Cotton Leaf Curl Multan Virus C4 Protein Suppresses Both Transcriptional and Post-transcriptional Gene Silencing by Interacting with SAM Synthetase

Author summary Geminiviruses are single-stranded DNA (ssDNA) viruses that infect a wide range of plant species and are responsible for substantial crop damage worldwide. However, how geminiviruses inhibit plant antiviral gene silencing defense is unclear. Here, we report that a single geminiviral protein CLCuMuV C4 inhibits both plant transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) to promote an effective viral infection. We show that CLCuMuV C4 protein interacts with SAMS, a core enzyme in methyl cycle, and inhibits SAMS activity. Overexpression of CLCuMuV C4 reduces the DNA methylation levels of both a transgene and an endogenous locus. Further, silencing of SAMS reduced both TGS and PTGS, and enhanced viral infection while CLCuMuV virus carrying a mutation in C4 that fails to interact with SAMS showed decreased infection. These findings reveal a novel mechanism by which the CLCuMuV C4 protein suppress SAMS mediated TGS and PTGS, leading to enhanced viral infection in plant

Suppression of Methylation-Mediated Transcriptional Gene Silencing by βC1-SAHH Protein Interaction during Geminivirus-Betasatellite Infection

DNA methylation is a fundamental epigenetic modification that regulates gene expression and represses endogenous transposons and invading DNA viruses. As a counter-defense, the geminiviruses encode proteins that inhibit methylation and transcriptional gene silencing (TGS). Some geminiviruses have acquired a betasatellite called DNA β. This study presents evidence that suppression of methylation-mediated TGS by the sole betasatellite-encoded protein, βC1, is crucial to the association of Tomato yellow leaf curl China virus (TYLCCNV) with its betasatellite (TYLCCNB). We show that TYLCCNB complements Beet curly top virus (BCTV) L2- mutants deficient for methylation inhibition and TGS suppression, and that cytosine methylation levels in BCTV and TYLCCNV genomes, as well as the host genome, are substantially reduced by TYLCCNB or βC1 expression. We also demonstrate that while TYLCCNB or βC1 expression can reverse TGS, TYLCCNV by itself is ineffective. Thus its AC2/AL2 protein, known to have suppression activity in other geminiviruses, is likely a natural mutant in this respect. A yeast two-hybrid screen of candidate proteins, followed by bimolecular fluorescence complementation analysis, revealed that βC1 interacts with S-adenosyl homocysteine hydrolase (SAHH), a methyl cycle enzyme required for TGS. We further demonstrate that βC1 protein inhibits SAHH activity in vitro. That βC1 and other geminivirus proteins target the methyl cycle suggests that limiting its product, S-adenosyl methionine, may be a common viral strategy for methylation interference. We propose that inhibition of methylation and TGS by βC1 stabilizes geminivirus/betasatellite complexes

A DNA sequence required for geminivirus replication also mediates transcriptional regulation.

Tomato golden mosaic virus (TGMV), a member of the geminivirus family, requires a single virus-encoded protein for DNA replication. We show that the TGMV replication protein, AL1, also acts during transcription to specifically repress the activity of its promoter. An earlier study established that AL1 binds to a 13-bp sequence (5'-GGTAGTAAGGTAG) that is essential for activity of the TGMV replication origin. Analysis of AL1 binding site mutants in transient expression assays demonstrated that the same site, which is located between the transcription start site and TATA box in the AL1 promoter, also mediates transcriptional repression. These experiments revealed that the repeated motifs in the AL1 binding site contribute differentially to repression, as has been observed previously for AL1-DNA binding and viral replication. Introduction of the AL1 binding site into the 35S promoter of the cauliflower mosaic virus was sufficient to confer AL1-mediated repression to the heterologous promoter. Analysis of a truncated AL1 promoter and of mutant AL1 proteins showed that repression does not require a replication-competent template or a replication-competent AL1 protein. Transient expression studies using two different Nicotiana cell lines revealed that, although the two lines replicate plasmids containing the TGMV origin similarly, they support very different levels of AL1-mediated repression. These results suggest that geminivirus transcriptional repression and replication may be independent processes