Chilli leaf curl virus infection downregulates the expression of the genes encoding chloroplast proteins and stress-related proteins

Abstract

Virus infection alters the expression of several host genes involved in various cellular and biological processes in plants. Most of the studies performed till now have mainly focused on genes which are up-regulated and later projected them as probable stress tolerant/susceptible genes. Nevertheless, genes which are down-regulated during plant-virus interaction could also play a critical role on disease development as well as in combating the virus infection. Hence, to identify such down-regulated genes and pathway, we performed reverse suppression subtractive hybridization in Capsicum annuum var. Punjab Lal following Chilli leaf curl virus (ChiLCV) infection. The screening and further processing suggested that majority of the genes (approximately 35% ESTs) showed homology with the genes encoding chloroplast proteins and 16% genes involved in the biotic and abiotic stress response. Additionally, we identified several genes, functionally known to be involved in metabolic processes, protein synthesis and degradation, ribosomal proteins, energy production, DNA replication and transcription, and transporters. We also found 3% transcripts which did not show homology with any known genes. The redundancy analysis revealed the maximum percentage of chlorophyll a-b binding protein (15/96) and auxin-binding proteins (13/96). We developed a protein interactome network to characterise the relationships between proteins and pathway involved during the ChiLCV infection. We identified that the most of the interaction occurs either among the chloroplast proteins (Arabidopsis proteins interactive map) or biotic and abiotic stress responsive proteins (Solanum lycopersicum interactome). Taken together, our study provides the first transcriptome and protein interactome of the down-regulated genes during C. annuum-ChiLCV interaction. These resources could be exploited in deciphering the steps involved in the process of virus infection.

Electronic supplementary material

The online version of this article (10.1007/s12298-019-00693-1) contains supplementary material, which is available to authorized users.

Introduction

Plant viruses are obligate parasites which require host proteins for multiplication and establishment of successful infection. Following infection, competition between viruses and hosts takes place to take control over cellular machinery. Host employs its defense system to prevent the virus domination and multiplication (Kushwaha et al. 2015). At the same time, viruses do utmost to weaken the host defense system (Sahu et al. 2010; Allie et al. 2014). A dominant pathogen successfully modulates the different cellular processes of the host and develops a conducive cellular environment (Sahu et al. 2010; Liu et al. 2014; Kushwaha et al. 2015). In most of the cases, pathogens accomplish cellular ambience by either suppressing or escaping from the host defense machinery (Allie et al. 2014). One of the ways to attain a favorable cellular environment is to suppress the expression of defense-related genes and boost the expression of genes whose products are required for virus multiplication. Therefore, a transcriptomic study in the host infected with a pathogen could reveal information about the genes expression pattern following pathogen attack (Sahu et al. 2010; Liu et al. 2014; Kushwaha et al. 2015).

Capsicum commonly called as pepper is one of the economically important crops grown throughout India and its production is affected by various factors including viruses. Chilli leaf curl virus (ChiLCV) poses serious threat to chilli production in India. Etiology of ChiLCV was first reported in 1960 (Mishra et al. 1963; Dhanraj and Seth 1968). In India, based on partial sequencing of viral genome, ChiLCV was first shown to be associated with the leaf curl disease by Senanayake et al. (2007), later on cloning of full-length genome and infectivity of the causal viruses were demonstrated (Chattopadhyay et al. 2008; Kumar et al. 2015). Symptoms of ChiLCV include leaf curling, thickening of veins, shortening of internodes and petioles, leaf puckering and stunted growth of plants, 100% crop loss in severe condition (Mishra et al. 1963; Kumar et al. 2006; Senanayake et al. 2007; Chattopadhyay et al. 2008; Kumar et al. 2015; Padhi et al. 2017). Several chilli-infecting begomoviruses have been characterized but the information about the host factors and genes that regulate viral infection is largely unknown. To identify such genes, we aimed to generate a transcriptomic profile of C. annum var. Punjab Lal (Kumar et al. 2006) infected with ChiLCV, belonging to the family Geminiviridae. The small (2.5–3.0 kb) single-stranded genome of geminiviruses replicate in the nuclei of host cells by rolling circle and recombination-dependent replication using host DNA polymerases and produce double-stranded DNA replicative form (Stanley et al. 1986; Hanley-Bowdoin et al. 2013). The dsDNA replicative form is used as a template for transcription of viral genes by host DNA dependent RNA polymerase II. The small genome of geminiviruses encodes multifunctional proteins to balance the limited coding capacity. ChiLCV has emerged as one of the major threats to chilli production in India. It is a monopartite geminivirus having DNA A-like viral genome and satellite DNA β is often associated with the genome (Chattopadhyay et al. 2008; Kumar et al. 2015). DNA A encodes for coat protein and pre-coat protein, transcribed by the right promoter located on the viral strand and replication initiator protein (C1), transcriptional activator protein (C2), replication enhancer proteins (C3) and C4 protein transcribed by the left promoter located on the complementary of the sense strand. Satellite DNA β encodes a single protein βC1 that enhances viral pathogenesis and symptom development.

