Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants.

Levente Kontra,Tibor Csorba,Alessandra Lucioli,József Burgyán,Raffaela Tavazza,Simon Moxon,Viktória Tisza,Massimo Turina,Mario Tavazza,Anna Medzihradszky

doi:10.1371/journal.ppat.1005935

Abstract

RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination.

Highlights

Viruses are among the most important plant pathogens that cause huge economic losses in many agriculturally important crops worldwide
We have shown that p19 can efficiently sequester endogenous small RNAs in mock-inoculated p19syn plants while it does not bind these sRNAs upon Cym19stop infection
We demonstrated that p19 preferentially sequesters positive:negative viral short interfering RNAs pairs and that the binding by p19 is independent of vsiRNA sequence or the type of the 5’-end nucleotide

Summary

Introduction

Viruses are among the most important plant pathogens that cause huge economic losses in many agriculturally important crops worldwide. RNA silencing is one of the most important mechanisms that serve to fight against viruses [1,2]. RNA silencing is a conserved eukaryotic pathway involved in almost all cellular processes like development, stress responses and antiviral defense. The siRNAs and the miRNAs (collectively named small RNAs, sRNAs) are processed by RNase III-type ribonucleases, the DICER (in plants Dicer-Like, DCL) enzymes [4,5] in collaboration with their partner DOUBLE-STRANDED RNA BINDING (DRB) proteins [6,7,8,9]. SRNAs associate with ARGONAUTE (AGO) proteins [12,13,14] the central effectors of RNA-induced silencing complex (RISC) [15,16]. Amplification of silencing occurs through double-stranded RNA synthesis by RNA-dependent RNA polymerases (RDRs) and secondary siRNA production [20,21,22]. sRNAs are non-cell autonomous, they can move within the plant to transmit gene silencing from cell-to-cell or systemically on long distance as mobile silencing signals [23,24,25]

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