recently that production of Tobacco rattle virus (TRV) derived vsiRNAs and antiviral silencing are strongly dependent on the combined activity of the host-encoded RDR1, RDR2, and RDR6 suggesting that they may convert viral ssRNAs into dsRNAs, which could serve as a substrate for vsiRNA production [11]. However, this model is not supported by previous observations indicating that the majority of vsiRNAs are derived from the plus (mRNA sense) viral strand [12,13,14]. A previous report also showed that RDR6 is not required for silencing the endogenous phytoene desaturase (PDS) gene using vectors based on the crucifer strain of tobacco mosaic virus (crTMV) and TRV [15]. These conflicting observations indicate that our knowledge about vsiRNA biogenesis is still limited, and that this process may depend on many factors including the replication strategy of the pathogen, the site of replication, and the nature of its genome. The generated siRNAs associate with distinct Argonaute (AGO)-containing effector complexes to guide them to their RNA target molecules [1,16,17]. In plants, loading of siRNAs into a particular AGO complex is preferentially -but not exclusively- dictated by their 59 terminal nucleotides [18]. AGO1 is presented as a major antiviral slicer, but other AGO paralogs are likely involved, potentially also mediating translational repression [19] or DNA methylation in a sequence-specific manner [6]. Viroids are the smallest plant infectious agents with a genome composed of a small non-protein-coding RNA that recruit host enzymatic machineries and redirect them to its replication and systemic movement (for a review see [20,21,22,23,24,25]). Viroid species have been classified into the families Pospiviroidae, type species Potato spindle tuber viroid (PSTVd), clustering viroids localized in the nucleus wherein they replicate by an asymmetric rolling- circle mechanism [26,27], and Avsunviroidae, type species Avocado sunblotch viroid (ASBVd), whose members accumulate and replicate in the chloroplast by a symmetric rolling circle mechanism wherein the cleavage steps are mediated by hammerhead ribozymes embedded in both (+) and (2) polarity strands [28,29]. In the absence of coding properties, (+) polarity is assigned conventionally to the most abundant viroid strand accumulating in vivo. Consistent with the proposed replication mechanisms, both (+) and (2) monomeric circular genomic RNAs accumulate in tissues infected by members of the family Avsunviroidae [30], whereas only circular forms of (+) polarity are detected in tissues infected by members of the family Pospiviroidae, whose (2) RNAs accumulate at low levels and mainly as oligomeric intermediates in the replication pathway [31]. Viroid-specific highly-structured ssRNA and dsRNA species accumulate in infected plants [26,32], suggesting that viroids similarly to plant viruses are potential activators of RNA silencing. Indeed, 21–24 nt viroid-derived small RNAs (vd-sRNAs) were identified in plant tissues infected by viroids of both families [33,34,35,36,37,38]. These findings raised several still controver- sial questions on the role of RNA silencing in plant-viroid interaction (for reviews see [22,23,39]) including: i) whether viroid RNAs are both triggers and targets of this defence mechanism [40,41,42,43], ii) if this is the case, how they may escape RNA silencing to systemically infect host plants, iii) whether vd-sRNAs are directly involved in viroid pathogenesis acting like microRNAs [33,44] or trans-acting siRNAs [45], two special classes of cellular small RNA (sRNAs) targeting endogenous mRNAs (for a review see [46]), and iv) which viroid RNA(s) serve as template(s) for vd- sRNAs and in which subcellular compartment they are generated. With respect to the last question, (+) circular mature viroid RNAs have been proposed as the prevalent RNA substrates of the DCLs generating the vd-sRNAs from two nuclear replicating viroids [38,40], but more complex scenarios can be also envisaged Small RNAs of Nuclear Viroids considering the nuclear co-localization of viroid replication and part of the RNA silencing machineries. In the present study we analyzed the composition and the molecular nature of vd-sRNAs in grapevine infected by Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1), two members of the family Pospiviroidae, using the high-throughput Solexa sequencing platform to get a better insight into the biogenesis of vd-sRNAs. We identified 21-, 22- and 24-nt vd- sRNAs of (+) and (2) polarities. In contrast to previous observation using low scale sequencing, our results show a prevalence of (2) vd-sRNAs. We also show that the majority of vd-sRNAs emerge from very narrow hotspots of viroid genomes. Finally, our findings highlight new aspects of RNA silencing in the highly complex plant-viroid interactions. Results High-Throughput Sequencing Revealed Infection by Two Viroids To establish the profile of the vd-sRNAs, four different cDNA libraries of sRNAs were generated from inflorescences (hereafter denoted flower), leaves, tendrils and berries of the Pinot noir grapevine clone ENTAV115. The cDNA libraries were sequenced on the Solexa high-throughput sequencing platform, yielding 3–6 million sequences for each library. Further processing of the raw deep sequencing data consisted of: i) removal of sequence tags with a non-matching 59 or 39-adapter or resulting from adapter self- ligation, ii) adapter trimming from the remaining tags, and iii) selection of sRNA sequences ranging in size between 16 and 26 nt. To improve the significance of comparisons among the four independent sequencing events, these data were normalized with respect to the 16–26 nt total sRNA reads from flower, the sample from which the most sRNAs were sequenced. Analysis of host- derived sRNAs will be described elsewhere. The presence of vd-sRNAs in the populations of sequenced sRNAs was first revealed by searching for sRNAs perfectly matching the reference sequences of all known viroid species. The output of this first screening revealed the presence of sRNAs derived from HSVd and GYSVd1 in the grapevine libraries (data not shown), strongly suggesting that the tested plant was infected by these two viroids. Subsequent analyses of total RNA by RT- PCR, followed by cloning and sequencing of the amplification products, conclusively confirmed HSVd and GYSVd1 infections. We failed to detect Citrus exocortis viroid (CEVd), Grapevine yellow speckle viroid 2 and Australian grapevine viroid (data not shown), which are the other viroids known to naturally infect grapevine [47]. Sequencing of several clones of HSVd and GYSVd1 full-length cDNAs showed limited sequence variability in the infecting viroid populations. These analyses identified the HSVd sequence variant already reported in databases with the accession number X06873 [48] as the most abundant (master sequence) in the infecting viroid population (Fig. S1). In contrast, a GYSVd1 master sequence was not identified because the sequenced variants of this viroid differed from each other at least in one position (Fig. S2). Sequence variability of the infecting viroid populations was taken into account to improve the search of vd-sRNAs (see below). HSVd- and GYSVd1-sRNAs Are Prevalently of (2) Polarity To find as many vd-sRNAs as possible from the sequenced sRNAs populations, we searched for the sRNAs perfectly matching the sequence variants characterized here (Fig. S1 and S2) and all the sequence variants of HSVd and GYSVd1 previously reported from grapevine and deposited in databases. HSVd- and GYSVd1-sRNAs fulfilling these criteria were identified in all tissues, with the highest PLoS ONE | www.plosone.org 2 November 2009 | Volume 4 | Issue 11 | e7686