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[This corrects the article DOI: 10.1371/journal.ppat.1006587.].
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[This corrects the article DOI: 10.1371/journal.ppat.1006587.].
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Geminiviruses constitute a group of plant viruses, with a ssDNA genome, whose replication in the nucleus of an infected cell requires the function of geminivirus-encoded replication initiator protein (Rep). Our results suggest that monoubiquitinated histone 2B (H2B-ub) promotes tri-methylation of histone 3 at lysine 4 (H3-K4me3) on the promoter of Chilli leaf curl virus (ChiLCV). We isolated homologues of two major components of the monoubiquitination machinery: UBIQUITIN-CONJUGATING ENZYME2 (NbUBC2) and HISTONE MONOUBIQUITINATION1 (NbHUB1) from N. benthamiana. ChiLCV failed to cause disease in NbUBC2-, and NbHUB1-silenced plants, at the same time, H2B-ub and H3-K4me3 modifications were decreased, and the occupancy of RNA polymerase II on the viral promoter was reduced as well. In further investigations, Rep protein of ChiLCV was found to re-localize NbUBC2 from the cytoplasm to the nucleoplasm, like NbHUB1, the cognate partner of NbUBC2. Rep was observed to interact and co-localize with NbHUB1 and NbUBC2 in the nuclei of the infected cells. In summary, the current study reveals that the ChiLCV Rep protein binds the viral genome and interacts with NbUBC2 and NbHUB1 for the monoubiquitination of histone 2B that subsequently promotes trimethylation of histone 3 at lysine 4 on ChiLCV mini-chromosomes and enhances transcription of the viral genes.
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Begomovirus/genética , Regulación Viral de la Expresión Génica/genética , Interacciones Huésped-Parásitos/genética , Proteínas Virales/metabolismo , Replicación Viral/genética , Animales , ADN Helicasas/genética , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Transferencia Resonante de Energía de Fluorescencia , Genoma Viral/genética , Immunoblotting , Inmunoprecipitación , Proteínas de Plantas/metabolismo , Reacción en Cadena de la Polimerasa , Nicotiana/virología , Transactivadores/genética , Transcripción Genética/genética , Técnicas del Sistema de Dos Híbridos , UbiquitinaciónRESUMEN
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.
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RNA silencing is an integral part of the cellular defense mechanisms in plants that act against virus infection. However, the specific role of RNA silencing and the interplay between host and virus components during recovery from geminivirus infection remains unknown. Hence, in this study we aimed to examine the mechanism behind the host-specific recovery of Nicotiana tabacum infected with Tomato leaf curl Gujarat virus (ToLCGV). Unlike Tomato leaf curl New Delhi virus (ToLCNDV), ToLCGV infection resulted in symptom remission in N. tabacum, and we found that this was mainly due to cross-talk between the pre-coat protein (encoded by the AV2 ORF) of the virus and the host RNA-silencing component RNA-dependent RNA polymerase 1 (encoded by NtRDR1) of N. tabacum. Moreover, apart from the AV2 mutant, other mutants of ToLCNDV developed severe symptoms on a transgenic NtRDR1-overexpression line of N. benthamiana. In contrast, inoculation with ToLCGV resulted in symptom remission, which was due to enhanced methylation of the ToLCGV promoter. Our study reveals a novel 'arms race' in which the pre-coat protein of ToLCNDV selectively blocks the recovery process through inhibiting host-specific RDR1-mediated antiviral silencing in tobacco.
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Proteínas de la Cápside/metabolismo , Geminiviridae/metabolismo , Nicotiana/enzimología , Enfermedades de las Plantas/virología , Proteínas de Plantas/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Begomovirus/fisiología , Proteínas de la Cápside/genética , Geminiviridae/genética , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Interferencia de ARN , ARN Polimerasa Dependiente del ARN/genética , Nicotiana/genética , Nicotiana/virologíaRESUMEN
Geminiviruses are the largest and most devastating group of plant viruses which contain ssDNA as a genetic material. Geminivirus-derived virus-induced gene silencing (VIGS) vectors have emerged as an efficient and simple tool to study functional genomics in various plants. However, previously developed VIGS vectors have certain limitations, owing to their inability to be used in tissue-specific functional study. In the present study, we developed a Chilli leaf curl virus (ChiLCV)-based VIGS vector for its tissue-specific utilization by replacing the coat protein gene (open reading frame (ORF) AV1) with the gene of interest for phytoene desaturase (PDS) of Nicotiana benthamiana. Functional validation of ChiLCV-based VIGS in N. benthamiana resulted in systemic silencing of PDS exclusively in the phloem region of inoculated plants. Furthermore, expression of enhanced green fluorescence protein (EGFP) using the same ChiLCV vector was verified in the phloem region of the inoculated plants. Our results also suggested that, during the early phase of infection, ChiLCV was associated with the phloem region, but at later stage of pathogenesis, it can spread into the adjoining non-vascular tissues. Taken together, the newly developed ChiLCV-based vector provides an efficient and versatile tool, which can be exploited to unveil the unknown functions of several phloem-specific genes.
