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. 2019 Jan 19;14(3):e1565595. doi: 10.1080/15592324.2019.1565595

Silencing of tomato CTR1 provides enhanced tolerance against Tomato leaf curl virus infection

Ravindra K Chandan a,b, Achuit K Singh c, Sunita Patel a, Durga Madhab Swain b, Narendra Tuteja d, Gopaljee Jha b,✉
PMCID: PMC6422369  PMID: 30661468

ABSTRACT

Tomato leaf curl virus (ToLCV) belonging to Begomovirus family of Geminivirus is known to cause one of the most destructive diseases in tomato. Amongst various ToLCVs, a monopartite Tomato leaf curl Joydebpur virus (ToLCJoV) is most prevalent in eastern part of India. In the present study, we observed induced expression of one of the negative regulators of ethylene signaling pathway gene (LeCTR1) in ToLCJoV infected plants. The Tobacco rattle virus (TRV) induced silencing of the LeCTR1 gene provided enhanced tolerance to ToLCJoV infections. The leaf curling as well as ROS accumulation was significantly reduced in the viral infected LeCTR1 silenced plants. Induction of several defense marker genes (NPR1, PR1, PR5, AOS2, EIN2, EIN3 and ERF5) reinforced enhanced tolerance against ToLCJoV infection in the LeCTR1 silenced tomato. Overall, the present study provides evidence that silencing of LeCTR1 can be deployed to protect tomato from ToLCJoV infections.

KEYWORDS: LeCTR1 gene, VIGS, marker defence gene, tomato, Tomato leaf curl virus

Introduction

Geminiviruses are a family of single-stranded DNA viruses which cause significant yield loss in economically important crops worldwide.1,2 On the basis of genome organization and insect vectors, geminiviruses have been classified into seven genera, namely Becurtovirus, Begomovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus and Turncurtovirus. Their genome generally comprised of either one (monopartite) or two (bipartite) DNA components. During infection process they are known to inject viral DNA into the host cell and interferes the host gene silencing machinery.3

Amongst various geminiviruses, the Begomovirus (Tomato leaf curl virus; ToLCV) causes devastating disease in tomato.4-7 The ToLCVs are known to be transmitted from one host to another host by white fly (Bemisia tabaci) insect vector and are either monopartite or bipartite in nature.8,9 Amongst Indian isolates of ToLCVs, a monopartite Tomato leaf curl Joydebpur virus (ToLCJoV) is prevalent in the eastern part of India. Besides severely damaging tomato cultivation, the ToLCJoVs are known to infect other important plants such as kenaf and chilli.10,11 Being monopartite in nature, the ToLCJoV infection is associated with Betasatellite, a subviral DNA molecule which encodes important pathogenicity determinants.12,13

Generally, ethylene (ET) plays a vital role in antiviral defense in various plants.14-16 Mutations in ET signaling genes, i.e., etr1 and ein2 alter plant susceptibility to viral infections. For example, etr1 and ein2 mutant Arabidopsis thaliana plants showed reduced susceptibility to Cauliflower mosaic virus (CaMV) infections.15,17 Previous reports suggested that, a Solanum lycopersicum ethylene responsive factor (SlERF) plays an important role in imparting resistance to one of the begomoviruses, i.e. TYLCV (Tomato yellow leaf curl virus) infection in tomato.18 However, role of ethylene in providing resistance against ToLCJoV infection has not been established.

