S-acylation mediates Mungbean yellow mosaic virus AC4 localization to the plasma membrane and in turns gene silencing suppression

Plants have developed small RNA (siRNA)-mediated post-transcriptional gene silencing as a defense mechanism against viruses. In response, plant viruses encode viral suppressors of RNA silencing (VSRs) that can interfere with various steps of the silencing pathway. Mungbean yellow mosaic virus (MYMV) is a plant bipartite geminivirus responsible for a devastating disease in some areas of tropics and sub-tropics where its natural host, Vigna mungo, is a staple food crop. We discovered that the MYMV-encoded AC4 protein is a determinant of pathogenicity, binds native 21-25 nt siRNAs in vitro, and counteracts virus induced gene silencing by strongly suppressing the systemic phase of silencing but not the local production of siRNA. MYMV AC4 undergoes palmitoylation, a post-translational modification never reported before for plant viral proteins that mediates specific localization of the protein to the plasma membrane (PM). Interestingly, palmitoylation and in turns PM localization is indispensable for direct VSR function of AC4. AC4 also binds the PM-located BAM1, a trigger of cell-to-cell spread of RNAi. Taken together our results suggest that AC4 has synergic mechanisms of action, based on the specific PM localization, to prevent spreading of antiviral RNAi silencing in not yet infected cells.

RNA silencing plays a critical role in plant resistance against viruses. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that interfere with the cellular silencing machinery through various mechanisms not always well understood. We examined the role of Mungbean yellow mosaic virus (MYMV) AC4 and showed that it is essential for infectivity but not for virus replication. It acts as a determinant of pathogenicity and counteracts virus induced gene silencing by strongly suppressing the systemic phase of silencing whereas it does not interfere with local production of siRNA. We demonstrate the ability of AC4 to bind native 21-25 nt siRNAs in vitro by electrophoretic mobility shift assay. While most of the known VSRs have cytoplasmic localization, we observed that despite its hydrophilic nature and the absence of trans-membrane domain, MYMV AC4 specifically accumulates to the plasma membrane (PM). We show that AC4 binds to PM via S-palmitoylation, a process of post-translational modification regulating membrane-protein interactions, not known for plant viral protein before. When localized to the PM, AC4 strongly suppresses systemic silencing whereas its delocalization impairs VSR activity of the protein. We also show that AC4 interacts with the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1), a positive regulator of the cell-to-cell movement of RNAi. The absolute requirement of PM localization for direct silencing suppression activity of AC4 is novel and intriguing. We discuss a possible model of action: palmitoylated AC4 anchors to the PM by means of palmitate to acquire the optimal conformation to bind siRNAs, hinder their systemic movement and hence suppress the spread of the PTGS signal in the plant.