Small RNAs of Nuclear Viroids Figure 5. HSVd-sRNAs and GYSVd1-sRNAs do not cover the same viroid structural domains. Sequence and computer-predicted secondary structure for the (+) strand of the HSVd (sequence variant X06873) (A) and the GYSVd1 (sequence variant GQ995473) (B), corresponding to the master and the consensus variants in the grapevine sequenced viroid populations, respectively. The viroid sequences covered by vd-sRNAs corresponding to hotspots (HS1, HS2 and HS3 for HSVd, and GY1, GY2 and GY3 for GYSVd1) are denoted by arrows whose sense indicates 59R39 orientation. The position of five structural domains proposed for PSTVd and closely-related viroids [80] are indicated (P: pathogenic; V: variable; C: central; T1: terminal left; T2: terminal right), although no data on the functional properties of these regions in HSVd and GYSVd1 are available. The secondary structures were obtained by the program Mfold [81]. doi:10.1371/journal.pone.0007686.g005 and (2) GYSVd1-sRNAs mapped to short regions covering only 22% and 17% of the genomic (+) and (2) viroid RNAs, respectively. Hotspots GY1 and GY2, containing (+) sRNAs, mapped to the left side of the central region and to the central region itself of the predicted GYSVd1 rod-like secondary structure, respectively, whereas hotspot GY3, containing (2) sRNAs, mapped to the terminal right domain including the terminal loop (Fig. 5B). Therefore, in contrast to HSVd, (+) and (2) GYSVd1-sRNAs mapped to distal and not overlapping regions, suggesting that localization of vd-sRNAs hotspots within the genome is an intrinsic property of the infecting viroid RNAs, thus differing between viroid species and between (+) and (2) strands of the same viroid RNA. Paralleling HSVd results, genome mapping of (+) and (2) GYSVd1-sRNAs from berry, flower and leaf tissues, generated distribution profiles essentially coincidental with those obtained from tendrils (Fig. S9 and S10). These data, besides confirming again the reproducibility of the deep sequencing method, also suggest that all tissue samples, including leaves from which the lowest level of vd-sRNAs was recovered, allowed exhaustive vd-sRNAs characterization. A further size distribution analysis within the hotspots showed that in most cases the 59 terminus of vd-sRNAs of different sizes mapped concurrently to the same position, although specific size- class sRNAs may largely prevail at certain genomic positions, as exemplified by Figure 6 showing details of HS3. In this case, positions 206, 210 and 212 correspond prevalently to the 59 termini of 21-nt sRNAs, whereas positions 208, 209 and 214 were almost exclusively occupied by the 59 termini of 24-nt sRNAs (Fig. 6). We do not know whether the differential sRNA distribution within the hotspots may have any significance, although the highly coincidental profiles of the four independent sequencing experiments are intriguing. Similar considerations can be extended to the other vd-sRNAs hotspots (data not shown). Mapping data of both HSVd- and GYSVd1-sRNAs were validated by Northern-blot hybridization using probes consisting of DNA oligonucleotides targeted against viroid genomic regions characterized by high (hotspots) and low vd-sRNA densities (Fig. 5). The obtained hybridization signals with hotspot-specific probes were much stronger than those generated by probes specific for viroid regions with low density of vd-sRNAs (data not shown), thus supporting the reliability of the deep sequencing data. Discussion In this work, high-throughput sequencing of sRNAs from different grapevine tissues has been applied to further characterize the vd-sRNAs of two nuclear-replicating viroids, HSVd and GYSVd1, and new data about their genesis and possible biological roles have been obtained. Previous efforts of characterizing vd- sRNAs by low-scale sequencing revealed that (+) vd-sRNAs of PSTVd [40] and CEVd [38] are the most abundant in tomato plants infected by these viroids. Based on these findings, it was proposed that vd-sRNAs mostly derive from direct DCL targeting of (+) viroid genomic RNA, likely its circular form, which is the PLoS ONE | www.plosone.org 7 November 2009 | Volume 4 | Issue 11 | e7686