RNA-dependent RNA polymerase (RDR2) and DICER-LIKE (DCL) are involved in the biogenesis of MiS small RNAs. Small RNA-enriched fractions isolated from wild type and RNAi lines were prepared and analyzed by Northern blot hybridization as described in Figure 3. Blots were hybridized with MiS and miRNA probes (see Supplemental Table S3.2 for a description of probes). (A) Detection of MiS5 (Sd3090 probe) small RNA and MIR171 in (St WT) wild-type S. tuberosum Katahdin; (St rdr2) S. tuberosum Katahdin RDR2 RNAi line; (Sl WT) wild-type S. lycopersicum VF36; (Nt WT) wild-type N. tabacum SR1; (At) wild-type Arabidopsis Col; and (Os) wild-type Oryza sativa Nipponbare. (B) Detection of MiS17 plus strand small RNA and MIR171 in wild-type tobacco SR1 and N. tabacum RNAi lines targeting (DCL2) DCL2-1, DCL2-2, and DCL2-3; (DCL3) DCL3-2 and DCL3-2; and (DCL4) DCL4-1 and DCL4-3 (for construction and characteristics of tobacco RNAi lines, see Supplemental Figs. S2.1 and S2.2; Supplemental Tables S4.1–S4.4). (A,B, bottom panels) The ethidium bromide (EtBr) staining of gels as loading controls. The numbers below the hybridization panels in A and B indicate the relative abundance (RA) of siRNA detected by Northern blot hybridization in each sample compared to StWT in A and NtWT in B. Relative siRNA abundance has been normalized (WT = 1.0) according to EtBr staining of gels in A and B.
