Endogenous RNA interference of chalcone synthase genes in soybean: Formation of double-stranded RNA of GmIRCHS transcripts and structure of the 5′ and 3′ ends of short interfering RNAs

In yellow soybean, seed coat pigmentation is inhibited via endogenous RNA interference (RNAi) of the chalcone synthase (CHS) genes. Genetic studies have shown that a single dominant gene, named the I gene, inhibits pigmentation over the entire seed coat in soybean. We previously isolated a candidate for the I gene from the yellow soybean genome with the I/I genotype, and designated it GmIRCHS. A structural feature of GmIRCHS is a perfect inverted repeat of the pseudoCHS gene lacking 5′-coding region. This suggests that the double-stranded RNA (dsRNA) structure of the pseudoCHS gene may be formed in the GmIRCHS transcript. RNAi is triggered by the dsRNA for a target gene, so the GmIRCHS transcript is likely to be a trigger for RNAi of CHS genes. In this study, we identified a 1087-bp dsRNA, including pseudoCHS region ranging from most of exon 2 to 3′-UTR, in the GmIRCHS transcript. Interestingly, this dsRNA was detected not only in the seed coat but also in the cotyledon and leaf tissues. Previously, CHS RNAi has been shown to be restricted to the seed coat, and we reported that endogenous short interfering RNAs of CHS genes (CHS siRNAs) are detected only in the seed coat and not in the cotyledon and leaf tissues. Taken together with these previous reports, our result suggests that seed-coat specificity of CHS RNAi may be determined in the amplification step of CHS siRNAs rather than dsRNA formation in the GmIRCHS transcript. Our studies further revealed that CHS siRNAs are modified at the 3′ ends and bear 5′ monophosphorylated ends, suggesting that CHS siRNA duplexes are generated by Dicer-like enzyme from CHS dsRNA and subsequently modified at the 3′ ends for stabilizing CHS siRNAs.