CTR1 (CONSTITUTIVE TRIPLE RESPONSE) is known to be a negative regulator of ethylene signaling and actively suppresses the ethylene signaling cascade.19-21 It is a Raf-like kinase consisting of a N-terminal regulatory domain and a C-terminal serine/threonine kinase domain.22 Previous study demonstrated that Arabidopsis thaliana CTR1 inhibits the ethylene responses by phosphorylating the EIN2 (ETHYLENE INSENSITIVE 2), a key regulator of ethylene signaling.22 Moreover, it had been demonstrated that ectopic expression of a tomato homolog of CTR1, i.e. Lycopersicon esculentum CTR1 (LeCTR1) can restore the ethylene signaling in ctr1-1 mutant A. thaliana.23 In this study, we observed that LeCTR1 is upregulated during ToLCJoV infections in tomato. The Virus induced gene silencing of LeCTR1, rendered tomato plants tolerant against ToLCJoV infection. Furthermore, inductions of various defense marker genes that include ethylene responsive genes were quite prominent in the LeCTR1 silenced tomato plants. Overall, the present study highlights the role of LeCTR1 during monopartite ToLCJoV infections in tomato.

Results

Expression of LeCTR1 is induced during ToLCJoV infection in tomato

In this study, the expression pattern of LeCTR1 was analyzed at different time points of ToLCJoV infections in tomato. Compared to uninfected tomato plants, the expression of LeCTR1 was ~4 fold upregulated at 12 dpi while more than ~7 fold upregulated at 21 dpi of ToLCJoV infection (Figure 1).

Figure 1.

Figure 1.

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The ToLCJoV infection induces the expression of LeCTR1 gene in tomato. Relative gene expression of LeCTR1 infected WT tomato plant at 12 and 21 dpi. The expression data were normalized with that of uninfected WT plants using tomato beta actin gene as an internal control. The experiments were independently repeated three times with minimum three technical replicates.

Virus-induced gene silencing (VIGS) of LeCTR1 gene in tomato

By using Tobacco rattle virus (TRV) based VIGS system, we silenced LeCTR1 gene in tomato (cv. Pusa ruby) (Supplemental Figure 1A). The LeCTR1 silenced (TRV:LeCTR1) plants as well as TRV:0 (EV) plants did not show any visual phenotypic alteration while the silencing of Solanum lycopersicum Phytoene desaturase (SlPDS) showed characteristic photo bleaching phenotype (Supplemental Figure 1B). The silencing of the target gene was further analyzed by qRT-PCR and semi-quantitative PCR (Supplemental Figure 1C and D). Interestingly, both the analysis revealed reduced LeCTR1 transcript accumulation in the LeCTR1 silenced plants (TRV:LeCTR1) compared to empty vector (TRV:0) and wild type (WT) plants (Supplemental Figure 1C and D).

Induced expression of ethylene marker genes were observed in LeCTR1 silenced tomato

We analyzed the expression of some of the key ethylene signaling marker genes (ERF5, EIN2 and EIN3) in different tomato plants (TRV:LeCTR1, TRV:0, and WT). The qRT-PCR analysis reflected these genes to be significantly up-regulated in the LeCTR1 silenced plants compared to WT and TRV: 0 plants (Supplemental Figure 2).

Silencing of LeCTR1 provided tolerance against ToLCJoV infection in tomato

Different tomato (TRV:LeCTR1; TRV:0 and WT) plants were inoculated with the infectious clone of ToLCJoV and monitored for visual appearance of disease symptoms. The leaf curling disease symptoms were less in the LeCTR1 silenced plants at 12 dpi, in comparison to that observed in case of infected TRV-0 and WT plants (Supplemental Figure 3A). The difference became more pronounced at 21 dpi, wherein the LeCTR1 silenced plants had limited leaf curling, while the WT and TRV-0 plants exhibited severe leaf curling symptoms (Figure 2(A)). Apart from this, the DAB staining revealed reduced ROS accumulation (seen as mild brownish color) in infected LeCTR1 silenced plants at both 12 and 21 dpi, compared to that observed in case of infected WT and TRV-0 plants (Figure 2(B) and Supplemental Figure 3B). The disease severity index also suggested enhanced disease tolerance in the LeCTR1 silenced plants (Figure 3A and B). Significantly higher chlorophyll content in the LeCTR1 silenced plants upon ToLCJoV infection further suggested it to have enhanced disease tolerance (Figure 3C and D). The semi-quantitative PCR-based estimation of viral DNA in the infected plants also revealed relatively less virus load in the LeCTR1 silenced plants than that observed in cased of infected WT and TRV-0 plants (Supplemental Figure 4).

