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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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Related in: MedlinePlus

RISC-mediated RNA silencing targets viral (+)RNA. (A) Schematic representations of the DI RNA variants DI-GFP1 and DI-GFP2 that contained the target sequence of the ‘gf698’ siRNA at different positions. The target sequence, which is indicated by a black line, corresponds to a short sequence of GFP mRNA and is complementary to the ‘gf698’ siRNA guide strand. The dashed line in DI-GFP2 represents a sequence that is complementary to the target sequence. (B) With both constructs shown in (A), the ‘gf698’ target sequence was introduced in either sense (s) or antisense (as) orientation. Accordingly, as it is shown in the schematic representation of DI RNA replication, RISC programmed with the ‘gf698’ siRNA was supposed to target either (+) or (−)RNA molecules. (C) ‘RISC formation/cleavage assay’ with DI-GFP RNAs. The assay was performed as described in Figure 1B using ‘gf698’ siRNA and 32P-labeled RNA transcripts of the respective (+) and (−)DI-GFP RNAs. The target RNA (indicated) and the cleavage products (asterisks) were analyzed by denaturing PAGE and autoradiography. Lanes 1, 3, 5 and 7; assays performed in the absence of siRNA (negative controls). Lanes 2, 4, 6 and 8; assays performed in the presence of ‘gf698’ siRNA. (D) ‘Replication inhibition assay’ with DI-GFP RNAs. The assays were performed as described in Figure 3B (variant 1), i.e. RISC programmed with ‘gf698’ siRNA was added to a translation/replication reaction performed with the (+)RNA of the different DI-GFP variants. RP and cleavage products (asterisks) are indicated. Lanes 1, 4, 7 and 10; assays in the absence of p92 (no replication). Lanes 2, 5, 8 and 11; assays in the absence of siRNA (negative controls). Lanes 3, 6, 9 and 12; assays in the presence of ‘gf698’ siRNA. (E) SiRNA-programmed RISC also interferes with ongoing viral replication. ‘Replication inhibition assays’ were performed with (+)DI-GFP1(s) and ‘gf698’ siRNA as depicted in Figure 3B following experimental variants 1 or 2. That is, the RISC- and replicase-forming reactions were combined either before the initiation of RNA replication (0 h) or 1 h (1 h) after starting the replication reaction. The RP and cleavage products (asterisks) are indicated. Lane 1; assay performed in the absence of p92 (no replication). Lanes 2 and 3; assays where the reaction mixtures were combined before the initiation of replication in the absence (lane 2) and presence (lane 3) of ‘gf698’ siRNA. Lanes 4 and 5; assays where the reaction mixtures were combined after 1 h of viral replication in the absence (lane 4) and presence (lane 5) of ‘gf698’ siRNA.
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gkt193-F4: RISC-mediated RNA silencing targets viral (+)RNA. (A) Schematic representations of the DI RNA variants DI-GFP1 and DI-GFP2 that contained the target sequence of the ‘gf698’ siRNA at different positions. The target sequence, which is indicated by a black line, corresponds to a short sequence of GFP mRNA and is complementary to the ‘gf698’ siRNA guide strand. The dashed line in DI-GFP2 represents a sequence that is complementary to the target sequence. (B) With both constructs shown in (A), the ‘gf698’ target sequence was introduced in either sense (s) or antisense (as) orientation. Accordingly, as it is shown in the schematic representation of DI RNA replication, RISC programmed with the ‘gf698’ siRNA was supposed to target either (+) or (−)RNA molecules. (C) ‘RISC formation/cleavage assay’ with DI-GFP RNAs. The assay was performed as described in Figure 1B using ‘gf698’ siRNA and 32P-labeled RNA transcripts of the respective (+) and (−)DI-GFP RNAs. The target RNA (indicated) and the cleavage products (asterisks) were analyzed by denaturing PAGE and autoradiography. Lanes 1, 3, 5 and 7; assays performed in the absence of siRNA (negative controls). Lanes 2, 4, 6 and 8; assays performed in the presence of ‘gf698’ siRNA. (D) ‘Replication inhibition assay’ with DI-GFP RNAs. The assays were performed as described in Figure 3B (variant 1), i.e. RISC programmed with ‘gf698’ siRNA was added to a translation/replication reaction performed with the (+)RNA of the different DI-GFP variants. RP and cleavage products (asterisks) are indicated. Lanes 1, 4, 7 and 10; assays in the absence of p92 (no replication). Lanes 2, 5, 8 and 11; assays in the absence of siRNA (negative controls). Lanes 3, 6, 9 and 12; assays in the presence of ‘gf698’ siRNA. (E) SiRNA-programmed RISC also interferes with ongoing viral replication. ‘Replication inhibition assays’ were performed with (+)DI-GFP1(s) and ‘gf698’ siRNA as depicted in Figure 3B following experimental variants 1 or 2. That is, the RISC- and replicase-forming reactions were combined either before the initiation of RNA replication (0 h) or 1 h (1 h) after starting the replication reaction. The RP and cleavage products (asterisks) are indicated. Lane 1; assay performed in the absence of p92 (no replication). Lanes 2 and 3; assays where the reaction mixtures were combined before the initiation of replication in the absence (lane 2) and presence (lane 3) of ‘gf698’ siRNA. Lanes 4 and 5; assays where the reaction mixtures were combined after 1 h of viral replication in the absence (lane 4) and presence (lane 5) of ‘gf698’ siRNA.

