begomoviral infections in susceptible host plants. Information from serological and/or molecular screenings can serve as a prelude to understanding dynamics and evolution of Leaf Curl Disease in economically important horticultural crops like papaya.

Post-resistance breakdown in cotton: Dominancy of a single strain of CLCuV on Indian subcontinent

Malik Nawaz Shuja^*, Muhammad Tahir

Email ID for Correspondence: maliknshuja@yahoo.com

Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad, Pakistan

Cotton leaf curl disease (CLCuD), a devastating disorder of cotton in Pakistan and north-western India, is caused by the complex of begomoviruses in association with alpha- and beta-satellite cognates. Sixty suspected cotton sample were collected on various spots in whole Punjab region-major cotton belt, during 2010–2013. Samples were confirmed with diagnostic PCR. Full-length begomovirus and their cognates were amplified using rolling cycle amplification (RCA) and PCR, sequenced and characterized. Sequences of the DNA As component were shown to be an isolate of Cotton leaf curl Kokhran virus-Bu (CLCuKoV-Bu) with >96% nucleotide sequence identity except one clone from Layyah with <93% nucleotide sequence identity. Isolates analysed exhibited an organization of old world monopartite begomovirus that contained six an overlapping open reading frames (ORFs). Sequence of viruses were determined to be 2759 bp except for DNA A component from Layyah was determined to be 2751 bp. In common with previous CLCuKoV-Bu isolates, the virus from Layyah was recombinant containing sequences derived from two virus species that were predominant in cotton pre-resistance breaking but with distinct recombination breakpoints, for which a name was proposed as cotton leaf curl Kokhran virus-Layyah. Associated betasatellites showed to be an isolate of Cotton Leaf Curl Multan Betasatellite (CLCuMB) with >97% identity-containing the recombinant fragment typical of this satellite post-resistance breaking. Besides a commonly associated alphasatellites, a distinct molecule was found recombinant, derived sequence of CLCuMA and its helper virus, containing both begomoviral and nanoviral nona-nucleotides. Agroinoculation of these tandem repeat constructs into Nicothiana benthamiana, N. glutinosa, N. tabacum and Cucurbits exhibited symptoms like vein thickening and leaf curling. Survey showed a wide spread CLCuKoV-Bu and highlights the dominance of resistant breaking CLCuD in this region.

Assessing genetic diversity of begomovirus population infecting papaya in India for stable RNAi mediated resistance

Sangeeta Saxena^1*, Priyanka Varun¹ and S.A. Ranade²

Email ID for Correspondence: dr_sangeeta_saxena@yahoo.com

¹Department of Biotechnology, Babasaheb Bhimrao Ambedkar University (A Central University), Vidya Vihar, Raebareli Road. Lucknow-226,025, India; ²PMB (Genomics) CSIR-National Botanical Research Institute., Lucknow, India

We have been working for some time in the area of in silico search for potential siRNA sequences to engineer resistance in papaya against Papaya leaf curl virus (PaLCuV) a geminivirus. siRNA known as short interfering sequences are 21–25 nucleotide long RNA duplex, which downregulates the gene expression of target viral mRNA it is homologous to. This process is highly homology dependent thus subject to extensive pre-study using various bioinformatics tools, which can result in selection of the lead molecules. Our preliminary results suggested that while designing the siRNA although algorithm and many stringent criteria are followed, the genomic variability at nucleotide level of PaLCuV isolates is often overlooked as it is not studied extensively till date. This is of great importance since a strategy developed against one virus strain might not function against a closely related virus or a strain if the region involved in siRNA design is even slightly non-homologous. Hence we have studied the sequence homology of several PaLCuV isolates to determine extent of variability among them. From this study, the identified conserved regions will be used not only as potential target for siRNA based gene silencing but also to assist in the prediction of stability of resistance. India, a leading producer of papaya in the world, faces a major problem of PaLCuV infected papaya crops. A strategy as above based on siRNA will therefore be invaluable in controlling PaLCuV in India.

Assessing the potential maintenance of TYLCV-betasatellite associations

D. Conflon¹, M. Peterschmitt¹, M. Granier¹, P. Gentit², S. Blanc³ and C. Urbino^1*

Email ID for Correspondence: cica.urbino@cirad.fr

¹CIRAD, UMR BGPI, Montpellier, France; ²ANSES Laboratoire de virologie/phytoplasmologie, Angers, France; ³INRA, UMR BGPI, Montpellier, France

Betasatellite-associated begomoviruses (family Geminiviridae) are reported from Asia and Africa but not from most of Mediterranean countries. The begomovirus/betasatellite association in natural conditions may not necessarily depend on an extended co-adaptation but merely on coinfection opportunities. Indeed, although the Mediterranean IL and Mild strains of Tomato yellow leaf curl virus (TYLCV) have never been reported in association with betasatellite in natural conditions, they readily transreplicate betasatellite of Asian and African origin in experimental conditions and always with a dramatic increase of symptom severity. Given the importance of tomato production and the prevalence of TYLCV viruses in the Mediterranean basin, the introduction of a betasatellite may have severe economic consequences. Because the probability of this scenario mainly depends on the putative maintenance of TYLCV-betasatellite association over time, we have studied various factors potentially determining this maintenance in tomato plants using a clone of Cotton leaf curl Gezira betasatellite (CLCuGB): (i) the relative intra-plant accumulation of the DNAs of TYLCV and CLCuGB, (ii) their intra-cellular co-occurrence and (iii) their co-transmission efficiency by the vector Bemisia tabaci. Using real time PCR we have shown that the DNA of CLCuGB accumulated at a similar or higher level than that of TYLCV, and that CLCuGB was efficiently co-transmitted with TYLCV by its whitefly vector. Consistently, at least 70% of the infected plant cells were detected positive by FISH for both TYLCV and CLCuGB. Very unexpectedly up to 25% of the infected cells were positive for CLCuGB only, which indicates at the least that CLCuGB can rely on a very low concentration of helper virus to persist within a cell, and further supports its potential maintenance with TYLCV. Hence, the risk of an association of TYLCV with a betasatellite cannot be ignored, all the more so as we showed that CLCuGB induce tolerance-breaking of tomato plants bearing the popular Ty-1 gene.

Seed borne nature of begomoviruses—a new threat to reckon with

V. G. Malathi^*, P. Renuka Devi, V. K. Sathya, T.K.S. Latha

Email ID for Correspondence: vgmalathi@rediffmail.com

Department of Plant Pathology, Tamil Nadu agricultural University, Coimbatore-641003, Tamil Nadu, India

The virus members belonging to the genus Begomovirus of the family Geminiviridae have a single stranded circular DNA as genome encapsidated within paired (geminate) para-icosahedral particles of size 22 × 38 nm. In India yellow mosaic disease caused by Mungbean yellow mosaic virus and Mung bean yellow mosaic India virus is the major challenge in improving productivity of grain legumes. The yellow discoloration of pods and seeds of infected plants and symptom emergence in the very first trifoliate leaf of the plants in the field were suggestive that the virus may be seed borne, which was investigated in the present study. The distribution of the virus Mungbean yellow mosaic virus (MYMV)in various parts of the seeds of black gram (Vigna mungo L. Hepper) plants naturally infected in the field in Tamil Nadu was determined by polymerase chain reaction (PCR), Southern blot analysis, and sequencing. Nucleotide sequencing of the PCR amplicons from the seed parts from groups of ten seeds revealed the presence of MYMV in the seed coat, cotyledon, and embryonic axes. From the perusal of the literature, it was speculated that amplification may also result from integrated geminivirus genome to test whether the amplicons are produced from the integrated segments of the viral genome or from native viral replicative forms, the DNA template was subjected to rolling circle amplification using φ29DNA polymerase. The results clearly reveal that full-length 2.7 kb replicative circular DNA is present in seed tissues from infected plants. The presence of virion particles was confirmed through double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and immunosorbent electron microscopy (ISEM) even in a single whole seed. However, in the grow-out tests performed with the same batch of seeds, there was no symptom development in the seedlings though the virus (both DNA A and B components) was detected in 32% of tested seedling. Investigations on leaf deformation and leaf curl disease of vegetables also indicate presence of begomovirus in seeds. Whether the seed borne virus will serve as inoculum contributing to disease development to a greater magnitude in the field will be discussed.

Isothermal amplification methods for efficient detection of banana bunchy top virus

R. Selvarajan^*, V. Balasubramanian and K. Prasanya Selvam

Email ID for Correspondence: selvarajanr@gmail.com

ICAR-NRC for Banana, Tiruchirapalli-620102, India

Bananas are affected by four relatively well-characterized viruses that cause severe economic losses to banana growers. Banana bunchy top disease caused by banana bunchy top virus (BBTV) which belongs to the genus Babuvirus, family Nanoviridae, is a major threat to banana production in many tropical and subtropical regions. Isothermal amplification method is becoming a user friendly, exploitable for on-site diagnostics of plant viruses in the field. In this study, total DNA was extracted using simple nucleic acid extraction method from banana leaf samples of plant showing bunchy top symptoms were collected during surveys. The amplified DNA products from banana infected with BBTV showed ladder-like bands, while no amplicons were detected in control. The color of LAMP products changed from orange to green when BBTV was detected with SYBR Green I, while the color remained orange for the healthy plant and water controls. Real-time turbidimetry was used to monitor the amplification result in the tubes, and it was shown that this real time LAMP assay was about 100-fold more sensitive and quicker than PCR. Rolling Circle Amplification (RCA) is better, easier, reliable and cheaper than polymerase chain reaction (PCR) or antibody-based detection of geminiviruses and presumably all viruses with small single-stranded circular DNA genomes. In our study we have developed sensitive RCA based detection for BBTV. Though this method does not require a thermal cycler, it is relatively time consuming than LAMP. Comparative analyses of both isothermal methods have been done for the detection of BBTVin survey samples. The assays developed in this study uses simple DNA extraction protocol hence it saves the cost, therefore the isothermal detection techniques would be useful for certification of tissue culture raised banana plants and also for survey and surveillance and disease forecasting system.

Diversity alphasatellites associated yellow vein mosaic and enation leaf curl disease of wild and cultivated species of okra in India

V. Venkataravanappa and M. Krishna Reddy^*

Email ID for Correspondence: mkreddy60@gmail.com

Indian Institute of Horticultural Research, Hessaraghatta Lake PO, Bangalore-560089, Karnataka State, India

Okra (Abelmoschus esculentus) is an important vegetable grown extensively in tropical, subtropical and warm temperate zones of the world. India ranks first in the world with a production of 6 million tonnes of okra from over 0.5 million ha area. Okra is one of India’s primary vegetable crops and is widely cultivated throughout the year in different regions. Yellow vein mosaic and enation leaf curl disease caused by whitefly transmitted begomoviruses associated with both wild and cultivated species of okra is a major constraint for its production in India. Presently none of the cultivated okra species are gave the complete resistance to yellow vein mosaic and enation leaf curl disease. The disease incidence of above two diseases ranged from 80 to 100% in different location of India. The leaf samples of wild and cultivated species of okra collected from different parts of the country were analyzed by PCR and complete genome sequence. The analysis indicated that the yellow vein mosaic and enation leaf curl disease of wild and cultivated species of okra are associated with different types of alphasatellites (NVLP, MYVD1, OLCuDD1, CyTLCuD1, GoDSLa, ToCSVD1, AEVD and CLCuBuVD1) respectively. Further recombination analysis showed that the alphasatellites isolated from wild and cultivated species of okra are showing symptom of yellow vein mosaic and enation leaf curl disease are highly recombinant in nature.

Identification of the nanovirus transmission helper protein and DNAs associated with coconut foliar decay disease

Tatiana Timchenko¹, John W. Randles² and Bruno Gronenborn^1*

Email ID for Correspondence: Bruno.Gronenborn@i2bc.paris-saclay.fr

1Institute for Integrative Biology of the Cell, I2BC, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France; ²Plant Pathology and Virology, School of Agriculture Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064

Nanoviruses require a virus-encoded helper factor for transmission by their vector aphids. We analyzed the role of nanovirus proteins for infection and aphid transmission taking advantage of virus reconstituted from its eight cloned genomic DNAs. That allowed us to describe disease determinants and to identify the so-called nuclear shuttle protein as the helper factor for aphid transmission. Using experimental reassortants between pea necrotic yellow dwarf virus and faba bean necrotic stunt virus genome components we demonstrated that the faba bean necrotic stunt virus helper factor protein is able to substitute that of pea necrotic yellow dwarf virus. Whereas nanoviruses are transmitted by aphids coconut foliar decay disease is transmitted by the plant hopper Myndus taffini. For decades however, the nature of a potential virus responsible for coconut foliar decay has been elusive. In an attempt to shed light on that still enigmatic virus we cloned and sequenced circular DNA molecules from particles associated with the disease. In addition, several deep sequencing experiments confirmed the presence of the identified DNAs in samples dating from 1989, 2013 and 2014. The sequences revealed features characteristic for both nanoviruses and geminiviruses. The nature of a potential coconut foliar decay virus will be discussed.

Cardamom bushy dwarf virus (genus Babuvirus) and aphid vector, Micromyzus kalimpongensis: Vector behaviour and dissemination of the virus

Bikash Mandal^* and Amalendu Ghosh

Email ID for Correspondence: leafcurl@rediffmail.com

Advanced Center for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi-110 012, India

Large cardamom (Amomum subulatum,family Zingiberaceae) is an economically important spice crop grown in the Northeast sub-Himalayan Mountains. Large cardamom production is seriously affected by foorkey disease, which results into excessive proliferation of small plantlets giving the bushy appearance at the base of the clump. The foorkey affected plants turn completely unproductive. Cardamom bushy dwarf virus (CBDV), a distinct species of the genus Babuvirus family Nanoviridae, was identified based on the complete genome sequence of the six major DNA components (DNA-R, -S, -M, -C, -N and -U3) and three additional components including one satellite Rep and two unknown components (DNA-U1 and -U2). Round the year surveys in the large cardamom plantation showed occurrence of three aphid species, Micromyzus kalimpongensis, Pentalonia nigronervosa and Aulacorthum solani. Transmission studies showed that only M. kalimpongensis was the vector of CBDV. M. kalimpongensis was observed to colonise in the underground plant parts and was prevalent throughout the year in the high altitude (>900 m) plantations. Under the natural plantation conditions significantly higher number of M. kalimpongensis was observed in CBDV-infected clumps compared to the healthy clumps. This observation was confirmed in a bioassay with the plant extracts, where significantly higher number of aphids migrated to the extract from the CBDV infected plant compared to the healthy plant. Aphids reared on CBDV infected plants showed altered biology having shortened nymphal period and increased longevity and fecundity compared to those grown on the healthy plants. In the small plot experiment, aviruliferous M. kalimpongensis migrated to the CBDV-infected plants, colonised and once the foorkey affected plants died, the viruliferous aphids migrated to healthy plants, which eventually became diseased. The results suggested that CBDV infection in large cardamom influenced the behaviour and biology of M. kalimpongensis, which favoured the dissemination of CBDV.

Studies on virus vector interaction, with reference to Bemisia tabaci and geminivirus

Raman Rajagopal^*, Vipin Singh Rana, Harpreet Singh Raina, Sonam Popli, Rohit Jamwal, Gunjan Kumar Saurav

Email ID for Correspondence: zoorajagopal@gmail.com

Department of Zoology, University of Delhi, New Delhi, India

Bemisia tabaci (Whitefly) is a worldwide agricultural pest with wide range of host plant preferences and is capable of transmitting Geminiviruses. B. tabaci is a species complex where in individuals though more morphologically similar are genetically distinct. This insect harbors two different types of bacterial endosymbionts the primary endosymbiont Portiera which alone is required for its survival since it provides essential non-dietary metabolites. The secondary endosymbionts are present in different combinations of Wolbachia, Rickettsia, Arsenophonus, Hamiltonella which provide resistance against parasitoids, thermal tolerance, virus transmission abilities etc. However, the detailed functional role of these endosymbionts is not known because of technical problems in culturing them in laboratory conditions. Gemniviruses are transmitted by the whitefly B. tabaci in a circulative and persistent manner. During feeding by B. tabaci, the geminivirus particle is ingested; enter the gut, thereafter the particles are transported to the salivary gland via haemolymph and are inoculated back into the plant during subsequent feeding. Endosymbionts of B. tabaci as well as other insect vector hosts have been shown to play a major role in virus transmission. The GroEL proteins of Portiera and Arsenophonus were purified and in vitro interaction studies were carried out using pull down and co-immunoprecipitationassays. In-vivo interaction was confirmed using yeast two hybrid system. In both in vitro and in vivo studies, the GroEL protein of Arsenophonus was found to be interacting with the CLCuV coat protein. These results suggest the involvement of Arsenophonus in the transmission of geminiviruses by B. tabaci. We then attempted to eliminate Arsenophonus from B. tabaci to study its effects with regard to development and other fitness parameters. Our results revealed that Arsenophonus negative (A−) whiteflies had more fecundity, increased juvenile developmental time, increased nymphal survival and increased adult life span as compared to control (A+) whiteflies. Thus, our results demonstrate that A+whiteflies have lesser fitness as compared to A−whiteflies. These observations give a new insight about the probable role of Arsenophonus in B. tabaci that need to be explored further.

Bemisia tabaci knottin-1 gene is implicated in regulating the quantity of Tomato yellow leaf curl virus associated with the insect

Hariton Shalev Aliza^a, SobolIris^a, Ghanim Murad^b, LiuShu-Sheng^c and Czosnek Henryk^a

Email ID for Correspondence: hanokh.czosnek@mail.huji.ac.il

^aThe Hebrew University of Jerusalem, Rehovot, Israel; ^bVolcani Center, Bet Dagan, Israel; ^cZhejiang University, Hangzhou, China

The whitefly Bemisia tabaci transmits begomoviruses such as Tomato yellow leaf curl virus (TYLCV) in a circular, persistent fashion. Examination of the B. tabaci transcriptome revealed that genes encoding knottin-like antimicrobial peptides were among thoseresponsive to various stresses. Comparing the whitefly transcriptome before and after ingestion of tomato begomoviruses showed that out of four knottin-like genes, two were upregulated, knot-1 (with the highest expression) and knot-3. We have applied RNAi-based gene silencing to examine the putative function of genes of the knottin family in relation to TYLCV circulative transmission. The knot-1 gene was silenced by feeding whiteflies with dsRNA via tomato leaves. Large amounts of knot-1 transcripts were present in the abdomen of whiteflies, an obligatory transit site of begomoviruses in their circulative transmission pathway; knot-1 silencing significantly depleted the abdomen from knot-1 transcripts. Knot-1 silencing led to an increase in the quantity of TYLCV ingested by the insects and transmitted to tomato test plants by several orders of magnitude. This effect was not observed following knot-3 silencing. Hence, knot-1 plays a role in restricting the quantity of virions an insect may acquire and transmit. Since TYLCV is associated with a decrease in the insect longevity and fertility and with the upregulation of stress-response genes, we have proposed that this virus is reminiscent of a whitefly pathogen. We suggest that knot-1 protects B. tabaci against TYLCV deleterious effects by limiting the amount of virions associated with the whitefly vector.

The whitefly Bemisia tabaci Cyclophilin B protein is implicated in Tomato yellow leaf curl virus transmission

Surapathrudu Kanakala^* and Murad Ghanim

Email ID for Correspondence: kanakalavit@gmail.com

Department of Entomology, Volcani Center, Bet Dagan 50250, Israel

Tomato yellow leaf curl virus (TYLCV) is an ssDNA begomoviruses that causes severe damage to tomato and several other crops worldwide. TYLCV is exclusively transmitted by the sweetpotato whitefly, Bemisia tabaci in a persistent circulative and propagative manner. Previous studies have shown the transmission, retention and circulation of TYLCV in its vector involves interaction with insect and endosymbiont proteins, which aid the transmission of the virus, or have a protective role in response to the presence of the virus in the insect body. However, only a handful number of proteins that have a role in TYLCV transmission by B. tabaci were identified. Several approaches are undertaken to search for such proteins including transcriptomics and proteomics, as well as functional validation of candidate proteins that are selected based on other pathogen-vector systems. Here, the role of B. tabaciCypB protein in the transmission of TYLCV was investigated. Cyclophilins (Cyps) are a large family of cellular prolyl isomerases that have many molecular roles including facilitating protein–protein interactions in the cell. We found that the expression of CypB is misregulated upon TYLCV acquisition and retention. Further experiments that employed immunocapture-PCR and co-immunolocalization showed specific interaction and co-localization between CypB and TYLCV in the midgut, eggs and salivary glands. Membrane feeding of anti-CypB antibodies and TYLCV virions showed a decrease in TYLCV transmission, suggesting a critical role that CypB plays in TYLCV transmission. Further experiments, which employed membrane feeding with the CypB inhibitor CyclosporinA (CsA) showed decrease in CypB-TYLCV co-localization in the midgut and virus transmission. Plant mediated RNAi was used to silence the midgut-expressed CypB in whiteflies by employing transgenic Solanum lycopersicum plants producing CypB dsRNA. The results showed that continuous ingestion of dsRNA triggered efficient silencing of the gene. Altogether, our results indicate that CypB plays an important role in TYLCV transmission by B. tabaci.

Identification and characterization of a receptor midgut protein for begomovirus in Bemisia tabaci

Vipin Singh Rana^1*, Sonam Popli², Gunjan Kumar Saurav², Harpreet Singh Raina², Rahul Chaubey³, V. V. Ramamurthy^c,R. Rajagopal²

Email ID for Correspondence: vipinsinghrana@gmail

¹Infectious Disease Immunology Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, New Delhi-110007, India; ²Gut biology laboratory, Department of Zoology, University of Delhi, Delhi 110007, India; ³Entomology division, Indian Agricultural Research institute, New Delhi-110012, India

Begomoviruses are the largest group of plant viruses, transmitted exclusively by Bemisia tabaci (Gennadius). B. tabaci contain several genetic groups which are morphologically identical and genetically differentiated. The information regarding molecular and cellular basis underlying begomovirus—whitefly interaction is very limited. Evidences from several studies have suggested that the B. tabaci gut possesses some crucial protein receptors that allow specific entry of virus into the insect haemolymph. We have performed yeast two hybrid (Y2H) gut cDNA expression library screening against coat protein of Tomato leaf curl New Delhi virus (ToLCV) and Cotton leaf curl Rajasthan virus (CLCuV) as bait. As a result of library screening, several candidate receptor proteins were identified. Midgut Protein (MGP) was the common protein found interacting with both ToLCV and CLCuV. Bioinformatical analysis revealed the absence of 5′ region of mgp gene in Y2H positive clone. Using 5′ RACE, complete sequence of mgp gene was retrieved. The full length mgp gene was over-expressed and purified in E. coli (BL-21). Purified MGP fractions were used for anti-MGP antibody generation and interaction studies. MGP was localized in whole mount B. tabaci as well as in dissected guts through confocal microscopy using fluorescent labelled LNA probe and antibody. Further, pull down and dot blot assays confirmed in vitro interaction between ToLCV/CLCuV coat protein and MGP. Immunolocalization analysis also showed co-localization of ToLCV/CLCuV particles and MGP within insect’s gut. In addition to this, immunoelectron microscopic analysis has also revealed the presence of MGP in ultrathin section (70 nm) of B. tabaci gut membrane. Finally, anti-MGP antibody fed B. tabaci, exhibited approximately 80 and 70% reduction in virus acquisition and ToLCV transmission respectively, suggesting a supportive role for MGP in virus transmission. These results will help in furthering the virus-vector interaction knowledge which could serve for future vector control strategies.

The roles of Arabidopsis RNA polymerases IV and V in defense against geminiviruses

Jessica M. Storer, Jamie N. Jackel, Jeffery B. Ostler, Tami Coursey, and David M. Bisaro^*

Email ID for Correspondence: bisaro.1@osu.edu

Department of Molecular Genetics, Center for Applied Plant Sciences, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210 USA

Epigenetic modifications play critical roles in chromosome organization and regulation of gene expression. An example of epigenetic control is repressive cytosine and histone methylation (e.g. dimethyl histone 3 lysine 9, H3K9me2) that leads to transcriptional gene silencing (TGS) of invasive DNAs, including transposable elements and DNA viruses. Geminiviruses have circular ssDNA genomes that replicate via dsDNA intermediates that associate with histones to form minichromosomes. As a host defense, minichromosomes can be repressed by epigenetic modification. Arabidopsis encodes two plant-specific RNA polymerases, Pol IV and Pol V, which are related to Pol II and known to carry out non-coding (nc) transcription resulting in cytosine methylation. We are using geminiviruses as models to define the roles of these polymerases in chromatin methylation. To date, we’ve found that Pol IV and V are not required to initiate viral DNA methylation, but are necessary for hypermethylation of viral promoters that accompanies host recovery from infection. Thus another polymerase, likely Pol II, can perform nc-transcription leading to initiation of cytosine methylation. However, Pol IV and V are individually required for establishment of H3K9me2 marks on viral chromatin. Interestingly, plants carrying a hypomorphic pol II mutation also are unable to deposit H3K9me2 on viral chromatin, suggesting that Pol II directly or indirectly recruits Pol IV and/or Pol V. In addition, we are using circular RT-PCR to characterize non-coding, intergenic region (IR) transcripts. A number of long nc-RNAs have been characterized to date, and all lack a poly-A tail and a 5′ cap. Transcripts are templated by both strands of the viral genome and lack conserved initiation sites, although the majority initiate with adenine. IR transcript levels are reduced in pol IV and pol V mutants, and work is in progress to identify transcripts produced by each polymerase. (Support: NSF-MCB-0743261 and NSF-MCB-1158262 to DMB).

Variation in host susceptibility to infection by Wheat dwarf virus

Jim Nygren¹, Elham Yazdkhasti¹, Nadeem Shad¹, Richard Hopkins², Anders Kvarnheden^1*, Anna Westerbergh¹

Email ID for Correspondence: anders.kvarnheden@slu.se

¹Department of Plant Biology, Uppsala BioCenter, Linnean Center of Plant Biology in Uppsala, Swedish University of Agricultural Sciences, Box 7080, SE-750 07 Uppsala, Sweden; ²Natural Resources Institute, University of Greenwich, Kent ME4 4TB, United Kingdom

Wheat dwarf virus (WDV; genus Mastrevirus; family Geminiviridae) is transmitted persistently by leafhoppers of the genus.

Psammotettix and it has a wide host range of grasses within the family Poaceae. The cultivated hexaploid bread wheat (Triticum aestivum spp. aestivum) is very susceptible to infection by WDV and infection early during the season may result in strong symptoms, such as dwarfing, and extensive crop losses. In contrast, cultivated forage grasses or wild grasses infected by WDV often do not develop symptoms after WDV inoculation. To study the variation in response to WDV infection, we have carried out controlled transmission tests to plants of domesticated wheat and its wild relatives as well as to ryegrass (Lolium spp.) and monitored their response and virus titres. Using leafhoppers, plants of four ryegrass cultivars (L. perenne and L. multiflorum) were inoculated with WDV. None of the plants developed any symptoms and they tested negative by ELISA. However, using qPCR it was possible to detect low WDV levels in the plants and leafhoppers could acquire virus for transmission to wheat. For wheat, plants of all 18 tested taxa of the genera Triticum, Aegilops and Amblyopyrum were susceptible to infection with virus being detectable by ELISA, but their responses varied. Three plant response patterns were found: (i) continuous reduction in growth over time, (ii) weak response at an early stage of plant development but a much stronger response at a later stage, and (iii) remission of symptoms over time. The results suggest that the observed differences are the consequence of shifting selection pressure rather than being effects of polyploidization or domestication. The donor of the D genome in wheat, Ae. tauschii, showed remission of symptoms and was identified as a potential genetic resource for improvement of resistance to WDV in bread wheat.

Deciphering the global landscape of molecular geminivirus-plant interactions

Tábata Rosas-Díaz^*, Xue Ding, Liping Wang, Tamara Jiménez-Góngora, Rosa Lozano-Durán

Email ID for Correspondence: tabatarosas@sibs.ac.cn

Shanghai Center for Plant Stress Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, 3888 Chenhua Rd, Shanghai 201602, China

Geminiviruses have reduced genomes, which imposes limitations in coding capacity. Remarkably, geminiviruses have compensated these restrictions by evolving overlapping open reading frames (ORFs) encoding four to eight multifunctional proteins able to effectively manipulate plant functions. Relying on these four to eight proteins, geminiviruses cause devastating diseases in staple and fiber crops worldwide, and currently pose a serious threat to food security. Using Tomato yellow leaf curl virus (TYLCV) as a model, our work aims at deciphering the multi-level interaction between virus and host. For this purpose, we are using complementary approaches to (i) define the TYLCV-host interactome, (ii) identify viral targets with a central role in the maintenance of cellular homeostasis, (iii) characterize these viral targets and their role in the presence or absence of the virus, and (iv) determine which cell functions are specifically modulated by the viral infection, either in the infected cells specifically or systemically. Through affinity purification of in planta expressed proteins followed by mass spectrometry analyses, we have systematically determined the interactome of all 6 proteins encoded by TYLCV with host proteins in two different plant species, Nicotiana benthamiana and Arabidopsis, uncovering a set of proteins targeted by the virus in both hosts. The dataset generated in this work offers an unprecedented global virus of the virus-host interface, facilitating a deeper understanding of the cellular re-shaping upon the viral infection.

A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana

Xueping Zhou^1,2

Email ID for Correspondence: zzhou@zju.edu.cn

¹State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China; ²State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310058, China

Calmodulin-like protein is an endogenous RNA silencing suppressor that suppresses sense-RNA induced post-transcriptional gene silencing (S-PTGS) and enhances virus infection, but the mechanism underlying calmodulin-like protein-mediated S-PTGS suppression is obscure. Here, we show that Nicotiana benthamiana calmodulin-like protein (NbCaM) interacts with Suppressor of Gene Silencing 3(NbSGS3). NbSGS3 is required for green fluorescent protein-triggered S-PTGS, geminivirus-induced gene silencing and defense against geminivirus infection. Deletion analyses showed that domains essential for the interaction between NbSGS3 and NbCaM are also required for the subcellular localization of NbSGS3 and the NbCaM suppressor activity. Overexpression of NbCaM reduced the number of NbSGS3 granules by degrading NbSGS3 protein accumulation in the cytoplasm. This NbCaM-mediated NbSGS3 degradation was sensitive to the autophagy inhibitor 3-methyladenine, and was compromised when key autophagy genes of the phosphatidylinositol 3-kinase (PI3K) complex were knocked down. Meanwhile, silencing of the key autophagy genes of the PI3K complex inhibited geminivirus infection. Taken together these data suggest that NbCaM acts as a suppressor of RNA silencing by degrading NbSGS3 through the autophagy pathway.

Mapping and identification of geminivirus genome sequences for efficient RNAi expression

Basavaprabhu L. Patil^1,2*, Basavaraj Bagewadi^2,3, Jitender Yadav^2,4 and Claude M. Fauquet^2,5

Email ID for Correspondence: blpatil2046@gmail.com

¹ICAR-National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi 110012, India; ²Donald Danforth Plant Science Center, St. Louis, MO 63132, USA; ³Current Address: Washington State University, Prosser, WA, USA; ⁴Current Address: BASF Plant Science, Research Triangle Park, North Carolina, USA; ⁵Current Address: Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia

Geminiviruses are among the most serious pathogens of many economically important crop plants and RNA interference (RNAi) is an important strategy for their control. Geminiviruses are known to infect large number of Legumes, vegetables and root & tuber crops. RNAi or PTGS (Post-Transcriptional Gene Silencing) is a sequence-specific gene inactivation mechanism. RNAi-mediated processes regulate the growth and development and protect the genome of eukaryotes from invading nucleic acids such as viruses. Central to these processes are the siRNAs generated from dsRNA substrates by the action of RNase III type endonuleases. Although any fragment of a viral genome can be used to generate a dsRNA trigger, the exact sequence and size requirements for efficient gene silencing and virus resistance have so far not been investigated in detail. Previous efforts to control geminiviruses by gene silencing mostly targeted AC1, the gene encoding replication-associated protein (Rep). In this study we made RNAi constructs for all the genes of both the genomic components (DNA-A and DNA-B) of a cassava mosaic virus (ACMV), one of the most devastating geminiviruses causing cassava mosaic disease (CMD). Using transient agro-infiltration studies, RNAi constructs were evaluated for their ability to trigger gene silencing against the invading virus, thus conferring protection against it. The results show that the selection of the DNA target is an important determinant for the amount of siRNA produced and the extent of resistance. The ACMV genes AC1, AC2, AC4 and BC1 were effective targets for RNAi-mediated resistance and their siRNA expression was higher compared to other RNAi constructs. We further extended this study to East African cassava mosaic virus (EACMV), by generating four RNAi constructs targeting AC1, AC2, AC4 and BC1. Our studies showed that the RNAi constructs targeting AC2 of both ACMV and EACMV-Ug gave highest level of resistance in the transient studies.

Geminiviral genome methylation: influenced by R genes and mitigated by viral AC4

Varsha Garg¹,Vipin Permar², Elangovan Mani², Peter Palukaitis³ and Shelly Praveen^1*

Email ID for Correspondence: shellypraveen@hotmail.com

¹Advanced Center for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi-110012, India; ²Advanta Ltd., Krishnama House, Road No. 7, Banjara Hills, Hyderabad-500034, India; ³Department of Horticultural Science, Seoul Women’s University, Seoul, 139-774, South Korea

Cytosine methylation is considered one of the epigenetic antiviral defenses. Influence of host factors having role in resistance, contribute towards activating methylation machinery towards viral genomes and viral factors tries to mitigate this effect. The efficiency by which these two processes operate, determines resistance versus susceptibility. The tomato leaf curl virus genome is methylated during infection in tomato. Here we show that cytosine methylation levels in viral genome get affected in presence of host resistant loci Ty-1/2/3 and viral protein AC4 in two different experiments. Among the three resistant loci, tomato genotypes harboring Ty-2 loci showed highest level of viral genome methylation. Two separate transgenic experiments; (1) Transgenic expression of viral protein AC4 and (2) Transgenic tomato producing viral derived siRNAs, suggest that viral protein AC4 and viral derived siRNAs influence viral genome methylation in opposite manner. The possible role of Ty-genes, AC4 protein and siRNAs in regulating de novo methylation is discussed.

A quantitative systems approach to understand differences in geminivirus-induced changes in Arabidopsis thaliana

Laura E. Herring¹, Sophia Yang¹, Inna Kulikova-Donovan¹, Elliott Wyatt ¹, Mariana Franco-Ruiz², Cyprian Rajabu³, Caroline Bryan¹, Elise Braswell¹, Lisa Rightmyer¹, Kevin Blackburn ¹, Michael B. Goshe¹ and Jose Trinidad Ascencio-Ibáñez^1*

Email ID for Correspondence: jtascenc@ncsu.edu

Departments of ¹Molecular and Structural Biochemistry and ²Horticultural Science, North Carolina State University, Raleigh, North Carolina 27695; ³Mikocheni Agricultural Research Institute, Dar es Salaam, Tanzania.

Previous microarray analysis of the Arabidopsis thaliana transcriptome in response to the Cabbage leaf curl virus showed an induction of genes involved in natural senescence. Cabbage leaf curl virus and Beet curly top virus infections cause severe symptoms in Arabidopsis. However, plants infected with these viruses differ in symptom timing and development. We used tubulin::GFP fusions to study how tubulin is affected during geminiviral infections. Furthermore, gene expression for all tubulin genes was assessed by quantitative RT-PCR in both infections with a concomitant comparison of the behavior of several proteins involved in senescence. Results showed a down-regulation of tubulin genes in Cabbage leaf curl infection while an up-regulation during Beet curly top infection. Also, different expression was found with other senescence markers, indicating that both geminiviruses impinge on different pathways during infection. Proteomics data revealed that many pathways are altered during infection with geminiviruses and that senescence is induced by Cabbage leaf curl virus but not by Beet curly top virus.

Distinct localization-specific virulence functions of C4 from Tomato yellow leaf curl virus

Tábata Rosas-Díaz¹, Liping Wang¹, Xue Ding¹, Tamara Jiménez-Góngora¹,Yuanyuan Zhang¹, Renyi Liu¹, Tan Li¹, Shingo Nagawa¹, Christine Faulkner², Eduardo R. Bejarano³ and Rosa Lozano-Durán^1*

Email ID for Correspondence: lozano-duran@sibs.ac.cn

¹Shanghai Center for Plant Stress Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China; ²John Innes Centre, Norwich, United Kingdom; ³Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora, University of Málaga – Consejo Superior de Investigaciones Científicas (CSIC) (IHSM - UMA – CSIC)/Department of Cell Biology, Genetics and Physiology, University of Málaga, Málaga, Spain

Geminiviruses have very limited coding capacity; in order to infect the host plant, geminiviruses rely on multifunctional proteins that interact with and manipulate the plant cell molecular machinery in order to create a cellular environment favorable to the infection. C4 is the smallest protein encoded by geminiviruses. In Tomato yellow leaf curl virus, C4 is essential for infectivity and has been suggested to act as movement protein; however, the exact molecular function of this viral protein remains unclear. In this work, we show that expression of C4 in arabidopsis plants triggers strong developmental abnormalities that resemble the symptoms of the viral infection. Surprisingly, C4 localizes to two distinct subcellular compartments, the plasma membrane (PM) and chloroplasts, and this localization is determined by the presence of two targeting signals in the protein sequence. Interestingly, the concomitant presence of these two signals is conserved in C4 from geminiviruses, and occurs in effector proteins of other plant pathogens and in a subset of plant proteins, many of which are known to function in defense responses. Using a combination of mutagenesis, in planta expression and affinity purification followed by mass spectrometry analysis, and functional studies, we have shown that C4 exerts distinct virulence function activities in each of these two specific localizations. The role of C4 at the plasma membrane is essential to cause developmental abnormalities in the plant. In this location, C4 interacts with a family of receptor-like kinases (RLKs) that regulates development, which may underlie the observed phenotypes. Indeed, the developmental phenotype of the C4-expressing plants is consistent with an inhibition of these RLKs. Our results suggest that the interaction of C4 with the kinase domain of these RLKs may displace more than 40% of their interactors in the plant cell, therefore providing a potential mechanism for the C4-mediated inhibition of the RLK function. Our work uncovers novel virulence functions for C4, an essential protein in geminiviruses, and unveils subcellular compartmentalization as a strategy to allow multifunctionality of proteins.

Tomato 26S proteasome subunit RPT4a regulates ToLCNDV transcription and activates hypersensitive response in tomato

Pranav Pankaj Sahu^1,2, Namisha Sharma¹, Swati Puranik¹, Supriya Chakraborty² and Manoj Prasad^1*

Email ID for Correspondence: manoj_prasad@nipgr.ac.in

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067, India; ²School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India

The role of 26S proteasomal subunits in plant pathogen-interactions has been remarkably underlined in past few years. The 26S proteasome have diverse functions in plants defense such as elicitation of defense responses, activation of defense proteasome and programmed cell death. In this regard, we aimed to functionally characterize a Solanum lycopersicum26S proteasomal subunit RPT4a (SlRPT4) gene, which was differentially expressed after Tomato leaf curl New Delhi virus (ToLCNDV) infection in the tolerant cultivar H-88-78-1. Molecular analysis revealed that SlRPT4 protein has an active ATPase activity and binding affinity towards ToLCNDV specific intergenic region (IR). Moreover, binding of SlRPT4 to IR inhibited the binding of RNA Pol-II, which subsequently reduced the transcription of ToLCNDV genome. Silencing of SlRPT4 gene revealed conversion of tolerant attributes of cultivar H-88-78-1 into susceptibility. Transient overexpression of SlRPT4 in tomato revealed the characteristic features of hypersensitive response (HR). SlRPT4 mediated programmed cell death involved action of caspase 3/9-like activity and action of antioxidant enzymes. Overall, present study highlights that SlRPT4 alters ToLCNDV transcription and activates programmed cell death mediated defense pathway. Overall, these results suggest that SlRPT4 is required for defense response against virus infection in tomato.