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Begomovirus/genética , Regulación de la Expresión Génica de las Plantas , Nicotiana/genética , Oxidorreductasas/genética , Floema/genética , Proteínas de Plantas/genética , Silenciador del Gen , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Nicotiana/virologíaRESUMEN
Chilli (Capsicum sp.) is one of the economically important spice and vegetable crops grown in India and suffers great losses due to the infection of begomoviruses. Conventional breeding approaches have resulted in development of a few cultivars of chilli resistant to begomoviruses. A severe leaf curl disease was observed on one such resistant chilli cultivar (Capsicum annuum cv. Kalyanpur Chanchal) grown in the experimental field of the Jawaharlal Nehru University, New Delhi. Four different viral genomic components namely, Chilli leaf curl virus (DNA A), Tomato leaf curl Bangladesh betasatellite (DNA ß), Tomato leaf curl New Delhi virus (DNA A), and Tomato leaf curl Gujarat virus (DNA B) were associated with the severe leaf curl disease. Further, frequent association of these four genomic components was also observed in symptomatic plants of other chilli cultivars (Capsicum annuum cv. Kashi Anmol and Capsicum chinense cv. Bhut Jolokia) grown in the experimental field. Interaction studies among the isolated viral components revealed that Nicotiana benthamiana and chilli plants inoculated with four genomic components of begomoviruses exhibited severe leaf curl disease symptoms. In addition, this synergistic interaction resulted in increased viral DNA accumulation in infected plants. Resistant chilli plants co-inoculated with four genomic components of begomoviruses showed drastic reduction of host basal (ascorbate peroxidase, thionin, polyphenol oxidase) and specific defense-related gene (NBS-LRR) expression. Our results suggested that synergistic interaction among begomoviruses created permissive cellular environment in the resistant chilli plants which leads to breakdown of natural resistance, a phenomenon observed for the first time in chilli.
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Begomovirus/crecimiento & desarrollo , Capsicum/inmunología , Capsicum/virología , Tolerancia Inmunológica , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , ADN Viral/aislamiento & purificación , Expresión Génica , India , Nicotiana/inmunología , Nicotiana/virología , Carga ViralRESUMEN
Chilli, which encompasses several species in the genus Capsicum, is widely consumed throughout the world. 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 on 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 one previously unreported species) with a diverse group of betasatellites found in crops and weeds. The associated 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 the begomoviruses were found to be intra-species recombinants. The betasatellites possess high nucleotide variability, and recombination among them was also evident. The nucleotide substitution rates were determined for the AV1 gene of begomoviruses (2.60 × 10- 3 substitutions site- 1 year- 1) and the ßC1 gene of betasatellites [chilli leaf curl betasatellite (ChiLCB), 2.57 × 10- 4 substitution site- 1 year- 1; tomato leaf curl Bangladesh betasatellite (ToLCBDB), 5.22 × 10- 4 substitution site- 1 year- 1]. This study underscores the current understanding of Indian ChiLCD-associated begomoviruses and also demonstrates the crucial role of betasatellites in severe disease development in Capsicum spp.
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Begomovirus/clasificación , Begomovirus/aislamiento & purificación , Capsicum/virología , Variación Genética , Enfermedades de las Plantas/virología , Virus Satélites/clasificación , Virus Satélites/aislamiento & purificación , Begomovirus/genética , India , Datos de Secuencia Molecular , Tasa de Mutación , Recombinación Genética , Virus Satélites/genética , Análisis de Secuencia de ADN , Nicotiana/virologíaRESUMEN
Geminivirus infection often causes severe vein clearing symptoms in hosts. Recently a betasatellite has emerged as a key regulator of symptom induction. To understand the host-betasatellite interactions in the process of symptom development, a systematic study was carried out involving symptoms induced by a betasatellite associated with radish leaf curl disease (RaLCB) in Nicotiana benthamiana. It has been found that ßC1 protein localized to chloroplasts of host cells, and RaLCB lacking ßC1, which failed to produce symptoms, had no effect on chloroplast ultrastructure. Vein flecking induced by transiently expressed ßC1 was associated with chloroplast ultrastructure. In addition, the betasatellite down-regulates expression of genes involved in chlorophyll biosynthesis as well as genes involved in chloroplast development and plastid translocation. Interestingly, the expression of key host genes involved in chlorophyll degradation remains unaffected. Betasatellite infection drastically reduced the numbers of active reaction centres and the plastoquinol pool size in leaves exhibiting vein clearing symptoms. Betasatellite-mediated impediments at different stages of chloroplast functionality affect the photosynthetic efficiency of N. benthamiana. To the best of the authors' knowledge, this is the first evidence of a chloroplast-targeting protein encoded by a DNA virus which induces vein clearing and structurally and functionally damages chloroplasts in plants.
