Fig. 6. CsWRKY70 specifically binds to the CsSAMT promoter and activates the expression of CsSAMTs.

a SDS–PAGE was conducted to detect the purification of the recombinant CsWRKY70 protein. b, c EMSA. Biotin-labeled probe or mutant probe was incubated with His-CsWRKY70 protein, and the mixtures were separated on a 6% native polyacrylamide gel. The symbols + and + + indicate increasing amounts of unlabeled probe used for competition with or increasing the amounts of mutant probe used to test the binding specificity. d Schematics of the reporter and effector constructs used in the DLR experiment. The CsSAMT promoter was inserted into a PGREENII 0800-LUC vector (KpnI and NcoI) as a reporter, and the full-length CsWRKY70 was fused to a PEAQ vector (AgeI and XhoI) as an effector. Each pair of effector and reporter plasmids was transformed into an A. tumefaciens strain and then transiently expressed in tobacco leaves (Nicotiana benthamiana). e CsWRKY70 transactivates the CsSAMT promoters. Activation was indicated by the ratio of LUC to REN. The ratio of LUC/REN of the empty vector plus promoter was used for calibration and set to 1. Each value represents the mean ± s.e.m. (n = 5). **Statistically significant differences at P < 0.01. f Effect of transient CsWRKY70 expression on endogenous MeSA biosynthesis genes (CsSAMTs). Error bars indicate standard errors (SEs) from three replicates