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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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Several AGO proteins support antiviral RNA silencing. (A) ‘RISC formation/cleavage assays’ performed with AGO1, AGO2 and AGO5 using different variants of ‘gf698’ siRNAs and 32P-labeled GFP mRNA as a target. The assay was performed essentially as described in Figure 1 (corresponding assays performed with AGO3, 4, 6, 7, 9 and 10 are provided as Supplementary Figure S3). That is, At AGO1, AGO2 or AGO5 were overexpressed in the BYL by in vitro translation of the corresponding mRNAs in the presence of 21 (lanes 2–5) or 22 nt (lanes 6–9) ‘gf698’ siRNA variants where the corresponding guide strands had different 5′-terminal nucleotides (as indicated). After addition of the target mRNA, the RISC cleavage products (marked by asterisks) were analyzed by denaturing PAGE and autoradiography. As a negative control, the reaction was carried out in the absence of siRNA (lane 1). (B) ‘Replication inhibition assays’ performed with (+)DI-GFP RNAs and RISC containing AGO1, AGO2 or AGO5 (corresponding assays performed with AGO3, 4, 7 and 10 are provided as Supplementary Figure S4). The assays were essentially performed as described in Figures 3B and 4D. Reactions where RISC were formed with AGO1 were performed with ‘gf698’ siRNA possessing a 5′-terminal U, reactions containing AGO2 or AGO5 were performed with ‘gf698’ siRNA variants that possessed a 5′-terminal A or C, respectively. The programmed RISC were added to a translation/replication reaction performed with (+)DI-GFP1(s) or (+)DI-GFP1(as). RP and cleavage products (asterisks) are indicated. Lanes 1 and 4; assays in the absence of p92 (no replication). Lanes 2 and 5; assays in the absence of siRNA (negative controls). Lanes 3 and 6; assays in the presence of ‘gf698’ siRNA.
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gkt193-F6: Several AGO proteins support antiviral RNA silencing. (A) ‘RISC formation/cleavage assays’ performed with AGO1, AGO2 and AGO5 using different variants of ‘gf698’ siRNAs and 32P-labeled GFP mRNA as a target. The assay was performed essentially as described in Figure 1 (corresponding assays performed with AGO3, 4, 6, 7, 9 and 10 are provided as Supplementary Figure S3). That is, At AGO1, AGO2 or AGO5 were overexpressed in the BYL by in vitro translation of the corresponding mRNAs in the presence of 21 (lanes 2–5) or 22 nt (lanes 6–9) ‘gf698’ siRNA variants where the corresponding guide strands had different 5′-terminal nucleotides (as indicated). After addition of the target mRNA, the RISC cleavage products (marked by asterisks) were analyzed by denaturing PAGE and autoradiography. As a negative control, the reaction was carried out in the absence of siRNA (lane 1). (B) ‘Replication inhibition assays’ performed with (+)DI-GFP RNAs and RISC containing AGO1, AGO2 or AGO5 (corresponding assays performed with AGO3, 4, 7 and 10 are provided as Supplementary Figure S4). The assays were essentially performed as described in Figures 3B and 4D. Reactions where RISC were formed with AGO1 were performed with ‘gf698’ siRNA possessing a 5′-terminal U, reactions containing AGO2 or AGO5 were performed with ‘gf698’ siRNA variants that possessed a 5′-terminal A or C, respectively. The programmed RISC were added to a translation/replication reaction performed with (+)DI-GFP1(s) or (+)DI-GFP1(as). RP and cleavage products (asterisks) are indicated. Lanes 1 and 4; assays in the absence of p92 (no replication). Lanes 2 and 5; assays in the absence of siRNA (negative controls). Lanes 3 and 6; assays in the presence of ‘gf698’ siRNA.

