Functional Analysis of V2 Protein of Beet Curly Top Iran Virus.

Geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top disease in Iran and the newly established Becurtovirus genus type species. Although the biological features of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, and no information is available on the function of their viral genes. In this work, we demonstrate that BCTIV V2, as the curtoviral V2, is also a local silencing suppressor in Nicotiana benthamiana and can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector also causes the induction of necrotic lesions in N. benthamiana, such as the ones produced during the HR, both at the local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant showed that V2 is required for systemic infection, but not for viral replication, in a local infection. Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 and plays a crucial role in viral pathogenicity and systemic movement.

Characterization of Curtovirus V2 Protein, a Functional Homolog of Begomovirus V2.

Geminiviruses are single-stranded DNA plant viruses with circular genomes packaged within geminate particles. Among the Geminiviridae family, Begomovirus and Curtovirus comprise the two best characterized genera. Curtovirus and Old World begomovirus possess similar genome structures with six to seven open-reading frames (ORF). Among them, begomovirus and curtovirus V2 ORFs share the same location in the viral genome, encode proteins of similar size, but show extremely poor sequence homology between the genera. V2 from Beet curly top virus (BCTV), the model species for the Curtovirus genus, as it begomoviral counterpart, suppresses post-transcriptional gene silencing (PTGS) by impairing the RDR6/SGS3 pathway and localizes in the nucleus spanning from the perinuclear region to the cell periphery. By aminoacid sequence comparison we have identified that curtoviral and begomoviral V2 proteins shared two hydrophobic domains and a putative phosphorylation motif. These three domains are essential for BCTV V2 silencing suppression activity, for the proper nuclear localization of the protein and for systemic infection. The lack of suppression activity in the mutated versions of V2 is complemented by the impaired function of RDR6 in Nicotiana benthamiana but the ability of the viral mutants to produce a systemic infection is not recovered in gene silencing mutant backgrounds. We have also demonstrated that, as its begomoviral homolog, V2 from BCTV is able to induce systemic symptoms and necrosis associated with a hypersensitive response-like (HR-like) when expressed from Potato virus X vector in N. benthamiana, and that this pathogenicity activity does not dependent of its ability to supress PTGS.

Integrated single-base resolution maps of transcriptome, sRNAome and methylome of Tomato yellow leaf curl virus (TYLCV) in tomato.

Geminiviruses are plant ssDNA viruses that replicate through dsDNA intermediates and form minichromosomes which carry the same epigenetic marks as the host chromatin. During the infection, geminiviruses are targets of the post-transcriptional and transcriptional gene silencing machinery. To obtain insights into the connection between virus-derived small RNAs (vsRNAs), viral genome methylation and gene expression, we obtained the transcriptome, sRNAome and methylome from the geminivirus Tomato yellow leaf curl virus-infected tomato plants. The results showed accumulation of transcripts just at the viral ORFs, while vsRNAs spanned the entire genome, showing a prevalent accumulation at regions where the viral ORFs overlapped. The viral genome was not homogenously methylated showing two highly methylated regions located in the C1 ORF and around the intergenic region (IR). The compilation of those results showed a partial correlation between vsRNA accumulation, gene expression and DNA methylation. We could distinguish different epigenetic scenarios along the viral genome, suggesting that in addition to its function as a plant defence mechanism, DNA methylation could have a role in viral gene regulation. To our knowledge, this is the first report that shows integrative single-nucleotide maps of DNA methylation, vsRNA accumulation and gene expression from a plant virus.

Geminivirus Replication Protein Impairs SUMO Conjugation of Proliferating Cellular Nuclear Antigen at Two Acceptor Sites.

Geminiviruses are DNA viruses that replicate in nuclei of infected plant cells using the plant DNA replication machinery, including PCNA (proliferating cellular nuclear antigen), a cofactor that orchestrates genome duplication and maintenance by recruiting crucial players to replication forks. These viruses encode a multifunctional protein, Rep, which is essential for viral replication, induces the accumulation of the host replication machinery, and interacts with several host proteins, including PCNA and the SUMO E2 conjugation enzyme (SCE1). Posttranslational modification of PCNA by ubiquitin or SUMO plays an essential role in the switching of PCNA between interacting partners during DNA metabolism processes (e.g., replication, recombination, and repair, etc.). In yeast, PCNA sumoylation has been associated with DNA repair involving homologous recombination (HR). Previously, we reported that ectopic Rep expression results in very specific changes in the sumoylation pattern of plant cells. In this work, we show, using a reconstituted sumoylation system in Escherichia coli, that tomato PCNA is sumoylated at two residues, K254 and K164, and that coexpression of the geminivirus protein Rep suppresses sumoylation at these lysines. Finally, we confirm that PCNA is sumoylated in planta and that Rep also interferes with PCNA sumoylation in plant cells.IMPORTANCE SUMO adducts have a key role in regulating the activity of animal and yeast PCNA on DNA repair and replication. Our work demonstrates for the first time that sumoylation of plant PCNA occurs in plant cells and that a plant virus interferes with this modification. This work marks the importance of sumoylation in allowing viral infection and replication in plants. Moreover, it constitutes a prime example of how viral proteins interfere with posttranslational modifications of selected host factors to create a proper environment for infection.

