Research Article: S‐acylation mediates Mungbean yellow mosaic virus AC4 localization to the plasma membrane and in turns gene silencing suppression

Date Published: August 1, 2018

Publisher: Public Library of Science

Author(s): Anna Vittoria Carluccio, Maria Isabella Prigigallo, Tabata Rosas-Diaz, Rosa Lozano-Duran, Livia Stavolone, Aiming Wang.

http://doi.org/10.1371/journal.ppat.1007207

Abstract

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.

Partial Text

Viruses are obligate intracellular parasites that exploit host machineries to propagate and spread in the host. Their presence and activity deploy diverse plant mechanisms to combat viral infections at both the cellular and the whole-organism levels. Double-stranded (ds)RNA forming during virus replication and self-complementary foldback RNAs from single-stranded viral RNAs or aberrant RNAs can trigger host defence responses via a mechanism of RNA interference (RNAi) that results in inhibition of target RNA expression [1, 2]. The RNase III-type DICER enzymes process these viral RNAs into small-interfering (si)RNAs (21–24 nucleotides) that accumulate in the infected cells and guide the RNA-induced silencing complex (RISC) to degradation of complementary viral RNA sequences [3, 4]. RNA silencing is a non-cell autonomous process thus, silencing signals spread from the site of induction to neighbouring cells and systemically to confer silencing of homologous targets in distant tissues of the host plant [5, 6]. However, the evidence that virus infection often induces symptom and damage in the host highlights the presence of a counter defence strategy that suppresses the host surveillance [7]. Viruses encode one or more proteins that can inhibit initiation (viral RNA recognition and the subsequent degradation), maintenance, or systemic spreading of silencing thus allowing efficient viral replication in single cells and spread of the infection. These virulence factors, called viral suppressors of RNAi (VSRs), share no obvious sequence homology with each other and follow distinct mechanisms of suppression by targeting different points of the RNA silencing pathway, such as viral RNA recognition, dicing, RISC assembly, RNA targeting, and amplification [2, 8]. To overcome the host silencing machinery, several virus species have developed a siRNA sequestration strategy that the different VSR apply in various manners by preventing the assembly of the RISC effector [8]. As siRNA duplexes act as mobile silencing signals moving ahead of the virus to activate antiviral silencing in not yet infected cells, by sequestering and inactivating siRNA VSRs can counter react this defense strategy and allow spreading of the viral infection in the plant [9].

AC4 is among the least conserved proteins of all geminiviruses and appears to have divergent biological functions among species of the family, being mostly involved in virus-plant interactions and in pathogenesis [48–51]. It was shown to play a role in the regulation of cell division [52] whereas, in other species, mutagenesis and/or transgenic expression of AC4 has no consequence on infection of several host plant [53].

 

Source:

http://doi.org/10.1371/journal.ppat.1007207