Analysis of African cassava mosaic virus Rep and AC4 proteins in fission yeast as a model organism

Katharina Hipp^1*, Peter Rau¹, Bruno Gronenborn², Jens Pfannstiel³, Holger Jeske¹

Email ID for Correspondence: katharina.hipp@bio.uni-stuttgart.de

¹University of Stuttgart, Institute of Biomaterials and biomolecular Systems, Department of Molecular Biology and Plant Virology, Pfaffenwaldring 57, 70569 Stuttgart, Germany; ²Institut des Sciences du Végétal, CNRS, 91198 Gif-sur-Yvette, France; ³University of Hohenheim, Mass Spectrometry Core Facility, August-von-Hartmann-Straße 3, 70599 Stuttgart, Germany

Fission yeast is a well-studied model organism with molecular mechanisms more closely related to higher plants than those of baker’s yeast or bacteria. The Rep protein of geminiviruses is essential for virus replication and induces host DNA synthesis in differentiated plant cells by interaction with the plant homologues of retinoblastoma proteins. The African cassava mosaic virus Rep protein was able to induce a cell division cycle phenotype in fission yeast accompanied with enlarged and less compact nuclei in comparison to control cells, although fission yeast lacks a retinoblastoma homologue. The DNA content of Rep expressing cells was increased beyond 2C, indicating ongoing replication without intervening mitosis. This effect was dependent on an intact, potential cyclin binding motif in the Rep sequence and may point to an alternative cell cycle control mechanism. The motif was essential for viral infection in plants, since its mutation in infectious clones prevented any infection. The AC4 protein is encoded within the open reading frame of Rep and has been implicated in intracellular transport and silencing for different geminiviruses. In African cassava mosaic virus, two in-frame start codons may be translated into a longer and a shorter variant of AC4. Both variants were expressed as fusion proteins in fission yeast. The longer variant formed distinct spots in the cytoplasm, whereas the shorter variant localized to the plasma membrane. The yeast-expressed shorter variant was myristoylated, a modification that has been detected biochemically for the first time, which may promote the plasma membrane targeting. Although both variants were translated in fission yeast, only the shorter variant is necessary for viral infection in plants.

The V2 protein encoded by a monopartite begomovirus is a suppressor of both post-transcriptional and transcriptional gene silencing

Muhammad Mubin^1*, Shah Nawaz ul Rehman¹, Rob W Briddon² and Shahid Mansoor²

Email ID for Correspondence: mmubin@uaf.edu.pk

¹Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture and Faisalabad, 38000, Pakistan; ²Plant Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), P O Box 577, Jhang Road, Faisalabad, Pakistan

Geminiviruses are small circular DNA viruses that occur as minichromosomes in the nucleus and are subjected to post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). In this report, using a green fluorescent protein-based transient suppression assay, V2 protein of Papaya leaf curl virus (PaLCuV), a monopartite begomovirus from the Old World, was shown to have suppressor of PTGS as well as TGS activity. The expression of V2 from potato virus X (PVX) vector in transcriptionally silenced transgenic 16cplants suppressed transcriptional gene silencing (TGS). A bimolecular fluorescence complementation assay was used to assess its interaction with metabolic regulators linked with host susceptibility to viral infection. The V2 interacted with itself while no interaction was found with adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle. We hypothesized that interaction of V2 with SGS3 may block gene silencing by preventing production of secondary siRNA that are required both for PTGS and TGS. Assays for suppressor of AV2 of Tomato leaf curl New Delhi virus (ToLCNDV) showed that the protein lack suppressor of either post transcriptional or transcriptional RNA silencing activity. The AV2 from ToLCNDV or V2 from PaLCuV were found to localize both in the nucleus and cell periphery and show that the mutation in the AV2 had no effect on cellular localization of the protein. Our data shows that the precoat protein of monopartite PaLCuV is a suppressor of RNA silencing, both at transcriptional and post-transcriptional levels. We conclude that PaLCuV can suppress both PTGS and TGS when it infects plant and V2 protein provides the virus with this function. This might also be concluded that ToLCNDV use some other protein to suppress RNA silencing activity.

A monopartite geminivirus encoded V2 protein exploits cellular pathways to traffic via endomembrane system

Nirbhay Kumar Kushwaha^*, Mansi and Supriya Chakraborty

Email ID for Correspondence: nirbhay002@gmail.com

Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi – 110067, India

Chilli leaf curl virus (ChiLCV) is a monopartite begomovirus (Geminivirideae) and infects chilli at several locations in India. Pre-coat protein (V2) of geminiviruses is a late gene product of approximately 13 kDa that plays a crucial role during establishment of successful infection and is known to be involved in viral transport, suppression of host defence. The pathway and mechanism of V2 trafficking through sub-cellular organelles are largely unknown. We performed a series of experiments to track ChiLCV V2 protein at cellular and subcellular level using Nicotiana benthamiana leaf epidermal cells through confocal microscopy. V2 protein was observed in the endosomes and found to be localized on the plasma membrane, endoplasmic reticulum (ER), golgi bodies (GB) and in the nucleus. Our results also demonstrated endosome mediated V2 trafficking between subcellular organelles and plasma membrane. We developed V2_1–49 deletion mutant and performed the localization assays that showed association of V2_1–49 with the plasma membrane, ER and Golgi bodies and nuclear membrane but absent in the nucleus. Our results also confirmed accumulation of the V2_1–49 proteinin multi vesicular structures and cytoplasmic aggregates that are produced through ER and GB system. We also report co-localization of V2 protein with plasmodesmata localization protein 1 (PDLP1) on the plasmodesmata that was further confirmed with callose assay. The current study provides a series of evidences that suggested pre-coat protein of ChiLCV exploits host endocytic and ER-GB pathways and transported to the plasmodesmata. Ability of the V2 protein to form a movement competent complex with host factor will be discussed.

Rep interfere with the soumoylation and ubiquitination of Proliferating cellular nuclear antigen (PCNA)

Manuel Arroyo-Mateos, Blanca Sabarit, Miguel A. Sánchez-Durán, Javier Ruiz-Albertand Eduardo R. Bejarano¹

Email ID for Correspondence: edu_rodri@uma.es

Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Universidad de Málaga, Campus Teatinos, 29071 Málaga, Spain

Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells using plant DNA polymerases. These viruses encode a protein designated as Rep that is essential for viral replication. Rep interacts with several host proteins including the host DNA replication protein PCNA (Proliferating cellular nuclear antigen), and the sumoylation enzyme that conjugates SUMO to target proteins (SUMO conjugating enzyme SCE1). Previously we have showed that ectopic Rep expression did not result in broad changes in the sumoylation pattern of plant cells, but specific changes were detected indicating that Rep modifies the sumoylation state of selected host proteins (Sanchez Durán et al. 2011). Using a reconstituted SUMOylation system in Escherichia coliwe confirm that the level of SUMO binding to PCNA is reduced in the presence of Rep but not when other geminiviral protein as Trap is expressed. Rep-mediated decrease of PCNA sumoylation does not require interaction with SCE1. However, SUMO binding to the CAT3, a plant protein that does not interact with Rep, it is not altered in the presence of the viral protein. Site directed-mutagenesis permit us to identify two lysine residues (lys 164 and lys 254 of plant PCNA that are sumoylated and confirm that Rep expression reduce the SUMO binding to both. Expression of truncated versions of Rep determine that the motif responsible for the interference of Rep PCNA-sumoilation overlaps with the PCNA binding motif (Bagewadi et al, 2004). Reduction in PCNA ubiquitination by Rep expression is also observed. Those results suggest that Rep is interfering with the SUMO and ubiquitin modification of PCNA lysine residues, post-translational modifications that play a key role in DNA recombination and repair.

Novel insight in the interactions between REP, PCNA and SCE1, key factors in Geminivirus replication

Francesca Maio^1*, Harrold A. van den Burg¹, Marcel Prins^1,2

Email ID for Correspondence: f.maio@uva.nl

¹Molecular Plant Pathology, Swammerdam Institute for Life Science, University of Amsterdam, Science Park 904 | 1098 XH Amsterdam, The Netherlands; ²KeyGene N.V. | P.O. Box 216 | 6700 AE Wageningen | The Netherlands

Whitefly-transmitted geminiviruses are one of the most serious threats to the cultivation of many crops worldwide. Natural sources of resistance are limited and, moreover, are being broken by novel (strains of) viruses. Therefore, novel ways to discover or generate sources of resistance against geminiviruses are required. Our strategy is to explore resistance that is based on altering plant susceptibility genes. The most potent susceptibility genes for geminivirus infection are those genes that encode plant proteins that are recruited by the viral replication initiator protein REP and are essential for viral DNA replication. Previous studies have already identified several host proteins that associate with the Rep protein. Among these are PCNA, the processivity factor for host DNA polymerase-δ, and the SUMO E2-conjugating enzyme SCE1. Our first aim is to identify additional plant proteins that associate with the viral replisome complex. We developed a new proteomic approach based on the co-expression of Tomato Yellow Leaf Curl virus Rep and tomato PCNA labeled to two different tags in tomato protoplasts, followed by pull-down assays and mass spectrometry to identify the co-precipitated proteins. Second, since the SCE1 protein is involved in regulation of some components of the viral replisome, we are interested in mapping the Rep-interaction surface on SCE1. We generated a series of SCE1 mutants that bear mutations in different sites of the protein and we screened the association of these mutants with Rep in yeast two-hybrid assays. Loss-of-interaction SCE1 mutants are confirmed by in planta protein–protein interaction complementation methods.

New insights into roles of the replication initiator protein in geminivirus pathogenesis

Supriya Chakraborty

Email ID for Correspondence: supriyachakrasls@yahoo.com

Molecular Virology Laboratory, School of life Sciences, Jawaharlal Nehru University, New Delhi-110067, India

Geminiviruses have evolved a structurally simple yet elegant potential to interact with a wide range of plant proteins and manipulate cellular processes to create an ambience for the benefit of the virus. Of the proteins encoded by geminivirus genome, the replication initiator protein (Rep, encoded by ORF AC1) is highly conserved both structurally and functionally across the Geminiviridae family and is indispensible for replication of the viral genome. However, the function of Rep during the post initiation stages is unknown. We have observed that Rep-ssDNA interaction depends strictly upon ATP binding. Results indicate that conserved residues in Rep that form a loop/β-hairpin structure interact with ssDNA while residues in the B′ motif located on the surface of the protein are involved in coupling ATP induced conformational change to DNA binding. Taken together, the role of B′ motif and β hairpin loop in ATP dependent ssDNA binding and DNA unwinding in a plant-infecting ssDNA virus was characterized. Further, we have identified contrasting roles of two paralogs of SlRPA32 in geminivirus replication. A hallmark of cells infected by DNA viruses is the synthesis of viral mRNA that must precede production of proteins. For efficient and orderly transcription of DNA viruses, the obligatory recruitment of machineries responsible for histone post translational modifications (PTMs) on viral promoter is essential. Although, multitasking roles of Rep in replication have been elucidated, information about its role in the activation of viral gene expression is lacking. Our results indicate that Rep influences the epigenetic control of geminivirus by modulating the recruitment of chromatin-modifying enzymes onto the geminiviral minichromosome. Optimal activation of viral transcription may favour synthesis of viral proteins that help to establish pathogenesis of the virus in a permissive host, and such a mechanism undoubtedly acts as an adaptive strategy employed by plant DNA viruses.

Characterization of an NSP-Interacting immune hub against begomoviruses

Iara P Calil, Joao Paulo B Machado, Anésia A. Santos, Elizabeth P B Fontes^*

Email ID for Correspondence: bbfontes@ufv.br

National Institute of Plant-Pest Interactions, Bioagro, Universidade Federal de Viçosa, Viçosa, MG, Brazil

The geminivirus nuclear shuttle protein (NSP) facilitates the intracellular transport of viral DNA from the nucleus to the cytoplasm and acts along with the movement protein (MP) to translocate the viral DNA to adjacent cells. However, the mechanism by which NSP mediates the nucleocytoplasmic movement of the viral DNA is unknown. A GTPase, designated NIG (NSP-interacting GTPase), which displays biochemical and structural properties consistent with a role in the nucleocytoplasmic transport of viral DNA, has been previously identified. NIG may act as a cellular cofactor for NSP function. To assess the potential role of NIG in general cellular nucleocytoplasmic transport of protein complexes, we performed yeast two-hybrid screenings with a Pro-rich domain of NIG as bait and we succeeded in isolating the NIG partners CSN5A and At2G41020, here designated as WWP1. We also showed that both CSN5A and WWP1 associate with NIK1 in vivo. CSN5A (COP9 Signalosome 5A) is one of the components of COP9 signalosome complex (CSN), which assembles in the nucleus to participate in proteasome-mediated degradation of nuclear proteins and acts as a key regulator in several basic cellular processes. CSN5A has also been shown to be a well-defined hub for interactions of the plant immune system and several effectors from divergent pathogens, including the C2 protein from geminiviruses. CSN5A resides in the cytoplasm as monomer and in the nucleus as part of the COP9 signalosome. NIG localizes in the cytoplasm and is associated with the nuclear envelope, but can shuttle between cytoplasm and nucleus because inhibition of nuclear export causes its nuclear accumulation. Binding of CSN5A and NIG occurs in the cytoplasm, which may not promote NIIG degradation. Nevertheless, the defense hormone salicylic acid promotes the translocation of NIG to the nucleus and its degradation via proteasome. Because the levels of NIG influence begomovirus infection, we propose that SA-induced degradation of NIG occurs in the nucleus via COP9 proteasome as part of plant defense against begomoviruses. With respect to the second NIG partner, WWP1 harbors two WW domains and a conserved C-terminal region with plant and animal homologous, such as mammalian PQBP1/NPW38. We showed that WWP1 is a nuclear protein capable of forming nuclear bodies via the WW domain and also interacts with CSN5A. Coimmunoprecipitation and BiFC assays demonstrated that AtWWP1 interacts in vivo with the cytoplasmic protein NIG, redirecting it to nuclear bodies. Colocalization assays demonstrated that nuclear bodies contained in AtWWP1 are distinct from those formed by proteins involved in RNA splicing, but colocalize with nuclear bodies containing CDKC2. We also found that WWP1-derived nuclear bodies do not colocalize with heterochromatin. Furthermore, AtWWP1 does not bind to RNA, but exhibits a binding activity to DNA and also binds to the C-terminus of RNA pol II. These characteristics imply that AtWWP1-derived nuclear bodies may be involved with transcription. As a component of a functional hub in geminivirus-host interaction network, WWP1 impairs begomovirus infection in a nuclear body formation-dependent manner. Collectively, our results identified a network of host interactions, such as NIK1, NIG and the interacting proteins WWP1 and CSN5A, which converges on NSP and forms an immune hub against begomoviruses.

Interaction between the geminivirus multifunctional AL2 protein and autophagy

Garry Sunter^1*, Maria Lockwood¹ and Jianhua Ruan²

Email ID for Correspondence: garry.sunter@utsa.edu

¹Department of Biology, University of Texas at San Antonio; ²Department of Computer Science, University of Texas at San Antonio

Resistance to infection depends on interactions between pathogen and host, and one initial response involves recognition of microbial or pathogen-associated molecular patterns (MAMPS/PAMPs) by pattern recognition receptors. This generally stimulates non-specific responses to molecules common to both pathogenic and non-pathogenic microbes, and results in PAMP-triggered immunity (PTI). For plant virus infections it has been proposed that RNA silencing is part of the PTI response, and as counter-defense viruses encode suppressors of silencing (VSRs). Geminiviruses encode small multifunctional proteins (AC2/C2), which act to counter plant immune responses including RNA silencing, and inactivation of SNF1-related protein kinase (SnRK1), and adenosine kinase (ADK). Inactivation of SnRK1 and ADK leads to enhanced susceptibility to infection and suppression of gene silencing respectively. AC2/C2-mediated inactivation of SnRK1 causes differential expression of autophagy-related genes, which can directly target intracellular pathogens, including viruses. A comparison of the Arabidopsis transcriptome when Spinach curly top virus C2 or antisense (as)SnRK1 was over-expressed revealed a number of genes differentially regulated by both treatments. This is interpreted to represent a response to SnRK1 inhibition. Using genes that were differentially regulated in response to both C2 and asSnRK1a novel network-based method for identifying gene functional modules was applied. A large complex network was identified, containing a smaller sub-network consisting of up-regulated genes with functions associated with autophagy and senescence. We measured significant increases in expression of genes within this sub-network using qPCR, which is interpreted to be a consequence of the inactivation/inhibition of SnRK1. A link between geminivirus-encoded VSRs and autophagy through the conserved SnRK1 pathway has the potential to provide information on geminivirus pathogenesis.

Minichromosomes in geminivirus infected plants: the case of PepGMV

Rivera-Bustamante Rafael Francisco^*, Ceniceros-Ojeda Adriana, Trejo-Saavedra Diana, Rodríguez-Negrete, Edgar

Email ID for Correspondence: rrivera@ira.cinvestav.mx

Department of Genetic Engineering, Centro de Investigación y de Estudios Avanzados del IPN, Cinvestav-Irapuato, Irapuato, Guanajuato, México

Geminiviruses are important plant pathogens consisting of circular, ssDNA genomes packed into icosahedral twined-shaped particles. However, in the nuclei of infected cells, viral dsDNA has been reported to associate with host histones to form minichromosomes. Geminiviruses are both inducers and targets of the host PTGS and TGS machinery. Studies on virus minichromosome structure and genome modifications have been hindered by the low concentration of these complexes due to the low number of infected cells especially in phloem-limited viruses. We have previously reported and characterized a “recovery” phenomenon observed in pepper plants infected with Pepper golden mosaic virus (PepGMV) that is associated with a reduction of viral DNA and RNA levels, the presence of virus-related siRNAs, and an increase in the levels of viral DNA methylation. Using our pepper-PepGMV system, we developed a methodology to obtain a viral minichromosome-enriched fraction. Using this procedure, we have characterized two populations of viral minichromosomes in PepGMV-infected plants. The minichromosomes were associated to nucleosome core histones indicating that the procedure does not disturb nucleosome integrity. Using sucrose gradient sedimentation, we observed that main population of viral minichromosomes from symptomatic and recovered tissues presented different sedimentation rates suggesting also differences in structure/conformation. In addition, the main fraction from symptomatic tissue presented a low level of DNA methylation, an association to Histone H3K4me3 (chromatin activation marker). On the other hand, the main fraction obtained from recovered tissue presented a high level of DNA methylation and an association to Histone H3K9me2 (chromatin repression marker). Interestingly, pepper plants infected with another begomovirus, Pepper huasteco yellow vein virus (PHYVV) also present recovery. However, there are some interesting biological differences between PepGMV and PHYVV infective cycles: viral DNA concentration, number of infected cells, etc. These differences might be important enough to modify the way the plant reacts against virus presence.

Cell to cell movement of monopartite begomoviruses

D. Guha², C. G. Poornima Priyadrshini³ and H. S. Savithri^1*

Email ID for Correspondence: bchss@biochem.iisc.ernet.in

¹Department of Biochemistry Indian Institute of Science, Bangaluru-560 012, ²St. Thomas College, Bhilai, ³CFTRI, Mysuru

Monopartite begomoviruses lack the DNA –B which encodes proteins for the movement of the viral genome namely BV1 and the BC1. It has been suggested that the proteins encoded by DNA-A such as the CP (V1), V2 and C4 might replace the functions of BV1 and BC1.The functional attributes of CP, V2 and C4 of the monopartite begomovirus, Cotton leaf curl Kokhran virus-Dabawali were analyzed in vitro and in vivo by their over-expression in E coli, and in the plant system respectively. Purified CP could bind to ssDNA via the conserved Zinc finger motif, with H85 of the motif being most crucial. The CP was shown to interact with V2 in vitro. GFP-V2 and YFP-CP transiently expressed in N. benthamiana plants by agroinfiltration revealed the localization of V2 to the cell periphery and CP predominantly to the nucleus. Interestingly, upon co-infiltration, CP was found both in the nucleus and in the cytoplasm along with V2 suggesting that the interaction of V2 with CP might be important for cell to cell movement. The purified GST-C4 protein exhibited metal ion dependent ATPase and inorganic pyrophosphatase activities. Interaction of V2 with GST-C4 resulted in an increase in both the enzymatic activities of GST-C4. Upon expression in planta, mCherry-C4 localized both in the nucleus and the cytoplasm. Interestingly, when GFP V2 and mcherry-C4 proteins were co-expressed, GFP-V2 re-localized to the nucleus. V2 lacking C-terminal 21 residues localized both in the nucleus and the cytoplasm suggesting that the C-terminal region of V2 promotes its retention in the cytoplasm and cell periphery. The interaction of V2 with C4 might mask this region and facilitate the transport of V2 into the nucleus. Thus, all the three proteins, CP, V2 and C4 might be involved in nuclear as well as cell to cell transport of viral genome.

Geminiviruses and viroids: it’s all about methylation

Mattia Pegoraro^1*, Enza Maria Torchetti², Beatriz Navarro², Marco Catoni³, Francesco Di Serio² and Emanuela Noris¹

Email ID for Correspondence: mattia.pegoraro@unito.it

¹Institute for Sustainable Plant Protection, National Research Council of Italy (IPSP-CNR),Torino, Italy; ²Institute for Sustainable Plant Protection, National Research Council of Italy, Bari Research Unit, Bari, Italy; ³The Sainsbury Laboratory, University of Cambridge, Cambridge, UK

Mixed infections are common in nature and can result in synergistic or antagonistic interactions. Studying mixed infections can be of help to untangle infection processes and defense responses. DNA methylation and post-transcriptional gene silencing are critical for the infection of geminiviruses and ssRNA viroids, respectively, and both pathogens can counteract these defense mechanisms to promote their infectivity. During a survey concerning the response of plants to double infection by geminiviruses and viroids, we found that when plants were experimentally co-inoculated with Tomato yellow leaf curl Sardinia virus (TYLCSV) and Potato spindle tuber viroid (PSTVd), TYLCSV infectivity and accumulation were strongly reduced, indicative of an antagonistic action of PSTVd. A similar antagonistic interaction also occurred towards other geminiviruses. Using methylation-sensitive restriction enzymes and bisulfite conversion assays, we discovered that PSTVd promoted a strong hyper methylation of TYLCSV DNA in tomato plants co-infected by both pathogens. Concomitantly, PSTVd alone or in double infection with TYLCSV significantly upregulated the expression of key genes governing DNA methylation in plants, thus supporting a mechanistic link with the antagonism of the viroid on the virus during co-infection. This study opens new perspectives concerning the outcome of multiple biotic stresses in plants and may reveal novel regulatory networks in the interplay between two nuclear-replicating pathogens and their host.

Unfinished business—the structural features of a begomovirus particle revealed by cryo-electron microscopy

Keith Saunders^1*, Emma Hesketh², Chloe Fisher², Neil Ranson² and George Lomonossoff¹

Email ID for Correspondence: keith.saunders@jic.ac.uk

¹Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK; ²Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK

We have subjected highly purified geminivirus preparations to cryo-electron microscopy. Images of geminate particles derived from reconstructed data sets, currently with a resolution of 4–5 Å will be presented. Geminate particle surface features and the density within particles will be discussed.

Functional analysis of an RNA silencing suppressor from Mulberry mosaic dwarf associated virus

Xiuling Yang^*, Shifang Li, and Xueping Zhou

Email ID for Correspondence: xlyang@ippcaas.cn

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China

Viruses encode RNA silencing suppressors to fine-tune host RNA silencing-mediated defense responses. In a previous study, Mulberry mosaic dwarf associated virus (MMDaV), a novel, highly divergent geminivirus, has been identified from a Chinese mulberry tree affected by a disease showing mosaic and dwarfing symptoms, but the functions of its proteins are unknown. In this study, we aimed to identify RNA silencing suppressors in MMDaV. All the five and two putative ORFs from the virion and complementary sense, respectively, were constructed under the control of 35S promoter and then tested for their abilities to suppress RNA silencing using green fluorescent protein (GFP). The results showed that V2 could inhibit local RNA silencing, but not the short distance spread of the GFP silencing signal in Nicotiana benthamiana 16c plants. Expression of the V2 protein from the heterologous Potato virus X vector induced leaf curling symptoms at 5 days post inoculation (dpi), and then severe necrosis at 9 dpi in N. benthamiana. Agroinfiltration transient expression of GFP-tagged V2 suggested that V2 forms aggregates and targets the cytoplasm of the epidermal cells of N. benthamiana leaves. Although the V2 protein encoded by begomoviruses has been previously shown to have suppressor activity, this is the first time a highly divergent V2 has been examined. Its mode of suppression of RNA silencing will be further elucidated.

Geminivirus-associated alphasatellites suppress transcriptional not post-transcriptional gene silencing

Qamar Abbas^1,2*, Imran Amin¹, Shahid Mansoor¹, Michael Wassenegger³ and Rob W. Briddon¹

Email ID for Correspondence: qamar92@gmail.com

¹Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan; ²Pakistan Institute of Engineering and Applied Sciences, Islamabad, Pakistan; ³RLP Agroscience: AlPlanta Institute for Plant Research, Neustadt, Germany

RNA silencing, also known as RNA interference, is a host plant defense mechanism against invasive nucleic acids such as viruses. It is homology based system that leads to the degradation of RNA sequences, so-called post-transcriptional gene silencing (PTGS), or methylation of DNA sequences, so-called transcriptional gene silencing (TGS). Alphasatellites are satellite-like molecules associated with viruses of the family Geminiviridae. The precise selective advantage for the virus of the association with an alphasatellite remains unclear. A previous study of the activity of the replication-associated protein (Rep) encoded by alphasatellites reported that this has suppression of PTGS activity. The study described here investigated the suppressor activity of the Rep proteins encoded by six alphasatellites-Cotton leaf curl Multan alphasatellite (CLCuMuA), Ageratum yellow vein India alphasatellite (AYVIA), Tomato leaf curl alphasatellite (ToLCA), Tomato leaf curl Pakistan alphasatellite (ToLCuPKA), Cotton leaf curl Gezira alphasatellite (CLCUGeA) and the Dragonfly associated alphasatellite (DfasA). For none of the alphasatellite-encoded Rep proteins was there any evidence of suppressor of PTGS activity. However, an analysis of the Rep protein encoded by CLCuMA showed this to have suppressor of TGS activity. The significance of this finding will be discussed.

Two Novel DNAs that Enhance Symptoms and Overcome CMD2 Resistance to Cassava Mosaic Disease

Linda Hanley-Bowdoin^1,*, Catherine D. Doyle¹, Cyprian A. Rajabu^1,2, Mary M. Dallas¹, Leandro de Leon^1,3, Jose T. Ascencio-Ibanez¹ and Joseph Ndunguru²

Email ID for Correspondence: linda_hanley-bowdoin@ncsu.edu

¹North Carolina State University, Raleigh, North Carolina, USA; ²Mikocheni Agricultural Research Institute, Dar es Salaam, Tanzania; ³I + D en Protección Vegetal, Madrid, Spain

Cassava mosaic begomoviruses (CMBs) cause cassava mosaic disease (CMD) in Africa and the Indian subcontinent. Like all members of the geminivirus family, CMBs have small, circular single-stranded DNA genomes. We report here the discovery of two novel DNA sequences designated as SEGS-1 and SEGS-2 (sequences enhancing geminivirus symptoms) that enhance symptoms and break resistance to CMD. Both SEGS enhanced CMD symptoms in cassava (Manihot esculenta Crantz) when co-inoculated with African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV) or East African cassava mosaic virus-Uganda (EACMV-UG). SEGS-1 also overcame resistance of a cassava landrace carrying the CMD2 resistance locus when co-inoculated with EACMV-UG. Episomal forms of both SEGS were detected in CMB-infected cassava, but not in healthy cassava. SEGS-2 episomes were also found in virions and whiteflies. SEGS-1 has no homology to geminiviruses or their associated satellites, but the cassava genome contains a sequence that is 99% identical to full-length SEGS-1. The cassava genome also includes three sequences with 84–89% identity to SEGS-2 that together encompass all of SEGS-2 except for a 52-bp region, which includes the episomal junction and a 26-bp sequence related to alphasatellite replication origins. These results suggest that SEGS-1 is derived from the cassava genome and facilitates CMB infection either as an integrated copy and/or an episome, while SEGS-2 was originally from the cassava genome but is now encapsidated into virions and transmitted as an episome by whiteflies. We are using Arabidopsis to characterize how the SEGS-1 and SEGS-2 enhance and break resistance to geminivirus infection.

Resistance breaking Chilli leaf curl virus and gene action governing plant resistance

Nivrutti Sonone, Suresh Kunkalikar, Surendra Reddy, Sharad Gulhane, Berad Patil, Mahesh Shekhar, Muktha Raghuram, Ravi Kankanallu, Gururaj Deshpande, Guruprasad Gaddikeri, Bharat Char, Radhamani Anandalakshmi^*

Email ID for Correspondence: radha.anandalakshmi@mahyco.com

Plant – Virus Interactions Lab, Mahyco Research Center, Maharashtra Hybrid Seeds Pvt Ltd, Jalna -431203, Maharashtra, India

The incidence of Chilli leaf curl disease caused by begomoviruses has been on the rise since last few years. Identification and analysis of the different begomoviruses causing leaf curl and response of chilli genotypes to different viruses was studied. Recently an epidemic of the leaf curl disease devastated the chilli crop in Madhya Pradesh. Results of the studies done to understand gene action governing resistance in Capsicum annuum to the resistance breaking strain will be discussed.

Proteomic analysis to identify resistant strategies adopted by host plants upon begomovirus infection

Bhavin S. Bhatt^1,2, Fenisha D. Chahwala², and Achuit K. Singh^3*

Email ID for Correspondence: achuits@gmail.com

¹Faculty of Applied Sciences, Parul University, Waghodia, Vadodara, Gujarat; ²School of Life Sciences, Central University Gujarat, Gandhinagar, Gujarat; ³Crop Improvement Division, ICAR-Indian Institute of Vegetable Research, Varanasi, UP

French bean (Phaseolus vulgaris L.) is one of the most important vegetable crops of India which is consumed widely. Begomoviruses have emerged as major constraint for vegetable production. We have cloned and sequenced begomoviral genomic components associated with bean dwarf mosaic disease (BDMD) manifested leaf samples from Varanasi, India. Sequence analyses revealed the presence of Mungbean yellow mosaic India virus (MYMIV). Further, Agro-inoculation studies on susceptible French bean cv. Arka Komal confirmed MYMIV is causal agent of BDMD. However, French bean cv. Arka Suvidha was found tolerant against MYMIV-Varanasi isolates. Therefore, proteomic analyses of French bean cv. Arka Suvidha leaf samples agro-inoculated with MYMIV were conducted for studying modulation of host proteome. Proteomics data showed that relative expression levels of most of identified proteins were upregulated. These proteins were mainly involved in defense (25%) signal transduction (15%), energy (15%) and metabolic regulation (20%). In our studies, molecular characterization in conjugation with proteomics approach was implemented to get information for molecular events occurring during plant virus interaction. Moreover, our analysis revealed that mock plants had no significant protein profile alteration as compared to virus inoculated plants demonstrating that virus infection initiates modulation of proteomic profile. Conclusively, this study showed that, most of the proteins involved directly or indirectly to defense, shoots up at higher level when both DNA A and DNA B components inoculated than DNA A components alone. This suggests an activation of SAR in inoculated plants, which restricts virus copy number accumulation. More in depth insights into host protein modulation will clear defense pathways taken up by host plants. In summary, all of the above proteins showed changed abundances induced by MYMIV infection on French bean leaves. SAR and induced resistance might be a combinatorial effect of altered interactome of these proteins.

Development of new tools and approaches for management of curly top disease of tomato caused by the beet leafhopper (Circulifer tenellus)-transmitted Beet curly top virus (genus Curtovirus)

Robert L. Gilbertson^1*, Li-Fang Chen¹, Ozgur Batuman¹, Sanwen Huang² and Willian J. Lucas³

Email ID for Correspondence: rlgilbertson@ucdavis.edu

¹Department of Plant Pathology, University of California-Davis, Davis, California, USA; ²Vegetable Genomics Lab, Chinese Academy of Agricultural Sciences, Beijing, China; ³Department of Plant Sciences, University of California-Davis, Davis, California, USA

In California, USA, curly top disease of tomato caused by Beet curly top virus (BCTV) is an economically important disease that caused ~US$ 100 million in losses to the processing tomato industry in 2013. Management options are limited to cultural practices and an annual insecticide spray program targeting overwintering populations of the beet leafhopper (Circulifer tenellus) vector. We have developed and applied molecular tools to improve management of BCTV, including PCR assays for detection of BCTV in leafhoppers and reservoir hosts and agroinoculation systems for screening tomato germplasm for resistance. The leafhopper PCR assay has been used to develop a model to predict curly top incidence in a given season (based on leafhopper populations and BCTV titer). Agroinoculation systems have been used to reveal resistance in tomato breeding lines with combinations of Ty genes (known to confer resistance to whitefly-transmitted geminiviruses, genus Begomovirus). A long-term management strategy is to interfere or block BCTV transmission at the level of the receptors of the beet leafhopper vector involved in the persistent transmission of the virus. On the virus side, the capsid protein (CP), in the form of virions, is required for leafhopper transmission. Mutational analysis of the CP has revealed domains involved in this process, and immunolocalization studies have detected the virus in the filter chamber and midgut and the principal salivary glands. This information is being used to identify leafhopper proteins involved in transmission. On the vector side, the genome of the beet leafhopper is being determined. The genome size of non-viruliferous females was estimated to be ~650 Mb based upon flow cytometry, and a transcriptome was generated. The determination of the complete genome sequence is underway.

A novel route controlling begomovirusresistance by the messenger RNA surveillance factor Pelota

Moshe Lapidot^1*, Dana Gelbart¹, Leah Chen¹, Zion Machbash¹, Silvia Schuster², Adi Avni², Moshe Reuveni¹ and Ilan Levin¹

Email ID for Correspondence: lapidotm@volcani.agri.gov.il

¹Institute of Plant Sciences, Volcani Center, ARO, P.O.Box 6, Bet Dagan 50250, Israel; ²Department of Molecular Biology and Ecology of Plants, Tel-Aviv University, 69978 Tel-Aviv, Israel

Tomato yellow leaf curl virus (TYLCV) is a major pathogen of tomato, causing extensive crop loss worldwide. Genetic resistance in the host plant is considered highly effective in the defence against viral infection in the field. Thus, the best way to reduce yield losses due to TYLCV is by breeding tomatoes resistant to the virus. Until now six major resistance loci, termed Ty-1 to Ty-6, have been characterized and mapped to the tomato genome using molecular DNA markers. We have shown that the TYLCV-resistant line TY172 carries a major recessive quantitative trait locus (QTL) and four additional minor QTL’s. The major QTL was mapped to chromosome 4 and was designated ty-5. Recently we were also able to identify the gene controlling TYLCV-resistance at this locus—the tomato homolog of the messenger RNA surveillance factorPelota (Pelo). A single amino acid change in the protein seems to be in charge of the resistance phenotype. Silencing of Pelo in susceptible plants rendered the transgenic plants highlyresistant, while in resistant plants had no effect on symptom development. In addition, over-expression of the susceptible allele of the gene in resistant plants rendered them susceptible, while over-expression of the resistant allele in these plants had no effect. These results confirm that Pelo is the gene controlling resistance at the ty-5 locus. Pelo, implicated in the ribosome recycling-phase of protein synthesis, offers an alternative route to promote resistance to TYLCV and other begomoviruses. Our hypothesis is that the resistant allele of Pelo is a “loss-of-function” mutant, and inhibits or slows-down ribosome recycling. This will negatively affect viral (as well as host-plant) protein synthesis, which may result in slower infection progression.

Resistance genes involved in tolerant cassava–South African cassava mosaic virus interaction that shows a recovery phenotype

Louis Bengyella and Chrissie Rey^*

Email ID for Correspondence: chrissie.rey@wits.ac.za

School of Molecular and Cell Biology, University of the Witwatersrand, 1, Jan Smuts, Ave, Braamfontein, Johannesburg, 2000, South Africa

Recovery from virus-induced symptoms as a RNA silencing defense is described in the literature, but the expression of disease resistance (R) genes that may play a key role in tolerance and recovery are not yet reported. We critically examined the involvement of R genes in tolerance and recovery from viral-induced symptoms. Transcriptome data of tolerant cassava TME3 (which exhibits a recovery phenotype) and susceptible cassava T200 infected with South African cassava mosaic virus (SACMV) was mined for putative candidates. Putative R proteins with amide-like indole-3-accetic acid–Ile-Leu-Arg (IAA-ILR) and leucine-rich repeat (LRR)-kinase conserved domains unique to TME3 were earmarked. Additionally, a cohort of 25 R proteins expressed solely during the recovery process in TME3 was identified. Common responsive R protein in TME3 and T200 were the dirigent-like protein (DIR), coil–coil (CC) nucleotide-binding site (NBS) and toll-interleukin-resistance (TIR), disease resistance zinc finger chromosome condensation-like protein (DZC) and NBS-apoptosis repressor with caspase recruitment (ARC)-LRR domains. A high level of autoactivating mutations in the MHD motif of the NBS-ARC2 subdomain were identified and novel insights into R proteins associated with the recovery phase in TME3 is discussed. Phylogenetic and expression analyses support that diverse R genes are differentially expressed during tolerance and recovery. We show that in cassava a perennial crop, autoactivated R genes participate in tolerance and differentially accumulate during recovery as a complementary defense mechanism to natural occurring RNA silencing to impair viral replication.

Is aphid transmission a distinguishing feature of the new capulavirus genus?

