Deep Sequencing of Viroid-Derived Small RNAs from Grapevine Provides New Insights on the Role of RNA Silencing in Plant-Viroid Interaction Beatriz Navarro1., Vitantonio Pantaleo1., Andreas Gisel2, Simon Moxon3, Tamas Dalmay4, Gyo¨ rgy Bisztray5, Francesco Di Serio1*, Jo´ zsef Burgya´ n1* 1 Istituto di Virologia Vegetale, Consiglio Nazionale delle Ricerche, Torino and Bari, Italy, 2 Istituto di Tecnologie Biomediche, Consiglio Nazionale delle Ricerche, Bari, Italy, 3 School of Computing Sciences, University of East Anglia, Norwich, United Kingdom, 4 School of Biological Sciences, University of East Anglia, Norwich, United Kingdom, 5 Corvinus University, Budapest, Hungary Abstract Background: Viroids are circular, highly structured, non-protein-coding RNAs that, usurping cellular enzymes and escaping host defense mechanisms, are able to replicate and move through infected plants. Similarly to viruses, viroid infections are associated with the accumulation of viroid-derived 21–24 nt small RNAs (vd-sRNAs) with the typical features of the small interfering RNAs characteristic of RNA silencing, a sequence-specific mechanism involved in defense against invading nucleic acids and in regulation of gene expression in most eukaryotic organisms. Methodology/Principal Findings: To gain further insights on the genesis and possible role of vd-sRNAs in plant-viroid interaction, sRNAs isolated from Vitis vinifera infected by Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1) were sequenced by the high-throughput platform Solexa-Illumina, and the vd-sRNAs were analyzed. The large majority of HSVd- and GYSVd1-sRNAs derived from a few specific regions (hotspots) of the genomic (+) and (2) viroid RNAs, with a prevalence of those from the (2) strands of both viroids. When grouped according to their sizes, vd-sRNAs always assumed a distribution with prominent 21-, 22- and 24-nt peaks, which, interestingly, mapped at the same hotspots. Conclusions/Significance: These findings show that different Dicer-like enzymes (DCLs) target viroid RNAs, preferentially accessing to the same viroid domains. Interestingly, our results also suggest that viroid RNAs may interact with host enzymes involved in the RNA-directed DNA methylation pathway, indicating more complex scenarios than previously thought for both vd-sRNAs genesis and possible interference with host gene expression. Citation: Navarro B, Pantaleo V, Gisel A, Moxon S, Dalmay T, et al. (2009) Deep Sequencing of Viroid-Derived Small RNAs from Grapevine Provides New Insights on the Role of RNA Silencing in Plant-Viroid Interaction. PLoS ONE 4(11): e7686. doi:10.1371/journal.pone.0007686 Editor: Ding Xiang Liu, Institute of Molecular and Cell Biology, Singapore Received July 30, 2009; Accepted October 7, 2009; Published November 5, 2009 Copyright: 2009 Navarro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by grants from the European Commission Sixth Framework Programme Integrated Project SIROCCO (Silencing RNAs: organisers and coordinators of complexity in eukaryotic organisms) contract number LSHG-CT-2006-037900, the bilateral research Program between the Consiglio Nazionale delle Ricerche (CNR, Italy) and Magyar Tudoma´nyos Akade´mia (MTA, Hungary), and the Dipartimento Agroalimentare of the CNR of Italy (A. Leone and D. Mariotti 2008 award for advanced research in agriculture to FDS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: f.diserio@ba.ivv.cnr.it (FDS); J.burgyan@ivv.cnr.it (JB) . These authors contributed equally to this work. Introduction RNA silencing-based antiviral plant response is one of the best- studied antiviral strategies of plant. The key element of this strategy is the virus-derived small interfering RNA (vsiRNA), which guides RNA induced silencing complex (RISC) to target viral genomes in plants and invertebrates [1]. VsiRNAs are processed from double-stranded RNAs (dsRNAs) or structured single-stranded RNAs (ssRNAs) by RNase III-like enzymes such as DICER [2,3] and, similarly to cell-derived small interfering RNAs (siRNAs), may guide the sequence-specific inactivation of target mRNAs by RISC [4]. Pathogenic RNAs like plant viruses are strong inducers as well as targets of RNA silencing and high levels of vsiRNAs accumulate during viral infection. However, despite extensive studies of siRNA biogenesis, the origin of vsiRNAs is still far from being fully understood. In virus infected plants, two distinct classes of vsiRNAs have been identified: the primary vsiRNAs, resulting from the cleavage of the initial trigger RNA by Dicer-like enzymes (DCLs)[5], and secondary vsiRNAs, whose biogenesis requires an RNA- dependent RNA polymerase (RDR) [6,7]. The vsiRNAs are thought to be processed from viral dsRNA replicative intermediates, from local self-complementary regions of a viral genome or from dsRNAs resulting from the action of RDRs on viral RNA templates [1,6]. DCL4 and DCL2 are the most important plant DICERs involved in ribovirus-induced RNA silencing and their products are vsiRNAs of 21 and 22 nt, respectively [8,9]. However, in the case of nuclear-replicating begomoviruses with DNA genomes, DCL3 (which produces vsiRNAs of 24 nt) is likely involved in addition to DCL4 and DCL2 [6,10]. Moreover, it has been shown PLoS ONE | www.plosone.org 1 November 2009 | Volume 4 | Issue 11 | e7686