Mentions: Having the ‘RISC formation/cleavage assay’ and the ‘replication inhibition assay’ in hands, we wanted to understand whether also other AGO proteins (besides AGO1) supported siRNA-directed cleavage of the TBSV RNA and potentially inhibited viral RNA replication in vitro. For this purpose, we cloned most AGO genes from A. thaliana (At). In fact, the subsequent experiments were performed with the At proteins, as comparative studies with the Nt AGO1 and At AGO1 yielded identical results (Table 1, and data not shown). In the BYL, all AGO proteins were expressed by in vitro translation of the corresponding mRNAs (Supplementary Figure S2). Using the 32P-labeled GFP mRNA as a target (Figure 1), we first performed ‘RISC formation/cleavage assays’ with the different AGO proteins and with 21 and 22 nt ‘gf698’ siRNAs, respectively. Moreover, considering that the sorting of siRNAs into AGO complexes was shown to be directed by the 5′-terminal nucleotide (21,50), we also tested ‘gf698’ siRNAs with different 5′-termini. Thus, we confirmed and extended earlier findings demonstrating that AGO1, 2, 3, 5, 7 and 10 had an evident slicer activity with 21 and 22 nt siRNAs. AGO4, 6 and 9 revealed no slicer activity with 21- and 22-nt siRNAs. RISC containing AGO1 or AGO10 were moreover confirmed having a clear preference for siRNAs with a 5′-U, AGO2 for siRNAs with a 5′-A and AGO5 for siRNAs with a 5′-C. AGO3 and AGO7 accepted the ‘gf698’ siRNAs only for cleavage if these had a 5′-terminal A. These data are summarized in Table 1, examples of cleavage assays are shown with AGO1, AGO2 and AGO5 in Figure 6A (cleavage data with AGO3, 4, 6, 7, 9 and 10 shown as Supplementary Figure S3). Next, we applied these findings to ‘replication inhibition assays’, which were performed with (+)DI-GFP1(s) and (+)DI-GFP1(as) RNAs (Figure 4), respectively. That is, RISC were reconstituted with the respective AGO proteins and programmed with the ‘gf698’ siRNA variant that turned out to be best-accepted by this AGO protein in the earlier cleavage assay (Table 1). We observed that those AGO proteins that had the most evident slicer activity, namely, AGO1, 2, 3 and 5, also had an inhibitory effect on replicating viral RNA (Figure 6B; replication data with AGO3, 4, 7 and 10 shown as Supplementary Figure S4). However, replication inhibition was observed only with the viral RNAs that contained the ‘gf698’ target site in sense orientation (Figure 6B and Supplementary Figure S4). This confirmed our initial findings and demonstrated that not only AGO1/RISC but also AGO2/RISC, AGO3/RISC and AGO5/RISC targeted viral replication exclusively on the level of the (+)RNA.Figure 6.


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)

Several AGO proteins support antiviral RNA silencing. (A) ‘RISC formation/cleavage assays’ performed with AGO1, AGO2 and AGO5 using different variants of ‘gf698’ siRNAs and 32P-labeled GFP mRNA as a target. The assay was performed essentially as described in Figure 1 (corresponding assays performed with AGO3, 4, 6, 7, 9 and 10 are provided as Supplementary Figure S3). That is, At AGO1, AGO2 or AGO5 were overexpressed in the BYL by in vitro translation of the corresponding mRNAs in the presence of 21 (lanes 2–5) or 22 nt (lanes 6–9) ‘gf698’ siRNA variants where the corresponding guide strands had different 5′-terminal nucleotides (as indicated). After addition of the target mRNA, the RISC cleavage products (marked by asterisks) were analyzed by denaturing PAGE and autoradiography. As a negative control, the reaction was carried out in the absence of siRNA (lane 1). (B) ‘Replication inhibition assays’ performed with (+)DI-GFP RNAs and RISC containing AGO1, AGO2 or AGO5 (corresponding assays performed with AGO3, 4, 7 and 10 are provided as Supplementary Figure S4). The assays were essentially performed as described in Figures 3B and 4D. Reactions where RISC were formed with AGO1 were performed with ‘gf698’ siRNA possessing a 5′-terminal U, reactions containing AGO2 or AGO5 were performed with ‘gf698’ siRNA variants that possessed a 5′-terminal A or C, respectively. The programmed RISC were added to a translation/replication reaction performed with (+)DI-GFP1(s) or (+)DI-GFP1(as). RP and cleavage products (asterisks) are indicated. Lanes 1 and 4; assays in the absence of p92 (no replication). Lanes 2 and 5; assays in the absence of siRNA (negative controls). Lanes 3 and 6; assays in the presence of ‘gf698’ siRNA.
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Related In: Results  -  Collection