M. Peterschmitt^1*, M. Granier¹, R. Deshoux¹, A. Ferdinand¹, Cœur d’acier² and P. Roumagnac¹

Email ID for Correspondence: michel.peterschmitt@cirad.fr

¹CIRAD-INRA-SupAgro, UMR BGPI, CIRAD, TA A -54K, Campus International de Baillarguet, F-34398 Montpellier, France; ²INRA, UMR 1062 CBGP (INRA, IRD, CIRAD, Montpellier SupAgro), Centre de Biologie pour la Gestion des Populations, Campus International de Baillarguet CS 30 016, F-34 988 Montferrier-sur-Lez, France

The genus Capulavirus has been proposed to accommodate two divergent geminiviruses, namely Euphorbia caput-medusae latent virus (EcmLV) from South Africa and French bean severe leaf curl virus from India (FbSLCV; Bernardo et al. 2013). Since then, two other capulaviruses have been discovered, Alfalfa leaf curl virus (ALCV) from France and Spain, and Plantago lanceolata latent virus (PlLV) from Finland (this conference, Bernardo et al. 2016). The Capulavirus genus was initially defined by genome relatedness and genome organization, and recently a potentially third distinguishing genus-level feature was added. Indeed, ALCV was the first geminivirus for which an aphid transmission was demonstrated (Roumagnac et al. 2015); the aphid vector belongs to the species Aphis craccivora. Using an agroinfectious clone of ALCV and a reared population of A. craccivora, the features of the aphid transmission are under investigation. To further validate aphid transmission as an additional feature to distinguish capulaviruses from other geminivirus genera, the potential aphid transmission of a second capulavirus, namely EcmLV is under investigation. In this attempt, we have collected and reared the most common aphids observed on Euphorbia caput medusae plants, which belong to the species Aphis tirucallis Hille Ris Lambers, 1954. A. tirucallis aphids have tiny differences of morphology with A. craccivora aphids and were not distinguishable from them according to CO1 sequences.

Deltasatellites, an emerging class of non-coding DNA satellites associated with begomoviruses

Jesús Navas-Castillo^1*, Elvira Fiallo-Olivé¹ and Rob W. Briddon²

Email ID for Correspondence: jnavas@eelm.csic.es

¹Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga – Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Algarrobo-Costa, Málaga, Spain; ²Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan

The genus Begomovirus is the largest in the family Geminiviridae in the entire virosphere. Begomoviruses are transmitted by the whitefly (Hemiptera: Aleyrodidae) Bemisia tabaci causing severe damage to important vegetable and fibre crops worldwide. Phylogenetically, two major groups of begomoviruses can be distinguished, the Old World and the New World begomoviruses. A group of phylogenetically distinct begomoviruses that infect plants of the family Convolvulaceae, including sweet potato, are designated sweepoviruses. Several classes of circular ssDNA satellites associated with begomoviruses have been described, the most wide spread being the betasatellites. Betasatellites depend on helper begomoviruses for their replication and movement within and between plants, and enhance the symptoms induced in some host plants. Rolling circle amplification using φ29 DNA polymerase has facilitated the recent discovery of a number of small non-coding DNA satellites that are a quarter of the size of the begomovirus genome components they are associated with. The name deltasatellites has recently been proposed for these satellites. Deltasatellites include the DNA satellite associated with tomato leaf curl virus in Australia (ToLCV-sat), DNA satellites associated with bipartite begomoviruses infecting wild malvaceous plants in the Caribbean, and DNA satellites associated with sweepoviruses in Spain and Venezuela. Deltasatellites contain a predicted stem-loop structure with the nonanucleotide sequence TAATATTAC conserved in the begomoviruses, a sequence with similarity to the satellite common region of betasatellites, an A-rich sequence and a second predicted stem-loop structure. Although the different sub-classes of deltasatellites share all the described genome features, they are not closely related phylogenetically. Based upon sequence and structural similarities with betasatellites, ToLCV-sat had been suggested to have originated as a defective betasatellite. However, although the delta satellites and betasatellites are clearly related, it is unclear whether one group evolved from the other or they have distinct origins.

Artificial small RNA mediated resistance against the deadly tomato virus ToLCV

Sunil Kumar Mukherjee

Email ID for Correspondence: sunilm@icgeb.res.in

Sr. Consultant, NER-BPMC, DBT, Govt. of India, New Delhi 110024, India

Tomato crop yield is devastated by a group of begomoviruses, generically termed as Tomato Leaf Curl Virus (ToLCV), which causes the leaf curl disease (TLD). ToLCVs have either bigenomic or monogenomic DNA components that are single stranded circles of ~2.7 kb. The RNAi suppressors encoded by these viruses are the most pathogenic factors. We made tomato plants that overproduce small RNAs targeted at these viral encoded RNAi factors (1, 2). A few of these designer tomato plants show immunity-level resistance to ToLCV. At least three RNAi-suppressors, commonly known as C4/AC4, C2/AC2 and V1/AV1, are encoded by the ToLCV genomes. Most of the available ToLCV accessions were aligned to identify the conserved regions of each of three suppressors. Each of the common regions contained at least two important viral ORFs. For example, the conserved C4/AC4 region overlapped with Rep (or C1/AC1) and the ORF (C4/AC4). From each of the conserved regions, a segment of 21 nucleotides was chosen and these sequences were bioinformatically predicted to have little or no off-target effects in the tomato as well as humans. The small RNAs were designed as artificial microRNAs and were expressed in tomato using the pre-miR backbones of anyone of ath-mir-319a, sly-mir-159a and sly-mir-168a vectors. Many different transgenics of three different types were thus developed and those expressing suitable amounts of artificial microRNAs were selected for advancing the generations. These were also tested for the abilities to resist the challenge by the infectious viral clones of ToLCV. The presented data will reveal that a few of the transgenics were extremely tolerant against ToLCVs while the challenged non-transgenics were either highly stunted or succumbed to death. Designing constructs that express small RNAs like si- or mi-RNAs in plants are generally not straight forward and often involve steps of difficult cloning. So we have developed a novel vector, which would produce siRNAs (technically tasi-RNAs), when the sequence of interest is directly cloned in the vector using suitable restriction sites. The design of this tasi-RNA producing vector will be described and we would present data to show its efficacy for production of tasiRNAs. This vector has been used to inhibit the growth of ToLCV in the engineered tomato lines. Pyramiding the above mentioned twoforms of designs might be necessary for a robust anti-viral strategy.

Field trial and molecular characterization of RNAi-transgenic tomato plants that exhibit resistance to tomato yellow leaf curl virus

Mikhail M. Pooggin

Email ID for Correspondence: pooggin@yahoo.com

University of Basel, Department of Environmental Sciences, Basel, Switzerland

RNA interference (RNAi) is a widely used approach to generate virus-resistant transgenic crops. However, issues of agricultural importance like the long term durability of RNAi-mediated resistance under field conditions and the potential side effects provoked in the plant by the stable RNAi expression remain poorly investigated. Here, we performed field trials and molecular characterization studies of two homozygous transgenic tomato lines, with different selection markers, expressing intron-hairpin RNA cognate to the Tomato yellow leaf curl virus (TYLCV) C1 gene. The tested F6 and F4 progenies of the respective kanamycin and basta-resistant plants exhibited unchanged field resistance to TYLCV and stably expressed the transgene-derived short interfering RNA (siRNAs) to represent 6–8% of the total plant small RNAs. This value outnumbered the average percentage of viral siRNAs in the non-transformed plants exposed to TYLCV-infested whiteflies. As a result of the RNAi transgene expression, a common set of up and down regulated genes was revealed in the transcriptome profile of the plants selected from either of the two transgenic events. A previously unidentified geminivirus causing no symptoms of viral disease was detected in some of the transgenic plants. The novel virus acquired V1–V2 genes from TYLCV and C1–C4 genes from a distantly-related begomovirus and thereby it could evade the repressive sequence-specific action of transgene-derived siRNAs. We deep-sequenced and compared siRNA populations for both viruses and assessed cytosine methylation of viral circular double-stranded DNA. Our findings shed light on the mechanisms of siRNA-directed antiviral silencing in tomato plants and highlight the applicability limitations of RNAi technology as it may alter the transcriptional pattern of non-target genes. This work has largely been a collaborative endeavor of Dr. Alejandro Fuentes’ team at Center for Genetic Engineering and Biotechnology, Habana, Cuba and my team at University of Basel.

Combining INPACT and RNAi strategies to confer resistance to the model geminivirus, Tobacco yellow dwarf virus

Ben Dugdale^*, Maiko Kato, Robert Harding and James Dale

Email ID for Correspondence: b.dugdale@qut.edu.au

Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Queensland, Australia

The In Plant ACTivation (INPACT) expression platform for plants is based on the deconstructed genomes of circular, single-stranded DNA viruses such as the geminiviruses. The organization of the INPACT expression cassette is unique and ensures that the gene of interest (GOI), when integrated into the host chromosome, is expressed only in the presence of the cognate virus-encoded replication protein (Rep). The inducible nature of the system is valuable in that it allows for the production of proteins with phytotoxic properties, an attribute that has been exploited in order to develop virus resistance in plants. For virus resistance, the GOI encodes a lethal protein, the ribonuclease barnase that is only expressed in plant cells in which the infecting virus replicates; the virus activates expression of the lethal gene product which in turn kills the infected cells. This essentially prevents further virus movement, limiting virus spread and minimising disease. Using this strategy alone, we have generated a number of tobacco lines with high levels of resistance to the model geminivirus, Tobacco yellow dwarf mastrevirus (TYDV). However, by super-transforming these elite INPACT lines with an RNAi cassette targeting the TYDV movement protein gene, we have increased the effectiveness of this resistance. Three lines containing both resistance cassettes remained symptomless and free of viral nucleic acids 6 weeks post challenge. This approach demonstrates the effectiveness of combining different molecular strategies in order to generate more robust geminivirus resistance. Importantly, the two strategies have different targets with INPACT targeting the cell and RNAi targeting the virus.

RNAi-mediated resistance against cotton leaf curl disease in Gossypium hirsutum

Mohd. Akmal, Jawaid A. Khan^*

Email ID for Correspondence: jkhan1@jmi.ac.in

Plant Virus Laboratory, Department of Biosciences, Jamia Millia Islamia University, New Delhi-110025, India

Cotton leaf curl disease (CLCuD) is a serious and complex disease causing enormous losses to cotton (Gossypium hirsutum) crop. It is caused by several distinct begomovirus species in association with a disease specific betasatellite essential for induction of disease symptoms. In this study, RNAi-mediated approaches were applied for generating resistance against Cotton leaf curl Multan virus (CLCuMV) and Cotton leaf curl Multan betasatellite (CLCuMB). In silico search was made to predict potential regulatory targets of G. hirsutum miRNAs against CLCuMV/CLCuMB genomes. Two potential miRNAs (Gh-miR398 and Gh-miR2950), targeting multiple genes of CLCuMV (AC1, AC4, AV1, and LIR) and CLCuMB were selected. Their efficacy against CLCuD was experimentally validated following expression in G. hirsutum plants. The nodal explants of G. hirsutum were used for Agrobacterium-mediated transformation with miRNA gene constructs. The presence of miRNAs in the transformed G. hirsutum plants was confirmed through molecular analysis and the level of virus resistance was evaluated following inoculation with viruliferous whitefly (Bemisia tabaci) insect vector. Though most of the transformed G. hirsutum plants remained healthy, a few of them showed mild symptoms at later stage of growth. In a similar approach, a hairpin RNAi construct capable of expressing dsRNA homologous to the Intergenic Region (IR) of CLCuMV was designed and produced. Several CLCuD-resistant G. hirsutum lines were generated following A. tumefaciens mediated transformation with the IR-based gene construct. PCR coupled with Southern hybridization confirmed the integration of IR in the transformed cotton plants. Following inoculation with viruliferous whiteflies, some of the transgenic cotton lines displayed high degree of resistance. The CLCuD symptoms were not observed even after 3 months post inoculation as compared to control plants. The transformed cotton plants showed the presence of siRNAs. The present study thus demonstrated that both miRNA as well as dsRNA mediated resistance strategies are effective means to control the devastating CLCuD in cotton.

Emergence of a new recombinant Mungbean yellow mosaic virus infecting blackgram in northern India and identification of resistance sources

Anirban Roy^1*, Manas Kumar Bag² and N.K. Gautam³

Email ID for Correspondence: anirbanroy75@yahoo.com

¹ICAR-Indian Agricultural Research Institute, New Delhi, India; ²ICAR-National Rice Research Institute, Cuttack; ³ICAR-National Bureau of Plant Genetic Resources, New Delhi, India

Yellow mosaic disease of Black gram (Vigna mungo L.) is caused by two predominant begomovirus present in Indian sub-continent, viz., mungbean yellow mosaic virus (MYMV) and mungbean yellow mosaic India virus (MYMIV). Predominantly, MYMIV is present in eastern, central and northern parts of India while MYMV is mainly restricted to western and southern part of the country. During the evaluation of germplasm resources of blackgram severe yellow mosaic disease was recorded in Delhi condition. Rolling circle amplification (RCA) was performed using $\mathit{\varphi }29$ DNA polymerase for detection and characterization of virus(es). Two distinct clones (MF1 and MF2) with different restriction profiles were sequenced. Sequence analyses showed that MF1 (DNA B) and MF2 (DNA A) shared highest sequence identity (92.8 and 88.2%) with two different MYMV isolates reported from Cambodia and Pakistan, respectively but had low sequence identity with MYMIV strains reported to be associated with the disease in northern India. In phylogenetic analysis both these components clustered in separate phylogenetic clad. This virus appeared to be a new isolate of MYMV and is the first report of MYMV in black gram from northern India. Further gene wise sequence analysis and recombination analysis indicated that this virus probably evolved due to recombination. In order to identify resistant sources, 344 germplasm were screened through robust method during 2011–2013 at National Bureau of Plant Genetic Resources, New Delhi, India. Eight accessions that have shown highly resistant (HR) or resistant (R) reaction (CI ≤ 9) consistently during field evaluation were further evaluated under challenged inoculation condition through viruliferous whiteflies. Response of IC144901 and IC001572 were categorized as HR and another two accessions viz. IC011613 and IC485638 were grouped into R category. The resistance of these four accessions was confirmed through agro-inoculation of the infectious virus construct of New Delhi isolate of MYMV and low accumulation of viral DNA were observed over a period of time in the resistant accessions. These four accessions could be directly as varieties resistant to YMD or could be used in future breeding programmes to develop black gram resistant cultivars.

Breeding tomatoes resistant to tomato leaf curl virus disease using marker assisted selection and genomic approaches

H C Prasanna^*, Sarvesh P. Kashyap, Geetika Bhagwat, Deepika Singh, Niranjan Shirgoankar, B Singh

Email ID for Correspondence: prasanahc@yahoo.com

Division of Crop Improvement, ICAR-Indian Institute of Vegetable Research, P B No. 01, P O-Jakhini, Shahashapur, Varanasi, 221305, UP, India

Breeding resistance to whitefly-transmitted begomoviruses is an important goal of tomato breeding programs in India. Series of Ty genes derived from Solanum chilense (Ty-1/Ty-3, Ty-4 and Ty-6), S. habrochaites (Ty-2) and possibly S. peruvianum (ty-5) provide valuable non-transgenic options to manage tomato leaf curl virus disease (ToLCVD) in tomato. However, these Ty genes confer differential response to different species of begomoviruses. For example, Ty-2 carrying tomato lines are known to be susceptible in northern India. In this background, we set to pyramid major Ty genes (Ty-2, Ty-3, ty-5 and Ty-6) for achieving broad spectrum resistance to both monopartite and bipartite begomoviruses of India. We determined the diagnostic value of markers linked to Ty genes for their suitability in gene pyramiding and used markers located on opposite ends of Ty introgressions to ensure selection of lines with an intact introgression. Using marker assisted selection we developed series of pyramided lines (Ty-2 + Ty-3) and developed lines carrying individual genes as resource for developing ToLCVD resistant hybrids. The response of pyramided lines (Ty-2 + Ty-3) and lines carrying individual Ty genes to ToLCV infection was tested by agroinoculation and field tests. The pyramided lines and Ty-3 carrying lines exhibited a high level of resistance to the monopartite and two bipartite begomoviruses in agroinoculation based screening. Tomato variety Kashi Aman carrying an allele of Ty-3 was identified from this program and released at the national level. Further, genomics- and marker assisted backcross program was initiated to pyramid Ty-2, ty-5 and Ty-6 in the background of Kashi Aman. In parallel, we have identified new pre-breeding lines conferring resistance to ToLCVD and generated interspecific backcross populations using S. chilense. We are deploying genomics based approaches to identify the regions associated with resistance and elucidate introgression borders and content in resistant lines and interspecific populations.

Resistance to geminiviruses in genetically engineered cowpea through RNA silencing

Sanjeev Kumar¹, Bhaben Tanti², Sunil Kumar Mukherjee³ and Lingaraj Sahoo^1*

Email ID for Correspondence: ls@iitg.ernet.in

¹Department of Bioscience and Bioengineering, Indian Institute of Technology Guwahati, Guwahati-781039, India; ²Department of Botany, Gauhati University, Guwahati-781014, Assam, India; ³International Center for Genetic Engineering and Biotechnology (ICGEB), New Delhi-110067, India

RNA interference (RNAi) plays an important role in natural antiviral defence mechanism and in genome maintenance and development in plants. Plant viruses encode RNAi suppressors to suppress the RNAi pathways to counter plant defence mechanism. In geminiviruses, AC2, AC4 and AV2 are major proteins known to act as plant RNAi suppressors. Geminiviruses transmitted by whiteflies cause yellow mosaic disease in cowpea (Vigna unguiculata). In this study, full-length genomic clones of 7 geminivirus isolates of cowpea, with yellow mosaic symptoms in India, were isolated and found to have 94–98% homology. We designed gene silencing vectors, to target either AC2 or AC4 or both AC2 and AC4 (two RNAi suppressor proteins) at a time of 7 geminivirus isolates of cowpea. Transgenic cowpea plants were raised using the AC2 or AC4 or both AC2 and AC4 siRNA-generating constructs. Presence and integration of transgenes were confirmed by Polymerase Chain Reaction and Southern hybridization. Plants were tested for resistance separately against Mungbean Yellow Mosaic India Virus (MYMIV) through agro-infiltration of a dimeric infectious clone of MYMIV (cowpea isolate) made in our lab. Northern hybridization showed accumulation of high level of siRNA in transgenic cowpea lines post 30 days post agroinfiltration. Most plants showed an absence of symptoms even after 3 months of agroinfiltration.

Banana bunchy top virus: control through resistance

Benard Mware¹, Anthony James, Misheck Soko², Robert Harding¹and James Dale^1*

Email ID for Correspondence: j.dale@qut.edu.au

¹Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Queensland, Australia; ²Bvumbwe Agricultural Research Station, Blantyre, Malawi

Banana bunchy top virus (BBTV) is the most serious virus of bananas and is widespread through Asia and Africa and parts of Australia. In some regions such as Australia, a level of control has been achieved through minimisation strategies such as removal of infected plants and using virus free planting material. However, eradication has proven to be elusive despite concerted efforts. Resistance in the longer term is likely to be the most appropriate strategy. We are taking two approaches. The first is to develop transgenic Cavendish bananas using either RNAi or our virus induced cell death strategy, INPACT. For RNAi, we first determined the ratios of the six different BBTV DNA components that occurred during BBTV infection in different cultivars as a pointer towards the most appropriate targets for RNAi. We subsequently designed inverted repeat RNAi constructs directed towards the South Pacific strain of BBTV targeting different BBTV genes and transformed these into Cavendish bananas. A large number of transgenic lines have been screened for resistance in the glasshouse. The most promising lines have subsequently been transferred to Malawi and will be planted in the field mid-2016. Further, we have designed an INPACT cassette which can be activated by the BBTV replication protein (Rep). This cassette contains the same “split” barnase gene that we have used to generate resistance to Tobacco yellow dwarf mastrevirus in tobacco. Cavendish transgenic lines have been generated with the construct and will undergo glasshouse challenge. We have also been developing a strategy to identify banana genes that provide resistance to BBTV that could be used in a cisgenic approach. As an initial step, we have screened a wide range of Musa genotypes and have identified two accessions with apparent immunity to BBTV as well as accessions with high levels of resistance.

Geminivirus problems in tropical tuber crops—Diagnosis and Management strategies

Thangaraj Makeshkumar

Email ID for Correspondence: makeshctcri@gmail.com

ICAR-Central Tuber Crops Research Institute, Thiruvananthapuram, India

Tropical tuber crops (cassava, sweet potato, yams and aroids) are rich in carbohydrates and serve as staple food for millions of people in different countries. These crops are the highest producers of calories per unit area and they are prone to many diseases caused by viruses. Among the viral diseases affecting tropical tuber crops, cassava mosaic disease and sweet potato leaf curl are important, which are caused by begomoviruses. Cassava (Manihot esculanta Crantz) is an important staple food and industrial crop in more than 80 different countries. It has been grown in India for more than a century mostly in Kerala, Tamil Nadu, Andhra Pradesh and a few north eastern states. Among the various biotic stresses, cassava mosaic disease (CMD) is one of the major constraints for cassava production in Indian sub continent resulting in yield loss of about 20–80%. The causative organism is Cassava mosaic virus which belongs to the family Geminiviridae and the disease spreads mainly through whitefly vector (Bemisia tabaci). About nine different viruses causing CMD were identified worldwide, yet of which Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV)are seen commonly in Indian subcontinent. In the span of one decade, SLCMV emerged as the major cause of CMD in Tamil Nadu and Kerala which virtually replaced the ICMV. SLCMV has occupied more than 90% of cassava growing areas, where ICMV was detected invariably earlier. Different diagnostic techniques like ELISA, PCR and NASH are being routinely employed. Various management approaches adopted to contain this disease includes, clean cultivation, use of disease free planting material from meristem derived plants and growing of CMD resistant cultivar Sree Padmanabha. CMD resistance breeding yielded a range of lines having resistance and recovery phenotypes which are on pipeline. Real time PCR studies showed reduction/absence of virus in recovery phenotypes. The development of transgenic cassava resistant against CMD is also on the way. Sweet potato (Ipomoea batatas) which is an important tuberous root crop is grown in many tropical and subtropical regions of the world. Among the various viral diseases affecting sweet potato, the whitefly-transmitted Sweet potato leaf curl virus (SPLCV) (Begomovirus, Geminiviridae) is one of the important viruses infecting sweet potato. PCR & NASH based diagnostics techniques were developed. Using Rolling circle amplification full genome of SPLCV were amplified, cloned and sequenced.

Geminivirus—an emerging threat to major cucurbits in western Maharashtra and their integrated management

Savarni Tripathi^* and Raj Verma

Email ID for Correspondence: savarni_pune@iari.res.in

ICAR-Indian Agricultural Research Institute, Regional Station, 125 ITI Road, Aundh, Pune, MH 411007, India

The family Cucurbitaceae includes many popular nutritious and highly remunerative vegetables and fruits. However, viral diseases infecting cucurbits are the major production constraints worldwide causing heavy economic losses. Recently, erratic climatic changes is having a greater impact on emergence of certain viral diseases especially the whitefly transmitted Geminiviruses. To assess the present status of whitefly transmitted viral diseases, surveys were conducted to determine the incidence of whitefly transmitted Geminiviruses infecting cucurbits naturally in different districts of western Maharashtra. Samples showing symptoms such as leaf curling, mosaic, vein banding, interveinal chlorosis, yellowing, puckering, rossetting, leaf reduction, shortening of internodes, stunting etc. were collected from cucurbits (Cucumis sativus, Momordica charantia, Cucumis melo, Cucurbita maxima, Luffa cylindrica, Lagenaria siceraria and Citrullus lanatus) grown in Pune, Solapur, Kolhapur, Nasik, Ahmadnagar and Satara districts. The presence of whitefly and symptoms indicated potential occurrence of Geminiviruses. Further association of Geminivirus in collected symptomatic samples was confirmed by polymerase chain reaction. The incidence of Geminivirus was higher in musk melon (10–30%) and bitter gourd (up to 100%) as compare to other cucurbits. Therefore, considering the potential threat of Geminiviruses in cucurbits, a field experiment was conducted to develop integrated management strategies to minimize the losses caused by these viral diseases. The silver colour mulching along with alternate foliar spray of agroneem (0.2%) or imidacloprid (0.01%) at fortnightly interval was found promising in minimizing the losses caused by whitefly transmitted geminivirus infections in cucurbits.

Genetic modification of cotton for resistance against whitefly and associated diseases

P. K. Singh

Email ID for Correspondence: pradhyumnasingh@hotmail.com; pksingh@nbri.res.in

Genetics and Plant Molecular Biology, Council of Scientific and Industrial Research (CSIR)-National Botanical Research Institute, Lucknow, India

Whitefly (Bemisia tabaci) is one of the highly invasive pests on several field crops across the world. High temperature, humidity and dense cropping in tropical and sub-tropical zones promote multiplication of this insect. Crops grown in polyhouses are also badly affected by this pest. The insect damages the crops by sucking phloem sap; causing fungal infection and spreading plant viruses that routinely leads to a loss of 10–30% in crop productivity, which may exceed to 100% in severe cases. Outbreak of whitefly caused widespread devastation in 1.5 million hectare of cotton crop in North India, in 2015. In terms of productivity, the revenue loss was estimated at US$ 2 billion. GM strategy for the control of whitefly is not available in world. We have identified a novel insecticidal protein (Tma12, 21.6 kDa) in a fern Tectaria macrodonta that kills whitefly (LC₅₀ 1.9 µg/ml). The protein is exclusively toxic to whitefly, and does not affect other crop insects and beneficial insects. Primary structure of the Tma12 was partially determined with N-terminal and MS based de novo sequencing that helped in cloning of the gene. The gene was found novel both in terms of protein sequence and function. The insecticidal protein was characterized as a chitin binding protein with minor chitinolytic activity. It is notable that a similar gene/protein is not present in any other terrestrial plant. The insecticidal gene was introduced in cotton for constitutive expression. The optimally expressing transgenic cotton lines showed excellent protection against whitefly though 6 consecutive generations. These lines control whitefly population by inhibiting its reproduction and hampering colonization. Defence in transgenic cotton by virtue of expression of Tma12 was equivalent to 3–4 sprays of chemical pesticides. Whitefly is also a vector of plant viruses. Absence of viral DNA in transgenic and presence in non-transgenic showed that the transgenic strategy also offers control over the viral diseases. This is for the first time a novel insecticidal protein is discovered from an untapped source. Whitefly and virus resistant transgenic crops have been a worldwide unmet need of agriculture biotechnology; our research has fulfilled the gap. This gene can be deployed in more than 30 crops to protect the yield without application of hazardous pesticides. The GM-cotton can also be used as an insecticidal trap to control whitefly population in polyhouses and protect field crops from viral disease.

Genetic characteristic and transmission vector of Cotton leaf curl Multan virus in China

He Zi-fu^*, Tang Ya-fei and She Xiao-man

Email ID for Correspondence: hezf@gdppri.com

Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China; Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou 510640, China

Cotton leaf curl Multan virus (CLCuMuV) is a whitefly transmitted Geminivirus endemic to the India subcontinent and is notorious as a causal agent of cotton leaf curl disease in Pakistan. In 2006, we firstly found an isolate of CLCuMuV infecting China rose (Hibiscus rosa-sinensis) to cause leaf curl disease in Guangdong, China. The objective of this study is to analyze the genetic characteristic of CLCuMuV and whitefly (Bemisiatabaci) cryptic species transmitting the virus in China, and to provide a theoretical basis for controlling the virus. All of the diseased samples collected from China were detected by PCR using specific primers pair of CLCuMuV and its betasatellite. The complete sequences of CLCuMuV isolates and betasatellite were cloned by rolling circle amplification. The results indicated that CLCuMuV had distributed in Guangdong, Guangxi, Hainan, Fujian, and Yunnan provinces of China, and infected okra (H. esculentus), Malvaiscusarboreus, cotton (Gossypiumhirsutum) and kenaf (H. cannabinus) to cause leaf curl diseases. Complete nucleotide sequences of 34 geographically and/or temporally distinct CLCuMuV isolates in China were determined to be 2737 nucleotides and shared more than 99% nucleotide sequence identity with each other. All of the CLCuMuV isolates were associated with a betasatellite and shared more than 98% nucleotide sequence identity with each other. The whitefly transmission experiment demonstrated that MEAM1, Asia7 and Asia1 cryptic species of whitefly were able to acquire CLCuMuV and betasatellite from the diseased cotton plants infected by CLCuMuV/betasatellite complex, but only Asia7 and Asia1 cryptic species were able to transmit CLCuMuV and its betasatellite to infect cotton, kenaf and okra. It is concluded thatthe genetic of CLCuMuV population in China is stable, and the indigenous cryptic species Asia7 and Asia1 but not MEAM1 are able to transmit CLCuMuV.

Studies on Virus-Vector relationship of Tomato leaf curl New Delhi virus and Bemisiatabaci (Gennadius) on Ash gourd BenincasahispidaCogn

T. Tamilnayagan^1*, M. Suganthy¹, P. Renukadevi², V.G. Malathi² and N. Ganapathy¹

Email ID for Correspondence: tamil.ento14@gmail.com

¹Department of Agricultural Entomology, ²Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India

Yellow vein mosaic disease caused by whitefly transmitted geminiviruses belonging to the genus Begomovirus of the family Geminiviridae is a major constraint in enhancing the production of ash gourd in India. Infected plants show interveinal chlorosis, yellow mottling, puckering and downward curling of leaves, poor fruit set and sterility. Investigations were carried out to identify the genotype of the vector, Bemisiatabaci and to study the virus–vector relationship. The genotype of B. tabaci was identified as Asia I group on the basis of mtCOI sequence. Laboratory experiments were conducted to determine the acquisition access period (AAP) and inoculation access period (IAP) along with back transmission study of Tomato Leaf Curl New Delhi Virus (ToLCNDV) infecting ash gourd. For assessing AAP, B. tabaci were allowed to feed on infected ash gourd leaves with different AAP of0.5, 1, 3, 6, 12 and 24 h. The results revealed that AAP of 30 min was sufficient to transmit ToLCNDV into healthy ash gourd. To determine the IAP, B. tabaci with 24 h AAP were allowed to feed on the healthy plants with different IAP of 0.5, 1, 3, 6, 12, 24 h. The results revealed that IAP of 30 min was sufficient to transmit ToLCNDV into healthy ash gourd. For back transmission studies, B. tabaci were allowed to acquire the virus from the infected plants after 24 h of AAP and IAP and allowed to feed on healthy ash gourd seedlings to test the virus transmission. The results revealed that the ToLCNDV was successfully transmitted to healthy plants. Presence of ToLCNDV in the vector was confirmed through PCR by specific primers of ToLCNDV (LH viral 1801-1818 and TOMR 2640-2622).

Generation of endosymbiont free lines of Bemisiatabaci and deciphering the affect of endosymbionts on its biological development and virus transmission

Harpreet Singh Raina^*,Guisuibou Daimei, RohitJamwal, PukhrambamPushpa Devi and Raman Rajagopal

Email ID for Correspondence: hsraina007@gmail.com

Gut Biology Lab, Room no. 117, Department of Zoology, University of Delhi, Delhi 110007, India

Bemisiatabaci is a worldwide invasive pest of agricultural crops with at least 34 genetic groups. They harbor obligatory as well as facultative endosymbionts which confer different ecological traits to their host. In this study we generated two different populations of Asia II-7 Genetic group of B. tabaci with the use of antibiotic tetracycline and then studied the effect of bacteria on different developmental traits. We used 50 µg/ml of tetracycline which was fed to whiteflies in 20% sucrose. The reduction frequency at 50 µg/ml was 0% for Portiera, 37.1% for Wolbachia, 37.8% for Rickettsia and 100% for Arsenophonus. Thus, tetracycline was able to eliminate Arsenophonus from B. tabaci at this concentration. Subsequently, we generated two insect lines, one with Arsenophonus (A⁺) and other without Arsenophonus (A⁻) for analyzing the effect on different biological traits. Oviposition rate was higher for A⁻ whiteflies as compared to A⁺ whiteflies (A⁻: 115.2 ± 6.65; A⁺: 69.4 ± 3.91; F_(1,8) = 35.25, P = 0.0003). Egg hatchability did not show significant difference between A⁺ and A⁻ whiteflies. There was no significant difference in sex ratio of F1 progeny between A⁺ and A⁻ whiteflies that developed to adults. The development time was significantly higher in the progeny of A⁻ parents than from A⁺ parents (F_(1,8) = 51.09, P = 0.0001). Further, the percentage of F1 offsprings that successfully completed development to adulthood was significantly higher among A⁻ group than those of A⁺ group (F_(1,8) = 9.99, P = 0.01). Thus, our results point towards the lesser fitness of A⁺ whiteflies as compared to A⁻ whiteflies. Moreover, the virus transmission of Tomato yellow leaf curl virus by A⁺ whiteflies was significantly higher as compared to A⁻ whiteflies, thus pointing towards the role of this endosymbiont in begomovirus transmission by B. tabaci.

Diversity of begomoviruses present in whiteflies (Bemisiatabaci) from different areas of Brazil

Erich Y. T. Nakasu^1*, Fernando L. Melo², Miguel MichereffFilho¹, Jose Luiz Pereira¹, Alice K. Inoue-Nagata¹

Email ID for Correspondence: erich.nakasu@embrapa.br

¹Embrapa Vegetables, Rodovia BR-060, km 09, 70351-970, Brasília, DF, Brazil; ²Department of Cell Biology, University of Brasilia, Campus Universitário Darcy Ribeiro, Brasília, DF, Brazil

The whitefly Bemisiatabacibiotype B is the most important horticultural pest in Brazil, vectoring several begomovirus species. With the development of high-throughput sequencing technologies, it is possible to identify and estimate the degree of begomovirus diversity occurring in cultivated areas. The aim of this study was to evaluate the diversity of begomoviruses in the whitefly B. tabaci, using an Illumina sequencer platform. Adult whiteflies were collected mainly in tomato crops and separated in two groups: the first consisting of samples from Goiás state, whereas the second was formed by whiteflies from Minas Gerais, São Paulo and Espírito Santo states. Whiteflies were macerated and treated with DNase/RNase before viral RNA and DNA extraction. DNA samples were further subjected to Rolling Circle Amplification (RCA). Nucleic acids (RNA, DNA and RCA) were fragmented, linked to adaptors and subjected to NGS. Reads were trimmed, assembled using the Velvet algorithm (71 kmer) and blasted against a Geminivirus Ref Seq database. The DNA library from group one generated 219,485 contigs, from which only three matched with Tomato severe rugose virus (ToSRV).Similarly, only nine contigs out of 334,051 generated for the DNA library from group two matched begomoviruses (Euphorbia yellow mosaic virus-EuYMV, Sidamicrantha mosaic virus-SiMMV and ToSRV). No matches were obtained for RNA libraries. For RCA libraries, 38,573 and 31,172 contigs were generated for groups one and two, respectively. In group one, from the 1484 matched contigs (3.8%), predominant species were ToSRV (63.4%), Tomato common mosaic virus-ToCmMV (8.8%) and Sweet potato leaf curl virus-SPLCV (6.8%). For group two, 6545 contigs (20.9%) matched begomoviruses, being SiMMV (44.9%), ToSRV (29.1%) and EuYMV (16.8%) the most prevalent. These results indicate that RCA greatly enriches libraries with begomovirus DNA. No putative novel begomovirus species were detected, suggesting that the viral diversity from sampled areas is fairly known.

ArsenophonusGroEL shows more binding affinity than HamiltonellaGroEL with coat protein of ToLCNDV

Gunjan Kumar Saurav^1*,Vipin Singh Rana^1,2, Sonam Popli¹, Kriti Khanna¹, N. C. Naveen³, Ambika Singh¹, Raman Rajagopal¹

Email ID for Correspondence: gunjanshukla24@gmail.com

¹Room No. 117, Gut Biology Laboratory, Department of Zoology, University of Delhi, Delhi 110007, India; ²Dr. B.R. Ambedkar Center for Biomedical Research (ACBR), University of Delhi, Delhi 110007, India; ³Division of Entomology, Indian Agriculture Research Institute, New Delhi, India-110012

Tomato leaf curl New Delhi virus (ToLCNDV) (Gemininiviridae: Begomovirus) is exclusively transmitted by Bemisiatabaci (Hemiptera: Alyrodidae). It has been reported that Tomato yellow leaf curl virus (TYLCV) transmission is dependent on 63 kDaGroEL protein of Hamiltonella bacterial endosymbiont of Vector B. tabaci. Cotton leaf curl virus (CLCuV) is reported to interact with GroEL protein of Arsenophonusbacterial endosymbiont of Asia II genetic group of B. tabaci. ToLCNDV interaction with GroEL protein of any endosymbiotic bacteria of vector has not been reported so far. In this study, we checked interaction of GroEL proteins of Hamiltonella and Arsenophonus with ToLCNDV coat protein and comparative binding affinity of different GroELs with ToLCNDV coat protein. We aligned GroEL protein sequence of Hamiltonella and Arsenophonus. We observed only 79% sequence similarities between GroEL proteins of Hamiltonella and Arsenophonus. We cloned, expressed and purified GroEL protein and coat protein in bacterial expression system. In-vitro interaction of GroEL and coat protein was carried out by pull-down assay. We observed that ArsenophonusGroEL protein was showing significantly more binding affinity than HamiltonellaGroEL protein for ToLCNDV coat protein. Later, we analyzedthe effect of pH on interaction of GroEL proteins with ToLCNDV coat protein. We were not able to observe any significant effect of pH on binding affinity of both GroEL proteins for ToLCNDV coat protein. These results suggest possible role of GroEL protein of bacterial endosymbionts in ToLCNDV transmission by B. tabaci and also indicate that GroEL-coat protein interaction can remain stable in various pH range in hostile environment of different organs of B. tabaci.

Flourescence in situ Hybridization (FISH) and Polymerase chain reaction (PCR) as diagnostic tools for detecting bacterial symbionts in Bemisiatabaci: a vector of begomoviruses

SonamPopli^*, Harpreet Singh Raina, Ambika Singh, Guisuibou Daimei, Raman Rajagopal

Email ID for Correspondence: sonampopli@gmail.com

Gut Biology Lab, Room no. 117, Department of Zoology, University of Delhi, Delhi 110007, India

Identification of bacterial endosymbionts in Bemisiatabaci is important for understanding the benefits or losses provided by them to the host. These interactions between bacterial endosymbionts and B. tabaciare important for host’s ecology and their efficiency to act as transmitters of hundreds of begomoviruses. In this study, we have used Flourescencein situ hybridization (FISH) and Polymerase reaction (PCR) for identification and localization of endosymbionts in B. tabaci. The identification of such endosymbionts is important as the proteins secreted by these bacteria have been shown to be interacting and helping the begomoviruses to survive inside the host. In this study, a comparative analysis has been performed for identification of endosymbionts using two techniques. The bacterial 16S rRNA gene specific primers were used for detection of bacteria by PCR while Locked Nucleic Acid (LNA) probes for specific bacterial endosymbionts were used for FISH. Primary endosymbiont Portiera was detected in 100% of the samples both by FISH and PCR. However, detection of secondary endosymbionts by FISH was significantly higher as compared to PCR [Wolbachia: (χ² = 3.25, P = 0.05), Rickettsia: (χ² = 6.13, P = 0.01) and Arsenophonus: (χ² = 6.85, P = 0.01)]. These results showed FISH to be more effective over PCR in detecting endosymbionts, with an advantage of localization within B. tabaci.