On the infected hosts, geminiviruses may induce leaf curl disease by interactions among array of viral and host proteins, eventually altering the cellular and physiological processes (Hanley-Bowdoin et al. 2013). In one of our previous studies, we identified the genes that were differentially expressed following ChiLCV infection (Kushwaha et al. 2015). Most of these genes were involved either in protein synthesis or degradation, DNA organization/replication/transcription and defense processes (Kushwaha et al. 2015). In another study, a tolerant variety of tomato infected with Tomato leaf curl New Delhi virus showed upregulation of defense-associated host gene expression (Sahu et al. 2010). The up-regulation in the expression of these genes was later correlated with the tolerant attribute of the host plant tomato. The present study provides the first transcriptome-based information of down-regulated genes, which could be further exploited to understand the geminivirus-host interaction, especially during the establishment of disease.

Materials and methods

Plant growth

Chilli (Capsicum annuum var. Punjab Lal) plants were grown in an insect-proof controlled glasshouse under the conditions of 16 h light and 8 h dark photoperiod, 25 ± 2 °C temperature and relative humidity of 60%.

Inoculation of the test plants

The infectious clones of ChiLCV-DNA A (GenBank accession no EF190217) and betasatellite (GenBank accession no EF190215) were available at the Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi India (Chattopadhyay et al. 2008). Agro-inoculation on chilli plants (32) was performed at 3–4 leaves stage following the procedure described by Kushwaha et al. (2015).

Total RNA and mRNA isolation

For isolation of total RNA, two uppermost leaves of either virus- or mock-inoculated plants were harvested. Total RNA was isolated following Kushwaha et al. (2015) and using Tri-reagent (SIGMA, St. Louis, USA). The quantity and integrity of total RNA were examined using Nanodrop (Nanodrop-2000, Thermo Fisher Scientific, Massachusetts, USA). In addition, RNA integrity was also examined by resolving the isolated RNA on 1.2% formaldehyde denaturing gel. Total Messenger RNA (mRNA) was purified from total RNA using the MagneSphere mRNA Purification Kit (Promega, Madison, USA) according to the manufacturer’s protocol.

Construction of suppression subtractive hybridization cDNA library

The subtracted cDNA library C. annuum var. Punjab Lal was constructed by suppression subtractive hybridization (SSH) approach, using the PCR-Select Subtractive Hybridization Kit, following manufacturer protocol (Clontech, California, USA). For the construction of the library, 2 µg of the total mRNA from ChiLCV-inoculated plants at 21 days post-inoculation (dpi) was used as the ‘Driver’ and similarly, for the ‘tester’ sample, 2 µg of total mRNA from mock-inoculated (vector pCAMBIA2300) plants at the same time point. The PCR products of the subtracted library were cloned into the pGEM-T easy vector (Promega, Wisconsin, United States), which were subsequently mobilized into Escherichia coli strain DH5α. A total of 96 colonies were screened and sequenced using M13 primer.

Data analysis and annotation of SSH ESTs

After sequencing, the vector sequence was identified and removed from raw sequence data using vec screen (https://www.ncbi.nlm.nih.gov/tools/vecscreen/) and annotation of the sequences was carried out by Blastx or Blastn tool on the NCBI database (https://www.ncbi.nlm.nih.gov/). Homologous gene, accession numbers, e-value (< 1e−5) and organism name were recorded with their molecular functions. A pie chart representing the percentage of ESTs on the basis of their predicted function was drawn using GraphPad Prism software (www.graphpad.com).

Protein-interactome network

The protein interactome network of putative proteins encoded by ESTs obtained from SSH analysis was constructed on the STRING database (www.string-db.org) using Arabidopsis thaliana and Solanum lycopersicum information.

cDNA preparation and qRT-PCR

A total of 5 µg total RNA was treated with DNase I for 45 min at 37 °C. DNase inactivation was done by addition of 5 mM EDTA at 72 °C for 10 min. A total of 1.5 µg was proceeded for cDNA preparation following Kushwaha et al. (2015). qRT-PCR was carried out using gene-specific primers mentioned in Table S1. The PCR reactions were carried out in 48 well (Illumina, California, United States) plates in 10 µl of reaction using SYBR green mix (Applied Biosystem, California, United States) under following PCR conditions—Initial denaturation at 94 °C for 10 min followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s and extension at 72 °C for 30 s. The obtained Ct value of the genes was normalized to the Ct value of the actin (internal control) and ∆∆Ct was calculated. The ∆∆Ct value was used to draw the graphs using GraphPad Prism software (www.graphpad.com).

