Rice yellow stunt rhabdovirus Protein 6 Suppresses Systemic RNA Silencing by Blocking RDR6-Mediated Secondary siRNA Synthesis

Abstract

The P6 protein of Rice yellow stunt rhabdovirus (RYSV) is a virion structural protein that can be phosphorylated in vitro. However its exact function remains elusive. We found that P6 enhanced the virulence of Potato virus X (PVX) in Nicotiana benthamiana and N. tabacum plants, suggesting that it might function as a suppressor of RNA silencing. We examined the mechanism of P6-mediated silencing suppression by transiently expressing P6 in both N. benthamiana leaves and rice protoplasts. Our results showed that P6 could repress the production of secondary siRNAs and inhibit systemic green fluorescent protein RNA silencing but did not interfere with local RNA silencing in N. benthamiana plants or in rice protoplasts. Intriguingly, P6 and RDR6 had overlapping subcellular localization and P6 bound both rice and Arabidopsis RDR6 in vivo. Furthermore, transgenic rice plants expressing P6 showed enhanced susceptibility to infection by Rice stripe virus. Hence, we propose that P6 is part of the RYSV's counter-defense machinery against the plant RNA silencing system and plays a role mainly in affecting RDR6-mediated secondary siRNA synthesis. Our work provides a new perspective on how a plant-infecting nucleorhabdovirus may counteract host RNA silencing-mediated antiviral defense.

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor

“…Inhibition of dsRNA synthesis and subsequent secondary siRNA synthesis can be another candidate for the general strategy employed by VSRs, because some VSRs have been supposed to be involved in that pathway. p2 of rice stripe tenuivirus and p6 of rice yellow stunt rhabdovirus interacted with SGS3 and RDR6, respectively (Du et al, 2011;Guo et al, 2013). V2 encoded by tomato yellow leaf curl geminivirus also interacted with and colocalized with SGS3 (Glick et al, 2008).…”

Alfalfa dwarf cytorhabdovirus P protein is a local and systemic RNA silencing supressor which inhibits programmed RISC activity and prevents transitive amplification of RNA silencing

“…The ability of ADV P protein to likely prevent transitive amplification of RNA silencing by suppressing the production of secondary siRNAs suggests its interference with components of amplification of RNA silencing, which involves the concerted activity of RDR6 and SGS3 (Glick et al, 2008;Guo et al, 2013). Therefore, we tested whether ADV P would co-localize and/or interact with RDR6 and SGS3 when transiently co-expressed in N. benthamiana leaves.…”

Tospovirus : induction and suppression of RNA silencing

“…Nucleorhabdovirus P6 (RYSV) is an RSS that inhibits systemic (but not local) silencing and this is likely achieved by interaction with RDR6 and blocking of RDR6-mediated secondary siRNA synthesis (Guo et al, 2013). (Zrachya et al, 2007;Glick et al, 2008;Fukunaga and Doudna, 2009) ssDNA Geminiviridae, Begomovirus TYLCCNV C1…”

“… interfere with production of secondary siRNAs, likely through interaction with RDR6 (Guo et al, 2013)  suppress systemic silencing, but NOT local silencing (Guo et al, 2013)  it does not bind 21-nt ds(siRNA) (Guo et al, 2013) (+)ssRNA Bromoviridae, Cucumovirus CMV 2b…”

“…In respect to this, it is interesting to highlight that suppression of systemic silencing (and not local silencing) is a strategy that has been analysed and identified with RSS proteins from other plant viruses as well. One example is P6 from rice yellow stunt virus (RYSV) (family Rhabdoviridae, genus Nucleorhabdovirus) which instead of sequestration of siRNAs is believed to target the RDR6 protein and through this the amplification step of RNA silencing (Guo et al, 2013). The amplification step of RNA silencing has been indicated to be essential for the systemic silencing, while the (secondary) siRNAs suggested to be involved in the mobile silencing signal (Molnar et al, 2011b).…”