title: The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative argonautes in Arabidopsis thaliana.
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.
authors.affiliation: Department of Plant Pathology and Microbiology, University of California, Riverside, California 92521, USA.
authors.forename: Xian-Bing
authors.initials: XB
authors.lastname: Wang
authors.forename: Juan
authors.initials: J
authors.lastname: Jovel
authors.forename: Petchthai
authors.initials: P
authors.lastname: Udomporn
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Qingfa
authors.initials: Q
authors.lastname: Wu
authors.forename: Wan-Xiang
authors.initials: WX
authors.lastname: Li
authors.forename: Virginie
authors.initials: V
authors.lastname: Gasciolli
authors.forename: Herve
authors.initials: H
authors.lastname: Vaucheret
authors.forename: Shou-Wei
authors.initials: SW
authors.lastname: Ding
mesh_terms: D000998:Antiviral Agents / Q000276:immunology; D017360:Arabidopsis / Q000235:genetics / Q000276:immunology* / Q000821:virology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D060565:Argonaute Proteins; D017799:Cucumovirus / Q000276:immunology*; D020868:Gene Silencing; D007113:Immunity, Innate / Q000235:genetics / Q000276:immunology; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000235:genetics*; D010935:Plant Diseases / Q000276:immunology / Q000821:virology; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000378:metabolism*
chemical_list: C117457:AGO1 protein, Arabidopsis; D000998:Antiviral Agents; D029681:Arabidopsis Proteins; D060565:Argonaute Proteins; D009711:Nucleotides; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C559855:argonaute 2 protein, Arabidopsis