Results

Construction of suppression subtractive hybridization library

Suppression subtractive hybridization (SSH) is one of the very sensitive techniques for transcriptome analysis and has previously been used for identification of differentially expressed transcripts following geminivirus infection (Sahu et al. 2010; Kushwaha et al. 2015). In the current study, we aimed to identify the down-regulated genes, following ChiLCV infection in C. annuum. Therefore, for the construction of the library, total mRNA from ChiLCV-inoculated plants at 21 dpi was used as the ‘driver’ whereas total mRNA from mock-inoculated plants, at the same time point, was used as ‘tester’. We screened 96 E. coli colonies harboring EST constructs and subsequently sequenced. We searched for the homology of all sequences on the NCBI database. After checking redundancy, we found 35 unique EST sequences, which were annotated with accession no, e-value and categorised on the basis of putative biological and molecular function. While screening the colonies, we found a large number of redundant sequences. This suggested the abundance of the specific category of genes which were mostly altered during this interaction.

Identification and classification of downregulated genes

Annotation of SSH ESTs was performed on the NCBI database and all ESTs were further categorized on the basis of their putative biological functions (Table 1). Most of the ESTs (35%) showed close homology with proteins either present in the chloroplast or actively involved in photosynthesis. Proteomic and transcriptomic studies have demonstrated that viral infection affects the expression of chloroplast and photosynthetic related genes (Mochizuki et al. 2014). Further, 16% of the ESTs showed sequence similarity with the genes involved in biotic and abiotic stress response. During plant-virus interaction, for the establishment of successful infection, virus attempts to down-regulate the expression of defense-related genes. We also encountered 14% genes involved in metabolic processes and 11% in protein synthesis and degradation (Fig. 1a). We identified 8% EST showing similarity with the genes that code for ribosomal proteins (8%), energy production (5%), replication and transcription (5%), and transporters (3%) (Fig. 1a). Interestingly in our experiment, we found ~ 3% genes, which did not show homology with any known protein (Fig. 1a).

Table 1.

Annotation of ESTs obtained through suppressive subtractive hybridization

	Gene	Accession no.	Organism	Accession no	E-value	Blast type	Biological function
1	Carbonic anhydrase, chloroplastic isoform X1	JZ979443	Capsicum annuum	XP_016561553	1E−68	Blastx	MAPK cascade, carbon utilization, defense response
2	Antifungal protein	JZ979444	C. annuum	AAL73184		Blastx	Metabolism of lipid, Patatin-related phospholipase A
3	Chloroplast chlorophyll a-b binding protein CP26	JZ979445	Gossypium hirsutum	ACO51066	7.00E−47	Blastx	Photosystem II encoding the light-harvesting chlorophyll a/b binding protein CP26
4	Curvature thylakoid 1B, chloroplastic	JZ979446	C. annuum	XP_016545573	3E−24	Blastx	Determines thylakoid architecture, Choloroplast biogenesis
5	X-hybrida s-adenosyl-l-homocystein hydrolase mrna, complete cds	JZ979447	Petunia	AY256915	3.00E−32	Blastn	Adenosylhomocysteinase activity
6	Ribulose bisphosphate carboxylase/oxygenase activase 2, chloroplastic	JZ979448	C. annuum	NP_001311536	6e-45	Blastx	5-bisphosphate carboxylase/oxygenase activator activity
7	60S ribosomal protein L7a-2-like	JZ979449	Solanum lycopersicum	XP_004241894	4E−78	Blastx	Cytosolic small ribosomal subunit
8	Protochlorophyllide reductase	JZ979450	C. annuum	XP_016555495	6E−102	Blastx	Porphyrin And Chlorophyll Metabolism.
9	Auxin-binding protein ABP19a-like	JZ979451	C. annuum	XP_016580306	8E−64	Blastx	Apoplast, biological process unknown, extracellular matrix
10	Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase, chloroplastic	JZ979452	C. annuum	XP_016544021	5E−43	Blastx	Oxidative cyclase, chlorophyll biosynthesis
11	Oxygen-evolving enhancer protein 3-2, chloroplastic	JZ979453	C. annuum	XP_016560066	2E−74	Blastx	Photorespiration, photosynthesis
12	40S ribosomal protein SA-like	JZ979454	C. annuum	XP_016578205	3E−90	Blastx	Cytosolic small ribosomal subunit
13	30S ribosomal protein prt S9	JZ979455	C. annuum	XR_001673095	1E−122	Blastn	Unknown
14	Catalase	JZ979456	C. annuum	AB007190	4.00E−26	Blastn	Catalase activity
15	Peroxisomal (S)-2-hydroxy-acid oxidase	JZ979457	C. annuum	XM_016697076	9E−87	Blastn	Photorespiration and photosynthesis
16	Elongation factor 2	JZ979458	Nicotiana tabacum	XP_016537814	4.00E−08	Blastx	GTP binding, GTPase activity, nucleic acid binding
17	Solanesyl-diphosphate synthase 3, chloroplastic	JZ979459	C. annuum	XP_016581059	3E−10	Blastx	Ubiquinone-9 biosynthesis
18	14 kDa proline-rich protein DC2.15-like	JZ979460	C. annuum	XP_016574952	4E−51	Blastx	Photosynthesis, photosynthesis, light harvesting
19	60S ribosomal protein L35a-3-like	JZ979461	Nicotiana tomentosiformis	XP_009587506	1E−46	Blastx	Cytosolic large ribosomal subunit
20	Serine hydroxymethyltransferase, mitochondrial	JZ979462	C. annuum	XP_016560852	8E−55	Blastx	l-serine metabolic process,
21	Hypothetical protein T459	JZ979463	C. annuum	PHT93715	2E−08	Blastx	Unknown
22	Glycine cleavage system H protein, mitochondrial-like	JZ979464	C. annuum	XP_016555439	1E−42	Blastx	Unknown
23	nifU-like protein 1, chloroplastic	JZ979465	C. annuum	XP_016539741	4E−54	Blastx	Iron ion binding, iron-sulfur cluster assembly
24	40S ribosomal protein	JZ979466	C. annuum	XP_016578205	1.00E−90	Blastx	Ribosome assembly, protein metabolosim
25	Thioredoxin-like protein CDSP32, chloroplastic	JZ979467	C. annuum	XP_016568326	4.00E−33	Blastx	Cell redox homeostasis, chloroplast, electron carrier activity
26	Peroxiredoxin-2B	JZ979468	C. annuum	XP_016540190	1E−91	Blastx	Antioxidant activity, Redox reaction
27	Peroxiredoxin Q, chloroplastic	JZ979469	Malus domestica	XP_008337814	8E−18	Blastx	Antioxidant activity, Redox reaction
28	28 kDa ribonucleoprotein, chloroplastic-like	JZ979470	C. annuum	XM_016708245.1	82–96	Blastn	RNA binding
29	Peroxiredoxin-2B	JZ979471	C. annuum	XP_016540190	8E−91	Blastx	Antioxidant activity, oxidation–reduction process
30	Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase, chloroplastic	JZ979472	C. annuum	XP_016544021	3E−73	Blastx	Chlorophyll syhtesis, chroroplast activity
31	Dicarboxylate transporter 1, chloroplastic	JZ979473	C. annuum	XP_016547657	5E−36	Blastx	Dicarboxylate transporter, 2-oxoglutarate/malate translocator
32	Ribulose-1,5-bisphosphate carboxylase, small subunit precursor	JZ979474	S. lycopersicum	AAA34192	9.00E−29	Blastx	Catalyzes the rate-limiting step of CO2 fixation in photosynthesis
33	Serine hydroxymethyltransferase, mitochondrial	JZ979475	C. annuum	XP_016560852	3E−57	Blastx	l-serine metabolic process, catalytic activity
34	1-aminocyclopropane-1-carboxylate oxidase	JZ979476	C. annuum	XP_016557813	4E−49	Blastx	1-Aminocyclopropane-1-carboxylate oxidase activity
35	FtsH-like protein precursor	JZ979477	S. lycopersicum	XP_002301927	9.00E−172	Blastx	ATP binding, ATP-dependent peptidase activity, ATPase activity

