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AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system.

Schuck J, Gursinsky T, Pantaleo V, Burgyán J, Behrens SE - Nucleic Acids Res. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Biotechnology (NFI), Martin Luther University Halle-Wittenberg, Halle/Saale D-06120, Germany.

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.

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RISC activity in BYL. (A) Quantity of AGO1 protein in BYL. Identical volumes of naive BYL (lane 1) and BYL that contained additional AGO1 (generated by in vitro translation; lane 2) were probed by immunoblot against AGO1. (B) Schematic representation of the ‘RISC formation/cleavage assay’ (47). AGO1 mRNA was translated in BYL in the presence of exogenous siRNA(s). Subsequently, a 32P-labeled target RNA was added and analyzed for cleavage. (C) ‘RISC formation/cleavage assay’ performed with ‘gf698’ siRNA and GFP mRNA as a target. The RISC cleavage products (indicated with asterisks) were analyzed by denaturing PAGE and autoradiography (lane 3). As negative controls, the reaction was carried out in the absence of additionally expressed (in vitro translated) AGO1 (lane 1) and in the absence of siRNA (lane 2), respectively.
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gkt193-F1: RISC activity in BYL. (A) Quantity of AGO1 protein in BYL. Identical volumes of naive BYL (lane 1) and BYL that contained additional AGO1 (generated by in vitro translation; lane 2) were probed by immunoblot against AGO1. (B) Schematic representation of the ‘RISC formation/cleavage assay’ (47). AGO1 mRNA was translated in BYL in the presence of exogenous siRNA(s). Subsequently, a 32P-labeled target RNA was added and analyzed for cleavage. (C) ‘RISC formation/cleavage assay’ performed with ‘gf698’ siRNA and GFP mRNA as a target. The RISC cleavage products (indicated with asterisks) were analyzed by denaturing PAGE and autoradiography (lane 3). As negative controls, the reaction was carried out in the absence of additionally expressed (in vitro translated) AGO1 (lane 1) and in the absence of siRNA (lane 2), respectively.

Mentions: By applying cytoplasmic extract of evacuolated Nicotiana tabacum BY2 protoplasts (termed BYL), a system which was earlier shown to support the replication of other plant (+)RNA viruses (45), we recently established an in vitro replication assay for TBSV. In one experimental variant, p33 and p92 are first synthesized by in vitro translation of separate mRNA transcripts in the BYL, and viral RNA replication is then initiated by the addition of a DI RNA or a gRNA template (46). BYL containing additionally expressed AGO1 protein was recently observed to also reproduce RISC activity on a target mRNA in vitro (47) (Figure 1).Figure 1.


AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system.

Schuck J, Gursinsky T, Pantaleo V, Burgyán J, Behrens SE - Nucleic Acids Res. (2013)

RISC activity in BYL. (A) Quantity of AGO1 protein in BYL. Identical volumes of naive BYL (lane 1) and BYL that contained additional AGO1 (generated by in vitro translation; lane 2) were probed by immunoblot against AGO1. (B) Schematic representation of the ‘RISC formation/cleavage assay’ (47). AGO1 mRNA was translated in BYL in the presence of exogenous siRNA(s). Subsequently, a 32P-labeled target RNA was added and analyzed for cleavage. (C) ‘RISC formation/cleavage assay’ performed with ‘gf698’ siRNA and GFP mRNA as a target. The RISC cleavage products (indicated with asterisks) were analyzed by denaturing PAGE and autoradiography (lane 3). As negative controls, the reaction was carried out in the absence of additionally expressed (in vitro translated) AGO1 (lane 1) and in the absence of siRNA (lane 2), respectively.
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gkt193-F1: RISC activity in BYL. (A) Quantity of AGO1 protein in BYL. Identical volumes of naive BYL (lane 1) and BYL that contained additional AGO1 (generated by in vitro translation; lane 2) were probed by immunoblot against AGO1. (B) Schematic representation of the ‘RISC formation/cleavage assay’ (47). AGO1 mRNA was translated in BYL in the presence of exogenous siRNA(s). Subsequently, a 32P-labeled target RNA was added and analyzed for cleavage. (C) ‘RISC formation/cleavage assay’ performed with ‘gf698’ siRNA and GFP mRNA as a target. The RISC cleavage products (indicated with asterisks) were analyzed by denaturing PAGE and autoradiography (lane 3). As negative controls, the reaction was carried out in the absence of additionally expressed (in vitro translated) AGO1 (lane 1) and in the absence of siRNA (lane 2), respectively.
Mentions: By applying cytoplasmic extract of evacuolated Nicotiana tabacum BY2 protoplasts (termed BYL), a system which was earlier shown to support the replication of other plant (+)RNA viruses (45), we recently established an in vitro replication assay for TBSV. In one experimental variant, p33 and p92 are first synthesized by in vitro translation of separate mRNA transcripts in the BYL, and viral RNA replication is then initiated by the addition of a DI RNA or a gRNA template (46). BYL containing additionally expressed AGO1 protein was recently observed to also reproduce RISC activity on a target mRNA in vitro (47) (Figure 1).Figure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Biotechnology (NFI), Martin Luther University Halle-Wittenberg, Halle/Saale D-06120, Germany.

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.

Show MeSH
Related in: MedlinePlus