V2 from a curtovirus is a suppressor of post-transcriptional gene silencing.

The suppression of gene silencing is a key mechanism for the success of viral infection in plants. DNA viruses from the Geminiviridae family encode several proteins that suppress transcriptional and post-transcriptional gene silencing (TGS/PTGS). In Begomovirus, the most abundant genus of this family, three out of six genome-encoded proteins, namely C2, C4 and V2, have been shown to suppress PTGS, with V2 being the strongest PTGS suppressor in transient assays. Beet curly top virus (BCTV), the model species for the Curtovirus genus, is able to infect the widest range of plants among geminiviruses. In this genus, only one protein, C2/L2, has been described as inhibiting PTGS. We show here that, despite the lack of sequence homology with its begomoviral counterpart, BCTV V2 acts as a potent PTGS suppressor, possibly by impairing the RDR6 (RNA-dependent RNA polymerase 6)/suppressor of gene silencing 3 (SGS3) pathway.

Functional analysis of gene-silencing suppressors from tomato yellow leaf curl disease viruses.

Tomato yellow leaf curl disease (TYLCD) is caused by a complex of phylogenetically related Begomovirus spp. that produce similar symptoms when they infect tomato plants but have different host ranges. In this work, we have evaluated the gene-silencing-suppression activity of C2, C4, and V2 viral proteins isolated from the four main TYLCD-causing strains in Spain in Nicotiana benthamiana. We observed varying degrees of local silencing suppression for each viral protein tested, with V2 proteins from all four viruses exhibiting the strongest suppression activity. None of the suppressors were able to avoid the spread of the systemic silencing, although most produced a delay. In order to test the silencing-suppression activity of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) proteins in a shared (tomato) and nonshared (bean) host, we established novel patch assays. Using these tools, we found that viral proteins from TYLCV were able to suppress silencing in both hosts, whereas TYLCSV proteins were only effective in tomato. This is the first time that viral suppressors from a complex of disease-causing geminiviruses have been subject to a comprehensive analysis using two economically important crop hosts, as well as the established N. benthamiana plant model.

Identification of host genes involved in geminivirus infection using a reverse genetics approach.

Geminiviruses, like all viruses, rely on the host cell machinery to establish a successful infection, but the identity and function of these required host proteins remain largely unknown. Tomato yellow leaf curl Sardinia virus (TYLCSV), a monopartite geminivirus, is one of the causal agents of the devastating Tomato yellow leaf curl disease (TYLCD). The transgenic 2IRGFP N. benthamiana plants, used in combination with Virus Induced Gene Silencing (VIGS), entail an important potential as a tool in reverse genetics studies to identify host factors involved in TYLCSV infection. Using these transgenic plants, we have made an accurate description of the evolution of TYLCSV replication in the host in both space and time. Moreover, we have determined that TYLCSV and Tobacco rattle virus (TRV) do not dramatically influence each other when co-infected in N. benthamiana, what makes the use of TRV-induced gene silencing in combination with TYLCSV for reverse genetic studies feasible. Finally, we have tested the effect of silencing candidate host genes on TYLCSV infection, identifying eighteen genes potentially involved in this process, fifteen of which had never been implicated in geminiviral infections before. Seven of the analyzed genes have a potential anti-viral effect, whereas the expression of the other eleven is required for a full infection. Interestingly, almost half of the genes altering TYLCSV infection play a role in postranslational modifications. Therefore, our results provide new insights into the molecular mechanisms underlying geminivirus infections, and at the same time reveal the 2IRGFP/VIGS system as a powerful tool for functional reverse genetics studies.

Geminiviruses subvert ubiquitination by altering CSN-mediated derubylation of SCF E3 ligase complexes and inhibit jasmonate signaling in Arabidopsis thaliana.

Viruses must create a suitable cell environment and elude defense mechanisms, which likely involves interactions with host proteins and subsequent interference with or usurpation of cellular machinery. Here, we describe a novel strategy used by plant DNA viruses (Geminiviruses) to redirect ubiquitination by interfering with the activity of the CSN (COP9 signalosome) complex. We show that geminiviral C2 protein interacts with CSN5, and its expression in transgenic plants compromises CSN activity on CUL1. Several responses regulated by the CUL1-based SCF ubiquitin E3 ligases (including responses to jasmonates, auxins, gibberellins, ethylene, and abscisic acid) are altered in these plants. Impairment of SCF function is confirmed by stabilization of yellow fluorescent protein-GAI, a substrate of the SCF(SLY1). Transcriptomic analysis of these transgenic plants highlights the response to jasmonates as the main SCF-dependent process affected by C2. Exogenous jasmonate treatment of Arabidopsis thaliana plants disrupts geminivirus infection, suggesting that the suppression of the jasmonate response might be crucial for infection. Our findings suggest that C2 affects the activity of SCFs, most likely through interference with the CSN. As SCFs are key regulators of many cellular processes, the capability of viruses to selectively interfere with or hijack the activity of these complexes might define a novel and powerful strategy in viral infections.