Bemisiatabaci: an invasive insect vector of begomoviruses

Pukhrambam Pushpa Devi^*, Rohit Jamwal, Harpreet Singh Raina, Raman Rajagopal

Email ID for Correspondence: ppsongye@gmail.com

Gut Biology Lab, Department of Zoology, University of Delhi, New Delhi-07, India

Bemisiatabacibelongs to the family Aleyrodidae. It is a polyphagous pest feeding on different crops like cotton, cowpea, tomato, tobacco, brinjal, potato, lettuce, cassava, etc. B. tabaci causes direct damage to the plants by sucking sap from the leaves which induces physiological damages. It also secretes a sticky substance known as honeydew that acts as substrate for fungal growth. There are more than 30 genetic groups of B. tabaci and are known to transmit viruses belonging to the genera Begomovirus (Geminiviridae) like Tomato yellow leaf curl virus, Cotton leaf curl virus etc. It is a group of icosahedral single stranded DNA virus transmitted in a persistent, circulative manner. In this mode of transmission B. tabaci acquires the virus while feeding over the infected plant and then the virus moves along theesophagus, foregut and reaches the midgut from where it gets absorbed into the heamolymph through the filter chamber. In the heamolymph, the begomoviruses interact with the proteins secreted by bacterial endosymbionts which help in the movement of the virus into salivary gland and thus transmission to the healthy plants via feeding. These viruses are numerous and are widely spread in warmer climatic conditions like tropical, sub-tropical and Mediterranean regions. They are responsible for causing devastating plant diseases leading to huge economic loses. Different symptoms of diseases caused by begomoviruses include yellow spots, yellow veins, leaf curling, stunting growth, vein thickening etc. Due to the invasive nature of B. tabaci, it has become a global threat. So, for understanding the viral transmission mechanisms of this pest, it is important to identify the molecular determinants of virus-vector interaction and also the role played by different bacterial endosymbionts.

Production and conservation of Tomato yellow leaf curl virus—free parthenocarpic tomato plants using tissue culture

Sota Koeda^1*, Rihito Takisawa², Tomoyuki Nabeshima³, Yuri Tanaka², Akira Kitajima², Shotaro Matsumoto¹, Yuki Matsumoto¹ and Shinya Kanzaki¹

Email ID for Correspondence: 818sota@nara.kindai.ac.jp

¹Facalty of Agriculture, Kindai University, Nara 631-8505, Japan; ²Graduate School of Agriculture, Kyoto University, Takatsuki 569-0096, Japan; ³Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Tomato yellow leaf curl virus (TYLCV) infections decrease plant growth and yield, and production is almost entirely lost if plants are infected during early growth. Cv. Kyo-temari is the commercial name for the parthenocarpic tomato MPK-1, which has been vegetatively propagated and distributed to local farmers in Kyoto City, Japan, for commercial cultivation. Parthenocarpy is a phenomenon in which fruit set and development proceed without pollination and fertilization. Parthenocarpic tomato cultivars produce normal fruits in conditions that are unfavorable for pollination and fertilization, such as high or low temperatures. Cultivating parthenocarpic tomatoes reduces fossil fuel consumption because the plants can be grown in unheated greenhouses during winter. The typical yellow leaf curl symptoms of TYLCV were observed in 10 parthenocarpic tomato cultivars, including MPK-1, maintained as mother stocks for vegetative propagation at Kyoto University during the winter of 2013. A microtissue direct polymerase chain reaction analysis revealed, a begomovirus-specific amplicon in the plants with yellow leaf curl symptoms. Sequencing and phylogenetic analyses clarified that a TYLCV-Mild isolate was infecting the parthenocarpic tomatoes. Because no TYLCV signal was detected in the shoot apical meristems (SAMs) of TYLCV-infected MPK-1 by in situ hybridization, TYLCV was eliminated by regenerating plants from leaf primordia (LP)-free SAMs of parthenocarpic tomato cultivars. LP-free SAM culture was combined with in vitro grafting to obtain TYLCV-free plants in approximately 3 months. Moreover, the TYLCV-free tomato plants were efficiently conserved in modified Murashige and Skoog medium at 10 °C for over 6 months without subculture. The techniques developed in this study will contribute in eliminating TYLCV from vegetatively propagated parthenocarpic tomatoes and those conservation by in vitro culture.

Okra infecting begomoviruses and evaluation of RNAi methods for developing resistance

MeeraKurulekar, KalyaniSarwadnya, AsawariGorane, Surendra Reddy, Suresh Kunkalikar^*, Sharad Gulhane, NileshDeshmukh, Satish Nalwade, VrushaliGawde, Narendran Nair, Bharat Char, RadhamaniAnandalakshmi

Email ID for Correspondence: suresh.kunkalikar@mahyco.com

Plant – Virus Interactions Lab, Mahyco Research Center, Maharashtra Hybrid Seeds Pvt Ltd, Jalna -431203, Maharashtra, India

Okra (Abelmoshchusesculentus) is an important vegetable in India. In the recent past, okra yellow vein mosaic, vein thickening and leaf curl diseases have affected okra productivity in all okra growing regions of India. These diseases are caused by several begomovirus species and their recombinants. Interaction between okra and different begomoviruses is complex and has to be understood to develop resistance strategies. In an experiment to develop proof of concept for the efficacy of RNA silencing in controlling infection of Okra yellow vein mosaic virus (OYVMV), antisense and hairpin constructs were introduced in okra. Transgenic plants inoculated with virus via viruliferous whiteflies showed significant tolerance to the OYVMV showing that RNAi has potential for the management of begomo viral diseases in okra.

Toxic effects on transformed friable embryogenic callus by a triple-stacked hpRNA silencing construct derived from three cassava geminiviruses

H. Walsh^1* and M. E. C. Rey

Email ID for Correspondence: 474725@students.wits.ac.za

¹University of the Witwatersrand

Cassava mosaic disease (CMD), caused by a number of begomoviruses within the Geminiviridae family, is the most important disease of cassava (ManihotesculentaCrantz) and can cause massive crop losses. The most viable method of controlling CMD is through the development of resistant crops using geminivirus-derived hpRNA to induce post-transcriptional gene silencing (PTGS). A triple stacked construct which targets the AC1/AC4 region or of South African cassava mosaic virus (SACMV), East African cassava mosaic virus (EACMV) and African cassava mosaic virus (ACMV)associated with CMD in southern Africa was used to transform cassava cv. 60444 and landrace T200 friable embryogenic callus (FEC). Transformation efficiency was found to be very low (>10%) due to necrosis of FECs. Virus-derived miRNAs have recently been demonstrated to target mRNA. It was hypothesised that the construct may be forming pre-miRNA structures which generate miRNAs, which possibly, along with off-target PTGS-associated siRNAs may affect hormones or signalling involved in FEC development, thereby eliciting programmed cell death. The aim of this study was to attempt to measure gene expression of possible hormones, transcription and signalling factors in transformed and non-transformed FECs, in addition to different tissues leading up to FEC production. Tissue was collected from cv. 60444, T200 and TME 3 (a non-regeneration control) auxiliary buds and somatic embryo, and non-transformed and transformed FECs at an early (define days or weeks after initiation) and later stages of the transformation process. Gene expression levels are currently being quantified using real time RT-qPCR, and results will be discussed. This research will help elucidate possible mechanisms of mRNA off-targets from transgene-derived sRNAs.

Environmental applications of ssDNA bacterial viruses

Swagata Karmakar^1*, Vandana Mishra¹, Radhey S. Sharma¹

Email ID for Correspondence: sway.swagata@gmail.com

¹Department of Environmental Studies, Bioresources& Environmental Biotechnology Laboratory, University of Delhi, India

Bacterial viruses or bacteriophages are prevalent in the environment; however a limited understanding of their ecological role poses a challenge for their environmental application. Among these, Inviruses belonging to family Inoviridae, are single stranded DNA viruses with a large cylindrical helical body. Owing to their shape, they are also known as filamentous phages. The phages follow two types of life cycles, lytic and lysogenic within a bacterial host. The viruses with the former type of life cycle are virulent and those with the later type of life cycle are non-virulent. In contrast, ssDNA viruses follow a combination of life cycles as, after infection, they produce viral progeny without killing their host. Such a chronic viral infection permanently alters bacterial physiology having major implication on the biology of its host and thereby on the environment. Besides the traditional use of filamentous phages in displaying proteins, their non-traditional use in carrying vaccine, therapeutic agents, bioconjugation scaffold and laboratory models for protein evolution has also been emphasized. The present analyses provide a scientific perspective on little explored non-traditional use of filamentous phages i.e. in the environment. The perspective uses evidence from laboratory studies and visualizes a bigger picture on the use of filamentous phages in influencing structure and function of the bacterial community. The challenges and future research prospect for practical realization of their environmental use are also highlighted.

RNAi-mediated regulation of Begomovirus vector Bemisiatabaci: an attempt to downregulate Cotton leaf curl virus infection

GazalWamiq, Jawaid A. Khan^*

Email ID for Correspondence: jkhan1@jmi.ac.in

Plant Virus Laboratory, Department of Biosciences, JamiaMilliaIslamia, New Delhi-110025, India

Whiteflies (Bemisiatabaci) are hemipterous insects of the family Aleyrodidae. They not only cause physical damage to crops by phloem feeding, and attracting other pests by excreting honey dew, but also act as carrier of many devastating plant viruses. Begomovirus (family Geminiviridae) is transmitted through whitefly vector. B. tabaci shows a very wide host range and is capable of feeding on over 600 plant species from 74 families. Cotton is an important commercial crop. The cotton productivity is severely affected by viruses belonging to genus Begomovirus, among others. Cotton leaf curl disease (CLCuD) is a complex disease caused by Cotton leaf curl virus, (genus Begomovirus; family Geminiviridae) in association with betasatellite. Begomoviruses are transmitted by B. tabaci in persistent manner. In the present study attempts were made to imply RNAi approach with a perspective of controlling the begomovirus vector, B. tabaci. Standalone software miRanda was used for in silico identification host miRNAs targets in the ESTs of B. tabaci. In planta expression of the desired miR gene was performed to produce stable and continuous generation of dsRNA. Transformed cotton (Gossypiumhirsutum) lines were checked for the integration of the transgene. The transcript level of the miR gene was quantified using qRT-PCR assay. Cotton plants showing significantly higher levels of the miR gene were selected. Significant level of whitefly mortality was achieved upon infestation of the insect on the transformed cotton lines. Indicating that miRNAs (or their precursor, dsRNAs) thus generated in the transformed cotton plants were capable to downregulate vital genes of B. tabaci. Thus, showing the potential for controlling insect pests.

Identification of siRNA hotspots in Begomoviruses causing leaf curl disease

Saurabh Verma^1*, Pradhyumna Kumar Singh², Shirish A. Ranade² and Sangeeta Saxena¹

Email ID for Correspondence: saurabhv1982@gmail.com

¹Department of Biotechnology, School of Biosciences and Biotechnology, BabasahebBhimraoAmbedkar University, Raebareli Road, Lucknow-226025, India; ²Molecular Biology and Genetics Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, India

Leaf curl disease caused by begomoviruses is a major threat to food productivity in Asian and American continents causing great economic losses to several cash crops including tomato, cotton, pepper, cucurbits and papaya. Apart from leaf anatomy, it alters the plant physiology and fruit production and can eventually lead to plant death. In last few years, small interfering RNA (siRNA) based RNA interference (RNAi) strategy has been successful to some extent in imparting resistance against leaf curl disease. Most of the strategies focussed on DNA A as the major siRNA target. The aim of this study is to identify siRNAs that could provide broad spectrum resistance i.e., effective against more than one strain of leaf curl causing begomovirus. The putative regions (HOT SPOTS) that could act as a source of efficient siRNAs were identified, which when incorporated in a hairpin construct could provide resistance against leaf curl causing begomoviruses. The DNA B component carries movement and nuclear shuttle protein open reading frames (ORFs) responsible for systemic spread in case of bipartite begomoviruses. Therefore, targeting this component may result in inhibition of systemic spread of virus particles in crop plants. A highly conserved HOT SPOT will confer resistance against more than one type of begomovirus causing leaf curl disease. Few HOT SPOTS that produce thermodynamically favourable and sequence specific siRNAs were identified. These putative siRNA HOT SPOTS could act as a cue for various RNAi strategies involving single or multiple siRNAs derived from DNA A and/or DNA B in a single construct. Thus, this study might help in developing begomovirus resistant crops for sustainable food production and security.

Cotton leaf curl virus bidirectional Promoter over Cauliflower mosaic virus 35S Promoter: a Comparative Study

Zainul A. Khan^*, Jawaid A. Khan

Email ID for Correspondence: zainulbiotechnology@gmail.com

Plant Virus Laboratory, Department of Biosciences, JamiaMilliaIslamia (Central University), New Delhi-110025, India

Cotton leaf curl virus belongs to the genus Begomovirus of family Geminiviridae and transmitted by whitefly (Bemisiatabaci). The intergenic region of begomoviruses possess bidirectional promoter. Bidirectional promoters possess the capability of expressing two genes simultaneously, thereby making them superior and functionally more efficient than the normal unidirectional promoter. Insufficient availability of such promoters arises a need and demand for designing and developing functional bidirectional promoters for use in plant molecular biology. In this study the bidirectional promoter representing complementary sense and virion sense gene of Cotton leaf curl Burewala virus (CLCuBuV) were isolated and characterized. The bidirectional promoter sequence was initially analyzed using PlanatCARE, PLACE, Cister and PlantPAN databases. The transcription strength of these promoters was assayed both in stable and transient expression systems in tobacco as well as cotton plants, and was compared with 35S promoter of Cauliflower mosaic virus (CaMV), which is more frequently used in plant genetic engineering. The bidirectional promoter of CLCuBuV and CaMV 35S promter were fused with GUS and GFP reporter genes and was quantified using fluorometric GUS assay, reverse transcription quantitative real-time PCR and confocal laser scanning microscopy (CLSM). The expression level of GUS driven by CLCuBuV complementary sense promoter in the transformed tobacco (Nicotiana tabacum) plants was shown to be fourfold higher than that of CaMV 35S promoter, while the expression by CLCuBuV virion sense promoter was slightly lower than that of CaMV 35S promoter. Further, the expression of GFP was monitored and compared in agro-infiltrated leaves of N. benthamiana, N. tabacum and Gossypiumhirsutum plants using CLSM. CLCuBuV complementary sense promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to CaMV 35S promoter. The strong constitutive CLCuBuV bidirectional promoter developed in this study could be very useful for high level constitutive expression of transgenes in a wide variety of plant cells.

Potential of a phage single-stranded DNA binding protein to control a geminivirus infection in transgenic plants

Ghulam Rasool,SumairaYousaf^¶, ShahidMansoor, Rob W. Briddon, Muhammad Saeed

Email ID for Correspondence: ghulamrasool3126@gmail.com

Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Jhang Road, Faisalabad 38000, Pakistan; ^¶Current address: Nuclear Institute for Agriculture and Biology, P O Box # 128, Faisalabad, Pakistan

Begomoviruses (family Geminiviridae) are a serious problem for the cultivation of crops in the warmer parts of the world. The study here has investigated the use of the sequence non-specific, ssDNA-binding protein G5, encoded by Escherichia coli phage M13, to obtain resistance to geminiviruses. Nicotiana benthamiana plants were transformed with G5 alone and with G5 fused to the maize opaque-2 nuclear localization signal (NLS) under the control of Cauliflower mosaic virus 35S promoter. The resistance was evaluate by inoculation with infectious clones of Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan betasatellite (CLCuMuB), the complex which causes cotton leaf curl disease across Pakistan and northwestern India, and the bipartite begomovirus Tomato leaf curl New Delhi virus (ToLCNDV). The transgenic plants transformed with G5 alone developed severe viral infection and accumulated large amount of virus DNA upon inoculation with CLCuKoV-CLCuMuB or with ToLCNDV. In contrast transgenic plants expressing G5 fused to NLS developed mild symptoms and accumulated low virus/satellite DNA. These results indicate that the G5 protein has the potential to impart broad-spectrum resistance against diverse ssDNA viruses in plants.

RNAi mediated broad spectrum resistance against chilli infecting begomoviruses

Nivedita Sharma,Veerendra Kumar Sharma and Supriya Chakraborty^*

Email ID for Correspondence: niveditaaau@gmail.com

Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

Chilli (Capsicum sp.) is an important vegetable crop in the Indian sub continent. Over the years, Chilli leaf curl virus (ChiLCV), a monopartite begomovirus (family Geminiviridae) has emerged as a serious threat to chilli production. Because of the social and economic importance of chilli as a rich source of vitamins in everyday diet of over a billion people worldwide, we attempted to generate transgenic chilli resistant to chilli-infecting begomoviruses using RNAi mediated gene silencing approach. For this purpose, two different hairpin RNAi constructs namely, TR1 (AC1/AC2) and TR4 (AV1/AV2) were used to generate transgenic plants of Nicotiana benthamiana and chilli (cv. Kasi Anmol) by Agrobacterium mediated transformation method. Two lines harboring TR1 construct (TR1-15 and TR1-8) and two lines harboring TR4 construct (TR4-1and TR4-2) showed resistance to the most predominant chilli-infecting begomoviruses. PCR analysis confirmed that the gene had been integrated and segregation analysis was further carried out to identify the lines (3:1 ratio) in T1 generation. Resistant lines of T1 generation showed no phenotypic abnormalities following infection with begomoviruses. Resistant lines accumulated transgene-specific siRNAs, confirming RNAi mediated resistance against begomoviruses. Furthermore, these resistant lines also displayed delayed and milder symptoms, as compared to virus inoculated non-transgenic plants. Average viral DNA accumulation in the resistant lines was reduced up to 90% as compared to non-transgenic plants. Thus, our studies demonstrate that the RNAi-mediated approach can confer resistance against diverse monopartite and bipartite begomoviruses associated with Chilli leaf curl disease.

Suppression of Chayote enation yellow mosaic disease in Chayote (Chow – chow) Sechiumedule.Sacq.sw

B. Sangeetha^1*, V. G. Malathi¹, D. Alice¹, P. Renukadevi¹ and M. Suganthy²

Email ID for Correspondence: sangeethaagri5@gmailcom

¹Department of Plant Pathology, ²Department of Agricultural Entomology; ²Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India

Chayote (Chow–chow-Sechiumedule.Sacq.sw, Family: Cucurbitaceae) fruits are rich in carbohydrates, minerals, vitamins, particularly vitamin A and C. Chayote is cultivated in Kodaikanal, Thandikudi and Sirumalai hills of Dindigul District and Kotagiri of Nilgiris District of Tamil Nadu. The crop is affected by a Chayote enation yellow mosaic disease which show symptoms such as characteristic yellow spots, mosaic, puckering, curling and occasional enations on the under surface of the leaf. This disease is caused by Chayote enation yellow mosaic virus (ChEYMV) which results in more than 90% yield loss. Since the yield loss was found to be heavy, an attempt has been made to manage the disease by using bioagents and antiviral principles (AVPs). In the management trial, uniformly in all the treatments, the seeds were treated with 0.5% of Bacillus amyloliquefaciens (Talc based formulation) for 20 min followed by foliar sprays of respective components (Bioagents and AVPs) at 60 DAS and at 90 DAS. Among ten treatments followed in various combinations, the foliar spray of root extract of Mirabilis jalapa (0.25%) recorded maximum yield of 34.7 tonnes per hectare followed by foliar spray of root extract of M. jalapa (0.25%) +Butter milk spray (10%) with 32.7 tonnes per hectare and foliar spray of Bougainvillea spectabilisleaf extract (10%) with 30 tonnes per hectare as against 18.7 tonnes per hectare in untreated control. Percent yield increase in treated plants over control was 46.0, 42.8 and 37.6% respectively as against control. The percent disease incidence was also found to be reduced in foliar sprays of Mirabilis jalapa, M. jalapa + Butter milk spray and Bougainvillea spectabilisas 1.0, 6.7 and 11.0% respectively as against 83.0% in untreated control.

In silico exploration of RNAi-mediated defence strategy against Cotton leaf curl Allahabad virus

Shweta^1,2*, Manoj Prasad² and Jawaid A Khan¹

Email ID for Correspondence: shweta.bioscience@gmail.com

¹Department of Biosciences, JamiaMilliaIslamia, New Delhi 110025, India; ²National Institute of Plant Genome Research, New Delhi 110067, India

Cotton leaf curl Allahabad virus (CLCuAV), a member of genus Begomovirus associated with cotton leaf curl disease transmitted by Whitefly (Bemisiatabaci). Its single stranded monopartite circular DNA genome, encodes V1 and V2 genes in sense direction while C1, C2, C3, C4 and C5 in complementary sense direction. MicroRNAs (miRNAs) are endogeneous small RNAs and play key role in plant development and stress resistance. miRNAs suppress expression of genes following cleavage or translational inhibition of target messenger RNAs (mRNA) based on the complementarity between miRNA and mRNA. In silico approach was applied in present study, to identify high scoring miRNA-target pairs satisfying rules of minimum free energy and maximum complementarity, followed by their further assessment of binding potential to the genome CLCuAV. Ghr-miR2950 emerged as most potential miRNA that can target all the viral genes. Overlapping transcripts of C1–C2 genes and C1–C4 genes found to be targeted by ghr-miR394, ghr-miR395 and ghr-miR408. Wet lab validation of identified miRNAs is under process.

RNAi mediated approach to combat the begomovirus causing cotton leaf curl disease

MeeraKurulekar^*, MukeshKharat, KalyaniSarwadnya, Deepratan Kumar, Ganapati Bhat, Paresh Auti, DipaliRane, Satish Nalawade, Sunil Kulkarni, SuyogTakte, Narendran Nair, Bharat Char, RadhamaniAnandalakshmi

Email ID for Correspondence: meera.kurulekar@mahyco.com

Plant – Virus Interactions Lab, Mahyco Research Center, Maharashtra Hybrid Seeds Pvt Ltd, Jalna-431203, Maharashtra, India

Cotton leaf curl disease caused by begomoviruses is a major limitation in cotton production in North West India. Prevalence of disease resistance breaking strains of the virus and lack of immunity against these viruses in cultivated Gossypiumhirsutum prompted us to look into transgenic approaches for developing resistance. For establishing proof of concept, truncated coat protein and transcription activator gene sequence of Cotton leaf curl Multan virus was used for the development of antisense and hairpin RNAi constructs. The constructs were introduced into Nicotiana benthamiana by Agrobacterium mediated transformation. N. benthamiana plants harboring CLCuMuV RNAi constructs when challenged with virus in controlled conditions showed significant level of virus tolerance. Encouraged by the results in N. benthamiana, different RNAi constructs were transformed in cotton and transgenic events were generated. The transgenic cotton lines (T1) were inoculated with virus in greenhouse using viruliferous whiteflies and plants were kept under observation for 60 days. RNAi constructs were able to impart significant tolerance to the virus. RNAi appears to be very promising in combating Cotton leaf curl.

Induction of resistance against tomato leaf curl Gujarat virus using external application of dsRNA molecules—initial observations

Tsewang Namgial^1,2, Athanasios Kaldis¹, Supriya Chakraborty², Andreas Voloudakis^1*

Email ID for Correspondence: avoloud@aua.gr

¹Laboratory of Plant Breeding and Biometry, Agricultural University of Athens, Athens 11855, Greece; ²School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India

Geminiviruses compose the largest plant infecting group of viruses, which are transmitted through insect vectors. Tomato leaf curl Gujarat virus (ToLCGV) is a monopartite geminivirus causing serious damage in tomato in various parts of India. RNA interference (RNAi) that is triggered by double stranded RNA (dsRNA) molecules has been established as a powerful means to control plant viruses in transgenic plants. Since transgenesis is not approved worldwide, a non-transgenic approach exploiting RNAi has been proposed to control plant viruses and is named ‘RNA-based vaccination’. Based on the developed methodology of Voloudakiset al, we designed the production of dsRNA molecules against specific target genes of ToLCGV and the 2b gene of Cucumber mosaic virus (CMV), since the later is a serious pathogen of tomato in India as well as worldwide. In the present study, dsRNAs were made for the overlapping regions of ToLCGV, namely AC1/AC4, AV1/AV2, AC1/AC4–AV1/AV2 (fusion construct) and the CMV_2b-ToCLV_AV1/AV2 (hybrid construct) and manufactured using a two-step PCR and in vitro transcription procedure. dsRNAs were manually applied by rubbing on the upper surface of carborundum-dusted leaves of 35-days old Nicotiana benthamiana plants. ToLCGV was introduced, the same day as dsRNA, in plants via agroinfiltration in the lower surface of the same leaf that dsRNA was applied onto. Our results indicated that dsRNAs for AC1/AC4 and AV1/AV2 did not induce any resistance against ToLCGV. In contrast, N. benthamiana plants that received dsRNAs for AC1/AC4-AV1/AV4 and CMV_2b-ToCGLV_AV1/AV2 exhibited a 4-day delay in ToLCGV symptom appearance. Experiments using tomato as the host for protection studies against ToLCGV are underway. If the hybrid construct CMV_2b-ToCLV_AV1/AV2 also induces resistance against CMV (experiments are underway), it will be the first time where a single dsRNA could induce resistance against two plant viruses (ToLCGV and CMV) infecting the same crop.

CRISPR/Cas9-mediated Immunity to Geminiviruses

Zahir Ali¹, Shakila Ali¹, Manal Tashkandi¹, Syed Shan-e-Ali Zaidi^1,2* and Magdy M. Mahfouz¹

Email ID for Correspondence: shan.e.ali@outlook.com

¹Laboratory of Genome Engineering, Division of Biological Sciences, 4700 King Abdullah University of Science and Technology, Thuwal, Saudi Arabia; ²Moleculaar virology and gene silencing lab, National Institute for Biotechnoloogy and Genetic Engineering, Faisalabad, Pakistan

Geminiviruses cause devastating crop losses that threaten food security. They evolve rapidly by recombination, component capture, and mutation, allowing these viruses to rapidly counter or evade introduced resistance strategies. Clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR associated 9 (Cas9) is a prokaryotic molecular immunity system against invading viruses and has been harnessed as a powerful tool for targeted genomic editing. Recently we have demonstrated that the CRISPR/Cas9 system could be harnessed to confer resistance against geminiviruses in plants by using sgRNAs designed to target viral genomic DNAs (Ali et al. 2015). We showed that CRISPR/Cas9 technology could impart molecular immunity against three geminiviruses [i.e., Tomato yellow leaf curl virus (TYLCV), Beet curly top virus (BCTV), and Merremia mosaic virus (MeMV)] in Nicotiana benthamiana plants, and revealed that a sgRNA designed to target a conserved sequence (TAATATTAC) in the viral intergenic region could be used to target multiple geminiviruses simultaneously. This sequence is conserved among geminiviruses and is also a hallmark of the betasatellites of begomoviruses. In a subsequent research spotlight we have highlighted that this approach could effectively impart resistance to multiple viruses under natural conditions, where mixed infections predominate (Zaidi et al. 2016). We have also evaluated the differential targeting and possible evasion of geminiviruses in plants having CRISPR/Cas machinery. I would like to present this recently developed technology accompanied with our latest data.

Screening for resistance to tomato yellow leaf curl disease-inducing viruses in different tomato varieties

Saeid Tabein^1*, Nafiseh Tousi^2*, Omid Eini², S. Ali Akbar Behjatnia¹, Gian Paolo Accotto³, Luca Laviano⁴, Nicola Pecchioni⁴, Emanuela Noris³, Laura Miozzi³

Email ID for Correspondence: saeid.tabein@ipsp.cnr.it

¹Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz, Iran; ²Department of Plant Protection, College of Agriculture, Zanjan University, Zanjan, Iran; ³Institute for Sustainable Plant Protection, National Research Council of Italy (IPSP-CNR), Torino, Italy; ⁴Department of Life Science, University of Modena and Reggio Emilia, Reggio Emilia, Italy

Developing cultivars resistant to tomato yellow leaf curl disease (TYLCD) is a big challenge for tomato breeders. TYLCD is induced by several begomoviruses and is a major constraint for tomato production worldwide. A screening survey was undertaken to evaluate the level of resistance to TYLCD-inducing viruses in several tomato cultivars, mostly cultivated in the Fars province. In our survey, plants were challenged under controlled greenhouse conditions with agroclones of Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), using the Moneymaker cultivar as susceptible control. Four weeks after inoculation, 4 of the 12 cultivars tested with both viruses developed disease severity indexes (DSI) ranging from 0 to 10%. Conversely, the other cultivars, including Moneymaker, resulted highly symptomatic. Molecular hybridization revealed that in the cultivars with low DSI%, virus incidence accounted to 0–70%, with the lowest values in the experiments where TYLCSV was tested. On the contrary, in all the cultivars highly symptomatic, virus incidence was greater than 70% and in most cases accounted to 100%. In some instances where virus could not be detected by molecular hybridization, quantitative PCR allowed us to evaluate that viral DNA varied from 4 × 10⁻² to 3.1 × 10⁻⁴ fold lower than in Moneymaker plants. The results will be discussed considering the presence of the resistance genes Ty-1, Ty-2 and Ty-3 in the tested cultivars in this survey.

RNAi mediated control of SLCMV in a commercially important Indian cassava cultivar

E. Bull Simon^1*, N. Abraham Leen², Kumar Vasudevan^3*, Thangaraj Makeshkumar², Gruissem Wilhelm¹ and Vanderschuren Hervé^1,3

Email ID for Correspondence: sbull@ethz.ch; kvasudevan@ulg.ac.be

¹Plant Biotechnology Group, Department of Biology, ETH Zurich, Universitaetstrasse 2, 8092 Zurich, Switzerland, ²Central Tuber Crops Research Institute, Thiruvananthapuram 695017, India, ³Current address: Gembloux Agro-Bio Tech, University of Liege, 5030 Gembloux, Belgium

Cassava mosaic disease (CMD) is a major constraint to food security and commercial production of cassava (ManihotesculentaCrantz) in southern India. In India, CMD is caused by Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV)—species of bipartite begomoviruses (family Geminiviridae) that are transmitted by whiteflies (Bemisiatabaci). Chimeric RNAi constructs targeting the replication protein coding sequence (AC1; Rep) of the recently sequenced SLCMV strains isolated from Kerala and Tamil Nadu regions have been designed and used for Agrobacterium-mediated stable transformation of cassava. In addition to the model cassava cultivar 60444, we assessed the capacity of the commercially important H165 to produce friable, embryogenic callus (FEC) required for transformation, as well as their ability to regenerate into healthy plantlets. Successfully transformed independent lines are being characterised using molecular techniques and screened via inoculation with infectious clones of SLCMV and ICMV. This project aims to generate commercially important cassava resistant/tolerant to ICMV and SLCMV in southern India.

Higher expression of isoflavonoid in soybean genotypes mitigate Mungbean yellow mosaic India virus (MYMIV) multiplication

Om Prakash Gupta^1*#, Sandeep Kumar¹, Bhagat S. Chouhan², S.V. Ramesh³, Shelly Praveen¹ and Anil Dahuja^1*

Email ID for Correspondence: opguptaiari@gmail.com

¹Division of Biochemistry, ICAR-Indian Agricultural Research Institute (ICAR-IARI), New Delhi, India; ²School of Life Sciences, Devi AhilyaVishwavidyalaya, Indore, India; ³ICAR-Indian Institute of Soybean Research (ICAR-IISR), Indore, India; ^#ICAR-Indian Institute of Wheat and Barley Research (ICAR-IIWBR), Karnal, India

Soybean is infected by numerous viruses causing severe yield losses. In Indian context, infection due to Begomoviruses, Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) causes yield reduction to the tune of 5–90%. We observed that the losses by these viruses are genotype dependent. Soybean genotypes showing higher expression of isoflavonoidphytoalexin pathway mitigate MYMV multiplication. Isoflavonoids play crucial role in plant defence, plant–microbe interaction, abiotic stresses, and in animals as nutraceuticals. Phytoalexins such as soybean glyceollin, are antimicrobial compound synthesized from universal phenylpropanoid pathway starting with phenylalanine in response to pathogen attack. It exerts its effect by cytoplasmic granulation, disorganization of the cellular contents, rupture of the plasma membrane etc. In the present study, we did isoflavone profiling in eleven commonly cultivated Indian soybean genotypes using HPLC and determined their total isoflavone content which showed a range of 155–1151 μg/g. Among all the genotypes examined, Bragg genotype showed maximum accumulation of isoflavonoid (~1151 μg/g) which incidentally also exhibited least MYMI virus infection load (3.0). Our results indicated that isoflavonoid content of soybean genotypes could be negatively correlated with MYMIV infection and could potentially be utilized as a marker for screening MYMIV-resistant soybean genotypes.

Screening Bhendi yellow vein mosaic virus genes for RNAi suppressor activity

Rashmi Rishishwar^*, LishaKhungar, Madhvi Naresh, FauziaZarreen and IndranilDasgupta

Email ID for Correspondence: rishishwar.rashmi@gmail.com

Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021

Bhendi/Okra, a common vegetable in the Indian subcontinent, is highly susceptible to a viral disease termed as Bhendi yellow vein mosaic disease (BYVMD), resulting in considerable yield loss to the crop. BYVMD has been shown to be caused by a complex of a monopartite begomovirus, Bhendi yellow vein mosaic virus (BYVMV) and a betasatellite. In a survey of BYVMD-affected okra from various parts of India, we found a recombinant viral DNA, having Mesta yellow vein mosaic virus and Malvastrum yellow vein Yunnan virus, to be associated with BYVMD. Plants employ RNA interference as defence mechanism to fight against viruses. Many plant viruses, in turn, are known to encode suppressors of RNA silencing called viral suppressors of RNA silencing (VSRs). In this study, we screened the V2, C2, C4 and C5 ORFs of BYVMV and the recombinant molecule, to check for suppressor activities. For this, GUS and GFP expression assays were performed. GUS was transiently expressed in wild type Nicotiana benthamiana using pBI121 (contains GUS under CaMV35S promoter), while co-infiltration of pBI121 and GUS hairpin construct (strong inducer of RNA silencing) silenced GUS expression. Co-infiltration of the leaves of N. benthamiana with pBI121, GUS hairpin and a silencing suppressor (test ORFs) would cause reversal of GUS expression. For GFP assay, GFP was transiently expressed (an inducer of s-PTGS) in transgenic GFP expressing N. benthamiana 16C plants using GFP-pBI121 (contains GFP under CaMV35S promoter). The GFP expression would transiently silence the endogenous GFP expression in 16C plants, while co-infiltration of GFP-pBI121 and a suppressor of silencing (2b-pBI121) construct would retain GFP expression. Co-infiltration of the leaves of N. benthamiana 16C with GFP-pBI121 and test ORFs allowed testing of OFRs for their RNAi suppression activity at local as well as systemic level.

The replication initiator protein (Rep) of a geminivirus recruits host mono-ubiquitination machinery on the viral promoter and stimulates transcription of the viral genome

Mansi^*, Nirbhay Kumar Kushwaha and Supriya Chakraborty

Email ID for Correspondence: mansi.botany@gmail.com

Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India

Geminiviruses are a group of plant viruses with ssDNA genome, which replicate in the nucleus of the infected cell. For efficient and orderly transcription from viral genomic DNA, recruitment of machineries responsible for histone post-translational modifications (PTMs) on viral promoter is essential. Replication initiator protein (Rep) encoded by a geminivirus is an early protein that is indispensible for viral replication to occur in a permissive host. Chilli leaf curl virus (ChiLCV) is a monopartite geminivirus that can replicate in permissive host Nicotiana benthamiana. The present study evidently proves that deposition of histone 2B monoubiquitination (ubH2B) promotes tri-methylation of histone 3 at lysine 4 (H3K4me3) on the promoter of ChiLCV. We isolated homologues of two major components of monoubiquitination machinery UBIQUITIN CONJUGATING ENZYME (NbUBC2) and HISTONE MONOUBIQUITINATION1 (NbHUB1) from N. benthamiana. ChiLCV failed to cause disease in NbUBC2 and NbHUB1 VIGS-silenced plants, concurrently, H2B-ub and H3-K4me3 modifications were also decreased, and the occupancy of RNA polymerase II on the viral promoter was reduced. As expected, accumulation of both transcripts and DNA of ChiLCV were also decreased in the silenced plants. Rep interacts and co-localizes with NbHUB1 and NbUBC2 in the nuclei of the infected cells. Furthermore, silencing of either NbUBC2 or NbHUB1 resulted in global cellular level of H3K4me3 and H2Bub. Rep binds specifically to the viral genome but not with actin DNA, suggesting recruitment of monoubiquitination machineries for specific stimulation of viral transcription. In sum, the current study reveals that ChiLCV Rep protein binds on the viral genome and recruits NbUBC2 and NbHUB1 for monoubiquitination of histones 2B that subsequently promotes H3K4me3 on ChiLCV mini-chromosomes and enhances transcription of the viral genes.

Changes in the Pepper golden mosaic virus minichromosome population after inoculation of recovered plants with Pepper huasteco yellow vein virus

Rodriguez-Gandarilla Myriam Guadalupe^1* and Rivera-Bustamante Rafael Francisco¹

Email ID for Correspondence: myrirod@gmail.com

¹Departamento de IngenieríaGenética, Centro de Investigación y de EstudiosAvanzadosdel IPN, Cinvestav-Irapuato; Irapuato, Guanajuato, México

In Mexico, since prehispanic days, pepper (Capsicum annuum) has been a very important crop, as a basic ingredient in Mexican cuisine, and as a main economic product. However, production of this crop is severely affected by diseases caused by two geminiviruses: Pepper golden mosaic virus (PepGMV) and Pepper huasteco yellow vein virus (PHYVV). Geminiviruses are important plant pathogens that affect crops around the world; geminiviral genome is a circular single-stranded DNA generates a replication intermediate double stranded DNA which is associated with nucleosomes to form a minichromosome, like cellular chromatin is subject to epigenetic regulation. Pepper plants in single infections under controlled conditions (light, temperature and humidity) present the phenomenon known as recovery. Recently our group showed that the PepGMVminichromosomes in recovered tissue were condensed and highly methylated compared to the minichromosomes obtained from symptomatic tissue (Ceniceroset al., 2016). However, if the recovered plants are inoculated with a second virus (PHYVV), symptoms appear again on the newly emerged tissue. In addition, the symptoms observed showed an increased severity compared with the ones observed in the initial symptom expression. This indicates a synergism. To characterize the changes in PepGMVminichromosome population caused by the second infection (PHYVV), pepper plants were first inoculated with PepGMV and when recovery was observed, the second inoculation with PHYVV was carried out. Leaf tissue was collected when the new symptomatic tissue emerged. The PepGMVminichromosome conformation (based in sedimentation rate) was determined by sucrose gradient centrifugation and subsequent Southern blot analysis. We found that most of PepGMV DNA is detected in the light fractions suggesting that the minichromosome population is predominantly in a relaxed conformation after reemergence of symptoms. Analysis of methylation level of viral DNA (bisulfite sequencing) will be presented and discussed.

