(about 135000 and 102500) and the lowest (about 5300 and 5800) reads resulting from tendril and leaf samples, respectively (Fig. 1A and 1B). Importantly, most of both HSVd- and GYSVd1-sRNAs were derived from their respective viroid (2) strand RNA: between 60 and 67% in HSVd-sRNAs, and between 70 and 75% in GYSVd1-sRNAs, with the highest (2)/(+) ratio being observed in leaf and flower samples in both cases (Fig. 1C and 1D). These findings, on the one hand, are in contrast with previous low-scale sequencing data from tomato infected by two members of the family Pospiviroidae, PSTVd [40] and CEVd [38], which revealed a prevalent accumulation of vd-sRNAs of (+) polarity. On the other hand, Machida et al. [37] identified similar levels of (+) and (2) PSTVd-sRNAs by low-scale sequencing in tomato infected by this viroid. In addition to a clear prevalence of (2) vd-sRNAs, our data also endow other questions related to their genesis and possible role in plant-viroid interaction (see below). HSVd- and GYSVd1-sRNAs Are Prevalently Composed of 21-, 22- and 24-nt Species When the HSVd-sRNAs of 20–24 nt were grouped according to their size, distributions with prevalent peaks of 21-, 22- and 24-nt species were obtained (Fig. 2A), with those of 21 nt being the most abundant in berry, tendril and leaf samples (52%, 38% and 43% HSVd-sRNAs, respectively). By contrast, the most prominent peak from the flower sample corresponded to 24-nt HSVd-sRNAs (41%) prevailing over the 21-nt (37%) and almost doubling the 24-nt HSVd-sRNAs from the other tissues (Fig. 2A). Interestingly, the size distribution of GYSVd1-sRNAs from the four grapevine tissues was similar (Fig. 2B). The possibility that the prevalence of 24-nt vd-sRNAs in flowers could derive from a technical bias Small RNAs of Nuclear Viroids seems unlikely because, in the same experiments, the sequenced host-derived sRNAs mostly belong to the 21-nt size class in all tested tissues, including flower (data not shown). These data point out that viroids infecting grapevine are targeted by DCLs generating different size classes of sRNAs, including the 24-nt sRNAs, and that this feature is not restricted to a single viroid species and/or to a single plant tissue. In line with the general major abundance reported above, (2) vd-sRNAs also prevailed in each size group and in all tissues (Fig. S3), indicating that they do not derive from a single DCL activity. The relative abundance of 24-nt vd-sRNAs is again in contrast with previous results obtained by low-scale sequencing of vd- sRNAs from PSTVd and CEVd infecting tomato, in which two prominent peaks of 21- and 22-nt RNAs and negligeble [38,40] or very low levels [37] of the 24-nt vd-sRNAs were detected. The tissues, developmental stages and different viroid-host combina- tions analyzed in each case could partially justify the divergent results reported above (see Discussion). In any case, the massive data generated by deep sequencing should allow to a more exhaustive retrieval than the limited datasets obtained by low-scale sequencing. Moreover, the existence in the infected tissues of vd- sRNAs of both polarity strands and ranging in size between 21- and 24-nt was confirmed by Northern-blot hybridization using leaf RNA preparations and digoxigenine-labeled full-length riboprobes for detecting HSVd (+) and (2) strands (data not shown). Uneven Distribution of the 59 Nucleotide in Grapevine vd-sRNAs Since the sorting process of sRNAs into effector Ago proteins is largely conditioned by their 59-terminal nucleotide [18], we Figure 1. HSVd- and GYSVd1-sRNAs from grapevine tissues are prevalently of (2) polarity. Histograms comparing the total reads and the ratio of (+) and (2) HSVd-sRNAs (A and C) and GYSVd1-sRNAs (B and D) obtained by deep sequencing from berry, tendril, flower and leaf grapevine tissues. doi:10.1371/journal.pone.0007686.g001 PLoS ONE | www.plosone.org 3 November 2009 | Volume 4 | Issue 11 | e7686