Transcriptional Activation and Yeast One-Hybrid Assay

In order to test the transcriptional activity of TaWRKY75-A, the full-length coding sequence (222 amino acids) and interceptive sequences including 1–44 amino acids (N-terminal), 1–102 amino acids, 44–222 amino acids, and 102–222 amino acids (C-terminal) were cloned into the pGBKT7 vector, respectively. The empty pGBKT7 vector was used as the negative control. The generated constructs were transformed into the AH109 yeast strain. The transformants were diluted into different concentrations and selected on SD/-Trp and SD/-Trp/-His/-Ade/X-α-gal deficiency media. The transcriptional activities were evaluated according to their growth status at 30°C for 3 days in darkness.

To analyze TaWRKY75-A binding to W-box motif, yeast one-hybrid assay was performed. The promoter sequence of Arabidopsis RD29A gene (AT5G52310), which contains triple tandem repeats of the TTGAC cis-acting element, was cloned into the yeast expression vector pHis2.1 as the reporter construct (pHis2.1-W-box). The full-length coding sequence of TaWRKY75-A was cloned into the pGADT7 vector and fused with the GLA4 domain to generate the effector pGADT7- TaWRKY75-A. Both recombinant vectors were transformed into yeast strain Y187 and selected on SD/-Trp/-Leu plates at 30°C for 2 days. The surviving colonies were then transferred to SD/-Trp/-Leu/-His medium containing 35 mM 3-AT. The interaction between TaWRKY75-A and W-box element was evaluated by the transformants’ growth performance. All the primers used here are listed in Supplementary Table 1.