Mentions: Antiviral RNA silencing with a virus-derived siRNA pool. (A) ‘RISC formation/cleavage assay’ with DI-R3.5 RNA. A pool of siRNAs was generated by RNase III (ShortCut®) cleavage of dsR3.5 RNA. Using BYL where AGO1 was overexpressed by in vitro translation, RISC was formed with this siRNA pool. 32P-labeled (+) or (−)DI-R3.5 RNA transcripts were added to the extract that contained the programmed RISC, and the reaction products were subsequently analyzed by denaturing PAGE and autoradiography (lanes 2 and 4). The analogous experiments performed with a non-related (‘gf698’) siRNA served as negative controls (lanes 1 and 3). The positions of the labeled target RNAs are indicated; most prominent cleavage products are indicated by asterisks. (B) Schematic representation of the in vitro ‘replication inhibition assay’. A BYL reaction mixture that contained in vitro translated AGO1 and RISC that was ‘programmed’ with the siRNA(s) of choice was added to a second BYL reaction mixture that contained the in vitro translated TBSV replicase proteins p33 and p92. In experimental ‘variant 1’, RNA replication was initiated by combining both reactions and by the subsequent addition of replication mix and DI-R3.5 RNA template. In experimental ‘variant 2’, the mixture that contained the programmed RISC was added at a later time point to the replication reaction (Figure 4E). (C) ‘replication inhibition assay’. The reaction described in (B) was performed with (+)DI-R3.5 RNA. Lane 1; in the absence of p92 (no replication). Lane 2; with an unrelated (‘gf698’) siRNA (negative control). Lane 3; with the dsR3.5-generated vsiRNA pool. The RNA replication products (RP) are indicated, as well as the most prominent RNA cleavage products (asterisks).


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-mediated RNA silencing targets viral (+)RNA. (A) Schematic representations of the DI RNA variants DI-GFP1 and DI-GFP2 that contained the target sequence of the ‘gf698’ siRNA at different positions. The target sequence, which is indicated by a black line, corresponds to a short sequence of GFP mRNA and is complementary to the ‘gf698’ siRNA guide strand. The dashed line in DI-GFP2 represents a sequence that is complementary to the target sequence. (B) With both constructs shown in (A), the ‘gf698’ target sequence was introduced in either sense (s) or antisense (as) orientation. Accordingly, as it is shown in the schematic representation of DI RNA replication, RISC programmed with the ‘gf698’ siRNA was supposed to target either (+) or (−)RNA molecules. (C) ‘RISC formation/cleavage assay’ with DI-GFP RNAs. The assay was performed as described in Figure 1B using ‘gf698’ siRNA and 32P-labeled RNA transcripts of the respective (+) and (−)DI-GFP RNAs. The target RNA (indicated) and the cleavage products (asterisks) were analyzed by denaturing PAGE and autoradiography. Lanes 1, 3, 5 and 7; assays performed in the absence of siRNA (negative controls). Lanes 2, 4, 6 and 8; assays performed in the presence of ‘gf698’ siRNA. (D) ‘Replication inhibition assay’ with DI-GFP RNAs. The assays were performed as described in Figure 3B (variant 1), i.e. RISC programmed with ‘gf698’ siRNA was added to a translation/replication reaction performed with the (+)RNA of the different DI-GFP variants. RP and cleavage products (asterisks) are indicated. Lanes 1, 4, 7 and 10; assays in the absence of p92 (no replication). Lanes 2, 5, 8 and 11; assays in the absence of siRNA (negative controls). Lanes 3, 6, 9 and 12; assays in the presence of ‘gf698’ siRNA. (E) SiRNA-programmed RISC also interferes with ongoing viral replication. ‘Replication inhibition assays’ were performed with (+)DI-GFP1(s) and ‘gf698’ siRNA as depicted in Figure 3B following experimental variants 1 or 2. That is, the RISC- and replicase-forming reactions were combined either before the initiation of RNA replication (0 h) or 1 h (1 h) after starting the replication reaction. The RP and cleavage products (asterisks) are indicated. Lane 1; assay performed in the absence of p92 (no replication). Lanes 2 and 3; assays where the reaction mixtures were combined before the initiation of replication in the absence (lane 2) and presence (lane 3) of ‘gf698’ siRNA. Lanes 4 and 5; assays where the reaction mixtures were combined after 1 h of viral replication in the absence (lane 4) and presence (lane 5) of ‘gf698’ siRNA.
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Related In: Results  -  Collection