Fig. 1.

Graphical representation of EST distribution and the frequency of their abundance. a The pie charts illustrating the categories of ESTs of C. annuum var. Punjab Lal identified in SSH. The EST sequences were searched for homology using BLASTN and BLASTX on the NCBI database. Genes were categorized on the basis of their putative biological functions. b The frequency of the transcripts encountered in the SSH library

Alteration in the genes encoding for chloroplast-associated and auxin-binding proteins expression during the infection

Sequencing and annotation results suggested frequent abundance of genes encoding for light-harvesting complex and auxin-binding proteins. The LHC-related genes includes, the gene encoding chlorophyll a-b binding protein (15), Chlorophyll a-b binding protein CP29.1 (4), Photosystem II oxygen-evolving (3). We also found that the genes encoding for auxin-binding protein (13) were highly abundant, along with the other key function genes such as Chloroplast rubisco activase (5), 60S ribosomal protein (4), Magnesium-protoporphyrin (4), P40-like protein (4) Serine hydroxymethyltransferase-5 (4), Thioredoxin peroxidise (4) (Fig. 1b). The frequency of genes encoding proteins was as Carbonic anhydrase (3), X hybrida S-adenosyl-L-homocysteine hydrolase (3), Photosystem II oxygen-evolving (3), Catalase (3), Peroxisomal (S)-2-hydroxy-acid oxidase (3) (Fig. 1b). Whereas frequency of other genes encoding proteins was as Elongation factor 2 (2), Uncharacterized protein (2), Peroxiredoxin 2B (2), 28 kDa chloroplast ribonucleoprotein (2), ATP synthase gamma chain (2), 14 kDa proline-rich protein (3) (Fig. 1b). The remaining ESTs Antifungal, Curvature thylakoid 1B, NADPH:protochlorophyllide oxidoreductase, LEFL1024DF10, Solanesyldiphosphate synthase, CP26, Glycine cleavage system h protein, Nitrogen fixation protein nifU, 40S ribosomal protein, Peroxiredoxin Q, Glycine cleavage system H, 1-aminocyclopropane-1-carboxylate oxidase, FtSH-like protein precursor were encountered once in 96 (Fig. 1b).

