Figure 7.

Binding of CrWRKY1 to W box elements in the TDC promoter. A, Autoradiographs show up-shifted bands of the EMSA using purified GST-CrWRKY1 protein and probe 1 (containing two W boxes), probe 2 (containing one W box), or mProbe 2 (containing a mutated W box). Gel 1 shows the products of DNA-binding reactions in the absence and presence of GST-CrWRKY1 or labeled and unlabeled probe 1. Gel 2 is the same as gel 1 except that labeled and unlabeled probe 2 were used. Gel 3 shows the reaction products of mProbe 2 in the absence and presence of GST-CrWRKY1 proteins. B, Yeast one-hybrid assays demonstrate CrWRKY1 activation of the C. roseus TDC promoter. The plasmid harboring a GAL4 activation domain/CrWRKY1 fusion (pAD-CrWRKY1) was cotransformed with the pTDC-HIS2 or pmTDC-HIS2 reporter plasmid. The transformants were grown in either double selection medium (SD-Leu-Trp) or triple selection medium (SD-His-Leu-Trp) with 100 mm 3-AT. Sector 1, pHIS2-TDC + pAD-CrWRKY1; sector 2, pHIS2-mTDC + pAD-CrWRKY1; sector 3, pHIS2-TDC + pAD; sector 4, pHIS2-mTDC + pAD. C, CrWRKY1 binds to W box elements of the TDC promoter in C. roseus protoplasts. The reporter plasmid (TDC), consisting of the Luciferase gene under the control of tandem repeats of W box elements upstream of a minimal 35S promoter, was either transformed alone or cotransformed with AD-CrWRKY1 into C. roseus protoplasts. Another reporter plasmid (mTDC), which is identical to the TDC reporter except that the W box sequence is mutated, was also transformed alone or together with AD-WRKY1 into the protoplasts. The relative levels of activation of the Luciferase gene are presented. Experiments were performed in triplicate.