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gkt193-F4: RISC-mediated RNA silencing targets viral (+)RNA. (A) Schematic representations of the DI RNA variants DI-GFP1 and DI-GFP2 that contained the target sequence of the ‘gf698’ siRNA at different positions. The target sequence, which is indicated by a black line, corresponds to a short sequence of GFP mRNA and is complementary to the ‘gf698’ siRNA guide strand. The dashed line in DI-GFP2 represents a sequence that is complementary to the target sequence. (B) With both constructs shown in (A), the ‘gf698’ target sequence was introduced in either sense (s) or antisense (as) orientation. Accordingly, as it is shown in the schematic representation of DI RNA replication, RISC programmed with the ‘gf698’ siRNA was supposed to target either (+) or (−)RNA molecules. (C) ‘RISC formation/cleavage assay’ with DI-GFP RNAs. The assay was performed as described in Figure 1B using ‘gf698’ siRNA and 32P-labeled RNA transcripts of the respective (+) and (−)DI-GFP RNAs. The target RNA (indicated) and the cleavage products (asterisks) were analyzed by denaturing PAGE and autoradiography. Lanes 1, 3, 5 and 7; assays performed in the absence of siRNA (negative controls). Lanes 2, 4, 6 and 8; assays performed in the presence of ‘gf698’ siRNA. (D) ‘Replication inhibition assay’ with DI-GFP RNAs. The assays were performed as described in Figure 3B (variant 1), i.e. RISC programmed with ‘gf698’ siRNA was added to a translation/replication reaction performed with the (+)RNA of the different DI-GFP variants. RP and cleavage products (asterisks) are indicated. Lanes 1, 4, 7 and 10; assays in the absence of p92 (no replication). Lanes 2, 5, 8 and 11; assays in the absence of siRNA (negative controls). Lanes 3, 6, 9 and 12; assays in the presence of ‘gf698’ siRNA. (E) SiRNA-programmed RISC also interferes with ongoing viral replication. ‘Replication inhibition assays’ were performed with (+)DI-GFP1(s) and ‘gf698’ siRNA as depicted in Figure 3B following experimental variants 1 or 2. That is, the RISC- and replicase-forming reactions were combined either before the initiation of RNA replication (0 h) or 1 h (1 h) after starting the replication reaction. The RP and cleavage products (asterisks) are indicated. Lane 1; assay performed in the absence of p92 (no replication). Lanes 2 and 3; assays where the reaction mixtures were combined before the initiation of replication in the absence (lane 2) and presence (lane 3) of ‘gf698’ siRNA. Lanes 4 and 5; assays where the reaction mixtures were combined after 1 h of viral replication in the absence (lane 4) and presence (lane 5) of ‘gf698’ siRNA.
Mentions: Antiviral RNA silencing with a virus-derived siRNA pool. (A) ‘RISC formation/cleavage assay’ with DI-R3.5 RNA. A pool of siRNAs was generated by RNase III (ShortCut®) cleavage of dsR3.5 RNA. Using BYL where AGO1 was overexpressed by in vitro translation, RISC was formed with this siRNA pool. 32P-labeled (+) or (−)DI-R3.5 RNA transcripts were added to the extract that contained the programmed RISC, and the reaction products were subsequently analyzed by denaturing PAGE and autoradiography (lanes 2 and 4). The analogous experiments performed with a non-related (‘gf698’) siRNA served as negative controls (lanes 1 and 3). The positions of the labeled target RNAs are indicated; most prominent cleavage products are indicated by asterisks. (B) Schematic representation of the in vitro ‘replication inhibition assay’. A BYL reaction mixture that contained in vitro translated AGO1 and RISC that was ‘programmed’ with the siRNA(s) of choice was added to a second BYL reaction mixture that contained the in vitro translated TBSV replicase proteins p33 and p92. In experimental ‘variant 1’, RNA replication was initiated by combining both reactions and by the subsequent addition of replication mix and DI-R3.5 RNA template. In experimental ‘variant 2’, the mixture that contained the programmed RISC was added at a later time point to the replication reaction (Figure 4E). (C) ‘replication inhibition assay’. The reaction described in (B) was performed with (+)DI-R3.5 RNA. Lane 1; in the absence of p92 (no replication). Lane 2; with an unrelated (‘gf698’) siRNA (negative control). Lane 3; with the dsR3.5-generated vsiRNA pool. The RNA replication products (RP) are indicated, as well as the most prominent RNA cleavage products (asterisks).

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