De Novo Reconstruction of Consensus Master Genomes of Plant RNA and DNA Viruses from siRNAs

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.

Perspectives on the Application of Next-generation Sequencing to the Improvement of Africa’s Staple Food Crops

“…In the wake of NGS technologies, powerful and high-throughput novel approaches, such as metagenomics, have been developed and widely used to analyze nucleotide sequence of microbial populations in plant samples (see section 2.8) [8,105,156]. In particular, deep sequencing of small RNA families such as short interfering RNAs (siRNAs) can be used to identify and reconstruct any DNA or RNA virus genome and its microvariants with the help of bioinformatics tools [155,157]. Furthermore, the application of NGS can be extended to insect vectors for discovery and characterization of insect viruses [109].…”

Viroid quasispecies revealed by deep sequencing

“…21,22 In this study, we have gone a step further; that is, we have used small RNA data to compare the mutational landscapes of different infecting viroid variants. These landscapes reflect a combination of replication efficiency, selection, and sequencing errors.…”

Identification and molecular characterization of a novel monopartite geminivirus associated with mulberry mosaic dwarf disease

“…A few highly divergent and still unclassified geminiviruses have recently been described from Euphorbia (Euphorbia caput-medusae-associated virus, EcmLV) (Bernardo et al, 2013), citrus (citrus chlorotic dwarf-associated virus, CCDaV) (Loconsole et al, 2012), grapevine (grapevine red blotch-associated virus, GRBaV) (Al Rwahnih et al, 2013;Krenz et al, 2012;Poojari et al, 2013). Viral RNAs, including those of geminiviruses (Seguin et al, 2014), are targeted by host defence mechanisms based on RNA silencing and, through the activity of Dicer-like enzymes (DCLs), become the source of virus-derived small RNAs of [21][22][23][24]. Recent developments in next-generation sequencing (NGS) of sRNAs and bioinformatics have supplied powerful tools for identifying hitherto unknown viral and subviral agents in plants (Al Rwahnih et al, 2009Giampetruzzi et al, 2012;Ito et al, 2013;Massart et al, 2014).…”