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. 2020 Apr 29;11(5):485. doi: 10.3390/genes11050485

Figure 5.

Figure 5

Identification of VviWRKY40 binding to the VviGT14 promoter using the yeast one hybrid (Y1H) (B) and electrophoretic mobility shift assay (EMSA) (C). A. The coding sequence of VviWRKY40 is introduced into the pGADT7 vector, and the three tandem w-box sequence into the pAbAi vector. B. Yeast one hybrid assay showing that yeasts carrying a combination of effector and reporter vectors can grow in the medium without or with 400 ng/mL antibiotic aureobasidin A (AbA), whereas yeasts with AD and 3× w-box could not grow on the media with 400 ng/mL AbA, indicating that the VviWRKY40 can bind with the 3 repeat w-box. C. Probes used for the EMSA. The w-box motif on the VviGT14 promoter is highlighted in red. D. EMSA showing that VviWRKY40 binds to the w-box motif of the VviGT14 promoter. The hot probe is a 3’ biotin-labledfragment of the VviGT14 promoter, while the cold probe is an unlabeled competitive probe. + and − represent the presence and absence of the VviWRKY40 or the probe, respectively, 50× and 200× indicate 50-fold and 200-fold excess of unlabeled competitive probes, respectively.