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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Contig Mapping
  • DNA Viruses / genetics*
  • High-Throughput Nucleotide Sequencing
  • Molecular Sequence Data
  • Mosaic Viruses / genetics
  • Plant Diseases / virology
  • Plant Leaves / virology
  • Plant Viruses / genetics*
  • Plants / virology*
  • Polymorphism, Single Nucleotide
  • RNA Interference
  • RNA Viruses / genetics*
  • RNA, Double-Stranded
  • RNA, Small Interfering / genetics*
  • RNA, Small Interfering / metabolism
  • Sequence Analysis, DNA
  • Viroids / genetics
  • Vitis / virology

Substances

  • RNA, Double-Stranded
  • RNA, Small Interfering

Associated data

  • GENBANK/KF137561
  • GENBANK/KF137562
  • GENBANK/KF137563
  • GENBANK/U65529
  • GENBANK/U65530

Grants and funding

The work was supported by European Cooperation in Science and Technology [grant SERI No. C09.0176 to L.F. and M.M.P.]; Swiss National Science Foundation [grant 31003A_143882/1 to M.M.P.]; Vinoculate, Inc. [contract 2010-744 to V.V.D.], USDA-NIFA [subcontract 2009-04401 to V.V.D.]; USDA-NIFA-SCRI [2009-51181-06027 subaward to R.R.M.]; and Bard [award IS-4314-10C to V.V.D.]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.