Validation of Reverse SSH by qRT-PCR

To validate the results of SSH in chilli, we accessed the expression of a few genes by qRT-PCR in the resistant variety C. annuum var. Punjab Lal and a susceptible variety C. annuum var Kashi Anmol. We analyzed the expression of Auxin-binding protein 1 (ABP1) from the group of ESTs involved in hormone signaling by qRT-PCR. The results showed a significant 3.2-fold down-regulation of ABP1 expression in C. annuum var Punjab Lal, whereas the expression of ABP1 was unaltered in C. annuum var. Kashi Anmol at 21 dpi (Fig. 2a). Further, we checked the expression of chlorophyll a-b binding protein CP26 and chlorophyll a-b binding protein CP29 genes involved in chloroplast structure and function. The results indicated that the expression of chlorophyll a-b binding protein CP26 reduces in C. annuum var. Kashi Anmol at 21 dpi. On the other hand, chloroplast chlorophyll a-b binding protein CP26 transcript level was reduced significantly (four fold) in the inoculated C. annuum var Punjab Lal as compared to mock-inoculated plants (Fig. 2b). Furthermore, chlorophyll a-b binding protein CP29 expression was also studied by qRT-PCR. We found no significant alteration in the transcript level of chlorophyll a-b binding protein CP29 in the susceptible variety C. annuum var. Kashi Anmol, whereas the expression was downregulated by 4.4-fold in the resistant variety (Fig. 2c). The relative abundance of FtSH was significantly reduced (4.15-fold) in C. annuum var. Kashi Anmol, whereas FtSH remains statistically unaltered in C. annuum var. Kashi Anmol (Fig. 2d). The transcript level of Peroxiredoxin, involved in a redox reaction, was reduced significantly by 11-fold in C. annuum var. Kashi Anmol, while C. annuum var. Punjab Lal showed 2.18-fold decreased in the expression of Peroxiredoxin by qRT-PCR (Fig. 2e). Results showed a reduction (2.86-fold) of the expression of the proline-rich protein transcripts in Kashi Anmol following infection, while the transcript level of proline-rich protein was not significantly altered in C. annuum var. Punjab Lal plants (Fig. 2f). The relative abundance of thioredoxin was down-regulated by three fold in C. annuum var. Kashi Anmol, whereas, we did not observe significant difference in the transcript level of thioredoxin in mock and inoculated C. annuum var. Punjab Lal plants (Fig. 2g). The expression of the genes coding for the protein Curvature thylakoid 1B remains unaltered in C. annuum var. Kashi Anmol whereas a significant reduction (5.2-fold) on the transcript level was noticed in C. annuum var. Punjab Lal (Fig. 2h).

Fig. 2.

Validation of reverse SSH by qRT-PCR. Expression analysis of aAuxin binding protein, bChlorophyll a-b binding protein CP26, cChlorophyll a-b binding protein CP29, dFtSH, ePeroxiredoxin, fProline rich protein, gThioredoxin, hCurvature thylakoid, in C. annuum variety Kashi Anmol and Punjab Lal

Protein interactome network

The EST sequences obtained in SSH were used to generate protein interactome network on a string database using Arabidopsis and tomato information (Table S21). Protein interactome using tomato genome information generated two major group of protein interactome. The first group consists of protein like thioredoxin-like protein (Solyc04g081970.2.1), thioredoxin peroxidase 1(TPx1), 2-Cys peroxiredoxin BAS1-like, chloroplastic-like (Solyc01g007740.2.1), glycolate oxidase (Solyc07g056540.2.1), catalase isozyme 2 (CAT2), peroxisomal (S)-2-hydroxyl-acid oxidase GLO4-like (Solyc03g122130.2.1), solanesyl diphosphate synthase (SppS). The second major group consists of proteins like Photosystem II oxygen-evolving complex protein 3 (PsbQ), chlorophyll a–b binding protein CP24 10A8 (CAP10A), Oxygen-evolving enhancer protein 2 (PSBP), ATP synthase gamma chain (Solyc02g080540.1.1) (Fig. 3a). Protein Interactome network using Arabidopsis information developed a single protein interactome that included proteins like EMBRYO SAC DEVELOPMENT ARREST 37 (EDA36), glycine cleavage system H protein (AT1G32470), solanesyl diphosphate synthase 2 (SPS2), ribulose bisphosphate carboxylase small chain 1A (RBCS1A), carbonic anhydrase 1 (CA1), catalase 2 (AT4G35090), photosystem I P subunit (PS-IP), photosystem II subunit P-1; (PSBP1), thioredoxin-like protein (CDSP32), chlorophyll a–b binding protein CP29.3 (LHCB4.3) (Fig. 3b).

Fig. 3.

