Figure 12.

BnWRKY15‐mediated modulation of BnWRKY33. (a) Transcriptional activation ability changes of BnWRKY33 were revealed by the relative LUC activity of the reporter. Arabidopsis protoplasts were cotransformed with pBD‐BnWRKY33 effector plasmids (Figure 7a) and 15SK plasmids (Figure 4a) together with reporter plasmids. Transcriptional activation ability produced by cotransforming the null plasmid pGreenII 62‐SK (Figure 4a) with the pBD‐BnWRKY33 effector into Arabidopsis protoplasts was used as a control. The GAL4 BD effector served as a negative control. The assays were repeated at least three times. The data represent the means ± standard errors (n ≥ 3). (b) Competitive binding of BnWRKY15 and BnWRKY33 to the promoter of BnWRKY33. Electrophoretic mobility shift assays were performed using recombinant BnWRKY15 or BnWRKY33 proteins and Cy5‐labelled W‐box probes. Equal amounts of recombinant BnWRKY15 and BnWRKY33 protein or a 2:1 ratio of BnWRKY15:BnWRKY33 fusion protein was incubated with Cy5‐labelled W‐box probes and separated using nondenaturing polyacrylamide gel electrophoresis. W‐box probes alone were used as controls. (c) Expression levels of BnWRKY15 in both the BnWRKY15‐overexpressing line and the control (Westar) after inoculation with Sclerotinia sclerotiorum. (d) Expression levels of BnWRKY33 in both the BnWRKY15‐overexpressing line and the control (Westar) after inoculation with S. sclerotiorum. Columns with different shades of grey indicate expression levels at different time points. Quantitative results represent three biological repeats. The error bars stand for standard error.