Figure 2.

Figure 2.

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Silencing of LeCTR1 provides tolerance against ToLCJoV infection. (A) Disease symptoms of ToLCJoV infected tomato at 21 dpi. The leaf curling like symptoms became more pronounced in WT and TRV-0 plants at 21 dpi, while the LeCTR1 silenced plants exhibited limited leaf curling symptoms, (B) ROS accumulation in the ToLCJoV infected tomato leaves visualized by DAB staining. Less number of brownish color (reflecting less ROS accumulation) was observed in LeCTR1 silenced plants, compared to WT, EV plants.

Figure 3.

Figure 3.

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LeCTR1 silencing provides tolerance against ToLCJoV infection in tomato. Disease severity index of ToLCJoV infected leaves of tomato, at 12 dpi (A) and 21 dpi (B). While (C) and (D) represent total chlorophyll content of the ToLCJoV infected leaves at 12 and 21 dpi, respectively. The infected LeCTR1 silenced (TRV: LeCTR1) plants retain high chlorophyll content compared to that of infected wild type (WT) and empty vector (TRV-0) plants at both the time points. Data are represented as mean ± SE of at least three biological replicates. Different letters represent significant difference between different samples (one-way ANOVA, p < 0.05; Student-Newman-Keuls test).

Several defense marker genes were induced upon ToLCJoV infection in the LeCTR1 silenced plants

Previous report suggested that, geminiviruses infection induces expression of Salicylic acid (SA), Jasmonic acid (JA) and ET-mediated defense marker genes.24-26 We analyzed expression pattern of some of the previously reported plant defense marker genes (NPR1, PR1, PR5, AOS2, ERF5, EIN2 and EIN3) upon ToLCJoV infection. Interestingly, the qRT-PCR analysis reflected most of these genes to be significantly up-regulated upon ToLCJoV infection (21 dpi) in silenced plants, compared to that in TRV-0 plants (Figure 4).

Figure 4.

Figure 4.

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LeCTR1 silencing enhances expression of some defense marker genes upon ToLCJoV infection. The extent of induction was significantly higher in infected LeCTR1 silenced (TRV: LeCTR1) plants compared to that of infected wild type (WT) and empty vector (TRV-0) plants at 21 dpi. The tomato beta actin (NM_001321306.1) gene was used as internal control during estimation of relative expression. Data are represented as Mean ± SE of at least three independent biological replicates. Different letters represent significant difference between different samples (One-way ANOVA, p < 0.05; Student-Newman-Keuls test).

