Seguin Jonathan, Rajeswaran Rajendran, Malpica-López Nachelli, Martin Robert R, Kasschau Kristin, Dolja Valerian V, Otten Patricia, Farinelli Laurent, Pooggin Mikhail M
University of Basel, Department of Environmental Sciences, Institute of Botany, Basel, Switzerland ; Fasteris SA, Plan-les-Ouates, Geneva, Switzerland.
University of Basel, Department of Environmental Sciences, Institute of Botany, Basel, Switzerland.
PLoS One. 2014 Feb 11;9(2):e88513. doi: 10.1371/journal.pone.0088513. eCollection 2014.
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.
病毒感染的植物会积累大量21 - 24个核苷酸的病毒小干扰RNA（viral siRNAs），这些小干扰RNA由进化上保守的RNA干扰（RNAi）机制产生，该机制调控基因表达并抵御入侵的核酸。在这里我们表明，与RNA病毒类似，DNA病毒的整个基因组序列在正义和反义方向上都被小干扰RNA密集覆盖。这意味着病毒DNA的编码和非编码区域在细胞核中都存在广泛转录，从而产生病毒小干扰RNA的双链RNA前体。与我们的发现和假设一致，我们证明了利用生物信息学工具对花椰菜花叶病毒科（Caulimoviridae）和双生病毒科（Geminiviridae）的DNA病毒的完整基因组进行深度测序和从头组装，可以重建它们的基因组。此外，我们证明这种“小干扰RNA组学”方法可用于可靠鉴定病毒准种中的共有主基因组及其微小变异体。最后，我们利用这种方法重建了一种与葡萄经济上重要的红斑点病相关的新兴DNA病毒和两种类病毒，并快速生成了代表油菜花叶病毒野生型主基因组的具有生物活性的克隆。我们的研究结果表明，深度小干扰RNA测序能够从头重建任何DNA或RNA病毒基因组及其微小变异体，使其适用于对不断进化的病毒准种进行全面表征，以及研究小干扰RNA生物合成机制和基于RNAi的抗病毒防御机制。
