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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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Identification and characterization of effective vsiRNAs. (A) Sequences of vsiRNAs1 and 2 that were identified to effectively target the TBSV R3.5 element. The siRNAs were deduced from cleavage products of ‘RISC formation/cleavage assays’ that applied the indicated AGO proteins, a pool of dsR3.5-derived siRNAs, and (+)DI-R3.5 as a target RNA. The position between the guide strand’s nucleotides 10 and 11 that is opposite the cleavage site in the target RNA (52, 53) is indicated by a triangle. (B) RNA secondary structure of the TBSV R3.5 region [modified from a previous study (38)]. Circles indicate the 5′-ends of RNA cleavage products, the cDNAs of which were cloned from ‘RISC formation/cleavage assays’ that applied AGO1 (white circles, Figure 3A, left panel) or AGO2 (black circles). Corresponding vsiRNAs were deduced via nucleotide 10 of the guide strand that should be complementary to these cleavage sites (indicated by the triangle in A). Arrows indicate the 5′-ends of the most frequently cloned cleavage products that led to the identification of vsiRNAs1 and 2. (C) ‘RISC formation/cleavage assays’ that were performed with AGO1, AGO2 and labeled (+)DI-R3.5 target RNA. The assays were performed as described in Figure 3A and tested the dsR3.5-derived siRNA pool side-by-side with the synthetic vsiRNA1 (left panel) and vsiRNA2 (right panel), respectively. Target RNA and cleavage products are indicated in the same way as in the previous figures. Lanes 1; assays performed with a non-specific (‘gf698’) siRNA (negative control). Lanes 2; assays performed with the dsR3.5-derived siRNA pool (siRNAs dsR3.5). Lanes 3; assays performed with vsiRNA1 or vsiRNA2. (D) ‘Replication inhibition assays’ with AGO1 and vsiRNA1 or AGO2 and vsiRNA2. The assays were carried out as described in Figure 3B (variant 1) using (+)DI-R3.5 RNA. RP and cleavage products (asterisks) are indicated. Lanes 1; assays performed in the absence of p92 (no replication). Lanes 2; assays performed in the presence of a non-specific (‘gf698’) siRNA (negative control). Lane 3; assays performed in the presence of vsiRNA1 or vsiRNA2.
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gkt193-F7: Identification and characterization of effective vsiRNAs. (A) Sequences of vsiRNAs1 and 2 that were identified to effectively target the TBSV R3.5 element. The siRNAs were deduced from cleavage products of ‘RISC formation/cleavage assays’ that applied the indicated AGO proteins, a pool of dsR3.5-derived siRNAs, and (+)DI-R3.5 as a target RNA. The position between the guide strand’s nucleotides 10 and 11 that is opposite the cleavage site in the target RNA (52, 53) is indicated by a triangle. (B) RNA secondary structure of the TBSV R3.5 region [modified from a previous study (38)]. Circles indicate the 5′-ends of RNA cleavage products, the cDNAs of which were cloned from ‘RISC formation/cleavage assays’ that applied AGO1 (white circles, Figure 3A, left panel) or AGO2 (black circles). Corresponding vsiRNAs were deduced via nucleotide 10 of the guide strand that should be complementary to these cleavage sites (indicated by the triangle in A). Arrows indicate the 5′-ends of the most frequently cloned cleavage products that led to the identification of vsiRNAs1 and 2. (C) ‘RISC formation/cleavage assays’ that were performed with AGO1, AGO2 and labeled (+)DI-R3.5 target RNA. The assays were performed as described in Figure 3A and tested the dsR3.5-derived siRNA pool side-by-side with the synthetic vsiRNA1 (left panel) and vsiRNA2 (right panel), respectively. Target RNA and cleavage products are indicated in the same way as in the previous figures. Lanes 1; assays performed with a non-specific (‘gf698’) siRNA (negative control). Lanes 2; assays performed with the dsR3.5-derived siRNA pool (siRNAs dsR3.5). Lanes 3; assays performed with vsiRNA1 or vsiRNA2. (D) ‘Replication inhibition assays’ with AGO1 and vsiRNA1 or AGO2 and vsiRNA2. The assays were carried out as described in Figure 3B (variant 1) using (+)DI-R3.5 RNA. RP and cleavage products (asterisks) are indicated. Lanes 1; assays performed in the absence of p92 (no replication). Lanes 2; assays performed in the presence of a non-specific (‘gf698’) siRNA (negative control). Lane 3; assays performed in the presence of vsiRNA1 or vsiRNA2.

Mentions: Because of their stability and high replication rate, TBSV DI RNAs were earlier shown to be most suitable to perform in vitro replication studies with BYL (46). Tombusvirus DI RNAs are suggested to attenuate infections by competing for viral and host replication factors and to modulate the antiviral immune response through the production of massive amounts of vsiRNAs. Thus, most wild-type (wt) DI RNAs turned out to be poor targets of antiviral RNA silencing (16). To obtain an RNA substrate that could be equally well used in replication and in silencing experiments, we constructed a modified version of the TBSV DI B10 (48) that included the so-called R3.5 region (DI-R3.5; Figures 2A and 7B). R3.5 is part of the TBSV gRNA’s 3′NTR; it is located between the RIII and RIV elements and contains the 3′CITE. R3.5 was chosen because in plants infected with the TBSV-related CymRSV, this region was found to be a hot spot for vsiRNA-mediated cleavage (19,37). The DI-R3.5 RNA showed the same replication competence as the DI B10 (Figure 2B, lanes 2 and 4).Figure 2.


