Roles and Programming of ArabidopsisARGONAUTE Proteins during Turnip MosaicVirus Infection

In eukaryotes, ARGONAUTE proteins (AGOs) associate with microRNAs (miRNAs), short interfering RNAs (siRNAs), and other classes of small RNAs to regulate target RNA or target loci. Viral infection in plants induces a potent and highly specific antiviral RNA silencing response characterized by the formation of virus-derived siRNAs. Arabidopsis thaliana has ten AGO genes of which AGO1, AGO2, and AGO7 have been shown to play roles in antiviral defense. A genetic analysis was used to identify and characterize the roles of AGO proteins in antiviral defense against Turnip mosaic virus (TuMV) in Arabidopsis. AGO1, AGO2 and AGO10 promoted anti-TuMV defense in a modular way in various organs, with AGO2 providing a prominent antiviral role in leaves. AGO5, AGO7 and AGO10 had minor effects in leaves. AGO1 and AGO10 had overlapping antiviral functions in inflorescence tissues after systemic movement of the virus, although the roles of AGO1 and AGO10 accounted for only a minor amount of the overall antiviral activity. By combining AGO protein immunoprecipitation with high-throughput sequencing of associated small RNAs, AGO2, AGO10, and to a lesser extent AGO1 were shown to associate with siRNAs derived from silencing suppressor (HC-Pro)-deficient TuMV-AS9, but not with siRNAs derived from wild-type TuMV. Co-immunoprecipitation and small RNA sequencing revealed that viral siRNAs broadly associated with wild-type HC-Pro during TuMV infection. These results support the hypothesis that suppression of antiviral silencing during TuMV infection, at least in part, PLOS Pathogens | DOI:10.1371/journal.ppat.1004755 March 25, 2015 1 / 27 OPEN ACCESS Citation: Garcia-Ruiz H, Carbonell A, Hoyer JS, Fahlgren N, Gilbert KB, Takeda A, et al. (2015) Roles and Programming of Arabidopsis ARGONAUTE Proteins during Turnip Mosaic Virus Infection. PLoS Pathog 11(3): e1004755. doi:10.1371/journal. ppat.1004755 Editor: Biao Ding, The Ohio State University, UNITED STATES Received: December 9, 2014 Accepted: February 19, 2015 Published: March 25, 2015 Copyright: © 2015 Garcia-Ruiz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and Supporting Information Files. Sequence data from this article can be found in the Gene Expression Omnibus (http://www.ncbi.nlm.nih. gov/geo) under accession number GSE64911. Funding: National Institutes of Health (www.nih.gov) grant AI43288 to JCC. National Science Foundation (www.nsf.gov) grant MCB-0956526 to JCC. Helen Hay Whitney (www.hhwf.org) Post-Doctoral fellowship (F-972) to HGR. USDA AFRI NIFA (www. csrees.usda.gov) Postdoctoral Fellowship (MOW2012-01361) to NF. NSF (www.nsf.gov) Graduate occurs through sequestration of virus-derived siRNAs away from antiviral AGO proteins by HC-Pro. These findings indicate that distinct AGO proteins function as antiviral modules, and provide a molecular explanation for the silencing suppressor activity of HC-Pro.