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 (TYLCV) functions as a TGS suppressor. However, how V2 mediates TGS suppression remains unknown. Here we show that V2 interacts directly with a histone deacetylase 6 (NbHDA6), a homolog of 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 expression resulted in enhanced susceptibility to TYLCV infection. In addition, methylation-sensitive PCR and bisulfite sequencing analysis showed that silencing of 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 pulldown and coimmunoprecipitation 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. Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses. In turn, geminiviruses encode proteins that inhibit methylation by TGS. Previous studies showed that TYLCV V2 can efficiently suppress TGS, but the mechanism remains unknown. We showed that V2 interacted with NbHDA6 but did not inhibit its enzymatic activity. As HDA6 is known to be involved in gene silencing in cooperation with MET1, we explored the relationship between V2, NbMET1, and NbHDA6. Our investigation showed that V2 did not interact but competed with NbMET1 for direct binding to NbHDA6. To our knowledge, this is the first report that viral proteins inhibit TGS by interacting with histone deacetylase but not by blocking the methyl cycle. This work provides an additional mechanism for TGS suppression by geminiviruses.

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.Cytosine DNA methylation is a conserved epigenetic silencing mechanism modulating many important biological processes (Bird, 2002;Goll & Bestor, 2005;Zhang et al., 2006) and a defence against biotic stresses such as geminivirus infection (Bisaro, 2006;Raja et al., 2010; Yang et al., 2011). Geminiviruses (family Geminiviridae) are a group of plant-infecting viruses containing circular, singlestranded DNA (ssDNA) genomes packaged into twinned particles (Dry et al., 1993;Fauquet et al., 2008;Zhang et al., 2009;Zhou, 2013). Geminiviruses are transmitted by whiteflies or leafhoppers and can cause significant damage to agronomically important crops (Navot et al., 1991;Hanley-Bowdoin et al., 2004;Rojas et al., 2005). Geminiviruses are classified into four genera (Mastrevirus, Curtovirus, Begomovirus, and Topocuvirus) and replicate in the plant cell nucleus using double-stranded DNA (dsDNA) intermediates as their replication and transcription templates (Pilartz & Jeske, 1992Fauquet et al., 2008).Recent studies have demonstrated that plants employ an RNA-directed methylation strategy to control transcriptional gene silencing (TGS) and use it as a defence mechanism against geminivirus infection (Raja et al., 2008;Rodríguez-Negrete et al., 2009). As a counter defensive measure, geminiviruses express unique proteins to serve as TGS suppressors. For example, the AC2/AL2 proteins encoded by cabbage leaf curl virus and tomato golden mosaic virus, the C2/L2 proteins encoded by beet curly top virus and beet severe curly top virus, and the bC1 protein encoded by tomato yellow leaf curl China betasatellite (TYLCCNB) can decrease DNA methylation and suppress TGS by interfering with the methyl cycle (Buchmann et al., 2009; Yang et al., 2011;Zhang et al., 2011). A recent report indicated that geminivirus replication-associated protein (Rep, also known as C1, AL1 or AC1) could also suppress TGS by reducing the expression of plant DNA methyltransferases (Rodríguez-Negrete et al., 2013).Tomato yellow leaf curl virus (TYLCV) is a member of the genus Begomovirus and contains a single genome component with six open r...