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Begomovirus/fisiología , Cloroplastos/virología , Nicotiana/virología , Fotosíntesis , Enfermedades de las Plantas/virología , Cloroplastos/metabolismo , Nicotiana/metabolismoRESUMEN
Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) that infects a wide range of plants. ToLCNDV has emerged as an important pathogen and a serious threat to tomato production in India. A comparative and molecular analysis of ToLCNDV pathogenesis was performed on diverse solanaceous hosts (Capsicum annuum, Nicotiana benthamiana, N. tabacum, and Solanum lycopersicum). N. benthamiana was found to be the most susceptible host, whereas C. annuum showed resistance against an isolate of ToLCNDV collected in New Delhi from tomato (GenBank accession no. U15015 and U15017). S. lycopersicum and N. tabacum developed conspicuous symptoms and allowed virus to accumulate to significantly high titers. The viral DNA level was concurrent with symptom severity. ToLCNDV-specific siRNA levels were directly proportional to the amount of viral DNA. To investigate the basis for the differences in response of these hosts to ToLCNDV, a comparative expression analysis of selected defense-related genes was carried out. The results indicated differences in expression levels of genes involved in the posttranscriptional gene silencing machinery (RDR6, AGO1 and SGS3) as well as basal host defense responses (nucleotide-binding site and leucine-rich repeat [NBS-LRR] proteins and lipid transfer protein [LTP]). Among these, expression of NBS-LRR genes was found to be significantly higher in C. annuum following ToLCNDV infection. Our analyses suggest that the expression of host defense responses determines the level of ToLCNDV accumulation and degree of symptom development.
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Begomovirus/fisiología , Enfermedades de las Plantas/virología , Interferencia de ARN/fisiología , Begomovirus/genética , Capsicum/virología , Regulación de la Expresión Génica de las Plantas/fisiología , Regulación Viral de la Expresión Génica/fisiología , Interacciones Huésped-Patógeno/fisiología , Solanum lycopersicum/virología , Reacción en Cadena de la Polimerasa , Nicotiana/virología , Carga ViralRESUMEN
Geminiviruses have evolved with tremendous potential of recombination and possess the ability to manipulate several cellular processes of hosts. Chilli leaf curl virus (ChiLCV) is a monopartite Begomovirus (family Geminiviridae) which has emerged as a serious threat to chilli production worldwide. To date, development of resistant chilli varieties through conventional plant breeding techniques remains the major antiviral strategy. To explore the potential resistance factors in Capsicum annuum var. Punjab Lal, we performed a transcriptome analysis in ChiLCV-infected plants by exploiting the advantage of sensitivity and efficiency of suppression subtractive hybridization (SSH). Out of 480 clones screened, 231 unique expressed sequence tags (ESTs) involved in different cellular and physiological processes were identified. An interactome network of ChiLCV responsive differentially expressed genes revealed an array of proteins involved in key cellular processes including transcription, replication, photosynthesis, and defense. A comparative study of gene expression between resistant and susceptible chilli plants revealed upregulation of several defense-related genes such as nucleotide-binding site leucine-rich repeat (NBS-LRR) domain containing protein, lipid transfer protein, thionin, polyphenol oxidase, and other proteins like ATP/ADP transporter in the ChiLCV-resistant variety. Taken together, the present study provides novel insights into the transcriptomics of ChiLCV-resistant chilli plants.