Identification of siRNA hotspots in Begomoviruses causing leaf curl disease

Saurabh Verma^1*, Pradhyumna Kumar Singh², Shirish A. Ranade² and Sangeeta Saxena¹

Email ID for Correspondence: saurabhv1982@gmail.com

¹Department of Biotechnology, School of Biosciences and Biotechnology, BabasahebBhimraoAmbedkar University, Raebareli Road, Lucknow-226025, India; ²Molecular Biology and Genetics Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, India

Leaf curl disease caused by begomoviruses is an imminent threat to food productivity in Asian and American continents causing great economic losses to tomato, cotton, pepper, cucurbits, papaya and other cash crops. Apart from leaf anatomy, it alters the plant physiology and fruit production which eventually leads to plant death. In last few years, small interfering RNA (siRNA) based RNA interference (RNAi) strategy has been successful to some extent in imparting resistance against leaf curl disease. Most of the strategies focussed on DNA A as the major siRNA target. The aim of this study is to identify siRNAs that could provide broad spectrum resistance i.e., effective against more than one strain of leaf curl causing begomovirus. The putative regions (HOT SPOTS) that could act as a source of efficient siRNAs were identified, which when incorporated in a hairpin construct could provide resistance against leaf curl causing begomoviruses. The DNA B component carries movement and nuclear shuttle protein open reading frames (ORFs) responsible for systemic spread in case of bipartite begomoviruses. Therefore, targeting this component may result in inhibition of systemic spread of virus particles in crop plants. A highly conserved HOT SPOT will confer resistance against more than one type of begomovirus causing leaf curl disease. Few HOT SPOTS that produce thermodynamically favourable and sequence specific siRNAs were identified. These putative siRNA HOT SPOTS could act as a que for various RNAi strategies involving single or multiple siRNAs derived from DNA A and/or DNA B in a single construct. Thus, this study might help in developing begomovirus resistant crops for sustainable food production and security.

A novel role of the βC1 protein in symptom induction by targeting chloroplast machinery

PrabuGnanasekaran^*, Dhriti Bhattacharyya, R Kishore Kumar, Nirbhay Kumar Kushwaha, and Supriya Chakraborty

Email ID for Correspondence: prabubc@gmail.com

Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India

Geminiviruses are known for their potency to interfere with plant physiology and cause huge economic losses to a wide range of vegetable crops like tomato, chilli, pumpkin, radish and okra. Vein clearing symptom is often associated with geminivirus-infected plants. Recently betasatellite, a group of DNA satellite has emerged as a major threat to crop production worldwide and acts as a key regulator for symptom induction. In this study, Nicotiana benthamiana—Radish leaf curl betasatellite (RaLCB) has been used as model system to understand the host-betasatellite interactions and symptom development. RaLCB initiates leaf curling, vein clearing and severe veinal chlorosis on infected plants; however, in the absence of βC1 protein, RaLCB failed to induce vein clearing symptoms. In the course of infection, βC1 protein localizes into chloroplasts of the host cells and damages chloroplast ultrastructure. In addition, the betasatellite downregulates expression of genes involved in chlorophyll biosynthesis, chloroplast development and plastid translocation. Interestingly, the expression of key host genes involved in chlorophyll degradation remains unaffected indicating selective suppression of host metabolism. Betasatellite infection drastically reduces the numbers of active reaction centres and plastoquinol pool size in leaves exhibiting vein clearing symptom. Betasatellite mediated impediments at different levels of chloroplast functionality affects photosynthetic efficiency of N. benthamiana. This study provides the first evidence of chloroplast localization of a DNA virus encoded protein which in turn affects photosynthesis. Further, the role of differentially expressed chloroplast protein in geminivirus pathogenesis has been studied.

Studies on the expression profiles and functions of micro RNA in begomoviruses-infected Abelmoschusesculentus plants

V. Kavitha^*, S. Nagesh and P. Gopal

Email ID for Correspondence: kavithabt28@gmail.com

Department of Plant Biotechnology, School of Biotechnology, Madurai KamarajUniversiy, Madurai-21

Bhendi (Abelmoschusesculentus) is an economically important vegetable crop. It is widely grown in Asia, Africa, Southern Europe and America for food. Begomoviruses belong to Geminiviridae family, having either bipartite or monopartite genomes. MicroRNAs are 21 nucleotides in size, known to play crucial role in pathogen invasion in various plants. The aim of this work is to study miRNAs function upon Bhendi yellow vein mosaic virus or Okra enation leaf curl virus infection and unravel how virus is exploiting the host machinery for its survival. Towards achieving the same, small RNA sequencing was performed from Bhendi plants by using Ion torrent and Illumina platforms. Data analysis revealed presence of 1768 miRNAs including 1505 novel candidates. In order to capture the precursor miRNA for known and novel candidates, we designed a suitable methodology and performed NGS by using Illumina platform. Through data analysis, we found 9 and 80 precursors for known and novel miRNAs respectively. In many cases, we found mature miRNA but not ^*miRNA suggesting that it may follow the non-canonical pathway for the biogenesis. We are in process to study the differential expression profiles of miRNA in Bhendi yellow vein mosaic virus or Okra enation leaf curl virus infected plants. During conference, these results will be presented.

Whole transcriptome profiling of Abelmoschusesculentus (Bhendi) for the effective control of yellow vein mosaic disease

P. Priyavathi, S. Nagesh and P. Gopal^*

Email ID for Correspondence: pgopal.biotech@mkuniversity.org, pgp79@yahoo.com

Department of Plant Biotechnology, School of Biotechnology and Madurai Kamaraj University, Madurai-625021

Bhendi (Abelmoschusesculentus) is an important vegetable crop widely cultivated in the developing countries of Asia and Africa for to its rich source of dietary fibre, vitamins (A, B, C, K) and antioxidants. India ranks first in the cultivation and contributes 72% of the total world production. Major constraint of bhendi production is due to the occurrence of yellow vein mosaic disease caused byBhendi yellow vein mosaic virus (BYVMV) results in the marketable yield loss of 50–94%. In order to develop a resistant cultivar either through breeding or genetic engineering approaches against BYVMV, sequence information of bhendi is essential. In addition, lack of genome sequence and low quality transcriptome data in the database hampers the understanding of virus-host interaction. In the present study, a comprehensive transcriptome assembly for bhendi has developed by analyzing 288 million Illumina Nextseq500 paired end reads from 4 samples. The resultant de novo transcriptome assembly comprised 3, 56, 271 contigs and 49,375 unigenes with the N50 value of 1657 and 1546 respectively. The unigenes were searched against Nr database. A total of 37,627 unigenes (76.2%) had BLAST hit. Annotated unigenes were also assigned to gene ontology and kytoEncylopedia of Genes and Genomes pathway database (KEGG). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis of selected transcripts further confirmed the quality of assembly from RNA-seq data. Similarly, transcriptome profiling will be performed with BYVMV-infected plants to identify the modulated transcripts upon infection. These results will be discussed in detail during the presentation.

AV2 protein of Tomato leaf curl Palampur virus contributes to virulence and interacts with F-box Kelch protein

Poonam Roshan^1,2* and Vipin Hallan^1,2

Email ID for Correspondence: pr4dreams@gmail.com

¹Plant Virus Lab, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh; ²Academy of Scientific and Innovative Research (AcSIR), New Delhi, India

Tomato leaf curl Palampur virus (ToLCPMV) is a bipartite begomovirus and DNA-A encodes ~14 kDa AV2 protein in sense orientation. It is well known that viral proteins associate with host proteins for proliferation, symptom development and suppression of defense system. Previously, AV2 has been worked out as pathogencity determinant protein via PVX based vectors. In the present study we have done the infectivity analysis of agroinfectiousToLCPMV virus clones without expressing AV2 ORF on N. benthamiana. We found that infectious clones lacking AV2 did not show typical virus symptoms and in southern blot analysis, coat protein accumulation was also reduced. In order to find host protein/s interacting with AV2, we performed yeast two hybrid assay using tomato cDNA library as prey and AV2 as bait. Interacting partners were able to grow on selective media (Gal/Raff-UHTL). On sequencing of the respective plasmid from positive colony, it showed maximum identity with F-box kelch like protein. F-box kelch protein belongs to E3 ligase; a component of Ubiquitin Proteasome Pathway (UPP). It consists of N-terminal F-box domain and C-terminal kelch domain (protein/substrate binding domain). AV2 associates with kelch domain of F-box keclh protein. Based on theses finding, we propose that AV2 is a key viral factor for symptom development and involved in the regulation/perturbation of UPP for virus spread.

Analysis of Nicotiana benthamiana degradome using high definition adapters reveals targets of artificial tasiRNAs against Tomato leaf curl New Delhi virus

Archana^1,2*, Irina-Ioana Mohorianu², Tamas Dalmay², Sunil Kumar Mukherjee³ and Indranil Dasgupta¹

Email ID for Correspondence: a.archana@uea.ac.uk

¹Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India; ²School of Biological Sciences, University of East Anglia, Norwich, UK; ³NER-BPMC, Department of Biotechnology, Government of India, New Delhi, India

Tomato leaf curl New Delhi virus (ToLCNDV), a begomovirus, causes Leaf curl disease of tomato in the Indian subcontinent, which can result in losses up to 100%. Nicotiana benthamiana, the model laboratory plant can be usedto understand the interactions between ToLCNDV and its host because the symptoms are similar to naturally infected tomato e.g. upward curling of the leaflet margins, reduction in the leaflet area, swelling of the veins. RNA interference (RNAi) is a conserved, RNA-mediated, gene regulatory mechanism in eukaryotes. In plants, it plays an important role in growth, development and resistance against viral infections. As a counter-defence, plant viruses encode RNAi suppressors; examples of such suppressors in begomoviruses include AC2, AC4 and AV2. We employed engineered artificial trans-acting siRNAs (ata-siRNAs) targeting the ToLCNDV AC2 and AC4, using miRNA390 dual target sites to obtain ToLCNDV resistant N. benthamiana plants. The mode of action of ata-siRNAs comprises of the cleavage of the target (similar to the miRNA targeting). Employing a degradome approach using High Definition sequencing adapters, the abundance of the resulting 3′ fragments of the cleaved transcript can be accurately quantified and the precise localization of the cleavage on the target mRNA can be identified. To determine the targets of the ata-siRNAs, we sequenced degradome libraries of N. benthamiana plants infected with ToLCNDV, which were treated with the atasi-RNA-AC2 construct. Mock treated plants were used as controls. Following quality checks, the abundance distributions of the degradation fragments were normalized. The transcript with different cleavage patterns was the AC2 supporting the conclusion that an efficient cleavage of the target occurred, without significant off-target effects.

Geminivirus infection in opium poppy renders metabolite biosynthesis and accelerates programmed cell death besides perturbing plant’s architecture

Susheel Kumar^*, Ashish Srivastava, Shri Krishna Raj

Email ID for Correspondence: susheelnbri@gmail.com

Plant Molecular Virology Laboratory, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, India

Opium poppy (Papaver sominiferum) plants exhibiting leaf curl, enation and stunting symptoms were observed in experimental plots of CSIR-NBRI with which the association of Ageratum enation virus (AEV) and Ageratum leaf curl betasatellite (ALCB) was identified. Virus infection renders structural anomalies as evident by the unorganized cell architecture, vascular bundles, more lignified cell wall and intensified leticifierous tissue in infected leaf and stem tissues. Blebs and membranous vesicles formed due to the disintegration of plasma membrane and intracellular organelles were also observed under TEM. Together with histological study, comet assay reveals nuclear DNA fragmentation suggesting an induced programmed cell death (PCD). Moreover, AEV infected plants lose synthesis potential of morphine, thebaine, codein and papaverine alkaloids except noscapine whose synthesis was increased which was well correlated with the expression analysis of their respective genes. Metabolomic analysis reveals perturbation in respiration cycle intermediates and organic acids resulting in decreased photosynthesis and respiration, while enhanced production of glucose and other carbohydrates was noticed revealing their role in virus induced stress signaling. This is the first extensive study unraveling the altered metabolic and alkaloid status and induced PCD in opium poppy plants due to begomovirus-(AEV) infection.

AC4 protein of Tomato leaf curl Palampur virus is a pathogenicity determinant and it interacts with Oxygen evolving enhancer protein 1 (OEE1) of Solanum lycopersicum

Aditya Kulshreshtha^1,2* and Vipin Hallan^1,2

Email ID for Correspondence: aditya.ihbt@gmail.com

¹Plant Virus Lab, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh; ²Academy of Scientific and Innovative Research (AcSIR), New Delhi, India

Tomato leaf curl Palampur virus was first characterized from Northern India infecting tomatoes. The virus was also found associated with several crop species. The virus has a bipartite genome. DNA-A of the virus encodes four proteins, AV1 and AV2 in sense orientation while AC1, AC2, AC3 and AC4 in antisense orientation. To find out the possible role of AC4 ORF in disease development, it was cloned in PVX based vector pGR106 at NotI and SalI restriction sites. The resulting construct (pGR-AC4) and empty vector (pGR106) were mobilised in agrobacterium strain GV3101. Nicotiana benthamiana plants were agro-inoculated with these constructs. After 10 days post inoculation, plants inoculated with pGR-AC4 developed leaf deformation, downward leaf curling, necrosis and leaf epinasty while vector inoculated plants showed typical symptoms of PVX infection. To understand the role of AC4 protein and to identify interactors, yeast two hybrid analysis was performed with tomato c-DNA library using AC4 as bait. AC4 protein was found to interact with tomato Oxygen evolving enhancer protein 1 (OEE1) on a selective nutritional growth media YNB (gal/raff)-UTHL. OEE1 is a part of Oxygen evolving complex (OEC) which is involved in oxidation of water during light reaction of photosynthesis. OEE1 is also essential for the stability of OEC. The results suggest that the virus effects photosynthesis via associating with OEE1 protein from the plant.

Biochemistry study of a bioactive peptide with specific binding on DNA of Tomato yellow leaf curl virus

J. S. Mendoza-Figueroa ^1*, M. Soriano-García¹, J. Méndez-Lozano², E. A. Rodríguez-Negrete², L. I. Cabrera-Lara³, A. Kvarnheden⁴, M. L. Moreno-Félix², N. Melendrez-Bojorquez²

Email ID for Correspondence: genotipoxy@gmail.com

¹Biomacromolecules Department, Chemistry Institute, Universidad Nacional Autónoma de México, México City,México; ²Agricultural Biotechnology Department, Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional Unidad Sinaloa, InstitutoPolitécnico Nacional, Guasave, Sinaloa, México; ³Physics-ChemistryDepartment, Centro Conjunto de InvestigaciónenQuímicaSustentable, Chemistry Institute, Universidad Nacional Autónoma de México, Toluca, México; ⁴Plant Biology and Forest Genetic Department, Biocenter, Swedish University of Agricultural Science, Uppsala, Sweden

Tomato yellow leaf curl virus is one of most important pathogens affecting tomatoes crops in worldwide. In Mexico, pathogen is distributed at north of country mainly in Sinaloa State. Current controls methods for this pathology are use of resistant varieties of tomato plants, chemical control of vector (whitefly) and transgenic plants, not having a direct control against virus. Biomolecules such as short peptides can binding to specific target and interfering with biological response of target. Pseudo cereals like amaranth, offers a big source of peptides due to high protein amount. The aim of work was the purification and selection of a peptide from amaranth globulin fraction with specific recognition site on DNA of Tomato yellow leaf curl virus with the goal to block the viral replication. Amaranth globulin was hydrolyzed and peptides were purified with HPLC, Surface Resonance Plasmon method was used like screening method to select peptides with specific affinity on viral DNA, then, peptide with best Kd value was used to in vitro assays. Rolling Circle Amplification was used to evaluate the effect of peptide in in vitro synthesis of viral genome. Mesophyll protoplast from Nicotiana benthamiana leaves were extracted and infected with Tomato Yellow Leaf Curl Virus, peptide was added post infection, after 12 h pi cells were fixed and hybridized with a specific probe against virus, cells were analyzed with Flow cytometry. General results show a peptide with a size of 2KDa and Kd value of 10 nM of binding Tomato Yellow Leaf Curl Virus DNA in specific recognition site. Peptide shows 70% inhibition of in vitro viral DNA synthesis. In protoplast assay, peptide decrease the TYLCV detection in cells, in N. benthamiana peptide application after 10 days post infection, delay symptoms in leaves infected.

Bioinformatics Protein–Protein Interaction Studies for Various Human and Plant Mastrevirus Proteins

Amandeep Kaur, Rajeshkumar, AvinashMarwal, R.K. Gaur^*

Email ID for Correspondence: gaurrajarshi@hotmail.com; rkgaur.fasc@modyuniversity.ac.

Department of Biosciences, College of Arts, Science and Humanities, Mody University, Lakshmangarh - 332311 Sikar, Rajasthan, India

Maize streak virus is a one of the members of the Mastrevirus genus in the family Geminiviridae. Out of the various members, maize streak virus (MSV) causes the most serious viral crop disease on the African continent. Virus proteins are found to be interacting with plant regulatory proteins. However, there is no rigid rule that plant virus could not interact with human or animals. It might have a probability that some plant families possesses direct or indirect role in human however, no such study is still available to consider them as human pathogen. Here we used protein–protein interaction studies for better understanding of the structural details of plant and human virus interacting proteins since structural detail is highly needed for comprehending biological mechanism. Here, we retrieved 9 human (beta hemoglobin, alpha hemoglobin, cyclins, cyclin dependent kinase, human keratin, Myoglobin, p21, p27, p53) and 4 maize streak virus protein structures (Rep A,Rep b, movement protein and capsid protein) by using phyre 2 server after putting their Fasta sequence in amino acid sequence box. Subsequently, each of the predicted human protein structure was docked with virus protein structuresusing Patch Dock and Fire Dock then we used Molegro molecular viewer 2.5 for viewing their interaction. Highest scoring was found during interactionof humanp21 proteinwith 4 plant virus proteins by patch dock server. Thereafter, fire dock server shows human cyclin and cdk possess highest global energy minimum after their interaction with virus Rep A and movement protein respectively. Human keratin also shows highest global energy minimum with both virus Rep B and full capsid protein. Our findings predictthe difference in the interaction abilityof some human protein with plant virus proteins.

Variation of small RNA population in Nicotiana benthamiana infected by Tobacco curly shoot virus and its betasatellite

Gentu Wu, Ke Li, Chenchen Jing, Jiangping Ren, Jiang Du, Xianchao Sun and Ling Qing^*

Email ID for Correspondence: qling@swu.edu.cn

Chongqing Key Laboratory of Plant Disease Biology, College of Plant Protection, Southwest University, Chongqing 400716, the People’s Republic of China

Tobacco curly shoot virus (TbCSV) belongs to genus Begomovirus. Some isolates of TbCSV are associated with betasatellites (TbCSB). In this study, the infectious clones of TbCSV isolate Y35 (Y35A) and its betasatellite (Y35β) were used to infiltrate Nicotiana benthamiana. The expression of microRNA in N. benthamiana was analyzed by small RNA sequencing. The results showed that there were more than 33 million clean reads in the two libraries of Y35A/Y35β infected plants and mock plants. Both in these two libraries, the length of predominant small RNA is 24 nucleotide (nt), and their frequency percentage are more than 44%. The number of 21 and 22nt long small RNA occupied 11.56 and 9.56% in total small RNA in the mock plant, respectively. However, in the infected plants, 13.71 and 14.96% of total small RNA were 21 and 22 nt length, respectively. These results indicated that more 22nt small RNA were produced in host plant induced by Y35A/Y35β infection. The results also showed that a total of 76 microRNAs were up-regulated, while a total of 69 microRNAs were down-regulated. In these differentially expressed small RNAs, 105 microRNAs were novel microRNAs. These 145 candidate microRNAs were identified using bioinformatics methods. All the known microRNAs can find their target genes in the repertoires of N. benthamiana genes set. Association studies indicate that most of target genes were categorized into DNA binding, nucleic acid binding and lipid metabolic process with reference to GO term. KEGG pathway studies showed that nearly 35% target genes were categorized into plant-pathogens interaction.

The genomes of several plant species contain endogenous geminiviral sequences

Denis Filloux^1*, Sasha Murrell,^2,3 Maneerat Koohapitagtam,^1,4 Michael Golden,² Charlotte Julian,¹ Serge Galzi,¹ Marilyne Uzest,¹ Marguerite Rodier-Goud,⁵ Angélique D’Hont,⁵ Marie Stephanie Vernerey,¹ Paul Wilkin,⁶ Stephan Winter,⁷ Ben Murrell,^2,8 Darren P. Martin,² and Philippe Roumagnac¹

Email ID for Correspondence: denis.filloux@cirad.fr

¹CIRAD-INRA-SupAgro, UMR BGPI, Campus International de Montferrier-Baillarguet, 34398 Montpellier Cedex-5, France; ²Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, Universityof Cape Town, Cape Town 4579, South Africa; ³Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA; ⁴Department of Pest Management, Faculty of Natural Resources, Prince of Songkla University, Hat Yai campus, Thailand 90120; ⁵CIRAD, UMR AGAP, TA A-108/03, Avenue Agropolis, F-34398 Montpellier Cedex 5, France; ⁶ Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK; ⁷DSMZ Plant Virus Department, Messeweg 11/12, 38102, Braunschweig, Germany; ⁸Department of Medicine, University of California, San Diego, La Jolla, CA

Endogenous viral sequences are essentially ‘fossil records’ that can sometimes reveal the genomic features of long extinctvirus species. Although numerous known instances exist of single-stranded DNA (ssDNA) genomes becoming stably integratedwithin the genomes of bacteria and animals, there remain very few examples of such integration events in plants. The best studied of these events are those which yielded the geminivirus-related DNA elements (GRD) and the geminivirus-like elements (EGV) found respectively within the nuclear genomes of several Nicotiana species and various Dioscorea spp. of the Enantiophyllum clade. Those two new classes of endogenous plant virus sequence are apparently derived from ancient geminiviruses in the genus Begomovirus. GRD and EGV sequences likely became integrated millions years ago. Interestingly, we found evidence of natural selection actively favouring the maintenance of EGV-expressed replication-associated protein (Rep) amino acid sequences, which clearly indicates that functional EGV Rep proteins were probably expressed for prolonged periods following endogenization. We recently found using in silico searches that other ssDNA virus-like sequences are included within complete or draft genomes of various plant species, including apple tree (Malus domestica), black cottonwood (Populustrichocarpa), various Coffeaspp, eggplant (Solanum melongena), lettuce (Lactucasativa), and Tepary bean (Phaseolusacutifolius), which suggests that endogenous geminiviruses may be more common in plant genomes than has previously been appreciated.

Global analysis of DEAD-Box ATP dependent RNA helicase family in tomato

Saurabh Pandey^*, Pranav Pankaj Sahu and Manoj Prasad

Email ID for Correspondence: pandey.saurabh784@nipgr.ac.in

National Institute of Plant Genome Research, New Delhi-110067

RNA helicases are highly conserved plant proteins contributing in all form of RNA metabolism. Based on sequence and comparative study all eukaryotic RNA helicases are categorized into Superfamily 1 and 2. DEAD-box belongs to the Superfamily 2. The DEAD-box helicase family is separated into three subfamilies, comprising of the DEAD-box, DEAH-box and DExD/H-box helicases, which are classified based on motif II variations. Microarray and expressed sequence tag data showed that RNA helicase protein family may play a crucial role in plant growth and development as well as in response to biotic and abiotic stresses. Though, contrasting to Arabidopsis, no in depth information about the RNA helicase family in biotic stress is currently available for tomato (Solanum lycopersicum) due to a restricted number of whole-genome sequences. In this study, we finda total of 103 RNA helicase genes in tomato genome. According to the structural features of the motif II region, we divided the tomato RNA helicase genes into DEAD-box (42), DEAH-box (18) and DExD/H-box (31) helicase genes. However, there were 12 RNA helicases which did not belong to either of three subfamilies, and further grouped into “other helicase”. We also aimed to study the role of DEAD-box helicase in the tomato against the Tomato leaf curl New Delhi virus (ToLCNDV). Based on our previous study; we selected 3 putative candidate DEAD-box helicase genes, belonging to same clade. Their differential expression pattern was confirmed with the real time PCR. Out of three genes, DEAD-box helicase SlDBP-12 showed upregulation in the tolerant cultivar H-88-78-1 during ToLCNDV infection. Further functional characterization of SlDBP-12 is in progress.

Role of C-terminal of the replication initiator protein of Tomato leaf curl New Delhi virus

RajraniRuhel,PrabuGnanasekaranand Supriya Chakraborty^*

Email ID for Correspondence: rajraniruhel@gmail.com

Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi – 110067, INDIA

Geminiviruses are plant infecting ssDNA virusesthat cause devastating diseases in a wide range of crop plants worldwide. It codes for the conserved and multifunctional replication initiator protein (Rep, AL1 or AC1) that is indispensible for viral replication. In addition, Rep is the only member of the SFIII helicase family reported to participate in rolling circle replication (RCR) in plants. Keeping in mind the importance of Rep protein in geminivirus replication, the present study was undertaken to understand nucleotide binding and hydrolysis coupled DNA unwinding activities of this protein. Tomato leaf curl New Delhi virus (ToLCNDV) Rep belongs to AAA+ (ATPase associated with various cellular activities) family of ATPases. Rep N-terminus (1–120 residues) confers endonuclease, ligase and sequence specific DNA binding activities while the ATPase and helicase activity is associated with the C terminus (120–361 residues). Residues within 120–180 form the oligomerization domain. Role of N terminus is well documented whereas information on role of C terminus is scarce. Amino acid sequence alignment withSV40 T antigen and Human Papilloma virus E1 protein resulted in identifying the conserved motifs in the C-Terminal of the ToLCNDV Rep (RepC). Based on this model, the positively charged amino acidsresiding within the predicted beta hairpin loop and B’ motif that have the potential to bind DNA were mutated to alanine by site directed mutagenesis. Circular-dichorismspectra of the mutants indicated that the mutations had not altered their overall structure. These RepC mutants were further checked for various activities such as ATPase, helicase and DNA binding. Mutational analysis revealed the importance of beta hairpin loop and B’ motif present in the C terminal of Rep in the ToLCNDV replication. This study provides insight into the critical roles of amino acids involved in unwinding viral DNA during post initiation stage of the RCR of geminivirus.

Multi-facet role of 26S Proteasome subunit RPT4a, in regulating ToLCNDV in tomato

Pranav Pankaj Sahu, Namisha Sharma^*, Swati Puranik, Supriya Chakraborty &Manoj Prasad

Email ID for Correspondence: namisha@nipgr.ac.in

National Institute of Plant Genome Research, New Delhi-110067

Ubiquitin/26S proteasome (UPS) pathway has been implicated in diverse aspects of eukaryotic cell regulation as it rapidly removes intracellular proteins. In addition to these functions, involvement of 26S proteasomal subunits in plant pathogen-interactions, and the roles of each subunit in independently modulating the activity of many intra- and inter-cellular regulators controlling physiological and defense responses of a plant were well reported. In this regard, we aimed to functionally characterize a Solanum lycopersicum 26S proteasomal subunit RPT4a (SlRPT4) gene, which was differentially expressed after Tomato leaf curl New Delhi virus (ToLCNDV) infection in tolerant cultivar H-88-78-1. Molecular analysis revealed that SlRPT4 protein has an active ATPase activity. SlRPT4 could specifically bind to the stem-loop structure of intergenic region (IR), present in both DNA-A and DNA-B molecule of the bipartite viral genome. Lack of secondary structure in replication associated gene fragment prevented formation of DNA–protein complex suggesting that binding of SlRPT4 with DNA is secondary structure specific. Interestingly, binding of SlRPT4 to IR inhibited the function of RNA Pol-II and subsequently reduced the bi-directional transcription of ToLCNDV genome. Virus-induced gene silencing of SlRPT4 gene incited conversion of tolerant attributes of cultivar H-88-78-1 into susceptibility. Furthermore, transient over-expression of SlRPT4 resulted in activation of programmed cell death and antioxidant enzymes system. Overall, present study highlights non-proteolytic function of SlRPT4 and their participation in defense pathway against virus infection in tomato.

Emergence of chilli-infecting begomoviruses in India and the role of associated satellites in pathogenesis

Neha Gupta^*, R. Vinoth Kumar, Achuit Kumar Singh, Ashish Kumar Singh, Divya Singh and Supriya Chakraborty

Email ID for Correspondence: g.neha92@gmail.com; vinobt06@gmail.com

School of Life Sciences, Jawaharlal Nehru University, New Delhi-110 067, India

The genus, Begomovirus includes a large number of viruses infecting a wide range of plant species across the continents. In the Indian subcontinent, production of chilli is constrained due to chilli leaf curl disease (ChiLCD) caused by begomoviruses. Despite the considerable economic consequences of ChiLCD on chilli cultivation in India, there have been scant studies of the genetic diversity and structure of the begomoviruses that cause this disease. Here we report a comprehensive survey across major chilli-growing regions in India. Analysis of samples collected in the survey indicates that ChiLCD-infected plants are associated with a complex of begomoviruses (including a previously unreported species) with a diverse group of alphasatellites (including a novel group) and betasatellites. The associated alphasatellite/betasatellites neither enhanced the accumulation of the begomovirus components nor reduced the incubation period in Nicotiana benthamiana. The ChiLCD-associated begomoviruses induced mild symptoms on Capsicum spp., but both the level of helper virus that accumulated and the severity of symptoms were increased in the presence of cognate betasatellites. Interestingly, most of these ChiLCD-associated begomovirus components were found to be recombinants. The nucleotide substitution rates were determined for the AV1 gene (ChiLCV), the βC1 gene (ChiLCB & ToLCBDB) and the rep gene (ChiLCA). This study underscores the current understanding of Indian ChiLCD-associated begomoviruses and also demonstrates the crucial role of betasatellites in chilli leaf curl disease development.

Agroinoculation of Tomato yellow leaf curl Kanchanaburi virus into solanaceous plants

Kanami Homma^1*, Yuri Tanaka², Elly Kesumawati³, Shinya Kanzaki¹, Sota Koeda¹

Email ID for Correspondence: 1633650006m@nara.kindai.ac.jp

¹Graduate School of Agriculture, Kindai University, Nara 631-8505, Japan; ²Graduate School of Agriculture, Kyoto University, Takatsuki 569-0096, Japan; ³Facalty of Agriculture, Syiah Kuala University, Banda Aceh 23111, Indonesia

Pepper (Capsicum spp.) and tomato (Solanam lycoperisicum) belong to the Solanaceae family, and are the most widely produced and consumed horticultural crops in the world. However, begomoviruses have caused economic damage in these crops during the last two decades in Indonesia. Our previous study clarified that Pepper yellow leaf curl Indonesia virus, Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), and Ageratum yellow vein virus infect peppers (Koeda et al. 2016). Because peppers and tomatoes are generally cultivated 2–3 times per year, begomoviruses appeared to be maintained by moving from one plant to another throughout the year. Therefore, breeding begomovirus-resistant peppers and tomatoes is of great importance for horticultural crop production. In the present study, an agroinoculation method to transfer TYLCKaV into tomato plants was established. The partial repeats of TYLCKaV DNA A or DNA B genomic components were cloned into pGreenII-p35S, and Agrobacterium were transformed with the constructs. First, Nicotiana benthamiana was used for agroinoculation to check construct infectivity. Buffer, empty vector, DNA A, or a mixture of DNA A and DNA B were agroinoculated from the abaxial side of leaves. In plants inoculated with DNA A and DNA B, the typical symptoms of begomovirus infection were observed 2 weeks after inoculation. In addition, both components were detected in the plants by polymerase chain reaction. Although no symptoms were observed, DNA A was detected in several plants infected with only DNA A. Because construct infectivity was confirmed, tomatoes were agroinoculated using the needle puncture method. Symptoms were observed 4 weeks after inoculation, and both components were detected in those plants. Tomato cuts were also agroinoculated using a soaking procedure, and virus infection was observed. These results indicate that these methods can be applied to screen for TYLCKaV resistant accessions.

Role of DNA B of Tomato leaf curl New Delhi virus in symptom development

Divya Singh^*, Prabu Gnanasekaran and Supriya Chakraborty

Email ID for Correspondence: divyasingh1987@gmail.com

Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi – 110067, India

Geminivirus infection results in developmental abnormalities leading to leaf curling, chlorosis and stunting of plants. Development of chlorotic symptoms in case of plant viral diseases could be attributed to different virus-induced physiological disturbances and virus-host interactions but predominantly it is due to reduced chlorophyll level. Infection of plants with Tomato leaf curl New Delhi virus leads to severe yellowing and chlorosis of leaf tissues, however, DNA A alone could not produce leaf yellowing symptom on plants. Therefore, it is hypothesized that DNA B is responsible for chlorotic symptom development. During infection of Nicotiana benthamiana plants with DNA B along with cognate DNA A, several photosynthetic parameters were altered which could be due to reduction in efficiency of PSII reaction centres to capture electron after viral infection. Chloroplast ultra structure was found to be severely damaged following DNA-B infection, however, when transiently expressed, the BC1 protein caused more severe damage of chloroplast leading to complete disappearance of thylakoid membranes. In addition DNA B was found to downregulate the key enzymes responsible for chlorophyll biosynthesis where as chlorophyll degradation pathway genes remain unaffected. Transcript levels of PsbP and PsbO, the major extrinsic protein components of the oxygen-evolving Complex (OEC) of the photosynthetic machinery were also found to be significantly downregulated upon DNA B infection. Role of DNA-B in affecting the components of OEC will be presented.

Yellow mosaic disease of legumes in Meghalaya is an association of Mungbean yellow mosaic India virus DNA A with a recombinant Mungbean yellow mosaic virus DNA B

Amrita Banerjee^1*, Rohit Mani Yadav², Yashi Umbrey³ and Somnath Roy²

Email ID for Correspondence: amrita.ars@gmail.com

¹ICAR Research Complex for North Eastern Hill Region, Umiam 793 103, Meghalaya, India;²ICAR-National Bureau of Plant Genetic Resources, Regional Station, Umiam 793 103, Meghalaya, India;³School of Crop Protection, College of Post Graduate Studies, Central Agricultural University

Yellow mosaic disease (YMD) of grain legumes is caused by four distinct begomoviruses collectively known as the yellow mosaic viruses (YMVs) such as, Mungbean yellow mosaic virus (MYMV), Mungbean yellow mosaic India virus (MYMIV), Dolichos yellow mosaic virus (DoYMV) and Horsegram yellow mosaic virus (HgYMV). However, MYMIV and MYMV are most important as it infect various legume crops in India. MYMIV is predominant in northern, central and eastern regions of India while, MYMV is prevalent in southern region. There is limited information on YMVs occurring in north-eastern region of India. During 2014–2015, typical yellow mosaic symptoms were observed in mungbean and black gram growing in mid-hill condition of Umiam, Meghalaya. The whole genome characterization following rolling circle amplification confirmed identical features in DNA A (KU950430:2741 bp) and DNA B (KU950431:2656 bp) as of Begomovirus. The nucleotide identity and phylogenetic analysis confirmed DNA A from Meghalaya as an isolate of MYMIV having 95–97% similarity. However, DNA B of Meghalaya isolate showed 90–96% identity with both MYMIV DNA B and MYMV DNA B. The highest identity of 96% was shared with MYMV DNA B from Tamil Nadu having a recombinant DNA B. The phylogenetic analysis of DNA B also showed the clustering of Meghalaya isolate along with MYMV and MYMIV reported to have recombinant DNA B. Further, one recombination event was identified in DNA B of Meghalaya isolate spanning the nucleotide region 2462–2729 nt (spanning the common region). Recombination signals detected by five different methods coupled with phylogenetic evidence confirmed MYMV DNA B as the major parent and MYMIV DNA B as the minor parent. Thus, DNA B of Meghalaya is the result of component exchange, commonly known as pseudo-recombination for begomoviruses. Therefore, the YMV isolate from Meghalaya is an association of MYMIV DNA A and recombinant MYMV DNA B.

Functional investigation of Bhendi yellow vein mosaic virus proteins in plant pathogenesis

K. S. Dinesh Babu, Ashirbad Guria, Kamlesh Kumari and P. Gopal^*

Email ID for Correspondence: pgp79@yahoo.com

Department of Plant Biotechnology, School of Biotechnology; Madurai Kamaraj University, Madurai – 625021

Bhendi yellow vein mosaic virus (BYVMV) causes yellow vein mosaic disease in Bhendi (Abelmoscus esculentus) and hampers the productivity of crop yield. BYVMV is a monopartite begomovirus associated with DNA β satellite. DNA A contains five complementary genes and two virion sense genes. DNA Beta encodes a single ORF called βC1 and responsible for symptom development. BYVMV C2 and C4 involved in transcriptional activation of host as well as viral genes and suppression of host mediated post-transcriptional gene silencing (PTGS) respectively. Being an ssDNA virus, BYVMV should able to interrupt transcriptional gene silencing (TGS) pathway which is not known currently. In order to identify whether BYVMV is having capability to interfere with TGS, we inoculated BYVMV into Nicotianabenthamiana and observed the leaf curl symptoms. The nature of infections is confirmed by PCR, qRT-PCR and Southern. We performed chop-PCR, Southern hybridization and bisulfite sequencing and found that BYVMV impairment on DNA methylation. In parallel experiments, we have introduced a stop codon in the N-terminal region of C2 and C4 ORFs in full-length DNA A and inoculated into N. benthamiana plants to find out whether C2 or C4 protein of BYVMV has role in suppression on DNA methylation. The mutation effect on the symptom development and DNA methylation will be discussed in the presentation.

Epidemiological studies of begomovirus diseases of tomato in Brazil

M. A. Macedo^1*, AK Inoue-Nagata¹, J. C. Barbosa², M. Michereff Filho¹, R. L. Gilbertson³ and A. Bergamin Filho⁴

Email ID for Correspondence: monica.macedo.unb@gmail.com

¹Embrapa Vegetables, Brasília, DF, Brazil; ²Bayer CropScience Vegetable Seeds, Uberlândia, MG, Brazil; ³Department of Plant Pathology, University of California-Davis and ⁴Department of Plant Pathology, Escola Superior de Agronomia Luiz de Queiroz, Piracicaba, SP, Brazil

The begomovirus disease is the viral disease with the highest incidence on the tomato crop in Brazil. A set of epidemiological studies was performed to understand the occurrence of begomovirus in the Brazilian agricultural system. The initial studies were done on the spatial and temporal spread of the begomovirus disease in processing tomato fields (24 plots of 15 × 15 plants). We observed a lower incidence and area under disease progress curve of the disease in plots located in the center (PC) than in the edge (PE) of the central pivot. The distribution of symptomatic plants in PC was more aggregated than in PE. These results suggest that the distribution of symptomatic plants in PC and PE is ruled out by different spreading mechanisms and that the primary dispersion assumes an important role in the spread of the disease. This observation was confirmed in a similar study performed in fresh-marked tomato fields. Then, we monitored the disease incidence in processing tomato fields in five growing regions during 3 years. Although the highest disease incidence was observed in the period immediately after the tomato-free-period (TFP) of 3 months (Nov–Jan), the first symptomatic plants were usually seen after 30 days of transplanting. The basic problem was the transplanting season coinciding with the harvesting period of soybean, i.e., with high whitefly population. Tomato severe rugose virus (ToSRV) is the predominant begomovirus in tomato in Brazil and this virus was found only in a few weed samples. During the TFP, ToSRV viruliferous whiteflies were not observed in the fields. Our studies suggest that the whitefly control measures should be performed to reduce the primary spread of the disease, the TFP has to be adjusted to avoid the high whitefly population periods, and the management measures need to be done in a regional basis.