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gkt193-F6: Several AGO proteins support antiviral RNA silencing. (A) ‘RISC formation/cleavage assays’ performed with AGO1, AGO2 and AGO5 using different variants of ‘gf698’ siRNAs and 32P-labeled GFP mRNA as a target. The assay was performed essentially as described in Figure 1 (corresponding assays performed with AGO3, 4, 6, 7, 9 and 10 are provided as Supplementary Figure S3). That is, At AGO1, AGO2 or AGO5 were overexpressed in the BYL by in vitro translation of the corresponding mRNAs in the presence of 21 (lanes 2–5) or 22 nt (lanes 6–9) ‘gf698’ siRNA variants where the corresponding guide strands had different 5′-terminal nucleotides (as indicated). After addition of the target mRNA, the RISC cleavage products (marked by asterisks) were analyzed by denaturing PAGE and autoradiography. As a negative control, the reaction was carried out in the absence of siRNA (lane 1). (B) ‘Replication inhibition assays’ performed with (+)DI-GFP RNAs and RISC containing AGO1, AGO2 or AGO5 (corresponding assays performed with AGO3, 4, 7 and 10 are provided as Supplementary Figure S4). The assays were essentially performed as described in Figures 3B and 4D. Reactions where RISC were formed with AGO1 were performed with ‘gf698’ siRNA possessing a 5′-terminal U, reactions containing AGO2 or AGO5 were performed with ‘gf698’ siRNA variants that possessed a 5′-terminal A or C, respectively. The programmed RISC were added to a translation/replication reaction performed with (+)DI-GFP1(s) or (+)DI-GFP1(as). RP and cleavage products (asterisks) are indicated. Lanes 1 and 4; assays in the absence of p92 (no replication). Lanes 2 and 5; assays in the absence of siRNA (negative controls). Lanes 3 and 6; assays in the presence of ‘gf698’ siRNA.
Mentions: Having the ‘RISC formation/cleavage assay’ and the ‘replication inhibition assay’ in hands, we wanted to understand whether also other AGO proteins (besides AGO1) supported siRNA-directed cleavage of the TBSV RNA and potentially inhibited viral RNA replication in vitro. For this purpose, we cloned most AGO genes from A. thaliana (At). In fact, the subsequent experiments were performed with the At proteins, as comparative studies with the Nt AGO1 and At AGO1 yielded identical results (Table 1, and data not shown). In the BYL, all AGO proteins were expressed by in vitro translation of the corresponding mRNAs (Supplementary Figure S2). Using the 32P-labeled GFP mRNA as a target (Figure 1), we first performed ‘RISC formation/cleavage assays’ with the different AGO proteins and with 21 and 22 nt ‘gf698’ siRNAs, respectively. Moreover, considering that the sorting of siRNAs into AGO complexes was shown to be directed by the 5′-terminal nucleotide (21,50), we also tested ‘gf698’ siRNAs with different 5′-termini. Thus, we confirmed and extended earlier findings demonstrating that AGO1, 2, 3, 5, 7 and 10 had an evident slicer activity with 21 and 22 nt siRNAs. AGO4, 6 and 9 revealed no slicer activity with 21- and 22-nt siRNAs. RISC containing AGO1 or AGO10 were moreover confirmed having a clear preference for siRNAs with a 5′-U, AGO2 for siRNAs with a 5′-A and AGO5 for siRNAs with a 5′-C. AGO3 and AGO7 accepted the ‘gf698’ siRNAs only for cleavage if these had a 5′-terminal A. These data are summarized in Table 1, examples of cleavage assays are shown with AGO1, AGO2 and AGO5 in Figure 6A (cleavage data with AGO3, 4, 6, 7, 9 and 10 shown as Supplementary Figure S3). Next, we applied these findings to ‘replication inhibition assays’, which were performed with (+)DI-GFP1(s) and (+)DI-GFP1(as) RNAs (Figure 4), respectively. That is, RISC were reconstituted with the respective AGO proteins and programmed with the ‘gf698’ siRNA variant that turned out to be best-accepted by this AGO protein in the earlier cleavage assay (Table 1). We observed that those AGO proteins that had the most evident slicer activity, namely, AGO1, 2, 3 and 5, also had an inhibitory effect on replicating viral RNA (Figure 6B; replication data with AGO3, 4, 7 and 10 shown as Supplementary Figure S4). However, replication inhibition was observed only with the viral RNAs that contained the ‘gf698’ target site in sense orientation (Figure 6B and Supplementary Figure S4). This confirmed our initial findings and demonstrated that not only AGO1/RISC but also AGO2/RISC, AGO3/RISC and AGO5/RISC targeted viral replication exclusively on the level of the (+)RNA.Figure 6.

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