Discussion

Plant defense response is a complex phenomenon and involves extensive cross talks between different defense proteins and signaling molecules.27,28 Many genes/proteins playing crucial roles in elaborating defense responses had been identified and some of them are being used to develop disease resistance against variety of pathogens.29,30 Also several negative regulators of plant defense responses had also been identified whose up-regulation dampen the host defense response.31 The CaWRKY1, AtWRKY11, AtWRKY17, OsWRKY62 are some notable example of negative regulator of plant defense response.31-33 During infection process, the phytopathogens may up-regulate expression of such negative regulators to avoid induction of host defense responses, which may otherwise can be deleterious for the pathogen. In a previous study, silencing of one of such negative regulators, i.e. GmMAPK4 had been found to enhance resistance to Soybean mosaic virus infections. Also, the silenced plants exhibited enhanced tolerance to downy mildew infections.34 Similarly, the silencing of a negative regulator gene OsMADS26 showed enhanced resistance to both bacterial and fungal pathogen (Xanthomonas oryzae and Magnaporthe oryzae) infections.35 Therefore silencing of negative regulator of plant defense is considered an important strategy to develop disease resistance plants. We observed that ToLCJoV infection leads to induction of one of the key negative regulators of ethylene signaling pathway, i.e. LeCTR1 gene in tomato. The induction of ethylene responsive genes in the LeCTR1 silenced tomato plants further validated role of the gene as negative regulator of ethylene signaling. Ethylene is a gaseous plant hormone involved in certain developmental stages such as senescence as well as the defense responses against necrotrophic pathogens.36 Apart from that, it plays a significant role in activating plant defense against invading pathogens such as bacteria, fungi, and viruses.37 Indeed we observed that the LeCTR1 silenced (TRV:LeCTR1) tomato plants have less curling like symptoms upon ToLCJoV infections. Overall the silenced plants exhibited less viral disease severity index compared to that in case of infected wild type and TRV-0 plants. Previous reports suggested that ToLCV imparts enhanced ROS accumulation and decreased chlorophyll content in the infected leaves of tomato.12,25,38 Our data revealed significantly lesser ROS accumulation and higher chlorophyll content in the ToLCJoV infected LeCTR1 silenced tomato plants, compared to infected control (WT and TRV-0) plants. The reduced ROS accumulation and higher chlorophyll content, also correlated with the reduced viral load in the silenced plants compared to the infected WT plants. Taken together these results confirm that silencing of LeCTR1 imparts enhanced tolerance against ToLCJoV infection in tomato. Interestingly beside induced expression of ethylene marker genes (ERF5, EIN2 and EIN3), the expression of Salicylic acid (NPR1, PR1, PR5) and Jasmonic acid (AOS) marker genes were also induced upon ToLCJoV infection in the LeCTR1 silenced tomato plants. Overall the induction of ET, SA and JA mediated defense responses in LeCTR1 silenced plants, reinforced enhanced resistance against ToLCJoV virus infections in tomato. In summary, our study demonstrates that the LeCTR1 acts as a negative regulator of ethylene response pathway and plays an important role during ToLCJoV infection in tomato.

Materials and methods

Plant materials and growth conditions

Tomato (Pusa ruby) plants were grown on soil rite under 26℃ with 16/8-h photoperiod, 70% relative humidity in growth chamber. Two week old tomato plants were used for VIGS experiment while 3 week old plants were used for ToLCJoV (Accession no: KF515609.) inoculation.

Silencing of LeCTR1 in tomato

TRV virus-induced gene silencing (VIGS) technique was used for silencing of LeCTR1 (Accession no: AF096250.1). The target sequence for silencing of LeCTR1 was predicted by sol genomics VIGS tool (http://vigs.solgenomics.net/) and gene-specific primer (LeCTR1_VIGS-Forward and LeCTR1_VIGS-Reverse) pair was used for PCR amplification of ~300 bp target region for silencing of LeCTR1 gene (Supplemental Table 1). The PCR product was first cloned into the multiple cloning sites (MCS) of pTRV2 vector and the recombinant plasmid was transformed into Agrobacterium (GV3101). Agrobacterium (GV3101) harboring pTRV1 was used as helper virus for Agro-inoculation into two-week-old tomato plant as described earlier.39 Mix culture of recombinant Agrobacterium strain (GV3101) harboring pTRV2 and pTRV1 was inoculated into two weeks old tomato plants by syringe infiltration method40,41 for silencing of LeCTR1 in tomato plants. Silencing of LeCTR1 was confirmed by semi-quantitative PCR as well as qRT-PCR using gene-specific primers (Supplemental Table 1). The tomato beta actin (Accession no: NM_001321306.1) gene was used as internal control for normalization of gene expression.

Virus (Tomato leaf curl Joydebpur virus) inoculation

The Agrobacterium strain (EHA105) containing infectious clone of Tomato leaf curl Joydebpur virus (ToLCJoV) and the associated betasatellite were used to infect 3-week-old tomato leaves. Both the agro bacterial strains were grown in YEP medium supplemented with 100mg/ml kanamycin and rifampicin (50mg/ml) until the OD (optical density) reached to 0.8. The pellets were individually re-suspended in infiltration medium (0.5 mM MES buffer, 100 mM MgCl2, 200 Mm Acetosyringone) followed by 2 h incubation at 28°C. Thereafter both the cultures were mixed (1:1 ratio) and inoculated in different tomato (TRV:LeCTR1, TRV-0 and WT) plants by syringe infiltration method. The disease symptoms were observed at 12 dpi and 21 dpi and photographed using DSLR camera (Nikon D3200). These samples were collected and stored at −80°C until used for further analysis.

