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      Roles and Programming of Arabidopsis ARGONAUTE Proteins during Turnip Mosaic Virus Infection

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          Abstract

          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, 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.

          Author Summary

          RNA silencing is a primary, adaptive defense system against viruses in plants. Viruses have evolved counter-defensive mechanisms that inhibit RNA silencing through the activity of silencing suppressor proteins. Understanding how antiviral silencing is controlled, and how suppressor proteins function, is essential for understanding how plants normally resist viruses, why some viruses are highly virulent in different hosts, and how sustainable antiviral resistance strategies can be deployed in agricultural settings. We used a mutant version of Turnip mosaic virus lacking a functional silencing suppressor (HC-Pro) to understand the genetic requirements for resistance in the model plant Arabidopsis thaliana. We focused on ARGONAUTE proteins, which have long been hypothesized to bind short interfering RNAs (siRNAs) derived from virus genomes for use as sequence-specific guides to recognize and target viral RNA for degradation or repression. We demonstrated specialized antiviral roles for specific ARGONAUTES and showed that several can bind viral siRNAs from across the entire viral genome. However, ARGONAUTE proteins are only loaded with virus-derived siRNAs in the absence of HC-Pro, which we showed binds siRNAs from the viral genome. This indicates that several AGO proteins, which collectively are necessary for full anti-TuMV defense, need to properly load virus-derived siRNAs to execute their antiviral roles.

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          Most cited references53

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          Genome-wide insertional mutagenesis of Arabidopsis thaliana.

          J M Alonso (2003)
          Over 225,000 independent Agrobacterium transferred DNA (T-DNA) insertion events in the genome of the reference plant Arabidopsis thaliana have been created that represent near saturation of the gene space. The precise locations were determined for more than 88,000 T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the approximately 29,454 predicted Arabidopsis genes. Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site bias at both the chromosome and gene levels. Insertion mutations were identified in genes that are regulated in response to the plant hormone ethylene.
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            Widespread translational inhibition by plant miRNAs and siRNAs.

            High complementarity between plant microRNAs (miRNAs) and their messenger RNA targets is thought to cause silencing, prevalently by endonucleolytic cleavage. We have isolated Arabidopsis mutants defective in miRNA action. Their analysis provides evidence that plant miRNA-guided silencing has a widespread translational inhibitory component that is genetically separable from endonucleolytic cleavage. We further show that the same is true of silencing mediated by small interfering RNA (siRNA) populations. Translational repression is effected in part by the ARGONAUTE proteins AGO1 and AGO10. It also requires the activity of the microtubule-severing enzyme katanin, implicating cytoskeleton dynamics in miRNA action, as recently suggested from animal studies. Also as in animals, the decapping component VARICOSE (VCS)/Ge-1 is required for translational repression by miRNAs, which suggests that the underlying mechanisms in the two kingdoms are related.
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              Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs.

              ARGONAUTE (AGO) RNA-binding proteins are involved in RNA silencing. They bind to short interfering RNAs (siRNAs) and microRNAs (miRNAs) through a conserved PAZ domain, and, in animals, they assemble into a multisubunit RNA-induced silencing complex (RISC). The mammalian AGO2, termed Slicer, directs siRNA- and miRNA-mediated cleavage of a target RNA. In Arabidopsis, there are 10 members of the AGO family, and the AGO1 protein is potentially the Slicer component in different RNA-silencing pathways. Here, we show that AGO1 selectively recruits certain classes of short silencing-related RNA. AGO1 is physically associated with miRNAs, transacting siRNAs, and transgene-derived siRNAs but excludes virus-derived siRNAs and 24-nt siRNAs involved in chromatin silencing. We also show that AGO1 has Slicer activity. It mediates the in vitro cleavage of a mir165 target RNA in a manner that depends on the sequence identity of amino acid residues in the PIWI domain that are predicted by homology with animal Slicer-competent AGO proteins to constitute the RNase catalytic center. However, unlike animals, we find no evidence that AGO1 Slicer is in a high molecular weight RNA-induced silencing complex. The Slicer activity fractionates as a complex of approximately 150 kDa that likely constitutes the AGO1 protein and associated RNA without any other proteins. Based on sequence similarity, we predict that other Arabidopsis AGOs might have a similar catalytic activity but recruit different subsets of siRNAs or miRNAs.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                25 March 2015
                March 2015
                : 11
                : 3
                : e1004755
                Affiliations
                [1 ]Donald Danforth Plant Science Center, St. Louis, Missouri, United States of America
                [2 ]Center for Genome Research and Biocomputing, Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, United States of America
                [3 ]Computational and Systems Biology Program, Washington University in St. Louis, St. Louis, Missouri, United States of America
                The Ohio State University, UNITED STATES
                Author notes

                The authors have declared that no competing interests exits.

                Conceived and designed the experiments: HGR JCC. Performed the experiments: HGR JSH AT AG MTGR MGM NL MTMB. Analyzed the data: HGR NF AC JSH JCC. Contributed reagents/materials/analysis tools: HGR AT AC NF JSH KBG JCC. Wrote the paper: HGR AC NF JSH JCC.

                [¤a]

                Current address: Department of Plant Pathology and Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America

                [¤b]

                Current address: Department of Biotechnology, Graduate School of Life Sciences, Ritsumeikan University, Noji-Higashi, Kusatsu, Shiga, Japan

                [¤c]

                Current address: Instituto per la Protezione Sostenible de la Piante del CNR, Bari, Italy

                [¤d]

                Current address: Recursos Genéticos y Productividad, Fisiología Vegetal, Colegio de Postgraduados, Montecillo, México

                [¤e]

                Current address: Molecular and Cellular Biology Program, School of Biological Sciences, Illinois State University, Normal, Illinois, United States of America

                [¤f]

                Current address: Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, United Kingdom

                Article
                PPATHOGENS-D-14-02961
                10.1371/journal.ppat.1004755
                4373807
                25806948
                d1b02280-df55-46cf-a8c4-29605cdff808
                Copyright @ 2015

                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

                History
                : 9 December 2014
                : 19 February 2015
                Page count
                Figures: 7, Tables: 3, Pages: 27
                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 (MOW-2012-01361) to NF. NSF ( www.nsf.gov) Graduate Research Fellowship (DGE-1143954) to JSH Japan Society for the Promotion of Science ( www.jsps.go.jp) Postdoctoral Fellowship to AT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Custom metadata
                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.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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