The protein interactome network. The ESTs obtained in SSH were used to develop protein interactome network using tomato and Arabidopsis information on the STRING database. a Protein interactome network developed on S. lycopersiucm revealed two major groups. One group of protein involved in basal non-specific resistance and proteins of another major group function in the chloroplast. b Protein interactome network on Arabidopsis showed a single protein interactome among chloroplastic proteins

Discussion

Viruses are equipped to modulate host gene expression, in order to achieve a permissive cellular environment for the multiplication (Arguello-Astorga et al. 2004; Hanley-Bowdoin et al. 2013). The degree of modulation of host gene expression by the viruses is dependent on the genetic specificity of host and virus. In susceptible host, viruses successfully create a conducive cellular environment (Sahu et al. 2010; Liu et al. 2014; Kushwaha et al. 2015). On the contrary, a resistant host expressed a set of genes that avert the virus propagation (Voinnet et al. 1999; Sahu et al. 2010; Allie et al. 2014). The transcriptomic profile of an infected host offers an excellent source for the information required to understand the mechanism of virus pathogenesis.

The present study provides the first transcriptomic profile representing downregulated genes following ChiLCV infection in C. annuum var. Punjab Lal. As a resistant variety, Punjab Lal activates the defense system (Kushwaha et al. 2015) to prevent ChiLCV multiplication. At the same time, the lower abundance of viral titer in C. annuum at 21 dpi (Kushwaha et al. 2015) may exert negative pressure on the host to down-regulate the expression of a set of genes that are required for healthy plants. In the current study, SSH was carried out to explore the expression of the genes, whose  expression remained higher in mock plants as compared to infected plants. The study revealed that the transcripts of chlorophyll a-b binding gene were encountered most suggesting the down-regulation of this gene following ChilCV infection. Chlorophyll a-b-binding gene encodes proteins that are the components of the light-harvesting complex of the photosystem II (PSII), and in association with chlorophyll and xanthophylls serves as the antenna complex (Nott et al. 2006). Previous studies have revealed that expression of the chlorophyll a-b-binding gene is regulated by multiple environmental factors (Thain et al. 2002; Yang et al. 1998; Chen et al. 2013; Xu et al. 2014; Staneloni et al. 2008) oxidative stress (Staneloni et al. 2008), chloroplast retrograde signal circadian clock (Thain et al. 2002) and the phytohormone abscisic acid (ABA) (Staneloni et al. 2008). The major function of chlorophyll a-b binding antenna complex is harvesting the light that ultimately is responsible for the conversion of photons into the biochemical energy. Plants have a wonderful mechanism of transforming solar energy into usable energy owing to the occurrence of light-harvesting antenna. Chlorophyll a-b binding protein constitutes an outer antenna. CP29 and CP26 belong to minor antenna complexes encoded by Lhcb4 and Lhcb5, respectively. CP29 gets phosphorylated in a reversible fashion in response to abiotic stresses (Chen et al. 2013). CP26 is minor antenna protein of LHCII. It binds to chlorophyll a and xanthophylls (Yakushevska et al. 2003).

Abscisic acid (ABA) regulates expression of LHCB proteins. ABA acts to fine-tune the LHCB expression through repressing WRKY transcription factors. The first co-relation between geminivirus and ABA was suggested in Arabidopsis inoculated with Beet severe curly top virus (BSCTV). ABA was shown to induce two members of homeodomain-leucine zipper family, ATHB12 and ATHB7. BSCTV infection also leads to induction of these transcription factors (Park et al. 2011). ABA acts as a key hormone in abiotic stress and also leads to defense against various pathogens. ABA has a role in antiviral defense. It acts by inducing callose deposition at plasmodesmata (De Storme and Geelen 2014) and the RNA silencing pathway (Alazem and Lin 2015; Alazem et al. 2017). We observed the level of CP26 to be down-regulated in both C annuum var. Kashi Anmol as well as Punjab Lal which can be explained on the basis that CP26 might act through ABA which possesses antiviral role. ChiLCV-mediated down-regulation of CP26 could be a strategy to dominate the host.

Role of CP29 is well known in case of abiotic stress in monocots, however its role in biotic stress is not yet known. CP29 role was emphasized in maize plants when exposed to cold stress condition. The expression of CP29 was found to be induced in thylakoid membrane upon chilling treatment to maize plants. In response to stress, CP29 was found to be phosphorylated that favours lateral migration of LHCII antenna complexes (Bergantino et al. 1995). Also, CP29 phosphorylation governs its movement from grana PSII core to the stroma to harvest light for PSI (Tikkanen and Aro 2012). Level of CP29 in the susceptible variety is unvarying in order to capture the additional light in order to sustain optimum photosynthesis rate to support the life of diseased plants. Whereas on the resistant variety, virus reduced the level of CP29 to make the host weaker in order to create the permissive cellular environment.

