Due to their nuclear replication and localization, certain viroid RNAs could enter this pathway at different levels. Qi and Ding (2003) showed that both (+) and (2) PSTVd RNAs localize in the nucleoplasm, whereas the (+) polarity strands prevalently accu- mulate in the nucleolus, wherein presumably they are cleaved and circularized [59]. However, it is unlikely that the highly-structured genomic (+) viroid RNA accumulating in the nucleolus is directly targeted by DCL3 because this scenario does not explain why both (+) and (2) 24-nt vd-sRNAs have been identified in infected grapevine tissues (Fig. S3). A more likely alternative is that (+) viroid RNAs migrating to the nucleolus could be recognized by RDR2, thus entering the DCL3 degradation pathway at this level. Identification of high levels of 24-nt vd-sRNAs in infected grapevine is consistent with the previous finding that replicating viroid RNAs induce de novo methylation of homologous transgenic DNA sequences, the first proof that DNA methylation is an RNA- mediated process [60]. Whether viroids may directly or indirectly interfere with host DNA methylation profiles, as well as whether 24- nt vd-sRNAs may target DNA methylation of host genes, is not known and remains an interesting challenge for next studies on viroid pathogenesis. However, it is noteworthy that the activity of an RNase-III like enzyme has been involved in the cleavage step of (+) viroid multimeric forms during replication of nuclear viroids [61], suggesting that interaction between viroid RNAs and RNA silencing pathways could be more complex than suspected before. In line with this considerations, it is important that a peak corresponding to 24-nt vd-sRNAs has not been identified in the size distribution profiles of vd-sRNAs from the chloroplast-replicating Peach latent mosaic viroid sequenced by low-scale [62] and deep sequencing [63]. Altogether these data support the notion that vd-sRNAs of members belonging to the families Pospiviroidae and Avsunviroidae arise, at least in part, from different pathways as a result of their diverse subcellular replication sites. In this same context, targeting of nuclear-replicating viroid RNAs by DCL3 supports that some vd-sRNAs are indeed generated in the nucleus. Pertinent in this respect is the observation that DCL2 and DCL4 can process Pol-IV derived dsRNAs when DCL3 is mutated in Arabidopsis [64], which suggests the possibility that 21- and 22-nt vd-sRNAs of nuclear replicating viroids could partially derive from a similar redundant activity of host DCLs. Further studies are needed to establish whether the 21- and 22-nt vd-sRNAs are actually produced in the nucleus before being exported to the cytoplasm wherein vd-sRNAs seem to accumulate [65], or directly in the cytoplasm or in both subcellular compartments. Thus, the possibility exists that the RNA silencing machinery targets nuclear viroids at several key points of their infectious cycle, including replication and cytoplasmic trafficking [66]. Mapping of vd-sRNAs to the viroid genomic RNAs has provided several insights on their genesis and biological function. The few number of hotspots in both (+) and (2) viroid genomic RNAs (Fig. 4), on the one hand, suggests that very limited regions of the viroid genome are potentially targeted by DCLs and, on the other hand, reinforces the view that the (+) circular forms cannot be the prevalent substrate for DCL-mediated generation of grapevine vd-sRNAs, as suggested before in other viroid-host combinations [38,40]. Therefore, besides the genomic (+) and (2) viroid RNAs, the viroid-derived dsRNAs synthesized during replication or by host RDRs could be proper substrates for generating both (+) and (2) vd-sRNAs. However, the distribution profiles of vd-sRNAs do not support the possibility that vd-sRNAs mapping to (+) and (2) hotspots derive directly from the same dsRNA molecule because they do not form duplexes with two 39- protruding nucleotides, the hallmarks of DCL activity. Therefore, (+) and (2) vd-sRNAs appear to be generated by two independent processes, additionally suggesting that the limited accessibility to Small RNAs of Nuclear Viroids DCLs is an intrinsic property of each viroid RNA. In line with this view, the hotspots of HSVd-sRNAs and GYSVd1-sRNAs do not cover the same viroid structural domains (Fig. 5). However, it is intriguing that, for each viroid strand, the vd-sRNAs of all size classes actually map to the same regions, indicating similar preferences of the different DCLs. Possible explanations for the hotspot profiles of vd-sRNAs from both viroids, which have been confirmed by Northern-blot hybridization, include: i) certain RNA-binding proteins [67] may protect genomic regions of (+) and (2) viroid RNAs from DCL digestion; ii) RDR(s) could have low processivity and synthesize short viroid dsRNAs; iii) vd-sRNAs might be differentially targeted by one or more exoribonucleases, like those acting upon mature miRNAs in Arabidopsis [68]; and iv) vd-sRNAs could have differential stability depending on whether they are or not incorporated into RISC complexes containing distinct AGO members [69]. In this respect, it is noteworthy that the 59 terminal nucleotide of most grapevine vd-sRNAs is C residue (Fig. 3). Sorting of Arabidopsis sRNAs is largely directed by the 59 terminal nucleotide [18] and sRNAs with a C at this position are preferentially recruited by AGO5, whose function has not been explored. Moreover, the second most abundant nucleotide at the 59 terminal position of grapevine 24-nt vd-sRNAs (the A residue) is preferentially found at the same position in the 24-nt sRNAs recruited by Arabidopsis AGO4, which acts in concert with DCL3, suggesting that 24-nt vd-sRNAs may enter the DNA methylation pathway. It should be mentioned that our protocol used for the preparation of sRNAs cDNA library is not apropriate to amplify and sequence sRNAs that are the products of unprimed RNA synthesis catalyzed by host RDR [70]. Since they are product of RNA synthesis they have 59-triphosphate, which does not allow the ligation of the 59- adaptor to sRNAs. Thus, if these sRNAs exist, they escaped from our analysis, although the existence of this type of sRNAs has not been reported from plants. In addition, very recent analysis of tombusvirus derived vsiRNAs failed to detect sRNAs produced by unprimed RDR synthesis (Szittya et al., unpublished). Viroid-derived small RNAs show remarkable similarites to as well as differences from plant virus-derived small RNAs. Plant viruses with RNA genome replicate in cytoplasm and generate predominantly 21- and 22-nt long vsiRNA [11,13,71,72], while the vsiRNAs deriving from plant DNA viruses replicating in the nucleus are mostly 21-, 22- and 24-nt long [73]. Interestingly, in contrast to a chloroplast replicating viroid [62,63], two members of nuclear-replicating viroids also generate vd-sRNAs of 21-, 22- and 24-nt, suggesting that the site of replication of invading nucleic acids is an important factor in the genesis of their small RNAs. Although both viruses and viroids show uneven sRNAs distribu- tion profiles along their respective genomes, the prevalent polarity of sRNAs varies remarkably depending on the specific system, the replication strategy and, very likely, the infection stage. Finally we would like to remark on the high potential offered by grapevine for studying plant-viroid interactions. For example, taking advantage of the available complete grapevine genome sequence, we searched for possible grapevine targets of the sequenced vd-sRNAs and identified only one perfectly maching 21-nt HSVd species, whereas several additional targets were identified with one or two mismatches. However, the frequency of vd-sRNAs having a putative target in grapevine genome was always extremely low and information on possible functional roles of these potential genomic targets are still lacking. Therefore, to understand whether vd-sRNAs may actually have any direct biological impact on host gene expression could be an interesting aim for future studies. PLoS ONE | www.plosone.org 9 November 2009 | Volume 4 | Issue 11 | e7686