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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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Composition and in vitro replication of TBSV DI RNAs. (A) Schematic representations of TBSV gRNA, DI B10 RNA and DI-R3.5 RNA. The NTRs of the viral genome are depicted as lines, coding regions as boxes. Arrows indicate the transcriptional start of the two subgenomic (sg) mRNAs that are generated in the course of the TBSV life cycle. DI B10 (48) that was used in this study is essentially composed of the regions RI-RIV. DI-R3.5 also encloses the genomic R3.5 region inserted between RIII and RIV. (B) DI-R3.5 RNA replicates with similar efficiency as the DI B10 RNA in vitro. Using the protocol of Gursinsky et al. (46), the viral proteins p33 and p92 were produced by in vitro translation of the corresponding mRNAs in the BYL. Viral RNA replication was then started by the addition of a replication mix that included [α-32P]CTP and DI B10 RNA (lanes 1 and 2) or DI-R3.5 RNA (lanes 3 and 4). Total RNA was isolated from the reaction and analyzed by denaturing PAGE and autoradiography. The replication products (RP) are indicated. Lanes 1 and 3; replication assays performed in the absence of p92 (negative controls). Lanes 2 and 4; replication assays performed in the presence of p33 and p92.
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gkt193-F2: Composition and in vitro replication of TBSV DI RNAs. (A) Schematic representations of TBSV gRNA, DI B10 RNA and DI-R3.5 RNA. The NTRs of the viral genome are depicted as lines, coding regions as boxes. Arrows indicate the transcriptional start of the two subgenomic (sg) mRNAs that are generated in the course of the TBSV life cycle. DI B10 (48) that was used in this study is essentially composed of the regions RI-RIV. DI-R3.5 also encloses the genomic R3.5 region inserted between RIII and RIV. (B) DI-R3.5 RNA replicates with similar efficiency as the DI B10 RNA in vitro. Using the protocol of Gursinsky et al. (46), the viral proteins p33 and p92 were produced by in vitro translation of the corresponding mRNAs in the BYL. Viral RNA replication was then started by the addition of a replication mix that included [α-32P]CTP and DI B10 RNA (lanes 1 and 2) or DI-R3.5 RNA (lanes 3 and 4). Total RNA was isolated from the reaction and analyzed by denaturing PAGE and autoradiography. The replication products (RP) are indicated. Lanes 1 and 3; replication assays performed in the absence of p92 (negative controls). Lanes 2 and 4; replication assays performed in the presence of p33 and p92.

Mentions: Members of the Tombusvirus genus of plant (+)RNA viruses, such as tomato bushy stunt virus (TBSV) and cymbidium ringspot virus (CymRSV) are intensively investigated to define the molecular determinants of viral replication (33) and of antiviral RNA silencing, respectively (19,34–37). The single-stranded ∼5–kb long TBSV genome (gRNA) consists of two non-translated regions (NTRs) at the 5′- and 3′-ends and several open reading frames (ORF; Figure 2A). Only the 5′-terminal ORF (5′-ORF) of the RNA is directly translated from the gRNA. Translation initiation is mediated by the so-called 3′-CITE (cap independent translational enhancer) element in the 3′-NTR and involves 5′–3′ interactions of the viral RNA (38,39). The 5′-ORF encodes the viral replicase proteins p33 and p92. p33 acts as an anchor of the viral replication complex and as an RNA chaperon; p92, which is generated by translational read-through of an ORF-internal stop codon, represents the RNA-dependent RNA polymerase. The residual ORFs are translated from two subgenomic mRNAs that are produced from the (−)RNA. They encode the coat protein p41, the viral movement protein p22 and the RNA silencing suppressor p19 (33).


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)

Composition and in vitro replication of TBSV DI RNAs. (A) Schematic representations of TBSV gRNA, DI B10 RNA and DI-R3.5 RNA. The NTRs of the viral genome are depicted as lines, coding regions as boxes. Arrows indicate the transcriptional start of the two subgenomic (sg) mRNAs that are generated in the course of the TBSV life cycle. DI B10 (48) that was used in this study is essentially composed of the regions RI-RIV. DI-R3.5 also encloses the genomic R3.5 region inserted between RIII and RIV. (B) DI-R3.5 RNA replicates with similar efficiency as the DI B10 RNA in vitro. Using the protocol of Gursinsky et al. (46), the viral proteins p33 and p92 were produced by in vitro translation of the corresponding mRNAs in the BYL. Viral RNA replication was then started by the addition of a replication mix that included [α-32P]CTP and DI B10 RNA (lanes 1 and 2) or DI-R3.5 RNA (lanes 3 and 4). Total RNA was isolated from the reaction and analyzed by denaturing PAGE and autoradiography. The replication products (RP) are indicated. Lanes 1 and 3; replication assays performed in the absence of p92 (negative controls). Lanes 2 and 4; replication assays performed in the presence of p33 and p92.
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Related In: Results  -  Collection

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

gkt193-F2: Composition and in vitro replication of TBSV DI RNAs. (A) Schematic representations of TBSV gRNA, DI B10 RNA and DI-R3.5 RNA. The NTRs of the viral genome are depicted as lines, coding regions as boxes. Arrows indicate the transcriptional start of the two subgenomic (sg) mRNAs that are generated in the course of the TBSV life cycle. DI B10 (48) that was used in this study is essentially composed of the regions RI-RIV. DI-R3.5 also encloses the genomic R3.5 region inserted between RIII and RIV. (B) DI-R3.5 RNA replicates with similar efficiency as the DI B10 RNA in vitro. Using the protocol of Gursinsky et al. (46), the viral proteins p33 and p92 were produced by in vitro translation of the corresponding mRNAs in the BYL. Viral RNA replication was then started by the addition of a replication mix that included [α-32P]CTP and DI B10 RNA (lanes 1 and 2) or DI-R3.5 RNA (lanes 3 and 4). Total RNA was isolated from the reaction and analyzed by denaturing PAGE and autoradiography. The replication products (RP) are indicated. Lanes 1 and 3; replication assays performed in the absence of p92 (negative controls). Lanes 2 and 4; replication assays performed in the presence of p33 and p92.
Mentions: Members of the Tombusvirus genus of plant (+)RNA viruses, such as tomato bushy stunt virus (TBSV) and cymbidium ringspot virus (CymRSV) are intensively investigated to define the molecular determinants of viral replication (33) and of antiviral RNA silencing, respectively (19,34–37). The single-stranded ∼5–kb long TBSV genome (gRNA) consists of two non-translated regions (NTRs) at the 5′- and 3′-ends and several open reading frames (ORF; Figure 2A). Only the 5′-terminal ORF (5′-ORF) of the RNA is directly translated from the gRNA. Translation initiation is mediated by the so-called 3′-CITE (cap independent translational enhancer) element in the 3′-NTR and involves 5′–3′ interactions of the viral RNA (38,39). The 5′-ORF encodes the viral replicase proteins p33 and p92. p33 acts as an anchor of the viral replication complex and as an RNA chaperon; p92, which is generated by translational read-through of an ORF-internal stop codon, represents the RNA-dependent RNA polymerase. The residual ORFs are translated from two subgenomic mRNAs that are produced from the (−)RNA. They encode the coat protein p41, the viral movement protein p22 and the RNA silencing suppressor p19 (33).

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