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)

Identification and characterization of effective vsiRNAs. (A) Sequences of vsiRNAs1 and 2 that were identified to effectively target the TBSV R3.5 element. The siRNAs were deduced from cleavage products of ‘RISC formation/cleavage assays’ that applied the indicated AGO proteins, a pool of dsR3.5-derived siRNAs, and (+)DI-R3.5 as a target RNA. The position between the guide strand’s nucleotides 10 and 11 that is opposite the cleavage site in the target RNA (52, 53) is indicated by a triangle. (B) RNA secondary structure of the TBSV R3.5 region [modified from a previous study (38)]. Circles indicate the 5′-ends of RNA cleavage products, the cDNAs of which were cloned from ‘RISC formation/cleavage assays’ that applied AGO1 (white circles, Figure 3A, left panel) or AGO2 (black circles). Corresponding vsiRNAs were deduced via nucleotide 10 of the guide strand that should be complementary to these cleavage sites (indicated by the triangle in A). Arrows indicate the 5′-ends of the most frequently cloned cleavage products that led to the identification of vsiRNAs1 and 2. (C) ‘RISC formation/cleavage assays’ that were performed with AGO1, AGO2 and labeled (+)DI-R3.5 target RNA. The assays were performed as described in Figure 3A and tested the dsR3.5-derived siRNA pool side-by-side with the synthetic vsiRNA1 (left panel) and vsiRNA2 (right panel), respectively. Target RNA and cleavage products are indicated in the same way as in the previous figures. Lanes 1; assays performed with a non-specific (‘gf698’) siRNA (negative control). Lanes 2; assays performed with the dsR3.5-derived siRNA pool (siRNAs dsR3.5). Lanes 3; assays performed with vsiRNA1 or vsiRNA2. (D) ‘Replication inhibition assays’ with AGO1 and vsiRNA1 or AGO2 and vsiRNA2. The assays were carried out as described in Figure 3B (variant 1) using (+)DI-R3.5 RNA. RP and cleavage products (asterisks) are indicated. Lanes 1; assays performed in the absence of p92 (no replication). Lanes 2; assays performed in the presence of a non-specific (‘gf698’) siRNA (negative control). Lane 3; assays performed in the presence of vsiRNA1 or vsiRNA2.
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gkt193-F7: Identification and characterization of effective vsiRNAs. (A) Sequences of vsiRNAs1 and 2 that were identified to effectively target the TBSV R3.5 element. The siRNAs were deduced from cleavage products of ‘RISC formation/cleavage assays’ that applied the indicated AGO proteins, a pool of dsR3.5-derived siRNAs, and (+)DI-R3.5 as a target RNA. The position between the guide strand’s nucleotides 10 and 11 that is opposite the cleavage site in the target RNA (52, 53) is indicated by a triangle. (B) RNA secondary structure of the TBSV R3.5 region [modified from a previous study (38)]. Circles indicate the 5′-ends of RNA cleavage products, the cDNAs of which were cloned from ‘RISC formation/cleavage assays’ that applied AGO1 (white circles, Figure 3A, left panel) or AGO2 (black circles). Corresponding vsiRNAs were deduced via nucleotide 10 of the guide strand that should be complementary to these cleavage sites (indicated by the triangle in A). Arrows indicate the 5′-ends of the most frequently cloned cleavage products that led to the identification of vsiRNAs1 and 2. (C) ‘RISC formation/cleavage assays’ that were performed with AGO1, AGO2 and labeled (+)DI-R3.5 target RNA. The assays were performed as described in Figure 3A and tested the dsR3.5-derived siRNA pool side-by-side with the synthetic vsiRNA1 (left panel) and vsiRNA2 (right panel), respectively. Target RNA and cleavage products are indicated in the same way as in the previous figures. Lanes 1; assays performed with a non-specific (‘gf698’) siRNA (negative control). Lanes 2; assays performed with the dsR3.5-derived siRNA pool (siRNAs dsR3.5). Lanes 3; assays performed with vsiRNA1 or vsiRNA2. (D) ‘Replication inhibition assays’ with AGO1 and vsiRNA1 or AGO2 and vsiRNA2. The assays were carried out as described in Figure 3B (variant 1) using (+)DI-R3.5 RNA. RP and cleavage products (asterisks) are indicated. Lanes 1; assays performed in the absence of p92 (no replication). Lanes 2; assays performed in the presence of a non-specific (‘gf698’) siRNA (negative control). Lane 3; assays performed in the presence of vsiRNA1 or vsiRNA2.
Mentions: Because of their stability and high replication rate, TBSV DI RNAs were earlier shown to be most suitable to perform in vitro replication studies with BYL (46). Tombusvirus DI RNAs are suggested to attenuate infections by competing for viral and host replication factors and to modulate the antiviral immune response through the production of massive amounts of vsiRNAs. Thus, most wild-type (wt) DI RNAs turned out to be poor targets of antiviral RNA silencing (16). To obtain an RNA substrate that could be equally well used in replication and in silencing experiments, we constructed a modified version of the TBSV DI B10 (48) that included the so-called R3.5 region (DI-R3.5; Figures 2A and 7B). R3.5 is part of the TBSV gRNA’s 3′NTR; it is located between the RIII and RIV elements and contains the 3′CITE. R3.5 was chosen because in plants infected with the TBSV-related CymRSV, this region was found to be a hot spot for vsiRNA-mediated cleavage (19,37). The DI-R3.5 RNA showed the same replication competence as the DI B10 (Figure 2B, lanes 2 and 4).Figure 2.

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