Small RNAs of Nuclear Viroids Figure 6. Specific size-classes vd-sRNAs may largely prevail at certain genomic positions. Histograms comparing location of the 59 termini, frequency and size distribution of (2) vd-sRNAs corresponding to the HSVd hotspot 3 (HS3) and recovered from the different grapevine tissues. Numbers are referred to HSVd sequence variant with the accession number X06873. 59R39 orientation is from right to left. doi:10.1371/journal.pone.0007686.g006 most abundant viroid RNA accumulating in vivo. In contrast, our data (Fig. 1 and Fig. S3) show that (2) vd-sRNAs largely dominate the sRNA populations of both HSVd and GYSVd1 in grapevine, strongly supporting that a more complex mechanism is involved in vd-sRNAs biogenesis. This consideration is in line with a similar prediction by Machida et al. [37], who identified relatively abundant (2) PSTVd-sRNAs in tomato leaves by low-scale sequencing. Whether the prevalent presence of (2)-vd-sRNAs as reported here is applicable to other viroids remains to be seen by deep sequencing vd-sRNAs in other viroid-host combinations. The (2) vd-sRNAs could derive from DCL(s) targeting dsRNAs generated during the nuclear replication or resulting from the activity of host RDRs recognizing some viroid RNA features (i.e. the lack of cap structure and poly-A tail) as aberrant traits [49,50]. Alternatively, DCL enzymes may preferentially target the (2) multimeric replicative intermediates, which are highly-structured ssRNAs. It is worth noting that RNA samples used in the present study were from a grapevine plant grown in the field. This plant was obtained by vegetative propagation from a mother plant presumably already infected by both HSVd and GYSVd1, which are the most widespread viroids in grapevine. Therefore, infection of both viroids in this woody plant, which did not show any symptoms, was very likely in stationary stage. We can assume that some of the above-mentioned studies with herbaceous host like tomato, performed at a logarithmic-stage of viroid infection, may show different vd-sRNA profiles, since the accessibility to the RNA silencing machinery of different viroid RNA species may be continuously changing. In line with this hypothesis, the results of Machida et al. [37] showed that the distribution profiles of PSTVd-sRNAs from symptomatic tomato leaves became more heterogeneous with time. Assuming that the four DCLs identified in the grapevine genome [51,52] have the same subcellular localization and biochemical activities as their homologous in Arabidopsis [53], the prevalence of 21-, 22- and 24-nt species observed in the size distribution profiles of the sequenced HSVd- and GYSVd1- sRNAs (Fig. 2 and Fig. S3) indicates that viroid RNAs are targeted by multiple DCLs. The 21-nt vd-sRNAs could derive from the nuclear activity of DCL1 targeting highly-structured genomic viroid RNAs of both polarity strands by a mechanism resembling miRNA biogenesis and/or from the activity of DCL4, which acts in concert with RDR6 in Arabidopsis [54]. Instead, the 22-nt vd- sRNAs likely derive from the activity of DCL2 that is hierarchically involved in antiviral defense together with DCL4 [8,9,55,56]. Identification of 24-nt vd-sRNAs in grapevine is consistent with the prediction that viroids are also targeted by a DCL homologous to the Arabidopsis DCL3 [51,52]. Because 24-nt sRNAs are involved in RNA directed-DNA methylation (RdDM), our finding opens new scenarios on both the biogenesis and possible role of vd- sRNAs in plant-viroid interaction. DCL3 acts in concert with ARGONAUTE 4 (AGO4), RDR2, and RNA polymerases IV and V (Pol IV and V) in a spatio-temporal regulated pathway. Current models propose that 24-nt sRNAs are loaded into AGO4 for targeting and methylating DNA by de novo cytosine methyltrans- ferase, DRM2, in concert with Pol V and other proteins [57,58]. PLoS ONE | www.plosone.org 8 November 2009 | Volume 4 | Issue 11 | e7686