Limits...
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.

Show MeSH

Related in: MedlinePlus

p19 counteracts antiviral RNA silencing in vitro. ‘Replication inhibition assays’ were performed with (+)DI-GFP1(s) and ‘gf698’ siRNA following experimental variant 1 (Figure 3B), and p19 was added at different time points of the RISC-forming reaction. Lane 1; assay performed in the absence of p92 (no replication). Lane 2; assay performed in the absence of siRNA and p19 (negative control). Lane 3; assay performed in the presence of ‘gf698’ siRNA and in the absence of p19 (positive control of silencing). Lane 4; assay performed in the presence of ‘gf698’ siRNA where p19 was added at the beginning(1) of the RISC formation reaction. Lane 5; assay performed in the presence of ‘gf698’ siRNA where p19 was added at the end(2) of the RISC formation reaction.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3643602&req=5

gkt193-F5: p19 counteracts antiviral RNA silencing in vitro. ‘Replication inhibition assays’ were performed with (+)DI-GFP1(s) and ‘gf698’ siRNA following experimental variant 1 (Figure 3B), and p19 was added at different time points of the RISC-forming reaction. Lane 1; assay performed in the absence of p92 (no replication). Lane 2; assay performed in the absence of siRNA and p19 (negative control). Lane 3; assay performed in the presence of ‘gf698’ siRNA and in the absence of p19 (positive control of silencing). Lane 4; assay performed in the presence of ‘gf698’ siRNA where p19 was added at the beginning(1) of the RISC formation reaction. Lane 5; assay performed in the presence of ‘gf698’ siRNA where p19 was added at the end(2) of the RISC formation reaction.

Mentions: As outlined, the Tombusvirus p19 is an effective antagonist of antiviral RNA silencing. To test for the suppression of antiviral RNA silencing by p19 in our system, we added a defined amount of the protein at different time points to the assay, i.e. at the beginning and at the end of the translation reaction that generated AGO1 and RISC (Figure 3B). Early addition of p19 effectively counteracted antiviral RNA silencing, i.e. RISC-mediated cleavage of the viral RNA was less prominent, and viral replication was only marginally inhibited (Figure 5, lane 4). In contrast, late addition of the suppressor to the RISC-forming reaction had nearly no effect (Figure 5, lane 5). We, therefore, deduced that p19 suppressed RNA silencing in a similar way in the plant BYL system as this was early found in a Drosophila in vitro system, namely, by sequestration of unbound siRNA molecules (49). In the experiment shown here, we applied a significant excess of the p19 protein as compared with the amount of ‘gf698’ siRNA. Reducing the amount of p19 (10 instead of 25 U) resulted in a partial inhibition of silencing (data not shown).Figure 5.


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)

p19 counteracts antiviral RNA silencing in vitro. ‘Replication inhibition assays’ were performed with (+)DI-GFP1(s) and ‘gf698’ siRNA following experimental variant 1 (Figure 3B), and p19 was added at different time points of the RISC-forming reaction. Lane 1; assay performed in the absence of p92 (no replication). Lane 2; assay performed in the absence of siRNA and p19 (negative control). Lane 3; assay performed in the presence of ‘gf698’ siRNA and in the absence of p19 (positive control of silencing). Lane 4; assay performed in the presence of ‘gf698’ siRNA where p19 was added at the beginning(1) of the RISC formation reaction. Lane 5; assay performed in the presence of ‘gf698’ siRNA where p19 was added at the end(2) of the RISC formation reaction.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643602&req=5

gkt193-F5: p19 counteracts antiviral RNA silencing in vitro. ‘Replication inhibition assays’ were performed with (+)DI-GFP1(s) and ‘gf698’ siRNA following experimental variant 1 (Figure 3B), and p19 was added at different time points of the RISC-forming reaction. Lane 1; assay performed in the absence of p92 (no replication). Lane 2; assay performed in the absence of siRNA and p19 (negative control). Lane 3; assay performed in the presence of ‘gf698’ siRNA and in the absence of p19 (positive control of silencing). Lane 4; assay performed in the presence of ‘gf698’ siRNA where p19 was added at the beginning(1) of the RISC formation reaction. Lane 5; assay performed in the presence of ‘gf698’ siRNA where p19 was added at the end(2) of the RISC formation reaction.
Mentions: As outlined, the Tombusvirus p19 is an effective antagonist of antiviral RNA silencing. To test for the suppression of antiviral RNA silencing by p19 in our system, we added a defined amount of the protein at different time points to the assay, i.e. at the beginning and at the end of the translation reaction that generated AGO1 and RISC (Figure 3B). Early addition of p19 effectively counteracted antiviral RNA silencing, i.e. RISC-mediated cleavage of the viral RNA was less prominent, and viral replication was only marginally inhibited (Figure 5, lane 4). In contrast, late addition of the suppressor to the RISC-forming reaction had nearly no effect (Figure 5, lane 5). We, therefore, deduced that p19 suppressed RNA silencing in a similar way in the plant BYL system as this was early found in a Drosophila in vitro system, namely, by sequestration of unbound siRNA molecules (49). In the experiment shown here, we applied a significant excess of the p19 protein as compared with the amount of ‘gf698’ siRNA. Reducing the amount of p19 (10 instead of 25 U) resulted in a partial inhibition of silencing (data not shown).Figure 5.

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