Differential interaction of Cassava mosaic geminivirus genomic components

Basavaprabhu L. Patil^1,2*, and Claude M. Fauquet^2,3

Email ID for Correspondence: blpatil2046@gmail.com

¹ICAR-National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi-12, India; ²Donald Danforth Plant Science Center, St. Louis, MO 63132, USA; ³Current Address: Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia

Cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses (CMGs) with bipartite genome organization, is a major constraint for production of cassava in the African continent and the Indian sub-continent. Currently there are eleven recognized species of CMGs and several diverse isolates represent them, with vast amount of sequence variability, reflecting into diversity of symptom severity/phenotypes. Here we make a systematic effort to study the infection dynamics of several species of CMGs and their isolates. We also try to identify the genomic component of CMGs contributing to the manifestation of diverse patterns of symptoms and the molecular basis for the differential behaviour of CMGs. The pseudo-recombination studies carried out by swapping of DNA-A and DNA-B components of the CMGs revealed that the DNA-B component significantly contributes to the symptom severity. Past studies had shown that the DNA-A component of Sri Lankan cassava mosaic virus (SLCMV) shows monopartite feature. Thus the ability of DNA-A component alone, to replicate and move systemically in the host plant with inherent monopartite features was investigated for all the CMGs. Geminiviruses are often associated with DNA satellites that require proteins encoded by the helper virus for their replication, movement and encapsidation. Hitherto, most of the DNA satellites are reported to be associated with monopartite begomoviruses. It is established that SLCMV can trans-replicate betasatellites and can cross host barriers. To extend these studies further, we carried out an exhaustive investigation of the ability of CMGs, to trans-replicate betasatellites (DNA-β) and to interact with alphasatellites (DNA-1). Each of the CMGs showed a contrasting and differential interaction with the DNA satellites, and differentially modulated the symptom phenotypes. Geminiviruses are known to trigger gene silencing and are also its target, resulting in recovery of the host plant from viral infection. In the collection of several different CMG species and isolates we had, there was a vast variability in their recovery and non-recovery phenotypes.

Transcript mapping of Sri Lankan cassava mosaic virusDNA-A

Fauzia Zarreen^* and Indranil Dasgupta

Email ID for Correspondence: fzarreen@gmail.com

Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi-110021, India

Sri Lankan cassava mosaic virus (SLCMV) is a bipartite begomovirus infecting cassava in India and Sri Lanka. SLCMV has a genome organizationresembling begomoviruses, encoding six ORFs in DNA-A and two ORFs in DNA-B molecule. DNA-A component of the genome has six ORFs, two in the viral-sense and four in the complementary-sense. The objective of this work was aimed at mapping of major transcripts of SLCMV to gain insight into the possible mechanism and control elements involved in the regulation of SLCMV gene expression. Nicotiana benthamiana plants were inoculated with an infectious clone of SLCMV DNA-A. The 3′ and 5′ ends of the transcripts were amplified (RACE) using cDNA from the above samples. Three clones for each 3′ and 5′ RACE products were sequenced and analyzed. 3′ RACE amplification was successfully obtained for all the six SLCMV ORFs. However, 5′ RACE amplification was obtained for both the virion sense ORFs (AV1 and AV2) and only two complementary sense ORFs (AC2 and AC3). 5′ ends of the AC1 and AC4 transcripts could not be amplified despite repeated attempts. Sequencing results indicate the presence of multiple polycistronic SLCMV transcripts with overlapping 5′ and 3′ ends diverging form the common intergenic region. In SLCMV multiple overlapping complementary sense transcripts are transcribed as compared to virion sense transcripts, which are just two in number. The 3′ ends of the complementary sense transcripts are co-terminal with the majority of the transcripts terminating at position 1064. The results of this study indicate the presence of multiple polycistronic SLCMV transcripts with overlapping 5′ and 3′ ends, majority of them being co-terminal, diverging form the common intergenic region of single stranded circular DNA genome.

Identification and molecular characterization of the yellow mosaic disease associated with ornamental plant Catharanthus roseus

Chitra Nehra, Avinash Marwal, R. K. Gaur^*

Email ID for Correspondence: gaurrajarshi@hotmail.com

Department of Bioscience, College of Arts, Science Huminities, Mody University, Lakshmangarh, Sikar-332311, Rajasthan, India

Begomovirus is one of the largest genus of the family Geminiviridae. Ornamentals are considered as a foundation of new viruses and reservoirs in the absence of the main crops. In the present report, we identified a begomovirus associated with an ornamental plant Catharanthus roseus commonly known as Madagascar periwrinkle which belongs to the family Apocynaceae. This is often cultivated in Indian gardens for decorative purposes and acknowledged for its medicinal values. Twenty symptomatic plant samples were collected from Sikar (Rajasthan) India and the complete viral genome was amplified by rolling circle amplification using TempliPhi DNA amplification kit, digested with EcoRI and SalI, resulted fragment were cloned in suitable pGEMT vector and sequenced (Gene Bank accession no clone C1 KP698312 and C2 KP698313). BLAST analysis of DNA-A of both clones C1 and C2 showed maximaum similarity 97 and 93% respectively with papaya leaf crumple virus. The phylogenetic analysis of begomovirus isolates clone C1 2736 bp and clone C2 2736 bpbased on DNA-A with corresponding sequences revealed its closest relationship papaya leaf crumple virus HM140367 and HM140368 respectively. To our knowledge this is the first report of natural occurrence of papaya leaf crumple virus infecting Catharanthus roseus in India. Thus, there is a pressing need for additional information on the diversity and distribution of begomoviruses in ornamental plants.

Field evaluation of recovery phenotypes of cassava for cassava mosaic disease based on symptom expression and virus load

D. C. Deepthi, J. Sreekumar, C. Mohan, S. K. Chakrabarti and T. Makeshkumar^*

Email ID for Correspondence: makeshctcri@gmail.com

ICAR-Central Tuber Crops Research Institute, Thiruvananthapuram, India

Geminiviruses are a large and important group of plant virus that affects a variety of crops worldwide. In plants, some virus–host interactions naturally lead to host recovery. Although Symptom remission is not common for geminiviruses, cassava infected with cassava mosaic virus exhibited recovery, in which newly emerged leaves of systemically infected plants show signs of reduction in symptom severity. In India cassava mosaic disease is caused by Indian Cassava Mosaic virus (ICMV) and Sri Lankan Cassava Mosaic Virus (SLCMV), which belong to begomoviruses transmitted by whitefly, Bemisia tabaci. SLCMV is the most prevalent virus in India now. Further understanding of the biology of these viruses is the most important prerequisite for their proper and effective control. The present study was designed to assess the correlation between disease symptom expression and virus load in different cassava genotypes considered as tolerant, susceptible and resistant. The genotypes were systematically evaluated under field conditions to quantify their response to virus infection and determine the relationship between virus load, symptom type and severity. To assess the virus concentration in susceptible, tolerant and resistant cassava cultivars, infected with SLCMV, the amount of DNA A and DNA B were analysed by quantitative PCR. Virus concentration was positively correlated with severity of symptoms. Virus concentration and symptom severity was much higher in susceptible breeding lines without much variation throughout the growth period and it is vice versa in resistant lines. Recovery types, initially responded with severe symptoms, but following the recovery, symptoms were reduced on upper leaves and eventually all newly developed leaves were symptom free. The differential assessment of DNA genomic components to reveal quantitative relations among the components showed that higher quantity of DNA A was present than DNA B throughout the crop growth period in resistant breeding lines. But the higher concentration of DNA B genomic component than DNA A was consistent in recovery and susceptible breeding lines. Since DNA-B is concerned in virus cell-to-cell spread and systemic movement, it can be postulated as a factor driving cassava mosaic disease epidemics.

Molecular diversity of cassava mosaic begomoviruses from Southern India

Akhilesh Kumar Kushawaha^1*, R. Rabindran² and Indranil Dasgupta¹

Email ID for Correspondence: akhilesh2203@gmail.com

¹Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021, and ²Center for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu-641003

Cassava (Manihot esculenta Crantz, Family: Euphorbiaceae) is the third largest source of dietary carbohydrates in the world. In India, only two begomoviruses are associated with Cassava mosaic disease (CMD); Sri Lankan cassava mosaic virus (SLCMV) and Indian cassava mosaic virus (ICMV). SLCMV being more common as compared to ICMV, in 2011 almost 100 cassava fields were visited in Tamil Nadu and cassava samples showing symptoms of CMD were collected from eighty locations in nine different districts. RCA-RFLP, cloning and sequence analysis were performed to study the genetic diversity of SLCMV and ICMV. Twelve complete CP genes were amplified from samples collected from eight locations and the sequence analysis was performed. Multiple sequence alignment showed only five single substitution mutations. There was no hot spot for mutation or no deletion or insertion in the CP. RCA-RFLP method was applied to characterize and to study the genetic diversity of all cassava-infecting begomoviruses. In most of the samples the RCA-RFLP band pattern was similar to expected pattern of SLCMV DNA-A and SLCMV DNA-B, reported earlier from India. For confirmation, RCA-RFLP generated DNA fragments showing unexpected and expected band patterns were cloned and partially sequenced and compared with a full-length SLCMV DNA-A, which was infectious on Nicotiana benthamiana. The sequence identities of 97–99% of cloned fragments indicated that SLCMV sequences contained only few single amino acid deletions and some substitution mutations. The RCA-RFLP pattern indicated the absence of ICMV from the samples analyzed.

Symptoms mimicking in tomato by mixed infection of begomovirus(es) and phytoplasma

Abhishek Sharma^*, Manmohan Dhkal¹, Nity Sharma², SI Kaur¹ and SS Kang¹

Email ID for Correspondence: abhishek@pau.edu

Department of Vegetable Science,¹Department of Plant Pathology and ²School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, India-141004

Tomato (Solanum lycopersicum), a member of family Solanaceae, is the second most important vegetable crop of the world after potato. This crop is vulnerable to diseases and insect pests due to its limited genetic variation. Among diseases phytoplasma and viral disease are common in tomato. Among virus diseases tomato leaf curl disease (ToLCD) caused by whitefly (Bemisia tabaci) transmitted begomoviruses is an important problem in all tomato growing areas. Beside ToLCD occurrence of Phytoplasma diseases of tomato (PDT) has also been reported in several countries around the world under different names. Recently under Punjab conditions symptoms plants showing typical symptoms imitating the ToLCD and PDT were observed on plants of rainy season tomato crop. The samples from most susceptible variety Punjab Chhuhara were collected and categorized into three categories (a) Plants showing typical ToLCD symptoms including leaf curling, stunted growth having normal stem girth with less or no flowering. (b) Plants showing typical ToLCD symptoms including leaf curling, stunted growth having normal stem girth with profuse flowering. (c) Plants showing symptoms including rudimentary leaves, stunted growth having very thick stem girth with less or no flowering. These samples were subjected to nucleic acid based assay using universal primers for begomovirus and phytoplasma. Further the begomovirus positive plants were subjected to virus species specific primers and satellite molecule (alpha and beta) specific primers. The PCR results indicated the association of Tomato leaf curl New Delhi virus (ToLCNDV) in all the samples. The sample b and c showed presence of Tomato leaf curl Karnataka virus (ToLCKV) with ToLCNDV. Nested PCR with phytoplasma specific primers resulted in an expected size amplicon of ~1.1 kb from sample a and b, whereas, sample c having thick stem and no flowering symptoms yielded a small size amplicon of ~800 bp. The samples a and b also showed the presence of beta satellite molecule. This shows the combination of helper virus along with satellite molecules and phytoplasma in mixed infection may influence the symptomatology of tomato plants.

Functional analysis of AV2 protein in Sri Lankan cassava mosaic virus

Akhilesh Kumar Kushawaha, Kanika Gupta^* and Indranil Dasgupta

Email ID for Correspondence: kanikagupta1903@gmail.com

Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021, India

Most geminiviruses have six proteins encoded by the DNA-A component in bipartite or its equivalent in monopartite viruses. Out of the above, Pre-coat protein (AV2) is present only in the “Old World” begomoviruses. AV2/V2 protein has been reported to have roles in pathogenicity and virus movement and also to act as RNAi suppressor in various begomoviruses. Functional domains have not been identified in this protein. In this study, we attempted to determine the function of AV2 in an infectious clone of Sri Lankan cassava mosaic virus (SLCMV), SLCMV-Attur (KC424490) by using site-directed mutagenesis to generate non-expressing or C-terminally truncated AV2 protein. SLCMV AV2 has 118 amino acid residues and shares 47, 56 and 70% amino acid identities with Mungbean yellow mosaic India virus, East African cassava mosaic Cameroon virus and Tomato leaf curl geminivirus from India respectively. Three mutants in AV2 were generated, one lacking the start codon and other two mutants forming first 39 and 54 amino acids. The effect of these mutations on viral DNA accumulation and symptom development was studied on the laboratory host Nicotiana benthamiana. A total of 60 N. benthamiana plants were biolistically inoculated with wild type and mutated SLCMV-Attur DNA-A clones in four sets. N. benthamiana inoculated with wild type SLCMV-Attur showed symptoms of leaf rolling and stunting and viral DNA accumulation in systemic leaves. None of the plants inoculated with mutants showed viral DNA accumulation or symptoms. The movement of the mutants from the inoculated to the systemic leaves is being investigated.

Genome diversity of begomoviruses in resistant and susceptible tomato cultivars

Camila de Moraes Rêgo^1*, Erich Yukio Tempel Nakasu², Alice Kazuko Inoue-Nagata^1,2

Email ID for Correspondence: camilamoraes.bio@gmail.com

¹University of Brasilia, ²Brazilian Agricultural Research Corporation, Brasília, Distrito Federal, Brazil

Among the diseases that affect tomato crops (Solanum lycopersicum) in Brazil, those caused by begomoviruses are particularly important due to their high incidence in growing areas. The use of resistant plants is the most efficient and cost-effective strategy to minimize losses caused by begomoviruses. However, the increasing cultivation of resistant cultivars may result in selection of specific viral isolates, accelerating changes in begomovirus population composition, and overcome resistance mechanisms. Since it is unknown whether this selection actually occurs in Brazilian conditions, the aim of this work was to compare the begomovirus species infecting processing begomovirus-resistant and -susceptible tomato cultivars (BRS Sena and Heinz-9553, respectively), and to evaluate the genome diversity within each species. Begomovirus diversity in samples from both cultivars was screened by RCA/RFLP. Positive samples were then subjected to PCR using species-specific primers, cloned and then sequenced. Two begomovirus species were detected in both the cultivars, Tomato severe rugose virus (ToSRV) and Tomato mottle leaf curl virus (ToMoLCV), being ToSRV the most prevalent. The presence of nucleotide insertions and deletions was only found in intergenic/non-coding regions of both ToSRV and ToMoLCV genomes, while nucleotide substitutions were observed throughout the ORFs in both species, mostly in AC1 (Rep) and AV1 (CP). Additionally, nucleotide substitutions were more common in the viral genome from resistant plants then in viruses from susceptible plants. Following phylogenetic analysis, begomoviruses from both samples were grouped together based on their geographical origin. Results indicate that most possibly viral isolates from the resistant cultivar are undergoing an initial process of genetic variation due to the selective pressure imposed by the use of resistant plants. However, to confirm this hypothesis, further analyses are needed on larger populations of ToSRV and ToMoLCV.

First report of Ageratum yellow vein China virus and Tomato leaf curl China virus infecting Oxalis corniculata L. in Guangxi, China

Zhanbiao Li, Bixia Qin, Huiting Xie, Lixian Cui and Jianhe Cai^*

Email ID for Correspondence: caijianhe@gxaas.net

Guangxi Key Laboratory of Biology for Crop Diseases and Insect pests; Institute of Plant Protection, Guangxi Academy of Agricultural Sciences, Nanning, 530007, China

Whitefly-transmitted Geminiviruses belong to Geminiviridae family which causes severe leaf curl diseases on many plants. In January 2012, Oxalis corniculata L. showed symptoms of dark green vein and enation were observed in the suburb of Nanning city, Guangxi. One sample with typical symptoms (OX1) was collected and total DNA was extracted. A 586 bp DNA fragment was amplified from OX1 Using the primer pair AV494 and AC1048, and the PCR product was cloned and three positive clones were sequenced. All of the sequences shared most closely related to the sequences of begomoviruses which available on Genbank. One sequence shared 99% identity with Ageratum yellow vein China virus (AYVCNV), and the other two sequences shared 98% identity with Tomato leaf curl china virus (ToLCCNV). Overlap primers were designed to amply the full-length DNA-A sequences, AYVCNV-F5′-GACTGCACCTTACACGGGCC-3′/AYVCNV-R 5′-GAGATCGGCGTCCGAGTGGTA-3′ for AYVCNV, and ToLCCNV-F 5′-GGACCCTCACAGCCTCGTGG-3′/ToLCCNV-R 5′-CAATATGCAGCTAAGGAACAGGCG-3′for ToLCCNV. The first complete DNA-A sequence from OX1 were determined to be 2744 nucleotides (Accession NO. JX972142), which showed 90–96% sequence identity with ToLCCNV; the second complete DNA-A sequences were 2739 nucleotides (Accession NO. JX972141), which showed high levels of sequence identity 92–99% with AYVCNV. Neither DNA-B component nor DNA-β could be detected using primers described previously. To demonstrate pathogenicity, virus-free adults of Bemisia tabaci biotype B were feed on the diseased Oxalis corniculata L. plants for 48-h, and then transferred to healthy Oxalis corniculata L., Ageratum and Tomato. Oxalis corniculata L. showed the original symptoms on the leaves (diseased rate 3/10), whereas Ageratum showed yellow vein symptoms (10/10), and tomato plants (10/10) showed mild yellow leaf curl symptoms. The infection plants was confirmed by PCR and the PCR products were cloned and sequenced, which confirmed the transmitted viruses were AYVCNV and ToLCCNV. To our knowledge, this is the first report of these two viruses infecting Oxalis corniculata L.

Diversity of geminiviruses associated with yellow vein disease of Bhendi/okra in India

Madhvi Naresh^*, Rashmi Rishishwar, Kanika Gupta, Mayur Saindane¹, Gaurav Dhande¹ and Indranil Dasgupta

Email ID for Correspondence: madhvinaresh79@gmail.com

Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi,¹Biotechnology Department, Nirmal Seeds Pvt. Ltd., Pachora, Jalgaon, Maharashtra

Okra/Bhendi (Abelmoschus esculentus L. (Moench), is an economically important vegetable crop, grown in several countries in Africa and Asia, mainly in the tropics. In India, yellow vein mosaic disease of Bhendi (BYVMD) is the most important constraint to its cultivation. Earlier reports have shown the monopartite begomovirus Bhendi yellow vein mosaic virus (BYVMV) and associated betasatellite to be the causative agent of BYVMD in southern India. Apart from BYVMV, Mesta yellow vein mosaic virus (MeYVMV), some recombinant begomoviruses and alphasatellites have also been reported to be associated with BYVMD in central India. With the aim of further studying the diversity and variability of geminiviruses associated with BYVMD, fifty-three symptomatic samples were collected from field locations from parts of central and western India and were used to amplify begomoviral sequences using PCR. Approximately 2.7 kb amplification products were obtained from a total of nine samples, representing four locations in Maharashtra and three in Gujarat. Partial nucleotide sequence analysis of the eight of the above clones showed 97% to 100% identity with sequences named as Okra enation leaf curl virus DNA A segment in sequence databases, while one sample showed 94% identity with Cotton leaf curl virus DNA A fragment. The progress towards analysis of full sequences of the above clones and their infectivities on okra and various laboratory plants will be presented.

The impact on health and disease of co-infection with single strand DNA viruses

Anne-Lie Blomström¹, Caroline Fossum², Per Wallgren³ and Mikael Berg¹

Email ID for Correspondence: anne-lie.blomstrom@slu.se

¹Department of Biomedical Sciences and Veterinary Public Health, Section of Virology, Swedish University of Agricultural Sciences, Uppsala, Sweden; ²Department of Biomedical Sciences and Veterinary Public Health, Section of Immunology, Swedish University of Agricultural Sciences, Uppsala, Sweden; ³National Veterinary Institute (SVA), Uppsala, Sweden

The development of high-throughput sequencing technologies has allowed the possibility to investigate and characterise the entire microbiome of individuals. This has not only lead to the discovery of novel viruses and identification of viruses behind disease outbreak but has also provided better insight to the complex interaction between different microorganisms co-existing in both healthy and sick individuals. We have applied viral metagenomics to investigate the virome of healthy pigs with the aim to increase knowledge of what influence the virome can have on the susceptibility of secondary infection. We show that in healthy pigs there is a complex co-infection with several viruses coexisting, many of which are ubiquitous single-stranded DNA viruses such as Porcine circovirus type 2, Torque tenosuis virus (TTSuV) 1 and 2 and different porcine bocaviruses (PBoV). These viruses are also found in pigs with complex multifactorial diseases such as Postweaning multi-systemic wasting syndrome but then the relative viral loadshave changed substantially. It is highly possible that some of these viruses found in healthy pigs could increase or decrease the susceptibility to secondary infection and or indirectly affect the pathogenicity of other viruses. We have also, in vitro, investigated if these viruses (in particular PBoV and TTSuV) modulate the immune system of the host and if so which genes are involved. We have investigated both modulatory sequence motifs in the genomes as well as the viral proteins in this respect.

New isolate of Pepper huasteco yellow vein virus (PHYVV) increase host range specificity by exhibiting differential symptoms severity patterns

N. Meléndrez-Bojórquez^1*, E. Camacho-Beltrán¹, E.A. Rodríguez Negrete ¹, N.E. Leyva-López¹ and J. Méndez-Lozano¹

Email ID for Correspondence: nataniel.melendrez@gmail.com

¹Instituto Politécnico Nacional, CIIDIR, Unidad Sinaloa. Departamento de Biotecnología Agrícola.Blvd.Juan de Dios Bátiz Paredes No. 250, Col. San Joachín. Guasave, Sinaloa, México C.P. 81101

Since 1993, Pepper huasteco yellow vein virus (PHYVV) was reported infecting pepper as serious pathogens in Mexico. Recently, emerging disease was observed in pepper crops cultivated in green house and open field in the state of Sinaloa, Mexico. The molecular evidence indicates the presence of PHYVV as a main actor in such samples. Full genome clones of DNA A and B were obtained designated as PHYVV-Sin. The complete sequence of the DNA A and B shows an identity of 94.1 and 83.6% respectively with the first reported sequence of PHYVV-Tam. The low homology of DNA B of the new isolate suggests a genetic evolution of PHYVV. To understand the biological effect of the DNA-B in the symptoms patterns expression and host range; both PHYVV isolates were evaluated by biological and molecular assays. The first step was to understand the role of the novel DNA B in symptoms severity and their response to interact within different host. Additionally, psedorecombination interaction was analyzed by swapping DNA-A and DNA-B of both isolates. In these sense N. benthamiana, pepper, tomato, petunia, cucumber and common bean seedlings were agroinoculated with PHYVV-Tam and PHYVV-Sin in single or pseudorecombination infection. The data suggest that genetic change of PHYVV-Sin DNA-B represent a positive evolutionary event occurring naturally and adapted to the new environmental circumstances. In this sense, host-virus interaction improves in a better manner for the virus to increase the pathogenicity and modifying the host rage. Finally, the new isolate of Pepper huasteco yellow vein virus became a pepper-infecting begomovirus with a significative biological effect to induce emerging diseases in Mexico.

Replication competition between malvastrum yellow vein virus and tobacco curly shoot virus associated with their cognate betasatellites

Chenchen Jing, Miao Sun, Yan Hu, Gentu Wu, Xianchao Sun and Ling Qing^*

Email ID for Correspondence: qling@swu.edu.cn

College of Plant Protection, Southwest University, Chongqing 400716, China

The cognate betasatellite (MaYVB) of Malvastrum yellow vein virus (MaYVV) is necessary for typical symptoms induction, while Tobacco curly shoot virus (TbCSV) can induce typical symptoms without its betasatellite (TbCSB). To investigate the interaction relations between the two kinds of geminivirus/betasatellite disease complex, the infectious clone combination of MaYVV isolate Y47 (Y47A), TbCSV isolate Y35 (Y35A), and their betasatellite (Y47β and Y35β) was used to inoculate Nicotiana benthamiana and the replication level of these two viruses and their cognate betasatellites were detected by PCR and real-time quantitative PCR (qPCR). The symptoms of shrinking, downward leaf curling, yellow vein, distorted stem, dwarf and enation were found in the early stage of infection, but yellow vein symptom disappeared in the later stage. Y35A and Y35β increasingly accumulated during the infection with a peak at 90 day post inoculation (dpi). However, Y47A and Y47β decreasingly accumulated during the process. Finally, Y47A and Y47β were undetectable at 180 and 120 dpi respectively. These results indicated that the competition probably occurred between these two disease complexes of Y47A/Y47β and Y35A/Y35β for the replication resources of the host and betasatellite is preferentially trans-replicated by its cognate helper virus.

Mungbean yellow mosaic India virus with Zucchini yellow mosaic virus infects soybean crop in India

Brijesh K. Yadav¹, Ritesh K. Jaiswal², Sangeeta¹, Swati Joshi¹ and Achuit K. Singh^3*

Email ID for Correspondence: achuits@gmail.com

¹School of Life Sciences, Central University Gujarat, Gandhinagar, Gujarat. 382030; ²Plant Protection, Krishi Vigyan Kendra Panna, Jawaharlal Nehru Krishi Vishwa Vidyalaya, M.P. 488001; ³Agricultural Biotechnology, Crop Improvement Division, Indian Institute of Vegetable Research, Varanasi, U.P. 221305

Soybean (Glycine maxL.) of Fabaceae family is one of the world’s most important sources of protein and vegetable oil, which is infected by many viruses among them begomoviruses and potyviruses are most important. Begomovirus, belonging to the family Geminivirideae, is single stranded DNA virus of ~2.7 kb that transmitted by white fly (Bemisia tabaci). Symptomatic leaf samples of soybean, showing yellow mosaic, leaf curling, and stunted growth, has collected from Madhya Pradesh, India and confirmed the presence of potyvirus and begomovirus infection by PCR with universal primers. For begomovirus, total genomic DNA was isolated and subjected to rolling-circle amplification (RCA) and 2.7 and 1.3 kb fragments were cloned in pBluescipt Vector (pKS+) cloning vector and sequenced. Obtained nucleotide sequences of clones (SY1KpnI2.7, SY1BamHI2.7 and SY11.3 kb) were blast (www.ncbi.nlm.nih.gov/BLAST/), which confirmed the presence of three kinds of molecules DNA-A, DNA-B and DNA-β. SY1 KpnI 2.7 kb clone showed maximum similarity with Mungbean yellow mosaic India virus (MYMIV) DNA-A (FM202445), SY1 BamHI 2.7 kb showed maximum similarity with Mungbean yellow mosaic India Virus (MYMIV) DNA-B (AY269992) and SY1 1.3 kb clone showed maximum similarity with Tomato leaf curl Gandhinagar betasatellite (ToLCGnB) DNA-β (KC952006). Potyvirus belonging to the family Potyviridae, is flexuous and filamentous virus having a genome of single-stranded positive-sense RNA (~9.7 kb) and transmitted by aphids (Aphis glycine). For potyvirus, total RNA was isolated by TRIzol method, cDNA was constructed and PCR amplified product was cloned in pTZ57R/T vector and sequenced. Sequencing results showed maximum similarity (97%) with Zucchini yellow mosaic virus (KF976713). This is the first kind of report that Mungbean yellow mosaic India virus with Zucchini yellow mosaic virus infecting soybean crop in India.

Replication competition between Malvastrum yellow vein virus and Tobacco curly shoot virus associated with their cognate betasatellites

Chenchen Jing, Miao Sun, Yan Hu, Gentu Wu, Xianchao Sun and Ling Qing^*

Email ID for Correspondence: qling@swu.edu.cn

College of Plant Protection, Southwest University, Chongqing 400716, China

The cognate betasatellite (MaYVB) of Malvastrum yellow vein virus (MaYVV) is necessary for typical symptoms induction, while Tobacco curly shoot virus (TbCSV) can induce typical symptoms without its betasatellite (TbCSB). To investigate the interaction relations between the two kinds of geminivirus/betasatellite disease complex, the infectious clone combination of MaYVV isolate Y47 (Y47A), TbCSV isolate Y35 (Y35A), and their betasatellite (Y47β and Y35β) was used to inoculate Nicotiana benthamiana, and the replication level of these two viruses and their cognate betasatellites were detected by PCR and real-time quantitative PCR (qPCR). The symptoms of shrinking, downward leaf curling, yellow vein, distorted stem, dwarf and enation were found in the early stage of infection, but yellow vein symptom disappeared in the later stage. Y35A and Y35β increasingly accumulated during the infection with a peak at 90 day post inoculation (dpi). However, Y47A and Y47β decreasingly accumulated during the process. Finally, Y47A and Y47β were undetectable at 180 and 120 dpi respectively. These results indicated that the competition probably occurred between these two disease complexes of Y47A/Y47β and Y35A/Y35β for the replication resources of the host and betasatellite is preferentially transreplicated by its cognate helper virus.

Molecular and serological detection of mungbean yellow mosaic virus (MYMV) infecting mungbean [Vigna radiata(l.) wilczek]

Swapnil Shahakar^1*, Shamprasad Phadnis¹, Anitha Peter¹, D. L.Savitramma², S. V. Keerthi³ and M. Sangeetha⁵

Email ID for Correspondence: swapnil.cbzr@gmail.com

¹Department of Plant Biotechnology, ²Department of Genetics and Plant Breeding, ³Department of Biotechnology College of Agriculture, GKVK, University of Agricultural Sciences, Bangalore

Mungbean [Vigna radiata (L.) Wilczek] is one of the most important nutritionally rich legumes widely cultivated in India. Yellow mosaic disease (YMD) caused by a Begomovirus is one of the most destructive and important diseases of mungbean as it can reduce seed yield up to 100% or even kill a plant infected at an early vegetative stage. The virus is transmitted by whitefly Bemisia tabaci and can infect mungbean at all growth stages. In the present study, identification of the virus strain infecting mungbean based on its coat protein gene sequence and standardization of Enzyme linked immunosorbent assay (ELISA) both indirect Plate and DOT for the detection of the virus was undertaken. The mungbean yellow mosaic virus isolate was collected from the field in GKVK, Bangalore based on the symptoms. The DNA from infected leaf was isolated and the 750 bp coat protein gene of the virus amplified using specific primers. Partial sequence of the gene obtained through Sanger sequencing and BLAST search showed 97% sequence similarity to the mungbean yellow mosaic India virus precoat protein gene (AV2) and coat protein gene (AV1). The antibodies were raised against the ultrapurified virus. Standardization of indirect plate ELISA gave optimum OD reading of 1.056 and 1.074 in 1:50 and 1:100 dilution of crude and ultrapurified antigen respectively and 1:1000 dilutions of antisera and secondary antibody alkaline phosphatase conjugate. Standardization of DOT-ELISA was done to fix the antibody titer raised against MYMV for infected plant sample (crude virus) and ultrapurified virus. In DOT-ELISA optimum color was observed in 1:1 dilution of crude and ultrapurified virus as antigen and 1:500 and 1:2000 dilution of antiserum for crude sample and ultrapurified sample respectively with 1:1000 dilution of secondary antibody.

Molecular characterization of ageratum enation virus and beta satellite associated with leaf curl disease of fenugreek in India

G. Swaranalatha, S. Jalali and M. Krishna Reddy^*

Email ID for Correspondence: mkreddy60@gmail.com; mkreddy@iihr.res.in

Division of Plant Pathology, ICAR-Indian Institute of Horticultural Research, Hessaraghatta Lake PO, Bangalore-560089,

Natural occurrence of leaf curl disease on Fenugreek (Trigonella foenumgraceum L) was observed at Pantnagar, Uttarakhand state of India, in the year 2012. Whiteflies (Bemisia tabaci) successfully transmitted the virus from diseased fenugreek to healthy seedlings of fenugreek and tomato, which indicated begomovirus infection. The begomovirus DNA-A and beta satellite fragments associated with leaf curl disease were amplified by rolling circle amplification using Ø-29 DNA polymerase from infected fenugreek and characterized by their sequence analyses. The virus isolate showed 93.7–98.7% nucleotide identities and close phylogenetic relationship with various isolates of Ageratum enation virus (AgEV); therefore the isolate under study was identified as AgEV. The beta component was also recognized to be associated with the ageratum yellow leaf curl betasatellite (AYLCB) based on their high sequence identities and close phylogenetic relationships. This is the first report of AgEV, and AYLCB beta satellite components associated with leaf curl disease of fenugreek from India.

Genetic diversity and interaction of DNA betasatellites associated with tomato leaf curl Gujarat virus

Sangeeta¹, Brijesh K. Yadav¹ and Achuit K. Singh^2*

Email ID for Correspondence: achuits@gmail.com

¹School of Life Sciences, Central University Gujarat, Gandhinagar, Gujarat. 382030; ²Agricultural Biotechnology, Crop Improvement Division, Indian Institute of Vegetable Research, Varanasi, U.P. 221305, India

Tomato leaf curl Disease (ToLCD) is a major economic constraint of tomato crop in the worldwide which is affected by begomoviruses (Family: Geminiviridae) ssDNA molecule, transmitted through whitefly (Bemisia tabaci). Mostly, Old world begomoviruses are associated with small circular ssDNA satellite molecule known as betasatellite. In this present study, infectivity assay was done to check the replication compatibility with Tomato leaf curl Gujarat virus (ToLCGV) mono/bipartite begomovirus association with four associated different betasatellites molecules: Tomato yellow leaf curl Thailand betasatellite (TYLCThB), Tomato leaf curl Karnataka betasatellite (ToLCKB), Tomato leaf curl Gandhinagar betasatellite (ToLCGnB) and Radish Leaf curl betasatellite (RaLCB). All different betasatellites molecule except ToLCKB were trans-replicated by helper begomovirus (ToLCGV) in Nicotiana benthamiana (model plant) and tomato plants (host). N. benthamiana plants agro-inoculated with ToLCGV + TYLCThB and ToLCGV + RaLCB express severe symptoms like curling, vein clearing and yellowing, while in tomato plant possess severe symptoms including leaf curling in newly emerged leaves along with stunted growth of the plant, twisting of petiole, vein enation and vein clearing. Whereas, ToLCGV co-inoculated with ToLCGnB express systemic symptoms downward leaf curling and stunted growth in both N. benthamiana and tomato plants. However, ToLCGV co-inoculated with ToLCKB induce mild symptoms in tomato as well as N. benthamiana. Further, the presence and accumulation level of viral DNA and associated DNA betasatellites were confirmed by PCR and Real Time-PCR respectively. Hence, it shows that ToLCGV has potentiality to trans-replicate a non-cognate betasatellite and to produce severe symptoms expression in host plants.

Novel association of cotton leaf curl Multan betasatellite with mesta yellow vein mosaic virus infecting Malvastrum coromandelianum in Pakistan

Hasan Riaz^1*, Muhammad Ali², Muhammad Ashfaq¹, Tariq Mukhtar¹, Saad Imran Malik³

Email ID for Correspondence: h.riaz@hotmail.com

¹Department of Plant Pathology, PirMehr Ali Shah Arid Agriculture University Rawalpindi (PMAS AAUR), Pakistan; ²Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad, Pakistan; ³Department of Plant Breeding and Genetics, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi (PMAS AAUR), Pakistan

Malvastrum coromandelianum is a widespread weed, belonging to the taxonomic family Malvaceae, known to host several begomoviruses. A plant sample displaying typical vein yellowing symptoms along with a non-symptomatic plant was collected to know the virus complex associated with the disease. A complete genome of a monopartite Begomovirus (the DNA-A) and an associated DNA β have been amplified (using RCA), cloned (using pBScript K +) and sequenced from the diseased plant only. Sequence pair wise comparisons of DNA-A showed 96.5% identity with Mesta yellow vein mosaic virus isolate MeYVMV-[PK-Lahore10-17-5-06] FR772081. Phylogenetic analysis also clustered the DNA-A, identified in this study, with the other isolates of Mesta yellow vein mosaic virus originating from Pakistan. DNA β proved to be Cotton leaf curl Multan Betasatellite with 84% identity with Cotton leaf curl Multan Betasatellite (CLCuMuβ) isolate Kashmir1 [LN610993] and grouped with CLCuMuβs reported from Pakistan. The sequences have been submitted under accession numbers KR013745 (DNA-A) and KR013746 (DNAβ). To the best of our knowledge, it is the first report of Mesta yellow vein mosaic virus associated with CLCuMuβ. This report adds with the CLCuMuβ to be compatibly trans-replicated under a diverse group of begomoviruses.

Virus isolates causing tomato leaf curl in three geographical locations in the Philippines are variants of two virus species, infectious and associated with DNA betasatellite

F. C. Sta. Cruz^1,2*, T. A. Melgarejo², M. R. Rojas², and R. L. Gilbertson²

Email ID for Correspondence: fcstacruz@up.edu.ph

¹Crop Protection Cluster, College of Agriculture, University of the Philippines Los Baños, College, Laguna 4031, Philippines; ²Department of Plant Pathology, University of California Davis, California 95616, USA

In the Philippines, tomato leaf curl was first identified to be caused by a monopartite Begomovirus species, the Tomato leaf curl Philippines virus (ToLCPV), an isolate from Los Baños, Laguna in southern Luzon. The ToLCPV is classified as strain B while the other isolates from various locations in Luzon and Mindanao islands that were reported later are variants of strains A, B or C; or new species such as Tomato leaf curl Cebu virus (ToLCCeV), Tomato leaf curl Mindanao virus (ToLCMinV) and Ageratum yellow vein virus (AYVV). In this study, we obtained the first full-length DNA-A sequence of the tomato-infecting Begomovirus isolate, (SB-Ilocos Norte) from Batac, Ilocos Norte in northern Luzon, new isolates from Los Baños, Laguna (LB-Laguna), and from Bukidnon (BK-Bukidnon) in Northern Mindanao. Pairwise sequence comparison with other Philippine isolates showed that SBN2-Ilocos Norte (KU946996) and LB4-Laguna (KX063715) have identity of 96.5 and 98.8% with ToLCPV strain B (AB050597), and thus considered as variants of ToLCPV strain B. The LB5-Laguna (KU946995) is a variant of ToLCPV strain A with 95.7% identity to ToLCPV-A [PH:Laguna3:06] (AB377113). The BK2-Bukidnon (KU946997) is a variant of ToLCCeV (EU487009) with 99.8% identity. The ToLCPV strains A and B have higher sequence identities in their V1, V2, C2 and C3, than C1 and C4 genes, and the intergenic region (IR). In this study, the infectivity of ToLCCeV (BK2-Bukidnon), ToLCPV strain A (LB5-Laguna) and SBN2-Ilocos Norte was first demonstrated by agroinonculation in Solanum lycopersicum L. and Nicotiana benthamiana. However, the plants infected with these isolates did not show any leaf curl disease symptoms suggesting that DNA betasatellite may be required for symptom expression. DNA betasatellite was associated with LB-Laguna, SB-Ilocos Norte and BK-Bukidnon isolates.

Cloning and analysis of satellite molecules associated with okra infecting begomoviruses in India

Asawari Gorane^1,2*, Suresh Kunkalikar¹, Radha Anandalakshmi¹

Email ID for Correspondence: asawarigorane@gmail.com

¹Plant – Virus Interactions Lab, Mahyco Research Center, Maharashtra Hybrid Seeds Pvt Ltd, Jalna -431203, Maharashtra, India; ²Department of Botany, University of Pune, Pune, Maharashtra, India

Okra is an important vegetable crop in India. Diseases caused by okra infecting begomoviruses are affecting productivity in all okra growing states. Satellite molecules are often associated with helper begomoviruses. Here, we report cloning and characterization of alpha satellite molecules associated with okra infecting begomoviruses.