ChiLCV infection also affected the expression of Curvature Thylakoid 1 (CURT1) gene, which is required for the chloroplast structural integrity. A. thaliana Curvature Thylakoid 1 (CURT1) protein modifies thylakoid architecture resulting in membrane curvature (Armbruster et al. 2013). Curvature thylakoid forms oligomer which is responsible for membrane curvature (Armbruster et al. 2013). It is present in grana margins and regulates grana formation. In the absence of CURT1 protein, chloroplasts consist of lobe-like thylakoid with reduced grana margins leading to impairment in photosynthesis (Armbruster et al. 2013). The expression of curvature thylakoid remains unaltered in Kashi Anmol, whereas it is down-regulated in Punjab Lal. Reduced level of CURT1 leads to fewer grana or flat lobe-like thylakoids. It suggests that ChiLCV has reduced the level of CURT1 to gain access to chloroplast for its own shield.

Our study also indicated the down-regulation of expression of other genes like Photosystem II oxygen-evolving complex protein (PsbQ), Oxygen evolving enhancer protein 2 (PsbP) involved in the photosynthesis at chloroplast. ChiLCV infection reduced the expression of chloroplast genes in order to make the host energetically weaker and negatively affect the cellular and physiological health of the host. Down-regulation of PsbP was also earlier observed during infection by a geminivirus and betasatellite complex (Bhattacharyya et al. 2015) and role of chloroplast in defense against plant viruses has been highlighted in a recent review (Bhattacharyya and Chakraborty 2018).

The transcripts of auxin-binding protein were also frequently screened in the SSH library revealing the down-regulation of expression of the auxin-binding protein gene. Early studies characterized the role of auxin-binding protein in rapid electrophysiological and cell expansion responses, cell cycle and cell division control,  modulation of  endocytic events at the plasma membrane, cytoskeletal rearrangements during asymmetric cell expansion (Geldner et al. 2001; Woo et al. 2002; David et al. 2007; Braun et al. 2008; Sauer and Kleine-Vehn 2011). Auxin binding protein has been implied as a receptor for auxin (Xu et al. 2014; Sauer and Kleine-Vehn 2011; Gaoa et al. 2015). According to the studies, ABP regulates cellular growth by activating plasma membrane GTPase (Xu et al. 2014). ABP1 was also known to reduce clathrin-dependent endocytosis to retain PIN-FORMED (PIN1) protein at membranes in order to increase auxin efflux (Geldner et al. 2001). ABP1 also regulates cytoskeleton by activating small GTPase ROP2 and ROP6 (Chen and Yang 2014). Later on, it was reported that ABP1 neither regulates auxin-mediated signaling nor is necessary for plant development (Xu et al. 2014). However, ZmABP1 has been implicated in conferring resistance against Sugarcane mosaic virus (SCMV) at an early stage (14dpi). ZmABP1 promotes SCMV resistance in a light-dependent manner owing to the occurrence of two light responsive cis-elements in the promoter of resistance allele (Leng et al. 2017). ZmABP1 lies in the Scmv2 locus which is the resistant allele against SCMV (Leng et al. 2017). In chilli variety, Kashi Anmol level of ABP1 remains unaltered. This could be because ABP1 of Kashi Anmol may not be a part of resistant allele, unlike ZmABP1. On the other hand, it is down-regulated in the resistant variety Punjab lal suggesting its role in resistance against ChiLCV. Possibly, ChiLCV infection decreased expression of auxin-binding protein in Punjab Lal to achieve a permissive environment in the host cell.

Thioredoxin-like protein, peroxiredoxin, glycolate oxidase, and catalase isozyme are involved in non-host resistance and govern defense responses to a broad range of potential pathogen species. The thioredoxin system is an important conserved system for protection against oxidative stress by reducing peroxides such as H₂O₂ to harmless products (Ross et al. 2000). Thioredoxins are antioxidant enzymes that catalyze reduction of disulfide bonds. Plant thioredoxins are involved in the photorespiration, lipid metabolism, membrane transport, hormone metabolism and ATP synthesis (Collet and Messens 2010). It was identified as an interacting partner of the E2 protein of Swine fever virus (CSFV). The interaction leads to the inhibition of replication of CSFV (Li et al. 2015). Zea mays thioredoxin type h (ZmTrxh) has been shown to impart resistance against Sugarcane mosaic virus (SCMV). ZmTrxh acts at an early stage of infection to limit the virus by acting as a molecular chaperone ZmTrxh showed upregulation following SCMV infection. Overexpression of ZmTrxh significantly down-regulates SCMV accumulation. ZmTrxh acts independent of both salicylic acid and jasmonic acid pathways (Liu et al. 2017). Also, h-type thioredoxin of Nicotiana tabacum confers resistance against Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) (Sun et al. 2010). Thioredoxin is about 3 times down-regulated in Kashi Anmol at 21dpi. Thioredoxin might be acting as resistance factor, therefore in order to overcome host defense, ChiLCV has down-regulated thioredoxin in Kashi Anmol whereas thioredoxin remains maintained at 21 dpi in Punjab Lal to combat virus infection.

