Structural features of GmIRCHS, candidate of the I gene inhibiting seed coat pigmentation in soybean: implications for inducing endogenous RNA silencing of chalcone synthase genes

Most commercial soybean varieties have yellow seeds due to loss of pigmentation in the seed coat. The I gene inhibits pigmentation over the entire seed coat, resulting in a uniform yellow color of mature harvested seeds. We previously demonstrated that the inhibition of seed coat pigmentation by the I gene results from post-transcriptional gene silencing (PTGS) of chalcone synthase (CHS) genes. Little is known about the structure of the I gene and the mechanism by which it induces PTGS of CHS genes. Here, we report a candidate of the I gene, GmIRCHS, which consists of a 5'-portion of a DnaJ-like gene containing a promoter region and a perfect inverted repeat (IR) of 1.1-kb truncated CHS3 sequences (5'-Delta CHS3 and 3'-Delta CHS3). RT-PCRs and RNase protection assay indicated the existence of the read-through product from 5'-Delta CHS3 to 3'-Delta CHS3 and the dsRNA region of Delta CHS3, suggesting that dsRNA of Delta CHS3 could be transcribed from GmIRCHS and could induce PTGS of CHS genes. Moreover, the IR structure of Delta CHS3 in GmIRCHS was lost in the soybean mutants in which I was changed to i, supporting the conclusion that GmIRCHS is the I gene.