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Begomovirus/inmunología , Capsicum/inmunología , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , Proteínas de Plantas/biosíntesis , Estrés Fisiológico , Begomovirus/crecimiento & desarrollo , Capsicum/genética , Capsicum/virología , Resistencia a la Enfermedad , Perfilación de la Expresión Génica , Homeostasis , Proteínas de Plantas/genéticaRESUMEN
Viruses are one of the most devastating plant pathogens causing severe economic losses worldwide. RNA silencing is a robust technology to knock down the expression of specific genes. This mechanism can be exploited to generate virus resistant plants through expression of the viral derived sequences. Viruses in turn have evolved to encode suppressors of RNA silencing to combat host defense. Mixed infection of plants is of common occurrence in nature and simultaneous targeting of suppressor(s) of multiple viruses offers an effective strategy. In this study, we have in silico designed siRNAs against suppressors of the two most devastating viruses of tomato, leaf curl causing tomato begomoviruses and Cucumber mosaic virus. Three different siRNA prediction programs were used to evaluate siRNAs generating capability of each sequence and common putative candidate siRNAs were selected fulfilling the stringent parameters. Our results indicated that in the case of each suppressor a particular region of 100-150 base pairs could be source of potent siRNAs referred as hotspots. Expression of these viral hot spots as a single construct in the plants would facilitate development of transgenic plants with a high degree of broad spectrum resistance against multiple viruses.
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Macroautophagy/autophagy is a conserved intracellular degradation pathway that has recently emerged as an integral part of plant responses to virus infection. The known mechanisms of autophagy range from the selective degradation of viral components to a more general attenuation of disease symptoms. In addition, several viruses are able to manipulate the autophagy machinery and counteract autophagy-dependent resistance. Despite these findings, the complex interplay of autophagy activities, viral pathogenicity factors, and host defense pathways in disease development remains poorly understood. In the current study, we analyzed the interaction between autophagy and cucumber mosaic virus (CMV) in Arabidopsis thaliana. We show that autophagy is induced during CMV infection and promotes the turnover of the major virulence protein and RNA silencing suppressor 2b. Intriguingly, autophagy induction is mediated by salicylic acid (SA) and dampened by the CMV virulence factor 2b. In accordance with 2b degradation, we found that autophagy provides resistance against CMV by reducing viral RNA accumulation in an RNA silencing-dependent manner. Moreover, autophagy and RNA silencing attenuate while SA promotes CMV disease symptoms, and epistasis analysis suggests that autophagy-dependent disease and resistance are uncoupled. We propose that autophagy counteracts CMV virulence via both 2b degradation and reduced SA-responses, thereby increasing plant fitness with the viral trade-off arising from increased RNA silencing-mediated resistance.
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Arabidopsis , Cucumovirus , Infecciones por Citomegalovirus , Arabidopsis/genética , Arabidopsis/metabolismo , Autofagia , Cucumovirus/genética , Enfermedades de las Plantas , Ácido Salicílico/metabolismo , Nicotiana/metabolismo , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismoRESUMEN
Autophagy is a conserved self-cleaning and renewal system required for cellular homeostasis and stress tolerance. Autophagic processes are also implicated in the response to 'non-self' such as viral pathogens, yet the functions and mechanisms of autophagy during plant virus infection have only recently started to be revealed. Compelling evidence now indicates that autophagy is an integral part of antiviral immunity in plants. It can promote the hypersensitive cell death response upon incompatible viral infections or mediate the selective elimination of entire particles and individual proteins from compatible viruses in a pathway similar to xenophagy in animals. Several viruses, however, have evolved measures to antagonize xenophagic degradation or utilize autophagy to suppress disease-associated cell death and other defence pathways like RNA silencing. Here, we highlight the current advances and gaps in our understanding of the complex autophagy-virus interplay and its consequences for host immunity and viral pathogenesis in plants.
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Autofagia/fisiología , Virus/patogenicidad , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Inmunidad Innata/inmunología , Inmunidad Innata/fisiología , Virus/inmunologíaRESUMEN
Geminiviruses are single-stranded DNA viruses that can cause significant losses in economically important crops. In recent years, the role of different kinases in geminivirus pathogenesis has been emphasized. Although geminiviruses use several host kinases, the role of phosphatidylinositol 4-kinase (PI4K) remains obscure. We isolated and characterized phosphatidylinositol 4-kinase type II from Nicotiana benthamiana (NbPI4KII) which interacts with the replication initiator protein (Rep) of a geminivirus, chilli leaf curl virus (ChiLCV). NbPI4KII-mGFP was localized into cytoplasm, nucleus or both. NbPI4KII-mGFP was also found to be associated with the cytoplasmic endomembrane systems in the presence of ChiLCV. Furthermore, we demonstrated that Rep protein directly interacts with NbPI4KII protein and influenced nuclear occurrence of NbPI4KII. The results obtained in the present study revealed that NbPI4KII is a functional protein kinase lacking lipid kinase activity. Downregulation of NbPI4KII expression negatively affects ChiLCV pathogenesis in N. benthamiana. In summary, NbPI4KII is a susceptible factor, which is required by ChiLCV for pathogenesis.