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. Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses, and geminiviruses respond by elaborating proteins that inhibit methylation and transcriptional gene silencing (TGS). Some geminiviruses have acquired a satellite called DNA β (betasatellite), which depends on the helper virus for replication and spread within and between hosts. In return, the sole betasatellite encoded protein, βC1, encodes a pathogenicity factor that enhances viral replication and is responsible for inducing disease symptoms. Geminivirus/betasatellite complexes are common and cause significant losses of food and fiber crops. Here, we explore the molecular basis of the association between Tomato yellow leaf curl China virus (TYLCCNV) and its betasatellite (TYLCCNB). We show that TYLCCNV by itself is unable to reverse TGS. However, co-inoculation of TYLCCNB, or expression of βC1 protein, results in reduced methylation of both the helper virus and host genome, and reversal of TGS directed against a transgene and an endogenous locus. We also present evidence that βC1 accomplishes this by interacting with and inhibiting the activity of S-adenosyl homocysteine hydrolase (SAHH), an enzyme needed to maintain the methyl cycle that generates the methyltransferase co-factor S-adenosyl methionine. Thus, we propose that inhibition of methylation-mediated TGS by βC1 drives geminivirus/betasatellite association.

A recently characterized calmodulin-like protein is an endogenous RNA silencing suppressor that suppresses sense-RNA induced post-transcriptional gene silencing (S-PTGS) and enhances virus infection, but the mechanism underlying calmodulin-like protein-mediated S-PTGS suppression is obscure. Here, we show that a calmodulin-like protein from Nicotiana benthamiana (NbCaM) interacts with Suppressor of Gene Silencing 3 (NbSGS3). Deletion analyses showed that domains essential for the interaction between NbSGS3 and NbCaM are also required for the subcellular localization of NbSGS3 and NbCaM suppressor activity. Overexpression of NbCaM reduced the number of NbSGS3-associated granules by degrading NbSGS3 protein accumulation in the cytoplasm. This NbCaM-mediated NbSGS3 degradation was sensitive to the autophagy inhibitors 3-methyladenine and E64d, and was compromised when key autophagy genes of the phosphatidylinositol 3-kinase (PI3K) complex were knocked down. Meanwhile, silencing of key autophagy genes within the PI3K complex inhibited geminivirus infection. Taken together these data suggest that NbCaM acts as a suppressor of RNA silencing by degrading NbSGS3 through the autophagy pathway. Post-transcriptional gene silencing (PTGS) is an elaborately regulated process for defense against virus infection in plants. To achieve effective infection, a betasatellite molecule associated with geminivirus induced high levels of an endogenous RNA silencing suppressor, calmodulin-like protein (CaM), to counter host defenses. However, although CaM is one of the first identified cellular suppressors of RNA silencing, the mechanism of PTGS suppression is still poorly understood. This study demonstrates that CaM interacts with and degrades Suppressor of Gene Silencing 3 (SGS3) in Nicotiana benthamiana. We found that domains essential for the interaction between NbSGS3 and NbCaM are also required for the subcellular localization of NbSGS3 and for NbCaM suppressor activity. Moreover, NbCaM mediated NbSGS3 protein degradation can be blocked using the autophagy inhibitors 3-methyladenine and E64d, and by knock-down of key autophagy-related genes within the phosphatidylinositol 3-kinase (PI3K) complex. Silencing of the PI3K complex also inhibited geminivirus infection, which is consistent with autophagy playing an important role in RNA silencing suppression pathway and geminivirus infection.

Cytosine DNA methylation is a conserved epigenetic silencing mechanism that defends against biotic stresses such as geminivirus infection. As a countermeasure, geminiviruses encode proteins that inhibit methylation and transcriptional gene silencing (TGS). Previous studies showed that V2 protein of Tomato yellow leaf curl virus (TYLCV) functions as a TGS suppressor. However, how V2 mediates TGS suppression remains unknown. Here we show that V2 interacts directly with a Nicotiana benthamiana histone deacetylase 6 (NbHDA6), a homolog of Arabidopsis HDA6 (AtHDA6), known to be involved in gene silencing in cooperation with METHYLTRANSFERASE 1 (MET1). NbHDA6 genetically complemented a late-flowering phenotype and restored histone deacetylation of an AtHDA6 mutant. Furthermore, our investigation showed that NbHDA6 displayed histone deacetylase enzymatic activity, which was not inhibited by V2. Genetic analysis revealed that silencing of NbHDA6 expression resulted in enhanced susceptibility to TYLCV infection. In addition, methylation-sensitive PCR and bisulfite sequencing analysis showed that silencing of NbHDA6 expression caused reduced DNA methylation of the viral genome in infected plants. HDA6 was previously shown to recruit and physically interact with MET1 to function in gene silencing. Using competitive pull down and co-immunoprecipitation assays, we demonstrated that V2 did not interact but competed with NbMET1 for direct binding to NbHDA6. These findings suggest that V2 interacts with host HDA6 and interferes with the recruitment of MET1 by HDA6 resulting in decreased methylation of the viral DNA genome by TGS with a concomitant increase in host susceptibility to TYLCV infection.

Tomato yellow leaf curl virus (TYLCV) is a DNA virus belonging to the genus Begomovirus. TYLCV replicates using double-stranded DNA intermediates that can become the target of plant transcriptional gene silencing (TGS). Here, we show that the V2 protein of TYLCV can suppress TGS of a transcriptionally silenced green fluorescent protein (GFP) transgene in Nicotiana benthamiana line 16-TGS. Through bisulfite sequencing and chop-PCR, we demonstrated that the TYLCV V2 can reverse GFP transgene silencing by reducing the methylation levels in the 35S promoter sequence. Both AtSN1 and MEA-ISR loci in Arabidopsis thaliana were previously reported to be strongly methylated, and we show that the methylation status of both loci was significantly reduced in TYLCV V2 transgenic Arabidopsis plants. We conclude that TYLCV can efficiently suppress TGS when it infects plants, and its V2 protein is responsible for the TGS suppression activity.