Correction: The replication initiator protein of a geminivirus interacts with host monoubiquitination machinery and stimulates transcription of the viral genome.

[This corrects the article DOI: 10.1371/journal.ppat.1006587.].

Salicylic acid and the viral virulence factor 2b regulate the divergent roles of autophagy during cucumber mosaic virus infection.

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.

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

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.

Nicotiana benthamiana phosphatidylinositol 4-kinase type II regulates chilli leaf curl virus pathogenesis.

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.

Autophagy-virus interplay in plants: from antiviral recognition to proviral manipulation.

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.

Correction: The replication initiator protein of a geminivirus interacts with host monoubiquitination machinery and stimulates transcription of the viral genome.

[This corrects the article DOI: 10.1371/journal.ppat.1006587.].

The replication initiator protein of a geminivirus interacts with host monoubiquitination machinery and stimulates transcription of the viral genome.

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.

Chilli leaf curl virus-based vector for phloem-specific silencing of endogenous genes and overexpression of foreign genes.

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.

A geminivirus betasatellite damages the structural and functional integrity of chloroplasts leading to symptom formation and inhibition of photosynthesis.

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.