Identification of amino acid residues of the coat protein of Sri Lankan cassava mosaic virusaffecting symptom production, viral titer and transmission

Vaishali Kelkar^*, Akhilesh Kumar Kushawaha and Indranil Dasgupta

Email ID for Correspondence: vaishalikelkar22@gmail.com

University of Delhi South Campus, Department of Plant Molecular Biology, New Delhi 110021, India

Sri Lankan Cassava Mosaic Virus (SLCMV),is bipartite begomovirus propagated vegetatively and main causative agent of Cassava Mosaic Disease (CMD) in the southern part of the Indian subcontinent. It is transmitted by Bemisia tabaci commonly known as whiteflies. The DNA-A component of the SLCMV is infectious to Nicotiana benthamiana by agroinoculation, causing symptoms of upward leaf rolling and stunting. To determine the importance of Coat Protein (CP) in both viral infectivity and transmission of the virus of the otherwise bipartite SLCMV, functioning as a monopartite virus in N. benthamiana, and to map the amino acid residues that can alter the extent of viral DNA accumulation and symptom development, mutations were introduced in the CP region of an infectious clone of SLCMV DNA-A. The mutated clones (bearing four single amino acid replacements and combinations of double, triple and quadruple mutations, total thirteen in all) along with two CP non-expressing (CP-null) mutants were tested in N. benthamiana for the accumulation of viral DNA and symptom development. A total of 11 out of 13 replacement mutants showed a decrease in the accumulation of viral DNA with varying symptoms and some totally abolishing the symptoms of leaf rolling and stunting otherwise shown by wild type SLCMV DNA. Mutations in amino acid residues 129 and 152 in SLCMV CP altered viral symptoms and severe reduction in viral DNA accumulation. When the whitefly transmission of the mutant clones were tested in Nicotiana tabacum, mutations in amino acid residues 134 and 152 showed the least transmission while mutations of 128 and 129 residues showed transmission similar to wild type. Triple mutations of residues 128, 138 and 152 resulted in lowest transmission of the virus. Yeast two hybrid studies confirmed the interactions between the wild type SLCMV CP protein and GroEL chaperone protein of Arsenophonus and Rickettsia, the secondary endosymbionts of whiteflies. However, SLCMV CP mutants showing low transmission retained their interactions with the GroEL proteins indicating that such interactions are not “the only essential” requirement for the virus transmission to occur. Our report reinforces the importance of certain critically positioned amino acids in the virus accumulation and the transmission of the virus and possibly the tool for targeting infectivity and transmission.

The biogenesis of Geminivirus related small RNAs and their role in virus infection

Qiuying Yang¹, Yuan Ming², Yuan Xi Lu²

Email ID for Correspondence: qiuying.yang@outlook.com; qyyang@ippcaas.cn

¹Chinese Academy of Agricultural Sciences, Institute of Plant Protection, ²Plants and Viruses Interaction Laboratory, Haidian District, Beijing, China. Zip code 100193

Small RNAs and small RNAs-mediated epigenetic modifications are key factors in Immune defense pathways against foreign nucleic acids, and key regulators in growth, development, differentiation, metabolism and reproduction pathways in Eukaryotes. Accompany the viral infection and anti-viral defense of plants, small RNAs are generated from and against the viral genomes. In defending against viruses, innate immune responses are activated and RNA interference pathways are boosted in plants. However, viruses encode silencing suppressors to inhibit the RNA silencing pathways in order to change the cellular environment to suit viral life cycle, the consequence of which manifested as viral symptoms in plants. Our study is to elucidate the mechanisms of small RNA biogenesis during viral infection, the influence of viral infection on the small RNA silencing pathways, plant morphology changes and viral symptoms development, the understanding of which will help in protecting the crops and contribute to the agricultural sciences.

Diversity and distribution of Cassava mosaic begomoviruses in Kenya

B. A. Muga¹, T. Makori², S. Mwaura², M. Ochwo-Ssemakula¹ and E. M. Ateka^2*

Email ID for Correspondence: eateka@agr.jkuat.ke

¹Makere university, Kampala, Uganda P.O. Box 7062, Kampala, Uganda; ²Jomo Kenyatta University of Agriculture and Technology P.O. Box 62000-00200, Nairobi, Kenya

Cassava mosaic begomoviruses are a major threat to cassava production in Kenya. However, their control is proving difficult because of the rapid spread by their vector, Bemisia tabaci and movement over long distances of infected planting materials from one area to another. In June–September 2015, a survey was carried out in major cassava growing areas of Kenya to determine the diversity and distribution of Cassava mosaic begomoviruses. A total of 250 cuttings were collected from symptomatic plants and grown in an insect proof screen house. Two months after planting, the fully expanded top leaves were used for detection of cassava mosaic begomoviruses using begomovirus specific primers. Results revealed unprecedented diversity of the begomoviruses. Three previously unreported begomoviruses namely East African cassava mosaic Malawi virus (EACMMV), South African cassava mosaic virus (SACMV) and East African cassava mosaic Cameroon virus (EACMCV) were detected in 1.6, 6.8 and 7.6% of the samples tested. The viruses were detected in samples collected from Coastal and Eastern regions, with EACMCV also being detected in samples collected from Western region. The other begomoviruses detected were African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), East African cassava mosaic virus-Ugandan variant (EACMV-Ug), East African cassava mosaic Kenya virus (EACMV-Ke), and East African cassava mosaic Zanzibar virus (EACMZV). Up to 7 begomoviruses were found co-infecting an individual plant. The study further revealed that viruses which were previously found in specific geographical locations have spread to new locations, for instance; EACMV-Ug which was earlier confined to the western region was now widely distributed in the eastern region. In addition, EACMZV, initially only found in the coastal region was found in all the major cassava growing areas in Kenya and is currently the most prevalent being detected in 66.8% of the samples tested. These findings highlight the importance of controlling the movement of planting materials and calls for further work to determine the genetic diversity of these begomoviruses.

PCR based detection and partial characterisation of Tomato leaf curl virus in Kerala

Arun Paul^*, Sally K. Mathew, Anita Cherian K. and Nazeem P.A

Email ID for Correspondence: aruntpaul@gmail.com

College of Horticulture, Kerala Agricultural University, Vellanikkara, Thrissur-680656, Kerala, India

Tomato leaf curl virus (ToLCV) a heterogeneous complex of whitefly vectored geminivirus has emerged as a serious production limiting factor in Kerala, results in severe crop loss during summer months. Tomato leaf curl disease (ToLCD) in tomato of Palakkad district, Kerala was surveyed and four distinct types of symptoms viz. upward curling and cupping (Type-I), curling and rolling (Type-II), curling with purple tint (Type-III) and yellowing and curling (Type-IV) were observed. The presence of ToLCV in four types of symptoms exhibiting plants were confirmed with the Polymerase Chain Reaction (PCR) amplification with coat protein specific primers CRv301F and CRc 1152R. The coat protein (CP) gene from the four types of samples were amplified and sequenced. The phylogenetic analysis of the four sequences revealed the existence of differences in their coat protein. Type-I had showed maximum homology to segment A of Ageratum yellow vein mosaic virus (AF314144.1) with a query coverage of 98% and Type-II with Pepper (Chilli)leaf curl Pakistan virus (AM491589.1) with query coverage of 95%. Type-III and Type-IV symptom producing strains were found identical to the DNA-A of three Tomato leaf curl Kerala virus isolates ToLCV K3 (EU910141.1), ToLCV K5 (EU910140.1) and ToLCV TC343 (KF551575.1). Sequence analysis of CP gene showed that, the four different types of symptoms associated with ToLCD in Kerala are caused by begomoviruses. This partial genome sequencing is indicating the existence of two more variants of ToLCV in Kerala in addition to ToLCV TC343, ToLCV K3 and ToLCVK5 which are already reported.

Suppressors of RNA silencing encoded by Velvet bean severe mosaic virus

Aruna Jangid¹, Vanita Chandel^1*, Yogesh Kumar² and Narayan Rishi¹

Email ID for Correspondence: vchandel@amity.edu

¹Amity Institute of Virology & Immunology, Amity University Uttar Pradesh, Noida-201313, India; ²Present Address: DAV University Jalandhar-144012 Punjab, India

RNA silencing is a conserved defence mechanism in eukaryotes against invading pathogens and viruses in counter-defence encodes proteins that suppress RNA silencing. These proteins are collectively known as RNA silencing suppressors (RSSs). With time viruses have acquired a variety of suppressors that work at different levels of the silencing pathways. Begomoviruses (Geminiviridae) cause serious losses in agricultural crops including few of having great medical importance in Indian medicine system. One such crop Mucuna pruriens (velvet bean) is called as magic bean, which is used to treat Parkinson’s disease, liver dysfunction and blood related diseases. Velvet bean severe mosaic virus (VbSMV) has been reported to cause a severe mosaic disease in M. pruriens in India. Velvet bean plants showing mosaic symptoms were analysed for presence of virus using rolling circle amplification (RCA) method. RCA products were restricted using endonucleases. Complete DNA-A and DNA-B were sequenced through primer walking. The infecting virus was identified as VbSMV. For identification of the candidate RSSs encoded by VbSMV, all viral genes were cloned in binary vector pCAMBIA1302 and transformed in Agrobacterium strain EHA105 through freeze–thaw transformation. These constructs were agro-inoculated in 3–4 weeks old Nicotiana benthamiana plants. After inoculation the plants were kept in an insect-free chamber at (28 °C ± 3) under 14–16 h lighting. Plants were photographed after 5dpi till 10 dpi. Agrobacterium co-infiltration assay was used to identify the candidate RSS encoded by VbSMV. Three genes were found to possess suppressor activity named as AC2, AV1, and AV2. Out of three genes AV2 was found to be strong suppressor of RNA silencing.

An unconventional satellite DNA in sugar beet infected by Beet curly top Iran virus

Gian Paolo Accotto^1*, Reianeh Soleimani², Slavica Matic¹, Nestor Vinals¹, S. Ali Akbar Behjatnia², Keramat Izadpanah², Emanuela Noris¹, Anna Maria Vaira¹

Email ID for Correspondence: gianpaolo.accotto@ipsp.cnr.it

¹Institute for Sustainable Plant Protection, National Research Council of Italy (IPSP-CNR), Torino, Italy; ²Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz, Iran

Begomoviruses are frequently associated with satellites, subviral agents composed of nucleic acid whose replication depends on the helper virus. Begomovirus satellites are about half the size of the virus genome and have distinct nucleotide sequences but share a genomic portion (the origin of replication) with their helper virus, necessary for their replication. Two main classes of satellites are associated with monopartite begomoviruses, alfa- and beta-satellites. Here, we describe a novel type of satellite found in Beta vulgar is infected by Beet curly top Iran virus (BCTIV), the type species of the new genus Becurtovirus, family Geminiviridae. This molecule (GenBank Acc. No. JX082260), 1315 nt in length, contains a 735-nt portion that does not derive from the helper virus, is rich in A/T, and does not encode any ORF. A BLASTN search revealed some similarities with B. vulgaris genomic sequences. Following co-agroinoculation of this satellite and BCTIV, we found that the satellite replicates not only in B. vulgaris, but also in Nicotiana benthamiana and Arabidopsis thaliana plants, without modifying symptomatology. Purified virion preparations from N. benthamiana plants co-inoculated with BCTIV and the satellite contained both the virus and satellite genomes, indicating that the satellite molecule is encapsidated. Other circular molecules isolated from plants infected by BCTIV are currently being investigated. Possible implications of this new type of satellite DNAs will be discussed.

Amaranthus and Verbesina new hosts of Croton yellow vein mosaic virus

Renuka Sharma¹, Vanita Chandel^1*, Pradeep Sharma² and Narayan Rishi¹

Email ID for Correspondence: vchandel@amity.edu

¹Amity Institute of Virology & Immunology, Amity University Uttar Pradesh, Noida-201313, India; ²Indian Institute of Wheat and Barley Research, Karnal-132001, India

Begomovirus is one of the largest genus of family Geminiviridae and the vector whitefly (Bemisia tabaci) is prevalent in the tropical and sub tropical regions of the world. Begomoviruses have very wide host range, ranging from cultivated crops to weed plants. Weeds are widely distributed throughout the world and have high environmental adaptability. They are considered as reservoirs of viruses. But for few exceptions there is no integrated study to identify viruses harbouring on weeds. Three weed species viz. Ageratum conyzoides, Verbesina encelioides, Amaranthus sp. growing in the vicinity of Chilly crop were showing prominent possible symptoms of Begomovirusi.e. yellow vein clearing, complete yellowing of leaves and chlorosis were collected from Yamuna bank, Amity University Uttar Pradesh, Noida Organic Crops farm. These weeds act as reservoir host of Begomoviruses and vectors can easily transmit viruses to cultivated crops. Total genomic DNA was isolated from each infected samples and was analyzed by PCR using begomovirus coat protein gene specific primers AV 494 and AC 1048 (Wyatt and Brown,1996). Following PCR confirmation of begomovirus infection, viral DNA was amplified by rolling circle amplification (RCA) using Phi-29 DNA polymerase. The RCA amplified DNA was cleaved with EcoR I, Bam HI, Pst I, Sph I, Hind III and Xba I, cloned in pUC 19 vector and were sequenced. Croton yellow vein mosaic virus was detected in Amaranthus and Verbesina the new hosts of these viruses. Agretum enation virus was detected on Ageratum.

Growing the tree-assembling of the genealogy of circular Rep-encoding ssDNA (CRESS) viruses

Li Li^*, Lele Zhao^*, Siobain Duffy

Email ID for Correspondence: lili.nott@gmail.com; lele.zhao@rutgers.edu

Department of Ecology, Evolution and Natural Resources, School of Environmental and Biological Sciences, Rutgers University, New Jersey, USA; 59 Dudley Rd. New Brunswick, NJ 08904

Circular Rep-encoding ssDNA (CRESS) viruses have been under the spotlight for theirdisplay of diversity in metagenomic data, in addition to their impact in agriculturaland medical fields. It will be helpful to have a clear understanding of the substitution patterns and to have access to a reliable genealogy of these viruses, as such knowledge will enhance identification and systematics of this diverse group that appears to be a clade distinct from other viruses. The most accurate CRESS genealogy requires an amino acid substitution matrix that accurately describes the evolutionary patterns of CRESS viruses. Although generalized amino acid substitution models are available, it has been shown that viruses of alternative lifestyles have being better delineated by specific amino acid substitution matrix, for example, implementing rtREV on retro-transcribing viruses. Thus, we have generated our own CRESS viruses specific matrix using published algorithms and tested its robustness against both general and other specific matrices. In total, 343 geminivirus replication initiator protein (Rep) reference sequences were extracted from NCBI GenBank as training dataset to generate geminiviruses specific matrix. This model outperformed all tested models at the genus level of begomoviruses and masterviruses. However, rtREV is best at describing circoviruses, another family of CRESS viruses. In general, the LG model ranked high when describing all test datasets. At species level, JTT model generally outperform REP derived model in describing sequence genealogy (19 out of 28 species tested). These results indicate that sequences with different divergence level are best modelled with different matrices. We intend to build the genealogy of all CRESS viruses using rtREV, LG and newly derived geminivirus REP specific model and compare these trees.

Molecular characterization of a novel bipartite begomovirus isolated from Hybanthus attenuatus and Rivina humilis in Colombia

Karina Lopez-Lopez, Frenyiline Jara-Tejada^* and Juan Carlos Vaca-Vaca

Email ID for Correspondence: fjarat@unal.edu.co

Facultad de Ciencias Agropecuarias, Universidad Nacional de Colombia sede Palmira.Palmira, Valle del Cauca, Colombia

In 2014, we detected begomoviruses in weeds Hybanthus attenuatus and Rivina humilisin Valle del Cauca (Colombia). In order to obtain the molecularly identify of those Begomovirus which were with these weeds, by PCR we obtain a 1.1 kb fragments corresponding to the A viral genome. Then it was cloned in pGEM T-Easy Vector (Promega), it was sequenced and then analyzed in the program Blastn. Comparison of the nucleotide sequences of the isolated 1.1 kb fragments of H. attenuatus and R. humilis indicated that belong to the same viral entity. After that the complete genome of Begomovirus detected in H. attenuatus was amplified by RCA, then was digested with HindIII and BamHI, then cloned into plasmid pBS and was sequenced and after that analyzed with the software CLC Main Workbench 7 and SDTv2.0. The complete genome sequence obtained of bipartite Begomovirus detected in H. attenuatus has 2584 and 2543 nt for genome A and B, respectively. The comparison of the A genome nucleotide sequence with other previously reported Begomovirus showed 87.2% identity with Bean mosaic dwarf virus (BDMV-CO-M88179.1). This value demarcated this virus as a new begomovirus species under the current classification criteria of these viruses, and we propose the name Hybanthus yellow mosaic virus (HybYMV) for this virus. Phylogenetic analyses showed HybYMV is more related with Abutilon mosaic virus clade isolated from north, central and South America plants. To our knowledge, HMYV is a new begomovirus for first time reported in H. attenuatus and R. humilis.

Replication competition between malvastrum yellow vein virus and tobacco curly shoot virus associated with their cognate betasatellites

Chenchen Jing, Miao Sun, Yan Hu, Gentu Wu, Xianchao Sun and Ling Qing^*

Email ID for Correspondence: qling@swu.edu.cn

College of Plant Protection, Southwest University, Chongqing 400716, China

The cognate betasatellite (MaYVB) of Malvastrum yellow vein virus (MaYVV) is necessary for typical symptoms induction, while Tobacco curly shoot virus (TbCSV) can induce typical symptoms without its betasatellite (TbCSB). To investigate the interaction relations between the two kinds of geminivirus/betasatellite disease complex, the infectious clone combination of MaYVV isolate Y47 (Y47A), TbCSV isolate Y35 (Y35A), and their betasatellite (Y47β and Y35β) was used to inoculate Nicotiana benthamiana and the replication level of these two viruses and their cognate betasatellites were detected by PCR and real-time quantitative PCR (qPCR). The symptoms of shrinking, downward leaf curling, yellow vein, distorted stem, dwarf and enation were found in the early stage of infection, but yellow vein symptom disappeared in the later stage. Y35A and Y35β increasingly accumulated during the infection with a peak at 90 day post inoculation (dpi). However, Y47A and Y47β decreasingly accumulated during the process. Finally, Y47A and Y47β were undetectable at 180 dpi and 120 dpi respectively. These results indicated that the competition probably occurred between these two disease complexes of Y47A/Y47β and Y35A/Y35β for the replication resources of the host and betasatellite is preferentially transreplicated by its cognate helper virus.

Molecular characterization of a novel bipartite begomovirus isolated from Croton hirtus in Colombia

Karina Lopez-Lopez, Frenyiline Jara-Tejada and Juan Carlos Vaca-Vaca^*

Email ID for Correspondence: jcvacava@unal.edu.co

Facultad de Ciencias Agropecuarias, Universidad Nacional de Colombia sede Palmira.Palmira, Valle del Cauca, Colombia

The aim of this research was to isolate the causative agent of golden mosaics present in Croton hirtus weed, first reported as host begomovirus agricultural systems in Valle del Cauca, Colombia. With this purpose, the viral genome was amplified by RCA from total DNA total isolated of C. hirtus. Then the RCA fragment was digested with appropriated restriction enzymes and cloning into pBS vector. Clones obtained were sequenced and then were analyzed using the CLC Main Workbench 7 and SDTv2.0 software. A typical bipartite Begomovirus was obtained: a 2613nt fragment corresponding to A genomic component and another 2551nt fragment corresponding to B genomic component. When the begomoviral A component were compared with its cognate components reported previously for others geminivirus, it showed 81.3% identity with Sida yellow mosaic Yucatan virus SiYMYuV-[MX-DQ875872.1]. According to the actual species criteria of demarcation (≤91% sequence identity for Begomoviruses), our begomovirus is a new virus for which we propose the name Croton golden mosaic Virus (CohGMV). The common region (CR) of CohGMV was 171 nt long and with a 100% identity with the B common region so that viral pairs in nature. In the CR, the iterated sequences in this virus were three: two direct copies ATTGGAGT and one inverted complementary CTCCAAAT and whose arrangement for the group in the clade Begomoviruses Virus Abutilon (AbMV). Phylogenetic, CohGMV is most closely related to AbMV clade, however, this virus not grouped with any virus specifically; this result could indicate its origin may be as a recombinant virus. To our knowledge a new geminiviral species CohGMV was detected in the alternative host C. hirtus and it is remarkable the growing number of alternative hosts which are describing as an alternative host for begomovirus at Valle del Cauca, Colombia.

Homology modeling and structural validation of coat protein (aey68281) of croton sparsiflorus yellow vein Lakshmangarh virus

Rajneesh Prajapat¹, Avinash Marwal² and Rajarshi Kumar Gaur^2*

Email ID for Correspondence: gaurrajarshi@hotmail.com

¹Rama University, Kanpur (U.P.) India; ²Department of Biosciences, Mody University of Science and Technology, Lakshmangarh, Sikar - 332311, Rajasthan, India

Research findings indicate that begomoviruses (family Geminiviridae) are geographically widespread viruses that cause destructive diseases in many crops. During an extensive survey for whitefly-transmitted geminiviruses, samples were collected from Lakshmangarh, Rajasthan (India) of stunted Croton sparsiflorusweed plants with small and mosaic yellow leaves. The DNA was extracted from infected leaves using CTAB method and PCR was carried out by using universal coat protein primer AC 1048 and AV 494. The PCR product was partially sequenced (JN998452) and its CDS sequence (AEY68281) was used for the in silico characterization. In the present study, the sequence coat protein sequence (AEY68281) of Croton sparsiflorus yellow vein Lakshmangarh virus was used to understand its structure, function and mechanism of the action of protein. The stereo chemical quality of the protein model was checked by using in silico analysis with PROCHECK, WHATIF, ProSA and QMEAN servers. The result of the study may be a guiding point for further investigations on coat protein (AEY68281) and its role in viral replication, infection and development of symptom. The 72.9% residue in the core region of Ramachandran plot showing high accuracy of protein model and the QMEAN Z-score of −7.07 indicates the overall model quality of coat protein (AEY68281).

Evaluation of infection efficiency of the two main begomovirus species in Brazil, tomato severe rugose virus and bean golden mosaic virus in different host

M. A. Macedo¹, M. R. Maliano², M. R. Rojas², J. O. Souza², E. Y. T. Nakasu¹, R. L. Gilbertson²; A. K. Inoue Nagata^1*

Email ID for Correspondence: alice.nagata@embrapa.br

¹Embrapa Vegetables, Brasília, DF, Brazil. ²Department of Plant Pathology, University of California-Davis, USA

In Brazil, tomato and bean crops are severely affected by the begomovirus disease. This is a viral disease caused by isolates of the begomovirus species, which are naturally transmitted by the insect vector, Bemisia tabaci. The predominant species in tomato and bean plants in Brazil are respectively Tomato severe rugose virus (ToSRV) and Bean golden mosaic virus (BGMV). The objective of this study was to evaluate the ability of BGMV and ToSRV to infect plants of the following species: Solanum lycopersicum, Phaseolus vulgariscv Topcrop and Carioca, Nicandra physaloides, Glycine max and Nicotiana benthamiana. Initially, they were agroinoculated with both viruses individually or simultaneously. Plants of S. lycopersicum, P. vulgariscv Topcrop, N. physaloides and N. benthamiana were excellent hosts of ToSRV, whereas BGMV was able to infect efficiently only P. vulgaris cv Topcrop. Plants of G. max and P. vulgariscv Carioca were not susceptible to ToSRV or BGMV by agroinoculation and a new inoculation assay was performed by biolistics. After inoculation, ToSRV and BGMV infected with a similar rate as the agroinoculation method the bean cv Topcrop, while only BGMV infected bean cv Carioca in a high infection. Soybean plants were not infected by any virus using this inoculation method. ToSRV infected bean cv Topcrop plants by agroinoculation were proven to be excellent inoculum sources for ToSRV under whitefly transmission tests to tomato plants cv Santa Clara. Quantification of viruses in infected plants demonstrated that bean and N. physaloides plants are good propagation hosts to ToSRV, and thus they are potential good inoculum sources of ToSRV for the tomato crop in Brazil.

An update of begomovirus infecting tomato and weeds in Argentine

C. G. Vaghi Medina¹, V. A. Bornancini¹, N. Puyané¹, V. Ranieri¹ and P. M. López Lambertini^1*

Email ID for Correspondence: lopezlambertini.pao@inta.gob.ar

¹Plant Pathology Research Institute, CIAP-INTA, Argentina

The begomovirus species diversity is wide as shown by the 288 species recognized by ICTV. In Argentine, begomoviruses are an important constrain for tomato production. Here, we reported the genetic diversity of begomoviruses infecting tomato crops and weeds in Argentine. The samples were selectedby RCA-RFLP patterns. Full-length fragments of the viral RCA products were cloned in bacteria and sequenced. We cloned and full sequenced a total of 47 DNA-A and 30 DNA-B. Blast analysis and a maximum-likelihood phylogenetic tree were constructed for DNA-A and DNA-B alignments using PhyML. Based on the current taxonomic criteria established for the genus Begomovirus we identified 9 species in tomato and 3 in weeds. Among the 9 species characterized in tomato, 5 were proposed as new species. A geographic distribution of specie was observed except for Tomato yellow vein streak virus that was the predominant species in all tomato production regions. Six species were found in different combination in mixed infections. The characterized weed infecting begomoviruses were: Sida mosaic Brazil virus infecting Malva sp and Malvastrum sp; Tomato yellow spot virus infecting a Coronopus sp and Leonurus sp; and a proposed new species named Papilla yellow mosaic virus in Pitraeacuneato-ovata. Interestingly, none of the begomoviruses identified in weed were found infecting tomato. The phylogenetic analysis revealed that Argentina begomovirus mainly clustered in three clades. Study the co-infections and the weed reservoirs of begomovirus are fundamental to understanding the disease dynamics in tomato crops.

Studies on evolutionary relationship of Papaya leaf curl virus beta-satellite DNA

Priyanka Varun^1*, S. A. Ranade² and Sangeeta Saxena¹

Email ID for Correspondence: priyanka.varun@yahoo.com

¹Department of Biotechnology, BBAU, Lucknow, India and ²Molecular Biology and Genetics, CSIR-NBRI, Lucknow, India

Papaya leaf curl viruses (PaLCuV), geographically widely prevalent disease causing agents of eponymous disease, are responsible for severe economic losses in papaya cultivation. They belong to Begomoviruses (whitefly transmitted geminiviruses with mono or bipartite genomes). The beta-satellites frequently associated with monopartite begomoviruses are responsible for symptom development but need viral DNA for replication, encapsidation and systemic spread in the host. Association of beta-satellites with papaya leaf curl viruses have already been reported. In natural habitats considerable variations have been observed among different symptom-based isolates of papaya leaf curl viruses. However, very few studies explain the variations among isolates in terms of viral genome sequence variations. Similarly, there are very few reports available about diversity in the sequences of DNA beta-satellites. In the present study, we have analysed diversity among all papaya leaf curl beta satellite DNA sequences available in sequence database using maximum composite likelihood model implemented in MEGA 6. The maximum substitution value of 4.14 was observed between beta-satellite sequences of KJ642219.2 (China: Hainan) and KC959933.1 (India: Coimbatore) isolates indicating the highest variation between these isolates whereas least value of substitutions was observed to be 0.00 (between accessions NC_004706 and AY244706.1 and two Iranian isolates KJ397536.1 and KJ397534.1). On the basis of the substitution matrix a phylogenetic tree was generated that clearly resolved all beta-satellite sequences into three clusters respectively including accessions with zero substitutions in one, 0.01 substitutions in another and all other isolates in the final large cluster. The tomato yellow leaf curl virus beta-satellite sequence (out-group) separated from all the papaya isolates. The study suggests phylogenetic significance of the beta-satellites in papaya leaf curl viral disease.

Rhynchosia golden mosaic Valle virus, a new bipartite begomovirus isolated from five weeds: Rhynchosia minima, Amaranthus dubius, Rivina humulis, Desmodium sp.and Caesalpinia sp

Karina Lopez–Lopez^*, Frenyiline Jara-Tejada and Juan Carlos Vaca–Vaca

Email ID for Correspondence: klopezl@unal.edu.co

Facultad de Ciencias Agropecuarias, Universidad Nacional de Colombia sede Palmira.Palmira, Valle del Cauca, Colombia

The goal of this research was characterized at molecular level Begomovirus previously detected in weeds Rhynchosia minima, Amaranthus dubius, Rivina humulis, Desmodium sp. and Caesalpinia sp, which were collected at southeast of Valle del Cauca, Colombia. A fragment of 1.1 kb belonging to the begomoviral component A genome was amplified by PCR, cloned into the vector pGEM T-Easy Vector, sequenced and analyzed in the program Blastn. The complete genome of Begomovirus found in Rhynchosia minima was obtained by RCA, then digested with EcoRI and EcoRV and cloned into plasmid pBS. This plasmid were sequenced and analyzed using software CLC Main Workbench 7 and SDTv2.0. Comparison of the nucleotide sequences of 1.1 kb fragments isolated of Rhynchosia minima, Amaranthus dubius, Rivina humulis, Desmodium sp. and Caesalpinia sp., indicated that all begomovirus detected were the same virus. The genome A of begomovirus found in Rhynchosia minima had a size of 2584 nt meanwhile the its B component had 2543 nt. The nucleotide sequence comparison of the A genome with begomovirus previously reported showed 90.9% identity with Rhynchosia golden mosaic Yucatan virus (KP641347.1). This value demarcated this virus as a new begomovirus species under the current classification criteria of these viruses. Based on the symptoms showed by the weed in which this begomovirus was detected, the name of Rhynchosia golden mosaic Valle virus (RhGMVV) is proposed. All evidence pointed that RhGMVV has the quality to adapt to various kinds of hosts as observed in this study.

Proteomic approaches to understand the bhendi yellow vein mosaic virus infection in plants

Ashirbad Guria, P. Priyavathi and P. Gopal^*

Email ID for Correspondence: pgopal.biotech@mkuniversity.org, pgp79@yahoo.com

Centre for Plant Molecular Biology, School of Biotechnology, Madurai Kamaraj University, Madurai-625021, India

Bhendi is an agronomically important crop grown in India and around the world. Unfortunately, the production is hampered significantly by the infestation of whiteflies carrying the Bhendi yellow vein mosaic virus and Okra enation leaf curl virus. Earlier reports concluded the extreme loss of up to 96% yield reduction by bhendi yellow vein mosaic disease alone. Effective disease management includes application of insecticides at timely intervals which concentrates the field and fruit with harmful chemicals. Since genome data is unavailable for bhendi, it is difficult to trace out its defence pathway and the genes responsible for such action. Keeping this point in mind, one approach would be to find out the protein profiling of plants at a particular time point. This would provide us with an insight of different types of proteins getting expressed in a stressed condition enabling us further to compare with the proteins in a normal state of the crop. Hence, the aim of the present work is to develop an on-site kit for detection of early infection against Bhendi yellow vein mosaic virus. Moreover, bhendi is already known to contain with high amount of mucilages and other phenolic compounds. Therefore, optimisation of different protein extraction protocols is of utmost necessity to carry out downstream analysis. These details will be explained further during the presentation.

Molecular characterization of virus causing yellow mosaic disease in chayote

B. Sangeetha, P. Renukadevi^*, V. G. Malathi, M. Suganthy and D. Alice

Email ID for Correspondence: renucbe88@gmail.com

Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu-641003

Chayote (Chow-chow-Sechium edule) is a cucurbitaceous vegetable crop grown in hilly regions of India in Karnataka, Tamil Nadu, West Bengal and Himachal Pradesh for its nutritious tasty fruits. It is grown in, Sirumalai, Thandikudi, Adallur, Kodaikanal hills of Dindigul District and Kothagiri of Nilgiris District of Tamil Nadu. The crop is infected by powdery mildew and downy mildew pathogens and a severe yellow mosaic disease. Among these, the yellow mosaic disease is widely prevalent and causes >90% yield loss. The leaves show mosaic mottling, yellowing and puckering. In extreme cases warts like dark green enations are observed on the under surface of the leaves. The fruits from symptomatic plants were found to be malformed and reduced in size. In the survey conducted it was observed that the seedlings do not express any symptom up to sixth month and only at the time of trailing when foliage gets exposed, symptoms are visible. Mandal et al. 2004 recorded a begomovirus closely related to ToLCNDV in yellow mosaic affected chayote in Sikkim and Darjeeling hills. An investigation was conducted on the etiological agent of the severe yellow mosaic disease in Tamil Nadu by analyzing the infected leaf, fruit samples from six locations in Dindugal district and kothagiri hills of Nilgiris district in Tamil Nadu. In dot immuno binding assay using antibody to African cassava mosaic virus and in PCR with Roja’s primers (PAR1v772 5′GGNAARATHTGGATGGA3′; PAL1c1960 5′ACNGGNAARACNATGTGGGC3′, Paximadis et al. 1997) positive results were obtained indicating the presence of a begomovirus in infected plants. Further characterization of the virus by Rolling circle amplification and cloning, the causal agent was identified as Tomato leaf curl New Delhi virus (ToLCNDV) since it showed 97.2% identity with ToLCNDV (GenBank accession no-KP191047) earlier described from chayote. However both the isolates from chayote showed only 92% identity with an isolate of ToLCNDV from ridge gourd (KJ1426903.1).Interestingly, the DNA B component exhibited very low identity with DNA B component of ToLCNDV (HM989846.1 and DQ169057.1), in fact only 65% of genome could be aligned and upstream of coding regions, ORFs BC1 and B1 did not find any identity with any of the begomoviruses. Preliminary study revealed the presence of virus in fruit tissues. Among seven seed samples analyzed virus was detected in seed coat, endosperm and in embryonic tissues. The virus was also detected in symptom free seedlings in 5 out of 17 seedlings studied in grow-out tests. From all the observations it can be inferred that ToLCNDV-Chayote, is seed borne and may play a role in disease spread. Accordingly efforts are being taken up to replace seed material with virus free seeds to ensure freedom from spread of the disease.

Identification of begomovirus and associated satellite components in Alcea rosea L. in India

Manish Kumar, R. Vinoth Kumar, D. Ragunathan, Supriya Chakraborty^*

Email ID for Correspondence: mannuranchi@gmail.com

School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India

Hollyhock (Alcea rosea L.) is an annual ornamental plant grown in the tropical and subtropical regions in India. Geminiviruses are encapsidated in twinned icosahedral particle which infect large variety of crops around the world and causes a new threat to global food security. During February 2016, begomovirus-like symptoms were noticed on the hollyhock plants grown in a garden at the campus of Jawaharlal Nehru University, New Delhi. Since the hollyhock plants exhibited typical symptoms of yellow vein mosaic, we anticipated the possibility of association of begomoviruses in these plants. Rolling circle amplification was performed to clone the associated begomoviral genomic components from the total DNA isolated from these infected plants. We have identified an isolate of the Cotton leaf curl Rajasthan virus, Luffa associated betasatellite and Okra leaf curl alphasatellite in association with yellow mosaic disease infected hollyhock samples. The phylogenetic and recombination analysis of the cloned begomoviral genomic components were analyzed and will be presented.

Association of a distinct bipartite begomovirus with the mosaic disease of Ivy gourd (Coccinia grandis L.) in India

G. Karthikeyan^1*, C. G. Balaji¹, V. K. Satya², S. Mohankumar³, V. G. Malathi¹, S. K. Manoranjitham¹, P. Renukadevi¹, and K. Nagendran^1,4

Email ID for Correspondence: agrikarthi2003@gmail.com

¹Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India – 641003; ²National Pulses Research Centre, Tamil Nadu Agricultural University, Vamban, Tamil Nadu, India; ³Department of Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India – 641003; ⁴Division of Crop Protection, ICAR – Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh – 221305, India

A distinct bipartite begomovirus was found to be associated with the mosaic disease on ivy gourd (Coccinia grandis L.) in Tamil Nadu, India. The complete DNA A and DNA B components were amplified by rolling circle amplification. The DNA sequences were determined for both the components and it comprised of 2749 and 2691 nucleotides respectively. The genome organization of the virus was found to be a representative of old world bipartite Begomovirus. There was no association of ß-satellite with this virus. Comparison of DNA A with the other begomoviruses shared only 73.4% nucleotide identity with Loofa yellow mosaic virus suggesting that it is distantly related to the other known begomoviruses infecting various crops. This leads to suggest for a new name for this virus as Coccinia mosaic virus (CoMoV). Similarly, the DNA B component has also shared a maximum identity of 55% with ToLCNDV. In phylogenetic analysis, CoMoV formed a separate cluster with the other known begomoviruses. The recombination analysis showed that there was no recombination events detected in the genome. This is a new begomovirus infecting ivy gourd with the mosaic symptoms in the world.

Molecular characterisation of Begomoviruses from ornamental plants in Tamil Nadu

S. Deepan, S. U. Mohammed Riyaz, T. Rajamuthuramalingam, R. Pandiyan, T. Kumaran and D. Michael Immanuel Jesse K. Kathiravan^*

Email ID for Correspondence: drkkathiravan@gmail.com

Plant Molecular Virology and Nanobiotechnology Laboratory, Department of Biotechnology, University of Madras, Maraimalai Campus, Guindy, Chennai- 600025

Begomovirus is one of the largest genera of the family Geminiviridae transmitted by vector whitefly (Bemisia tabaci). During 2014 in Tamil Nadu state, very high disease incidence (up to 90%) was observed in major vegetable crops such like tomato, chilli, squash and bitter guard. In recent years, geminiviruses infection randomly spreading towards other crops, weeds and also in ornamental crops. In this study, we investigated begomovirus infection on ornamental plants cultivated in northern districts of Tamil Nadu. At northern districts, Jasmine (Jasminum) plants are predominantly cultivated and the production is hampered by few known factors including virus infection. The field was identified and the begomovirus characteristics field symptoms were observed such like upward severe curling, and reduced size of leaves. Severely affected plants showed stunted growth and produced small and no flowers. For begomovirus infection confirmation, Infected jasmine leaves samples were collected and subjected to polymerase chain reactions (PCR) using the coat protein and Replicase gene specific primer designed using Bio edit software, which gave an approximately 500 bp and 850 bp amplified products respectively. The same amplified products were subjected to Sanger sequencing method. A BLAST search with obtained sequence, revealed close (96%) similarity of the sequence of Squash leaf curl china virus (SLCCV) from the NCBI database. Hence, it confirmed that Jasmine (Jasminum) is one among the host and acts as a reservoir for Begomoviruses population at all seasons in the agriculture field of northern districts of Tamil Nadu.