Peroxiredoxin belongs to the family of peroxidases present in chloroplast and mitochondrion. Peroxiredoxin functions as housekeeping antioxidant metabolism or in abiotic stress leading to oxidative stress. Its functions include protection of photosynthesis, protection of thylakoids, and also contribute to signaling in the cell. Peroxiredoxin level shoots up in oxidative stress to counteract the situation. In animals, Peroxiredoxin II serves as a marker for identification of hepatitis B virus-induced liver fibrosis. This is owing to upregulation of Peroxiredoxin II upon hepatitis B virus infection (Lu et al. 2010). Also, Peroxiredoxin I has been reported to be indispensable for replication and transcription of measles virus (MeV) (Watanabe et al. 2011). Peroxiredoxin levels increases in incompatible reaction owing to the occurrence of HR leading to oxidative burst accompanied by the production of hydrogen peroxide (Delledonne et al. 2001). On the contrary, Peroxiredoxin levels fall off following compatible reaction as there is no need for detoxification (Rouhier et al. 2004). ChiLCV infection caused a significant reduction of peroxiredoxin gene expression in the susceptible variety Kashi Anmol and the resistant variety Punjab Lal. Since the ChiLCV infection does not induces HR, hence the level of Peroxiredoxin was relatively low. Peroxiredoxin also functions to protect photosynthetic machinery, thus down-regulation of Peroxiredoxin could be one of the ways to weaken host defence.

Filamentation temperature sensitive (FtSHs) are proteases involved in housekeeping proteolysis of membrane proteins. These proteins are generally membrane-bound and contain AAA type and Zn²⁺metalloproteases domain (Wagner et al. 2012). FtSH is, highly expressed in photosynthetic tissues (Gustavsson et al. 2002) and targeted to the chloroplast and mitochondrion. It is involved in chromoplast vesicle fusion in Capsicum sp. (Summer and Cline 1999), and HR caused by TMV infection (Seo et al. 2000). FtSH complex located in thylakoid functions to degrade photodamaged D1 protein. In the current study, FtSH is significantly reduced in the susceptible variety Kashi Anmol suggesting, during pathogenesis, ChiLCV hampered the FtSH-mediated repairing mechanism of chloroplast to make the host weaker. Since the accumulation of damaged proteins leads to stress, making it vulnerable to infection, this could be one of the factors contributing to susceptibility of Kashi Anmol to ChiLCV. In contrast, ChiLCV failed to alter the expression of FtSH in the resistant chilli variety Punjab Lal. The down-regulation of expression of protease FtSH occurs following infection, which is involved in biogenesis and maintenance of PSII (Silva et al. 2003). In one of the earlier reports, TMV  infection resulted in reduction of FtSH transcripts levels in tobacco leading to HR (Silva et al. 2003). FtSH functions as a chaperone (Akiyama et al. 1994). Lower levels of FtSH retard cell growth and export of protein in E. coli. In eukaryotes, FtSH functions both to degrade protein (Nakai et al. 1995) as well as protein assembly (Akiyama et al. 1994). The chloroplast FtSH acts to degrade unassembled Rieske Fe-S protein (Ostersetzer and Adam 1997). Likewise, FtSH is involved in clearing the products of proteolysis caused by photodamage (Seo et al. 2000). PVX-mediated virus-induced gene silencing of FtSH in N. benthamiana caused bleaching of systemic leaves, suggesting its role in protecting photosynthetic machinery (Saitoh and Terauchi 2002). Also, silenced plants exhibited increased susceptibility to TMV and Botrytis cinerea. Low level of FtSH in Kashi Anmol could be correlated with the susceptibility of the plant to the ChiLCV. On the other hand, unaltered level of FtSH in Punjab Lal (compared to the mock) can be attributed to the resistant nature of the plant. FtSH levels are maintained to combat ChiLCV infection. Overall, FtSH seems to be a common target for the viral pathogens.

Furthermore, proline-rich proteins (PRPs) are a group of proteins characterized by the presence of proline repeats. The expression of PRPs is stimulated by abiotic stress and are involved in a number of cellular processes like root hair development, abscission and senescence, flower development abiotic stress responses (Zhan et al. 2012; Kavi Kishor et al. 2015). Because of the reduced expression of PRPs in the susceptible variety, Kashi Anmol was possibly unable to recover from the ChiLCV infection. In Punjab Lal, level of PRPs was not significantly altered, implying that the defense-related protein is supporting the plant during infection.

The current study showed that, for establishment of successful pathogenesis, ChiLCV attempts to down-regulate expression of various key genes of host cellular pathways, in order to create a permissive cellular ambience in host plants.

Electronic supplementary material

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Funding

Funding was provided by University Grants Commission [Grant Nos. UGC-RNW (SLS/SC/2016, UGC-SAP (SLS/SAP/SC/2016)], Ministry of Science and Technology [Grant No. DST-FIST (JNU/SLS/SC/FIST-16)].

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Conflict of interest

The authors declare that they do not have any conflict of interest.