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De novo reconstruction of consensus master genomes of plant RNA and DNA viruses from siRNAs.

Seguin J, Rajeswaran R, Malpica-López N, Martin RR, Kasschau K, Dolja VV, Otten P, Farinelli L, Pooggin MM - PLoS ONE (2014)

Bottom Line: Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids.This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs.Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies.

View Article: PubMed Central - PubMed

Affiliation: University of Basel, Department of Environmental Sciences, Institute of Botany, Basel, Switzerland ; Fasteris SA, Plan-les-Ouates, Geneva, Switzerland.

ABSTRACT
Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense.

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Related in: MedlinePlus

Test of the original and the corrected ORMV clones for infectivity.The plasmid containing the full-length ORMV genome sequence (original or corrected) behind the T7 promoter is depicted schematically: the restriction site Pst I or Nsi I, respectively, just downstream of the genome (located in multiple cloning site; MCS) was used for linearization of the plasmid, followed by run-off transcription by T7 polymerase in the presence of a cap analog. The resulting in vitro transcript (ORMV genomic RNA) was taken for mechanical inoculation of N. benthamiana plants. The picture shows the inoculated plants at 10 days post-inoculation.
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pone-0088513-g003: Test of the original and the corrected ORMV clones for infectivity.The plasmid containing the full-length ORMV genome sequence (original or corrected) behind the T7 promoter is depicted schematically: the restriction site Pst I or Nsi I, respectively, just downstream of the genome (located in multiple cloning site; MCS) was used for linearization of the plasmid, followed by run-off transcription by T7 polymerase in the presence of a cap analog. The resulting in vitro transcript (ORMV genomic RNA) was taken for mechanical inoculation of N. benthamiana plants. The picture shows the inoculated plants at 10 days post-inoculation.

Mentions: To determine if such ‘siRNA omics’ (siRomics) approach is applicable for identification of a master genome in viral ‘quasispecies cloud’, we sequenced sRNAs from Arabidopsis infected with Oilseed rape mosaic virus (ORMV), the RNA virus for which an available cDNA clone was not infectious [20] because of potential cloning errors or because it represented a defective microvariant from the ORMV quasispecies. Using Oases followed by Seqman, the 6,303 nt ORMV genome was reconstructed de novo as a single contig from two independent sRNA libraries (Dataset S1C and Dataset S2). This reconstructed genome differed from the available cDNA sequence at three positions (G-to-A at position 12, A-to-T at position 231, and G-to-T at position 5612; Figure 1). SNP calling using the two sRNA libraries confirmed these mismatches in 96.5–97.7% (A12), 99.5–99.9% (T231) and 88.6–93.8% (T5612) viral redundant reads and highlighted the overall variation in the ORMV quasispecies (Dataset S3A). We corrected these mismatches (presumably cloning errors) in the cDNA clone and tested it for infectivity. Strikingly, the resulting clone was fully biologically active, causing the disease symptoms indistinguishable from those of the wild type ORMV sap (Figure 3). Thus, a common problem of virology, often taking years to overcome [21], was solved in one step. Our unpublished results suggest that G at position 12 in the original cDNA clone had a drastic impact on ORMV infectivity, possibly because it affects initiation of positive stand synthesis during the viral replication process; the nucleotide substitutions at the positions 231 and 5612 likely represent viable variants in the virus quasispecies (N.M.L., R.R., and M.M.P., in preparation).


De novo reconstruction of consensus master genomes of plant RNA and DNA viruses from siRNAs.

Seguin J, Rajeswaran R, Malpica-López N, Martin RR, Kasschau K, Dolja VV, Otten P, Farinelli L, Pooggin MM - PLoS ONE (2014)

Test of the original and the corrected ORMV clones for infectivity.The plasmid containing the full-length ORMV genome sequence (original or corrected) behind the T7 promoter is depicted schematically: the restriction site Pst I or Nsi I, respectively, just downstream of the genome (located in multiple cloning site; MCS) was used for linearization of the plasmid, followed by run-off transcription by T7 polymerase in the presence of a cap analog. The resulting in vitro transcript (ORMV genomic RNA) was taken for mechanical inoculation of N. benthamiana plants. The picture shows the inoculated plants at 10 days post-inoculation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3921208&req=5

pone-0088513-g003: Test of the original and the corrected ORMV clones for infectivity.The plasmid containing the full-length ORMV genome sequence (original or corrected) behind the T7 promoter is depicted schematically: the restriction site Pst I or Nsi I, respectively, just downstream of the genome (located in multiple cloning site; MCS) was used for linearization of the plasmid, followed by run-off transcription by T7 polymerase in the presence of a cap analog. The resulting in vitro transcript (ORMV genomic RNA) was taken for mechanical inoculation of N. benthamiana plants. The picture shows the inoculated plants at 10 days post-inoculation.
Mentions: To determine if such ‘siRNA omics’ (siRomics) approach is applicable for identification of a master genome in viral ‘quasispecies cloud’, we sequenced sRNAs from Arabidopsis infected with Oilseed rape mosaic virus (ORMV), the RNA virus for which an available cDNA clone was not infectious [20] because of potential cloning errors or because it represented a defective microvariant from the ORMV quasispecies. Using Oases followed by Seqman, the 6,303 nt ORMV genome was reconstructed de novo as a single contig from two independent sRNA libraries (Dataset S1C and Dataset S2). This reconstructed genome differed from the available cDNA sequence at three positions (G-to-A at position 12, A-to-T at position 231, and G-to-T at position 5612; Figure 1). SNP calling using the two sRNA libraries confirmed these mismatches in 96.5–97.7% (A12), 99.5–99.9% (T231) and 88.6–93.8% (T5612) viral redundant reads and highlighted the overall variation in the ORMV quasispecies (Dataset S3A). We corrected these mismatches (presumably cloning errors) in the cDNA clone and tested it for infectivity. Strikingly, the resulting clone was fully biologically active, causing the disease symptoms indistinguishable from those of the wild type ORMV sap (Figure 3). Thus, a common problem of virology, often taking years to overcome [21], was solved in one step. Our unpublished results suggest that G at position 12 in the original cDNA clone had a drastic impact on ORMV infectivity, possibly because it affects initiation of positive stand synthesis during the viral replication process; the nucleotide substitutions at the positions 231 and 5612 likely represent viable variants in the virus quasispecies (N.M.L., R.R., and M.M.P., in preparation).

Bottom Line: Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids.This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs.Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies.

View Article: PubMed Central - PubMed

Affiliation: University of Basel, Department of Environmental Sciences, Institute of Botany, Basel, Switzerland ; Fasteris SA, Plan-les-Ouates, Geneva, Switzerland.

ABSTRACT
Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense.

Show MeSH
Related in: MedlinePlus