Materials and Methods Plant Material All plant material was from Vitis vinifera, cultivar ‘‘Pinot noir’’, clone ENTAV115 grown in collection fields. Young leaves and tendrils were collected from 1st to the 3th internode from the shoot apex. The inflorescences were collected at their appearance, whereas the small fruits used were 1–4 mm in size. Extraction, Fractionation and Sequencing of Grapevine sRNAs Vitis vinifera total RNA was extracted following the method reported by Turturo et al. [74] except for the silica particle absorption. Low molecular weight RNA (LMWR) was further enriched by using Quiagen RNA/DNA midi kit and following the manual procedures. LMWR was used to generate short RNA libraries as indicated by German et al. [75]. Deep-sequencing was done on Illumina Solexa platform using the standard protocol of manufacturer. Characterization of Infecting Viroids Total RNA preparations were obtained from grapevine leaves as reported by Turturo et al. [74]. cDNAs were synthesized by using the high-capacity cDNA Reverse Transcription kit as suggested by the supplyer (Applied Biosystems). PCR amplification reactions were carried out with the following primer pairs: HSVd-83M-Rev: 59- AACCCGGGGCTCCTTTCTCA-39 and HSVd-78P-For: 59-AA- CCCGGGGCAACTCTTCTC-39, specific for full-length HSVd cDNA amplification [76]; GYSVd1-Rev: 59-GCGGGGGTTC- CGGGGATTGC-39 and GYSVd1-For: 59-TAAGAGGTCTC- CGGATCTTCTTGC-39, specific for full-length GYSVd1 cDNA amplification; GYSVd2-C2-Rev: 59-CCGAGGTGTAACCACAG- GGAACC-39 and GYSVd2-H1-For: 59-TTGAGGCCCGGC- GAAACGC-39, specific for partial (194 bp) GYSVd2 cDNA amplification [74]; CEVd-C1Rev: 59-CGAAAGGAAGGAGAC- GAGCTCCTG-39 and CEVd-H3For: 59-TTCAGGGATCCCC- GGGGAA-39, specific for partial (115 bp) CEVd cDNA amplifica- tion [77]; AGVd-Rev: 59-CTCGACGACGAGTCGCCAGGT- GAG-39 and AGVd-For: 59-GTCGACGAAGGGTCCTCAGCA- GAG-39, specific for full-length (375 bp) AGVd cDNA amplification. Full-lenght HSVd and GYSVd1 monomeric cDNAs were cloned in p-GEM-T- easy vector (Promega) and sequenced. Multiple alignments of viroid sequence variants were performed with the CLUSTAL W program [78]. Sequence Analysis of HSVd- and GYSVd1-sRNAs After trimming the adapters from the resulting sequences, they were sorted into separate files according to the length. Sequences between 20 and 24 nt were pooled and each set of sequences was analyzed by BLAST [79] against the nucleotide sequence of HSVd and GYSVd1 variants cloned and sequenced in this study and of variants of these viroid species previously reported on grapevine and deposited in databases (Materials and Methods S1). No mismatch was allowed and the circularity of the viroid genome was taken into consideration. A set of perl scripts to analyze and visualize the mapping data search for hot spots was developed. Northern-Blot Hybridization of vd-sRNAs with Oligodeoxyribonucleotides LMWR were electrotransferred and fixed by UV irradiation to nylon membranes (Hybond-N, Amersham). The membranes were hybridized at 42uC in DIG-hybridization buffer (Roche) with riboprobes corresponding to the full length HSVd genome RNAs Small RNAs of Nuclear Viroids of both polarities. In additional experiments the membranes prepared as reported were hybridized at 37uC in Perfect-Hyb buffer (Sigma) with each of the following 59-radiolabeled probes: Phs-1 (59-GATGCCACCGGTCGCGTCTCATCGGAAG-39) and Phs-2 (59-CTTCTTTACCTTCTTCTGGCTCTTCCGAT- GAGACG-39) complementary and identical to positions 201–229 and 180–214, respectively, and Phs-3 (59-CAAAAGCAGGTTG- GGACGAACCGAGAGGTGATGCC-39) and Phs-4 (59-GGC- ATCACCTCTCGGTTCGTCCCAACCTGCTTTTG-39) com- plementary and identical to positions 223–257, respectively, of the HSVd variant X06873; Pgy-5 (59-GCACTCGGAATGCACCC- CTTCGTCGACGACGAG-39) and Pgy-6 (59-GCCTATTCAG- CATCGCGTCCTTGAGGC-39) complementary and identical to positions 96–128 and 198–224, respectively, and Pgy-7 (59-GA- GCTTGTACCAACGCGCCCCGCGAGTGCAATC-39) and Pgy-8 (59-GATTGCACTCGCGGGGCGCGTTGGTACAAG- CTC-39) identical and complementary to positions 315–346, respectively, of the GYSVd1 variant GQ995473. After overnight hybridization, the membranes were washed twice with 2X SSC plus 0.1% SDS for 10 min at room temperature, and once with 0.1X SSC plus 0.1% SDS at 55uC for 15 min, and examined with a bioimage analyzer (Fujifilm FLA-5100). Supporting Information Figure S1 Multiple sequence alignment of HSVd cDNA variants from grapevine Pinot noir ENTAV115 identified in this study. Dashes and stars denote gaps and nucleotide identity, respectively. HSVd variants PN.1, PN.3, PN.7, PN.8, PN.10, PN.13 and PN.14 are identical to HSVd variant with accession number X06873, which is the master sequence in the infecting viroid population. Accession numbers for variants PN.9, PN.11 and PN.12 are GQ995464, GQ995465 and GQ995466, respec- tively. Nucleotides in red correspond to changes with respect to the master sequence. Numbers at the end of each line indicate nucleotide positions of each variant in the multiple alignment. Found at: doi:10.1371/journal.pone.0007686.s001 (1.69 MB TIF) Figure S2 Multiple sequence alignment of GYSVd1 cDNA variants from grapevine Pinot noir clone ENTAV115 identified in the present study. Dashes and stars denote gaps and nucleotide identity, respectively. Nucleotides in red correspond to changes with respect to the consensus sequence, which corresponds to the sequence variant GYSVd1.PN.22 (accession number GQ995473) in the alignment. Numbers at the end of each line indicate nucleotide positions of each variant in the multiple alignment. Found at: doi:10.1371/journal.pone.0007686.s002 (1.16 MB TIF) Figure S3 Size distribution (20–24 nt) of vd-sRNAs from different grapevine tissues. Histograms comparing the size distribution (20–24 nt) of HSVd- (upper panels) and GYSVd1- sRNAs (lower panels) isolated from berry (A and E), tendril (B and F), flower (C and G) and leaf (D and H). Vd-sRNAs of 21 nt of both viroids were the most abundant in berry, tendril and leaf samples, whereas the most prominent peak from the flower samples corresponded to 24-nt HSVd-sRNAs and GYSVd1- sRNAs. Found at: doi:10.1371/journal.pone.0007686.s003 (8.98 MB TIF) Figure S4 Frequency of the 59-terminal nucleotide in vd-sRNAs. Histograms comparing the size distribution (20–24-nt) and nucleotide at the 59 termini of HSVd-sRNAs (upper panels) and GYSVd1-sRNAs (lower panels) from berry (A and D), flower (B and E) and leaf (C and F). Found at: doi:10.1371/journal.pone.0007686.s004 (10.22 MB TIF) PLoS ONE | www.plosone.org 10 November 2009 | Volume 4 | Issue 11 | e7686