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PLoS One. 2014 Feb 11;9(2):e88513. doi: 10.1371/journal.pone.0088513. eCollection 2014.

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

Author information

1
University of Basel, Department of Environmental Sciences, Institute of Botany, Basel, Switzerland ; Fasteris SA, Plan-les-Ouates, Geneva, Switzerland.
2
University of Basel, Department of Environmental Sciences, Institute of Botany, Basel, Switzerland.
3
United States Department of Agriculture-Agricultural Research Service, Horticultural Crops Research Laboratory, Corvallis, Oregon, United States of America ; Oregon State University, Department of Botany and Plant Pathology, Center for Genome Research and Biocomputing, Corvallis, Oregon, United States of America.
4
Oregon State University, Department of Botany and Plant Pathology, Center for Genome Research and Biocomputing, Corvallis, Oregon, United States of America.
5
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.

PMID:
24523907
PMCID:
PMC3921208
DOI:
10.1371/journal.pone.0088513
[Indexed for MEDLINE]
Free PMC Article
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