Identification and characterization of begomovirus species infecting pumpkin or squash (cucurbita moschata) in the Philippines

Cherry Relevante-Belagantol^1,2*, Filomena C. Sta. Cruz¹, Jaira T. Lorenzo², Maristil I. Pinote², Hatthaya Arunothayanan³ and Conrado H. Balatero²

Email ID for Correspondence: cherry.relevante@eastwestseed.com

¹Crop Protection Cluster, College of Agriculture, University of the Philippines Los Baños, College Laguna, Philippines; ²East-West Seed Company, Inc., Hortanova Research Center, Purok 3, Pagoligin Bata, Lipa City, 4217 Batangas, Philippines; ³Hortigenetics Research (S.E. Asia) Limited, Station 7 Moo9, Tambol Maefak Mai Sansai, Chiang Mai 50290 Thailand

The leaf curl disease is becoming a major constraint of squash production in the Philippines. Surveys and sample collections were conducted in 2014 and 2015 cropping seasons covering a total of 20 locations in the squash growing regions of Luzon, Visayas and Mindanao islands in the country. All 227 squash samples collected had leaf curl and yellowing symptoms. We describe here the identification and characterization of Begomovirus species infecting squash in the Philippines, based on the sequence analysis of the coat protein (CP) gene and replication-associated protein (Rep) gene. Begomoviruses infecting squash were detected by indirect enzyme-linked immunosorbent assay (I-ELISA) using commercial mouse monoclonal antibody (MAb D2) against whitefly transmitted geminiviruses and confirmed by polymerase chain reaction (PCR) using degenerate and universal primers targeting the begomovirus CP gene and Rep gene. BLAST analysis of the CP nucleotide sequences of representative samples revealed4/4 samples (2 Luzon, 1 Visayas and 1 Mindanao) sharing 97–99% identity with Squash leaf curl Philippines virus (SLCuPHV), and 96–97% identity with Squash leaf curl Philippines virus from Taiwan (SLCuPHV-TW) and Squash leaf curl China virus-Philippines (SLCCNV-PH). BLAST analysis of the Rep nucleotide sequences revealed 3/8 samples (2 Luzon, 1 Mindanao)sharing 97–98% identity with SLCuPHV isolates, 1 sample (Visayas) sharing 96% identity with isolates of SLCuPHV-TW, 1 sample (Luzon) sharing 97% identity with SLCCNV-PH and 1 sample (Mindanao) sharing 97% identity with SLCCNV isolate from Coimbatore, India (AY184487). Phylogenetic analysis of the Rep nucleotide sequences showed that all Luzon samples clustered with previously reported SLCuPHV isolates sharing 96–98% nucleotide identity, while the Visayas and Mindanao samples clustered withthe reported SLCCNV isolates sharing 97% nucleotide identity. Based on the CP nucleotide sequences, however, all samples were most closely related to SLCuPHV isolates with 97–99% nucleotide identity.

Molecular and functional analysis of disease resistance breaking tomato leaf curl Bangalore virus

Ganapati Bhat^*, Suresh Kunkalikar, Yogita Deshmukh, Muktha Raghuram, Ravi Kankanallu, Prem Rajagopalan, Radhamani Anandalakshmi

Email ID for Correspondence: ganapti.bhat@mahyco.com

Plant – Virus Interactions Lab, Mahyco Research Center, Maharashtra Hybrid Seeds Pvt Ltd, Jalna -431203, Maharashtra

Leaf curl disease is a destructive viral disease of tomato caused bydifferent begomoviruses. Several resistance loci (Ty1 to Ty6) have been identified in different wild relatives and introgressed into cultivated tomato-Solanum lycopersicon for getting broad spectrum resistance to begomoviruses around the world. Tomato leaf curl Bangalore virus (ToLCBV) is a type monopartite virus that is prevalent in India and resistance to this virus is important for developing tomato varieties and hybrids. In recent years, an outbreak of resistance breaking strain of ToLCBV is causing losses in tomato production in South India. In this study, full length virus genome of three ToLCBV isolates that are virulent on tomato carrying Ty2 locus were cloned and sequenced. Mutations in the virus responsible for disease resistance breaking and the response of tomato harboring different Ty genes will be discussed.

Characterization of strain of Tomato leaf curl New Delhi virus associated with yellow mosaic disease of ridge gourd in southern India

Chandrakant V. Patil, S. V. Ramdas, K. S. Shankarappa^*, and M. S. Kulkarni

Email ID for Correspondence: ksshankarappa@gmail.com

Department of Plant Pathology, K.R.C College of Horticulture, Arabhavi-591218; University of Horticultural Sciences, Bagalkot, Karnataka, India

Ridge gourd is an important vegetable crop in Karnataka and is affected by several biotic and abiotic factors. Of the different biotic factors, yellow mosaic disease is a newly emerging problem affecting the yield to a considerable extent. Incidence of the disease varied from 43 to 100% among six districts of northern Karnataka, India with the highest incidence (100%) in Belgaum district. The infected plant showed initial chlorosis, mosaic, upward and downward curling and cupping of leaves, blistering, reduction in leaf size and stunted growth. Transmission characteristics were determined and the disease was confirmed to be transmitted by whitefly Bemisia tabaci. Sponge gourd, ash gourd, pumpkin, summer squash, cluster bean, tobacco, datura and cucumber were identified as reservoir hosts for the virus. Polymerase chain reaction was carried out by amplifying the fragments of full length DNA-A and DNA-B components of begomovirus and it was found to be bipartite. Sequence analysis revealed that causal virus shared highest nucleotide sequence identity of 92.3% with Tomato leaf curl New Delhi virus infecting sponge gourd from India. Sequence and phylogenetic analysis of both DNA-A and DNA-B components showed that the begomovirus associated with yellow mosaic disease of ridge gourd is found to be strain of Tomato leaf curl New Delhi virus in southern India.

Molecular diversity of begomoviruses infecting cucurbitaceous crops in Tamil Nadu

K. Nagendran^1,2*, R. Aravintharaj², S. Mohankumar³ and G. Karthikeyan²

Email ID for Correspondence: krishnagendra@gmail.com

¹Division of Crop Protection, ICAR – Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh, India – 221305; ²Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India – 641003; ³Department of Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu – 641003, India

The leaf samples of cucurbitaceous crops showing characteristic symptoms of Begomoviruses were collected from all the seven agro-climatic zones of Tamil Nadu. The symptoms include mild to severe mosaic mottling, stunting, shoe-string, chlorotic spots on leaves, enations, vein banding and clearing, chlorosis and reduction of leaf and fruit sizes, blistering and deformations of leaves and fruits. The begomoviruses were detected in 98% of the samples through PCR using the coat protein (CP) gene specific primer pair (GK ToLCV F-5′ATGKYGAAGCGACCAGCMGA3′; GK ToLCV R-5′CGCCCKCMGAYTGGGMTTTTCTT3′). The samples were also subjected for DAS-ELISA using polyclonal antibody ofSquash leaf curl virus. In the provisional identification of begomoviruses based on the sequences of coat protein revealed that the Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl China virus (SLCCNV) were the begomovirus species associated with the cucurbitaceous crops in Tamil Nadu with an identities of around 88–98% with the isolates of India, Pakistan, Bangladesh and Spain. The phylogenetic analyses based on nucleotide sequence of CP revealed that SLCCNV formed a separate cluster away from the ToLCNDV. There was also a large diversity in ToLCNDV isolates infecting cucurbitaceous crops in Tamil Nadu and they were distributed throughout the state irrespective of geographical locations, crop specificity and symptoms. The sequences of the present isolates of ToLCNDV infecting cucurbitaceous crops were distantly related to the isolates infecting tomato and other crops whereas SLCCNV were found to be specific in the cucurbitaceous crops.

Viability of Mungbean yellow mosaic virus and evidence for the indirect seed transmission of virus in mungbean through seeds

Mahatma Lalit^1*, I. Patel Mitalkumari, M. Pawar Dyneshwar, P. Patel Harshal, R. Pandya Jaimin, K. Mahatma Mahesh, and K. Solanki Ramesh³, A. Solanki Vikram

Email ID for Correspondence: mahatmalalit@yahoo.co.in

¹Department of Plant Pathology, NM College of Agriculture, NAU, Navsari-396 450, Gujarat, INDIA; ²ICAR-Directorate of Groundnut Research, Ivnagar Road, P. Box # 5, Junagadh-362 001, Gujarat, INDIA; ³ICAR-National Research Centre on Seed Spices, Tabiji, Ajmer, 305 206, Rajasthan, India

Presence of the begomovirus, particularly Mungbean yellow mosaic virus (MYMV) in the mungbean (cv GM-4) seeds, specifically in the entire seed, cotyledon and seed coat was first time detected in 2010 from the Navsari, Gujarat by our group and subsequently demonstrated the indirect role of seeds in the epidemiology of MYMV in mungbean in 2015. Within a short span, the seed transmission of Sweet potato leaf curl virus (SPLCV) in sweet potato, Tomato yellow leaf curl virus (TYLCV) in tomato and Okra yellow vein mosaic in okra have been reported. Presence of virus can be detected by the molecular tool, however, information on the virus viability in the seeds is the most important criteria for the adjudging the virus as seed borne in nature. Attempts were made to study the viability by acquisition of the virus by its vector, whitefly, Bemisiatabaci on the cotyledon and subsequently transferring the same on the susceptible cultivar (GM-4). Seeds were collected from the mungbean plant showing characteristics symptoms within 25 days of sowing. Before, collection of the seeds, the symptoms on the pods were also observed. Seeds from the plant were pooled and randomly 2 seeds were taken for the detection of the MYMV by the PCR to assure the presence of virus. After assuring the seeds were incubated at the room temperature (~28 °C) for a month and used for the experimentation. Whiteflies were allowed to feed on cotyledon for 12 h to acquire the virus from the water soaked turgid and spongy cotyledon. Mortality of the whiteflies on the cotyledon was more (60%). After acquisition the cotyledon were tested by the PCR for the presence of virus. Eight, out of the 20 seeds cotyledon used for the acquisition showed the presence of MYMV. Ten whiteflies were released on the 10 days old seedling of susceptible cultivar (GM-4) in the insect proof chamber. Out of the 9 plants inoculated, two plants showed characteristic symptoms of the MYMV after the 15 days of inoculation. The result indicated that the MYMV survive for at least 1 month in the susceptible cultivar (GM-4). The findings strongly support the hypothesis of indirect seed transmission of the MYMV in mungbean.

Chickpea infecting geminiviruses

Suresh Kunkalikar, Ramesh Karnawar^*, Asawari Gorane, Mukesh Kharat, Radhamani Anandalakshmi

Email ID for Correspondence: ramesh.karnawar@mahyco.com

Plant – Virus Interactions Lab, Mahyco Research Center, Maharashtra Hybrid Seeds Pvt Ltd, Jalna -431203, Maharashtra, India

Chickpea (Cicer arietinum L.) is one of the important commercial legumes grown in India. Analysis of chickpea plants showing yellowing, bronzing, chlorosis of leaves and stunting and virus transmission showed that the plants were infected by a virus that is not transmitted via whiteflies or mechanical sap inoculation. However, when shoots of infected chickpea were side grafted onto healthy chickpea plants, transmission of virus was observed and plants developed mild chlorosis and leaf curling. The characterization of virus showed it is a Mastrevirus. Prevalence of this virus in India and its host range will be presented.

Study of radish leaf curl viruses and their host range

Kalyani Sarwadnya^*, Suresh Kunkalikar, Sharad Gulhane and Radhamani Anandalakshmi

Email ID for Correspondence: kalyani.sarwadnya@mahyco.com

Plant – Virus Interactions Lab, Mahyco Research Center, Maharashtra Hybrid Seeds Pvt Ltd, Jalna -431203, Maharashtra, India

Radish and okra plants showing leaf curl symptoms were studied for identification of the casual organism. Whitefly transmission of virus from symptomatic samples to healthy radish and okra produced typical leaf curling symptoms 12–15 days post inoculation of plants. Total DNA isolated from infected plants was used for amplification of virus genome with degenerate primers. The amplicons were cloned and plasmid DNA isolated from two individual colonies was sequenced. A comparative analysis of these sequences with corresponding gene sequences of other Geminiviruses in the GenBank revealed highest sequence identity with Radish leaf curl virus (family: Geminiviridae, genus: Begomovirus). Transmission of RaLCV to commercial vegetable crops, symptoms, putative recombination of virus with other Geminiviruses and host range will be discussed.

Consequences of synergistic interaction among chilli-infecting begomoviruses

Ashish Kumar Singh^*, Nirbhay Kumar Kushwaha and Supriya Chakraborty

Email ID for Correspondence: aks.ibt@gmail.com

Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

Plants are continuously exposed to several pathogens. Multiple virus infections may lead to either synergistic or antagonistic interaction among the invading pathogens. During synergistic interaction, at the cellular level viruses exert positive influence on the replication, movement and provide suitable environment for each other which eventually favours virus accumulation and pathogenesis. We observed breakdown of natural resistance in the chilli cultivar (Capsicum annuum cv. Kalyanpur Chanchal) due to the association of four genomic components of begomoviruses such as DNA-A of Tomato leaf curl New Delhi virus, DNA-B of Tomato leaf curl Gujarat virus, DNA-A of Chilli leaf curl virus and DNA β of Tomato leaf curl Bangladesh betasatellite. Further, frequent association of these four genomic components was also observed in the symptomatic plants of other resistant and susceptible chilli cultivars (Capsicum chinense cv. Bhut Jolokia and Capsicum annuum cv. Kashi Anmol) grown in the experimental field. The nature of molecular interactions among these four molecules of begomoviruses was analyzed in laboratory host N. benthamiana as well as in natural host chilli. Symptom severity was proportional with the higher viral DNA accumulation in plants infected with more than one begomovirus. Semi-quantitative assessment revealed presence of high viral titer in mixed infected resistant chilli plants. Analysis of the host basal (ascorbate peroxidase, thionin, polyphenol oxidase) and specific defense-related gene (NBS-LRR) expression revealed that mixed infection of chilli-infecting begomoviruses suppress the expression of defense genes that finally leads to the breakdown of resistance and establishment of pathogenesis in hitherto known resistant chilli cultivars.

Identification and molecular characterization of begomoviruses infecting bean crops in Northwestern Region of Argentina (NOA)

Gonzalo Varela, Vera Ávalos, Irma G. Laguna, Patricia Rodriguez Pardina^*

Email ID for Correspondence: rodriguez.patricia@inta.gob.ar

INTA-Instituto de Patología Vegetal.-IPAVE. Camino 60 Cuadras Km 5 ½ X5020ICA, Córdoba, Argentina

The subtropical Northwestern region (provinces of Tucumán, Salta, Jujuy, Santiago de Estero and Catamarca) is the main bean producing area of Argentina. It has a high incidence of the whitefly Bemisia tabaci, therefore the presence of begomoviruses is common in bean, causing up to 100% yield losses and seriously diminishing the quality of seeds. Three geminiviruses were previously found infecting bean in Argentina: Bean golden mosaic virus, Tomato yellow spot virus and Soybean blistering mosaic virus, which were also found in soybean crops. Specific nucleic acid hybridization probes were developed to differentiate these three begomoviruses, and were used to assay field samples. Some of the tested symptomatic plants didn’t react with either of the probes, so the presence of other viral species was suspected. Total DNA, of two of these samples: one from Jujuy Province collected during 2010 agricultural season (P160), and the other form Tucumán, collected in 2013 (PRCA4) was extracted by CTAB method: DNA was amplified by Rolling circle amplification using TempliPhi Kit (GE Healthcare) and DNA fragments obtained after digestion with Apa I, Sac I and Pst I were cloned into Bluescript II, SK. Four clones were fully sequenced by primer walking. Nucleotide similarity comparisons demonstrated that clone P160 Sac I. had highest nucleotide identity (93%) with DNA-A of Sida brazil virus (SiBrMV), P160 Apa I with DNA-B of the same virus (94%), PRCA4.6 PstI shared 94% identity with DNA-A of Tomato mottle wrinkle virus and PRCA4.17 PstI 95% identity with the DNA-A of Tomato yellow vein streak virus,, indicating a mixed infection. To our knowledge this is the first report of these three virus infecting bean, on the other hand, it is important to remark the diversity of begomoviruses found in bean in our country, since up to the present six different species have been detected.

New isolate of Pepper huasteco yellow vein virus (PHYVV) increase host range specificity by exhibiting differential symptoms severity patterns

N. Meléndrez-Bojórquez^*, E. Camacho-Beltrán, E. A. Rodríguez Negrete, N. E. Leyva-Lópezand J. Méndez-Lozano

Email ID for Correspondence: nataniel_xy@hotmail.com

InstitutoPolitécnico Nacional, CIIDIR, Unidad Sinaloa. Departamento de Biotecnología Agrícola.Blvd.Juan de Dios Bátiz Paredes No. 250, Col. San Joachín. Guasave, Sinaloa, México C.P. 81101

Since 1993, Pepper huasteco yellow vein virus (PHYVV) was reported infecting pepper as serious pathogens in Mexico. Recently, emerging disease was observed in pepper crops cultivated in green house and open field in the state of Sinaloa, Mexico. The molecular evidence indicates the presence of PHYVV as a main actor in such samples. Full genome clones of DNA A and B were obtained and designated as PHYVV-Sin. The complete sequence of the DNA A and B shows an identity of 94.1 and 83.6% respectively with the first reported sequence of PHYVV-Tam. The low homology of DNA B of the new isolate suggests a genetic evolution of PHYVV. To understand the biological effect of the DNA-Bin the symptoms patterns expression and host range; both PHYVV isolates were evaluated by biological and molecular assays. The first step was to understand the role of the novel DNA B in symptoms severity and their response to interact within different host. Additionally, psedorecombination interaction was analyzed by swapping DNA-A and DNA-B of both isolates. In these sense Nicotiana benthamiana, pepper, tomato, petunia, cucumber and common bean seedlings were agroinoculated with PHYVV-Tam and PHYVV-Sin in single or pseudorecombination infection. The data suggest that genetic change of PHYVV-Sin DNA-B represent a positive evolutionary event occurring naturally and adapted to the new environmental circumstances. In this sense, host-virus interaction improves in a better manner for the virus to increase the pathogenicity and modifying the host rage. Finally, the new isolate of Pepper huasteco yellow vein virus became a pepper-infecting begomovirus with a significative biological effect to induce emerging diseases in Mexico.

Watermelon, a new host for Chickpea chlorotic dwarf virus

T. Zaagueri¹, M. Mnari-Hattab¹, S. Zammouri¹, M. R. Hajlaoui¹, A. M. Vaira², G. P. Accotto^2*

Email ID for Correspondence: emanuela.noris@ipsp.cnr.it

¹Laboratory of Biotechnology Applied to Agriculture, INRAT,University of Carthage, 1004 El Menzah, Tunis, Tunisia; ²Institute for Sustainable Plant Protection (IPSP-CNR)– Strada delle Cacce 73, 10135 Torino, Italy

Watermelon (Citrullus lanatus) plants showing mild curling and size reduction of young leaves, and ripe fruits exhibiting chlorotic mottling on rind, white hard portions inside the flesh and unpleasant taste, were observed in different areas of Tunisia during several growing seasons (years 2005–2015). Leaves, fruit rind and fruit flesh were collected and total DNA was extracted. Rolling-circle amplification (RCA) using ϕ29 DNA polymerase was performed from a few representative samples. Several restriction enzymes were tested on the amplified DNA and a single DNA fragment of about 2500 bp was obtained following SpeI digestion from three different samples. The SpeI-restricted DNA molecule was cloned in pBluescriptKS(+) and fully sequenced on both strands. The newly cloned DNA was 2571 nucleotides long and showed a typical Mastrevirus genomic organization. Sequence alignment analysis by BLASTN showed high nucleotide sequence identity values with several isolates of Chickpea chlorotic dwarf virus (CpCDV). Specific primers are currently used to test incidence of CpCDV infection in watermelon in Tunisia. Data regarding CpCDV infection in watermelon and association with the “hard watermelon syndrome” will be presented and discussed.

Finding the needle in the haystack: Composition of begomovirus populations in cultivated and non-cultivated hosts

Camila G. Ferro^1,2*, Márcio T. Godinho^1,2, César A. D. Xavier^1,2, Vitor B. Pinto², J. P. Silva^1,2, J. C. F. Silva², F. Murilo Zerbini^1,2

Email ID for Correspondence: camila.ferro@ufv.br, cgfufv@hotmail.com

¹Departamento de Fitopatologia, ²National Institute for Plant-Pest Interactions, Universidade Federal de Viçosa, Viçosa, MG, 36570-900, Brazil

Tomato-infecting begomoviruses emerged in Brazil in the mid-1990s following the introduction of Bemisia tabaci Middle East-Asia Minor 1 (MEAM1). Several lines of evidence indicate that these viruses evolved from indigenous viruses infecting non-cultivated hosts. However, tomato-infecting viruses are only rarely found in non-cultivated hosts, and vice versa. It is possible that viral populations in a given host are composed primarily of viruses which are better adapted to this host, but also include a small proportion of viruses which are poorly adapted. Then, after transfer to a different host by the whitefly vector, the composition of the viral population changes rapidly, with the viruses which are better adapted to the new host predominating. To test this hypothesis, we collected tomato and Sida sp. plants growing next to each other, as well as whiteflies, at two locations (Coimbra and Florestal, both in Minas Gerais state, Brazil). After confirmation of viral infection using PCR with degenerate primers and RCA, total DNA from one tomato and one Sida sp. sample from each location, and from pools of whiteflies from each location, was sequenced in the Illumina HiSeq 2000 platform. Following a highly stringent set of criteria, reads were mapped to a data set including all DNA-A and DNA-B New World begomovirus sequences. For each read, the three best hits were recorded and three files were prepared, with reads mapping to (i) Tomato severe rugose virus (ToSRV), (ii) Sida micrantha mosaic virus (SiMMV) and (iii) any other begomovirus. The results indicate that > 98% of the reads from Sida sp. mapped to SiMMV, but 0.01% of the reads mapped to ToSRV. Conversely, more than 99% of the reads from tomato mapped to ToSRV, with 0.001% mapping to SiMMV. These results are consistent with the hypothesis that the composition of viral populations shifts after transfer to a different host.

Evolution of Tomato severe rugose virus during infection of a single plant

V. B. Pinto^1,2, M. T. Godinho^1,2*, J. C. F. Silva², F. M. Zerbini^1,2

Email ID for Correspondence: marcio.godinho@ufv.br, marcioslash@gmail.com

¹Departamento de Fitopatologia, ²National Institute for Plant-Pest Interactions, Universidade Federal de Viçosa, Viçosa, MG, 36570-900, Brazil

To evaluate the mutational dynamics of the bipartite begomovirus Tomato severe rugose virus (ToSRV), plants of tomato and Nicandra physaloides were biolistically inoculated with an infectious clone and the leaves sampled at 30, 75 and 120 days after inoculation. Total DNA was extracted and sequenced in the Illumina HiSeq 2000 platform. The datasets were trimmed with the quality score limit set to 0.01, and the assembly was performed using the infectious clone sequence as reference. We inferred high rates of nucleotide substitution for both DNA components in both hosts: 3.06 × 10⁻³ and 2.03 × 10⁻³ sub/site/year for the DNA-A and DNA-B, respectively, in N. physaloides, and 1.38 × 10⁻³ and 8.68 × 10⁻⁴ sub/site/year, respectively, in tomato. Substitution rates in the range of those described for other begomoviruses were found for the CP, Rep, MP and NSP genes in both hosts. We quantified synonymous and non-synonymous substitutions, transversions and transitions, deletions and insertions in the CP, Rep, MP and NSP genes. A decrease in the number of variable sites was observed during the course of the experiment for the DNA-A in both hosts, with a corresponding increase in the number of identical sites to the reference genome. The opposite was observed for the DNA-B, indicating distinct evolutionary pathways for the two components. Furthermore, suppression of the stop codons of the MP and NSP genes was observed in the N. physaloides libraries, providing additional evidence of an adaptive strategy. Determination of Shannon entropy indicated mutation hotspots in the 5′-region of Rep, in the common region and in the long intergenic region between the MP and NSP genes in the DNA-B (LIR-B). Overall, the results indicate that ToSRV evolves as a quasispecies, with a high degree of genetic variability which could be partly responsible for its prevalence in the field.

Replication competition between Malvastrum yellow vein virus and Tobacco curly shoot virus associated with their cognate betasatellites

Chenchen Jing, Miao Sun, Yan Hu, Gentu Wu, Xianchao Sun and Ling Qing^*

Email ID for Correspondence: qling@swu.edu.cn

College of Plant Protection, Southwest University, Chongqing 400716, China

The cognate betasatellite (MaYVB) ofMalvastrum yellow vein virus (MaYVV) is necessary for typical symptoms induction, while Tobacco curly shoot virus (TbCSV) can induce typical symptoms without its betasatellite (TbCSB). To investigate the interaction relations between the two kinds of geminivirus/betasatellite disease complex, the infectious clone combination of MaYVV isolate Y47 (Y47A), TbCSV isolate Y35 (Y35A), and their betasatellite (Y47β and Y35β) was used to inoculate Nicotiana benthamiana and the replication level of these two viruses and their cognate betasatellites were detected by PCR and real-time quantitative PCR (qPCR). The symptoms of shrinking, downward leaf curling, yellow vein, distorted stem, dwarf and enation were found in the early stage of infection, but yellow vein symptom disappeared in the later stage. Y35A and Y35β increasingly accumulated during the infection with a peak at 90 day post inoculation (dpi). However, Y47A and Y47β decreasingly accumulated during the process. Finally, Y47A and Y47β were undetectable at 180 dpi and 120 dpi respectively. These results indicated that the competition probably occurred between these two disease complexes of Y47A/Y47β and Y35A/Y35β for the replication resources of the host and betasatellite is preferentially transreplicated by its cognate helper virus.

Metagenomic analysis of begomoviruses infecting pepper in the northeast of Argentine

V. A. Bornancini^1*, C. G. Vaghi Medina¹, C. R. Flores², A. Amadio³, D. A. Ducasse¹ and P. M. López Lambertini¹

Email ID for Correspondence: lopezlambertini.pao@inta.gob.ar

¹Plant Pathology Research Institute, CIAP-INTA, ²Yuto Experiment Station, INTA, ³Rafaela Experiment Station, INTA, Argentina

Horticultural crops are threatened by the emergence of new begomoviruses in Latin America. One of the main production areas where begomoviruses cause economic losses is the northeast of Argentina (NOA). In this region, pepper is an important crop mostly grown under greenhouse condition. Begomoviruses incidence is increasing probably due to the dissemination of its vector (Bemisia tabaci). The genetic diversity of begomoviruses infecting pepper has not been determined in Argentine and the aim of this study was to establish that diversity by metagenomic analysisin crops of NOA region. We analyzed by Multiplex-PCR 101 symptomatic peppers plants sampled on three locations named Pichanal, Oran, and Yuto during the period 2005–2014. From 52 begomovirus positive samples, 21 were selected to be amplified by RCA and gel purified. These 21 samples were sequencedusing Illumina Hiseq1500 system (INDEAR). Bioinformatics analysis of the sequences resulted in the specific identification of Soybean blistering mosaic virus, Tomato yellow spot virus, Tomato yellow vein streak virus, and Tomato dwarf leaf virus. Soybean blistering mosaic virus, Tomato yellow vein streak virus and Tomato dwarf leaf virus were also found infecting tomato in the same years and production area. Besides, Soybean blistering mosaic virus was identified in soybean and bean crops while Tomato yellow spot virus was reported in soybean, bean and chia crops in Argentine showing the great adaptation of these viruses to different hosts.

Prevalence of begomoviral diseases in crop plants of Chhattisgarh state in India

P. N. Sivalingam^*, K. C. Sharma, S. K. Jain and V. K. Choudhary

Email ID for Correspondence: pnsivalingam@gmail.com

ICAR-National Institute of Biotic Stress Management, Baronda, Raipur – 493 225, Chhattisgarh, India

Chhattisgarh is one of the agriculturally important states of central India. It comes under eastern plateau and hilly region of the 15 agro-climatic zones of India and represented by three agro-geographical regions viz. northern hill, Chhattisgarh plain and Bastar plateau. Information on the prevalence of virus diseases caused by the genus Begomovirus in this region is lacking. Recently, two surveys have been conducted in the farmers’ field of two districts of Chhattisgarh; Raipur and Durg (plain) to understand the prevalence of begomoviral and other viral diseases in agriculturally important crop plants. No viral disease has been observed in rice, wheat and maize. Mungbean, Dolichus bean and pegion pea were found to be affected with 21–94% yellow mosaic disease, vegetable crops such as bhendi (91–100% yellow vein mosaic and 78–100% leaf curl disease), sponge gourd (78–98% leaf crinkle disease), bitter gourd (52–100% leaf crinkle and 43% mosaic disease) and fruit crop; papaya (100% leaf curl disease) were found likely to be infected with whitefly transmitted Begomovirus. Other viral diseases such as leaf crinkle disease in mung bean (98%), ring spot disease in papaya (97%) and mosaic disease in cucumber (3%) were also noticed in this region. Preliminary information suggests that among the diseases caused by different viruses, Begomovirus are the most important and causing huge economic losses to the growers. Host-pathogen interaction studies and identifying the management practices are underway to attend the control of diseases.

Development of infectious clones of cotton leaf curl virus to study mutations that affect virulence and systemic spread of the virus

Deepratan Kumar^*, Meera Kurulekar, Surendra Reddy, Mukesh Kharat, Prem Rajagopalan, Radhamani Anandalakshmi

Email ID for Correspondence: deepratan.kumar@mahyco.com

Plant – Virus Interactions Lab, Mahyco Research Center, Maharashtra Hybrid Seeds Pvt Ltd, Jalna -431203, Maharashtra, India

Cotton leaf curl disease (CLCuD) causes huge yield loss in cotton in Northwestern India. The disease is caused by different monopartite begomoviruses and associated satellite molecules. Since CLCuD is whitefly transmitted, often it is difficult to decipher the response of plants to viruses alone. Hence infectious clones were made to understand plant responses to the different species/strains of cotton leaf curl viruses. Full length genome of Cotton leaf curl Rajasthan Virus (CLCuRV), Cotton leaf curl Burewala Virus with intact C2 gene and CLCuBuV with truncated C2 gene were cloned and partial dimers constructed along with Cotton leaf curl Multan DNA beta, associated with CLCuD. All partial dimer clones were mobilized in Agrobacterium and infiltrated either individually and along with DNA-beta into the leaves of Nicotiana benthamiana and cotton. The infectivity of agro infectious clones on N. benthamiana and cotton and related molecular characterization will be described.

Mechanical Transmission of Bean Golden Mosaic Virus and Cucumber Mosaic Virus on Six Soybean (Glycine max L.) Cultivars

Suleiman Abdullahi Suleiman^1*, N. Rajput² and Mishra Vikas²

Email ID for Correspondence: suleiman.ab@fud.edu.ng

¹Department of Crop Production, Federal University Dutse, P.M.B. 7156, Jigawa State, Nigeria; ²Department of Plant Protection, Sam Higginbottom Institute of Agriculture, Allahabad 211 007, Uttar Pradesh, India

The study was carried out to determine the comparative pathogenic response of six cultivars of soybean; TGx 14-08E, TGx 18-21E, TGx 19-26F, TGx 10-22F, TGx 16-18N and TGx 12-29E to single and mixed infections with bean golden mosaic virus and cucumber mosaic virus. The experiment was conducted in the screenhouse at the Crop Production Department, Faculty of Agriculture, Federal University Dutse, Jigawa State Nigeria. The results of the experiment revealed that all soybean cultivars were susceptible to single and mixed infection of the two viruses but to seemingly different extent. The single infection with bean golden mosaic virus (BGMV), however, caused the most severe symptoms on the soybean cultivars. Cucumber mosaic virus (CMV) alone was not as severe as the BGMV. The mixed infection of BGMV and CMV did not cause higher severity than BGMV alone indicating that there was little or no synergistic effect between the two viruses on soybean.

Characterization of gemycircularviruses associated with non-cultivated plants in Brazil

Rafael R. Rezende^1,3*, Talita B. Mar^2,3, Lina M. Paes¹, Jesus Navas-Castillo^3,4, F. Murilo Zerbini^2,3, Poliane Alfenas-Zerbini^1,3

Email ID for Correspondence: rafaelrezende@outlook.com.br

¹Departamento de Microbiologia, ²Departamento de Fitopatologia, and ³National Research Institute for Plant-Pest Interactions, Universidade Federal de Viçosa, Viçosa, MG, 36570-900, Brazil; ⁴Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas, 29750, Algarrobo-Costa, Málaga, Spain

In recent years, new groups of circular replication-associated protein encoding single-stranded (CRESS) DNA viruses have been described infecting animals (including humans), plants and fungi. One of these groups is the gemycircularviruses, which have been detected in insects, in the faecal matter of various animals, and in fungi-infected cassava leaves. It has not yet been unequivocally demonstrated that gemycircularviruses can infect plants, as the virus isolated from cassava is more related to mycoviruses and infectivity tests were not carried out. Here, we describe gemycircularviruses associated with two non-cultivated hosts which are important invasive species in field crops in Brazil: Euphorbia heterophylla (two samples showing yellow mosaic, leaf curling, and stunting collected in Rio Grande do Sul state) and Momordica charantia (samples collected in Minas Gerais state). Viral genomes (aprox. 2100 nt) were amplified by RCA, cloned and sequenced. Samples of E. heterophylla were also infected by Euphorbia yellow mosaic virus, but begomoviruses were not detected in M. charantia. A Bayesian-inferred tree based on full-length sequences clustered Euphorbia heterophylla-associated circular virus (EHasCV) and Momordia charantia-associated circular virus (MCasCV) in a well-supported clade with Sclerotinia sclerotiorum hypovirulence-(SsHADV), Hypericum japonicum-(HJasCV), Cassava-(CasCV) and Dragonfly-(DfaCV)associated circular viruses, with EHasCV more closely related to DfaCV and MCasCV to HJasCV. Comparing the genome organization of EHasCV with that of SsHADV we identified a putative coat protein (298 amino acids, 37% identity with the SsHADV CP), two domains of a putative replication-associated protein probably expressed from spliced transcripts (126 aa, 63% identity with the helicase domain, and 205 aa, 64% identity with the catalytic domain SsHADV Rep), and the nanonucleotide TAATATTAT (the genome of MCasCV contains a TAATGTTAT nonanucleotide) within a potential stem-loop sequence. Infectivity tests are being performed in E. heterophylla, M. charantia and Nicotiana benthamiana plants.

Understanding persistence and evolution of begomoviruses infecting feral papaya and solanaceous weeds by epidemiological screenings

S. Hamsa, Tabasum Akhter, Parul Bhardwaj, Aashima Mehra, Sunil Mukherjee and Sudeshna Mazumdar-Leighton^*

Email ID for Correspondence: smceres21@yahoo.co.in

Plant Biotic Interactions Lab, Department of Botany, University of Delhi, New Delhi-7.

Epidemiological screens and molecular surveys are essential to understand invasion, prevalence and persistence of Begomovirusesin any given region. Weeds and other susceptible hosts growing in the vicinity of papaya plants showing typical symptoms of Leaf Curl Disease were surveyed from 2011 to 2016 in the National Capital Region of Delhi by post-graduate students of the Department of Botany, University of Delhi. Plant samples collected from gardens, fields and parks were tested by DAS-ELISA using commercial kits used for detection of Tomato yellow leaf curl virus and other begomoviruses. Samples were also screened using antibodies raised against a recombinant coat protein from Tomato leaf curl New Delhi virus isolate infecting papaya cultivar Pusa Dwarf reported from the same area. PCR screens were performed for coat protein genes, as well as satellite molecules to identify plants infected by these viruses. Sequence analyses of selected sample were used to evaluate persistence, emergence and predominance of diverse begomoviruses infecting papaya, and other plant species in the region. Results underline the importance of continuous monitoring of leaf curl diseased plants in order to understand dynamics of begomovirus evolution and diversity.

Characterization of capulaviruses recovered from cultivated and uncultivated plants from Africa, Asia and Europe

Pauline Bernardo,¹ Brejnev Muhire,² Sarah François,^1,3,4 Maëlle Deshoux,¹ Penelope Hartnady,² Kata Farkas,⁵ Simona Kraberger,⁵ Mikko J. Frilander,⁶ Denis Filloux,¹ Emmanuel Fernandez,¹ Serge Galzi,¹ Romain Ferdinand,¹ Martine Granier,¹ Armelle Marais,⁷ Pablo Monge Blasco,⁸ Thierry Candresse,⁸ Fernando Escriu,^8,9Anna-Liisa Laine,¹⁰ Arvind Varsani,^5,11,12 Gordon W. Harkins,¹³ Darren P. Martin,² and Philippe Roumagnac^1*

Email ID for Correspondence: philippe.roumagnac@cirad.fr

¹CIRAD-INRA-SupAgro, UMR BGPI, Campus International de Montferrier-Baillarguet, 34398 Montpellier Cedex 5, France; ² Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town 4579, South Africa; ³INRA, UMR 1333, DGIMI, Montpellier, France; ⁴CNRS-IRD-UM1-UM2, UMR 5290, MIVEGEC, avenue Agropolis, Montpellier, France; ⁵School of Biological Sciences and Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch, New Zealand; ⁶Institute of Biotechnology, University of Helsinki, Helsinki, Finland; ⁷UMR 1332 Biologie du Fruit et Pathologie, INRA, Univ Bordeaux, CS20032, 33882 Villenave d’Ornon cedex, France; ⁸Unidad de Sanidad Vegetal, Centro de Investigacion y Tecnologıa Agroalimentaria de Aragon (CITA), Av. Montañana 930, 50059 Zaragoza, Spain; ⁹Unidad de Sanidad Vegetal, Instituto Agroalimentario de Aragón IA2 (CITA—Universidad de Zaragoza), Av. Montañana 930, 50059 Zaragoza, Spain; ¹⁰Metapopulation Research Centre, Department of Biosciences, University of Helsinki, Helsinki, Finland; ¹¹Department of Plant Pathology and Emerging Pathogens Institute, University of Florida, Gainesville, USA; ¹²Structural Biology Research Unit, Department of Clinical Laboratory Sciences,University of Cape Town, Observatory, South Africa; ¹³South African National Bioinformatics Institute, MRC Unit for Bioinformatics Capacity Development, University of the Western Cape, Cape Town, South Africa

The family Geminiviridae comprises seven genera differentiated by genome organization, sequence similarity and insect vector. Capulavirus, a novel genus-level geminivirus lineage, was discovered between 2010 and 2011 in Europe, Asia and Africa. Presently, three distinct capulavirus species have been described: Euphorbia caput-medusae latent virus (EcmLV) from South Africa, French bean severe leaf curl virus (FbSLCV) from India and Alfalfa leaf curl virus (ALCV) from France and Spain. This latter geminivirus was found to be transmitted by Aphis craccivora. We have recently discovered a fourth capulavirus species, Plantago lanceolata latent virus (PlLV), from Plantago lanceolata in Finland, from which we determined two full genome sequences. In contrast to EcmLV and PlLV isolates that were found within asymptomatic uncultivated host plants, the presence of ALCV in cultivated alfalfa plants was always strongly associated with severe disease symptoms. Large sampling surveys carried out in Southern Africa and France has revealed that the prevalence of ALCV and EcmLV in their respective hosts exceeded 10%. Besides using patterns of detectable negative selection to identify ORFs that are probably functionally expressed, we show that both ALCV and EcmLV display evidence of inter-species recombination and the presence of biologically functional genomic secondary structures. Finally, we show that whereas the EcmLV likely experiences restricted geographical dispersal, ALCV is probably freely moving across the French Mediterranean region.