title: AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system.
abstract: AGO/RISC-mediated antiviral RNA silencing, an important component of the plant's immune response against RNA virus infections, was recapitulated in vitro. Cytoplasmic extracts of tobacco protoplasts were applied that supported Tombusvirus RNA replication, as well as the formation of RNA-induced silencing complexes (RISC) that could be functionally reconstituted with various plant ARGONAUTE (AGO) proteins. For example, when RISC containing AGO1, 2, 3 or 5 were programmed with exogenous siRNAs that specifically targeted the viral RNA, endonucleolytic cleavages occurred and viral replication was inhibited. Antiviral RNA silencing was disabled by the viral silencing suppressor p19 when this was present early during RISC formation. Notably, with replicating viral RNA, only (+)RNA molecules were accessible to RISC, whereas (-)RNA replication intermediates were not. The vulnerability of viral RNAs to RISC activity also depended on the RNA structure of the target sequence. This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC. These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC. The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing.
authors.affiliation: Institute of Biochemistry and Biotechnology (NFI), Martin Luther University Halle-Wittenberg, Halle/Saale D-06120, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Schuck
authors.forename: Torsten
authors.initials: T
authors.lastname: Gursinsky
authors.forename: Vitantonio
authors.initials: V
authors.lastname: Pantaleo
authors.forename: Jozsef
authors.initials: J
authors.lastname: Burgyán
authors.forename: Sven-Erik
authors.initials: SE
authors.lastname: Behrens
mesh_terms: D060565:Argonaute Proteins / Q000378:metabolism*; D001483:Base Sequence; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology / Q000378:metabolism; D017864:Tombusvirus / Q000235:genetics*; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D060565:Argonaute Proteins; C493885:P19 protein, tomato bushy stunt virus; D010940:Plant Proteins; D034741:RNA, Small Interfering; D012367:RNA, Viral; D034743:RNA-Induced Silencing Complex; D014764:Viral Proteins
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