Geminiviral V2 Protein Suppresses Transcriptional Gene Silencing through Interaction with AGO4

In plants, RNA-directed DNA methylation (RdDM)-mediated transcriptional gene silencing (TGS) is a natural antiviral defense against geminiviruses. Several geminiviral proteins have been shown to target the enzymes related to the methyl cycle or histone modification; however, it remains largely unknown whether and by which mechanism geminiviruses directly inhibit RdDM-mediated TGS. In this study, we showed that Cotton leaf curl Multan virus (CLCuMuV) V2 directly interacts with Nicotiana benthamiana AGO4 (NbAGO4) and that the L76S mutation in V2 (V2^L76S) abolishes such interaction. We further showed that V2, but not V2^L76S, can suppresses RdDM and TGS. Silencing of NbAGO4 inhibits TGS, reduces the viral methylation level, and enhances CLCuMuV DNA accumulation. In contrast, the V2^L76S substitution mutant attenuates CLCuMuV infection and enhances the viral methylation level. These findings reveal that CLCuMuV V2 contributes to viral infection by interaction with NbAGO4 to suppress RdDM-mediated TGS in plants.IMPORTANCE In plants, the RNA-directed DNA methylation (RdDM) pathway is a natural antiviral defense mechanism against geminiviruses. However, how geminiviruses counter RdDM-mediated defense is largely unknown. Our findings reveal that Cotton leaf curl Multan virus V2 contributes to viral infection by interaction with NbAGO4 to suppress RNA-directed DNA methylation-mediated transcriptional gene silencing in plants. Our work provides the first evidence that a geminiviral protein is able to directly target core RdDM components to counter RdDM-mediated TGS antiviral defense in plants, which extends our current understanding of viral counters to host antiviral defense.