Tomato Yellow Leaf Curl Virus V2 Interacts with Host Histone Deacetylase 6 To Suppress Methylation-Mediated Transcriptional Gene Silencing in Plants

Abstract

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.

Alterations of Rice (Oryza sativa L.) DNA Methylation Patterns Associated with Gene Expression in Response to Rice Black Streaked Dwarf Virus

“…Tomato Ty-1 , an RNA-dependent RNA polymerase from Solanum chilense , confers resistance to tomato yellow leaf curl virus (TYLCV) by increasing cytosine methylation of viral genomes [ 11 ]. Silencing of Nicotiana benthamiana histone deacetylase 6 ( NbHDA6 ) expression results in a reduced DNA methylation level of the viral genome and enhanced host susceptibility to TYLCV infection [ 12 ]. Unlike DNA viruses, RNA viruses possess scarcely methylated RNA genomes for replication [ 10 ].…”

Superinfection by PHYVV Alters the Recovery Process in PepGMV-Infected Pepper Plants

“…In African cassava mosaic virus (ACMV), the suppressor of silencing is C4 like Tomato leaf curl New Delhi virus (ToLCNDV) [26,59], while in CLCuDV both TrAP and C4 have suppressor functions [24]. Another protein associated with suppression is V2 from Mulberry mosaic dwarf-associated virus (MMDAV), Apple geminivirus (AGV), Tomato yellow leaf curl virus (TYLCV), and Beet curly top virus (BCTV) [29,48,[60][61][62]. Therefore, despite belonging to the same viral family each virus has different suppressor silencing proteins, so this function cannot be generalized in all geminiviruses and it is necessary to analyze each individual virus.…”

“…Identified geminivirus-encoded VSRs included TrAP, L2, C4, V2, Rep, and C5. These viral proteins can disrupt both PTGS and TGS defensive pathways, interfering at different points: reduction in siRNA synthesis, sequestration of siRNA molecules, interaction with AGO proteins, inhibition of enzymes of the methyl cycle, reduction of the expression of DNA methyltransferases, and interaction with histone methyltransferase KYP [13,[23][24][25][26][27][28][29][30][31].…”

Geminiviral V2 Protein Suppresses Transcriptional Gene Silencing through Interaction with AGO4

“…To counterattack, geminiviruses encode viral suppressors of RNA silencing (VSRs) that are able to suppress TGS. However, it is unclear whether and by what mechanism VSRs could directly inhibit RdDM-mediated TGS, although several geminiviral proteins can suppress TGS by targeting methyl cycle-related enzymes, histone deacetylase, and H3K9 histone methyltransferase in plants (20,(23)(24)(25)29). In addition, the 2b protein from Cucumber mosaic virus (CMV), an RNA virus, is reported to bind to AGO4-related small RNAs and impair AGO4 slicing activity (33).…”

“…In addition, C2/TrAP from Beet severe curly top virus (BSCTV), TGMV, and Cabbage leaf curl virus (CbLCV) inhibit TGS by interacting with the H3K9 histone methyltransferase SUVH4/KYP and inhibiting its activity (27,28). TYLCV V2 suppresses TGS by interacting with host histone deacetylase HDA6 (29).…”