The 21-Nucleotide, but Not 22-Nucleotide, Viral Secondary Small Interfering RNAs Direct Potent Antiviral Defense by Two Cooperative Argonautes inArabidopsis thaliana

Xian-Bing Wang,Qingfa Wu,Juan Jovel,Petchthai Udomporn,Herve Vaucheret,Shou-Wei Ding,Ying Wang,Wan-Xiang Li,Virginie Gasciolli

doi:10.1105/tpc.110.082305

Xian-Bing Wang, Qingfa Wu + Show 7 more

Open Access

https://doi.org/10.1105/tpc.110.082305

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Abstract

Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Here, we describe a mutant of Cucumber mosaic virus (CMV-Δ2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. We show that production of the viral secondary siRNAs targeting CMV-Δ2b requires SUPPRESSOR OF GENE SILENCING3 and DICER-LIKE4 (DCL4) in addition to RDR6. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-Δ2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-Δ2b. Possible mechanisms for the observed qualitative difference in RNA silencing between 21- and 22-nucleotide secondary siRNAs are discussed.

Highlights

RNA silencing provides protection against diverse RNA viruses in many eukaryotic organisms (Ding, 2010; Llave, 2010; Qu, 2010)
These results show that the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)/SGS3 pathway mediates the Arabidopsis resistance to cucumber mosaic virus (CMV)-D2b in an RDR1-independent manner
It should be pointed out that the accumulation of CMV-D2b was higher in dcl2-dcl4 plants than in dcl4, rdr6, and sgs3 plants (Figure 2A, see below), indicating that silencing of CMV-D2b occurs in dcl4, rdr6, and sgs3 mutants, likely through primary viral small interfering RNAs (siRNAs). These results show that production of 21-nucleotide viral siRNAs by DCL4 is sufficient to confer resistance to CMV-D2b in the absence of the 22-nucleotide viral siRNAs produced by DCL2, whereas 22-nucleotide viral siRNAs produced by DCL2 do not guide efficient silencing of CMV-D2b in the absence of the 21-nucleotide viral siRNAs produced by DCL4

Summary

Introduction

RNA silencing provides protection against diverse RNA viruses in many eukaryotic organisms (Ding, 2010; Llave, 2010; Qu, 2010). Production of viral siRNAs in plants involves multiple Dicer-like proteins (DCLs) (Blevins et al, 2006; Boucheet al., 2006; Deleris et al, 2006; Fusaro et al, 2006; Moissiard and Voinnet, 2006; Diaz-Pendon et al, 2007). 24-nucleotide siRNAs targeting positive-strand RNA viruses are difficult to detect in wild-type plants, unable to guide antiviral silencing independently, and enhance virus resistance only in certain conditions, such as a compromised DCL4 function (Boucheet al., 2006; Deleris et al, 2006; Fusaro et al, 2006; Diaz-Pendon et al, 2007)

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