Detection of ToLCJoV in infected tomato plants

Leaf samples of ToLCJoV infected LeCTR1 silenced (TRV: LeCTR1), TRV-0 (EV) as well as WT tomato plants were used for total RNA isolation using RNeasy plant mini kit (Qiagen; Cat. No. 74904). One microgram of total RNA was used for cDNA synthesis by using Verso cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Cat.no. AB1453A), as per the manufacturer’s protocol and the ToLCJoV gene (AC4; accession no: AGV02032.1) specific primer pairs (AC4_f and AC4_r) were used for PCR amplification. The tomato beta actin gene was used as internal control.

Determination of chlorophyll content

The infected leaves of TRV: LeCTR1, TRV-0 (EV) as well as WT plants were used for total chlorophyll estimation. For this the leaves were incubated overnight in 1:1 ratio of DMSO and acetone solutions. Thereafter absorbance was recorded at 645 and 663 nm by using Shimadzu UV 1800 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). The total chlorophyll content was estimated using Arnon’s method42 i.e. total chlorophyll (μg/cm2) = 20.2 (A645) + 8.02 (A663)/leaf area(mm/square).

DAB assay for ROS detection

To visualize ROS accumulation in the ToLCJoV infected as well as control leaves, DAB (3,3′-Diaminobenzidine) staining was performed. The leaves of infected TRV: LeCTR1, TRV-0 (EV) as well as WT plants (both 12 and 21 dpi) were excised and stained with 1 mg/mL DAB (prepared in 50 mM Tris acetate buffer, pH 5.0) followed by incubation at 25°C for 16 hrs in dark. Upon destaining (in a solution of 3:1 ratio of ethanol:glacial acetic acid), ROS was visualized as a brown color (due to DAB polymerization) and photographed using DSLR camera (Nikon D3200)

Expression analysis of defense marker genes using qRT-PCR

Total RNA was isolated from both infected and uninfected leaves of TRV: LeCTR1, TRV-0 (EV) as well as WT tomato plants using RNeasy plant mini kit (Qiagen; Cat. No. 74904). One microgram of the total RNA was used for cDNA synthesis using verso cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Cat.no. AB1453A). Relative expression of the defense marker genes (NPR1, PR1, PR5, AOS2, ERF5, EIN2, EIN3) in ToLCJoV infected plant samples were calculated with respect to that of uninfected plants (TRV: LeCTR1, TRV-0 and WT) using tomato beta actin gene as internal control. The experiment was repeated at least three times, with three independent biological as well as technical replicates. The relative fold change was calculated by using 2−∆∆Ct method.43

Funding Statement

This work was supported by NIPGR core research grant.

Acknowledgments

We acknowledge Ms Sangeeta Rathore from Central University of Gujarat (India) for providing ToLCJoV infectious construct. RKC acknowledges research fellowship from Central university of Gujrat (India). DMS acknowledges fellowship from Department of Biotechnology (DBT), Govt. of India. Research in GJ lab is supported by funding from Department of Biotechnology, Govt. of India and NIPGR core research grant.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors’ contributions

GJ conceived and planned the work and coordinated its progress, AKS and SP helped in ToLCJoV related experiments. RKC carried out the detail characterization of LeCTR1 and established its role on plant defense against ToLCJoV infections. RKC, DMS and GJ had written the manuscript. GJ, AKS, SP, DMS and NT critically evaluated the manuscript. All authors have read and approved the manuscript.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.

Supplemental Material

References

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