Figure 3. Post-translational regulation of OsWRKY53 transactivation activity by OsMKK4^DD.

A, Diagrams of effector, reporter, and reference plasmids used in transient reporter gene assays. B, Regulation of transactivation activity of OsWRKY53 by OsMKK4^DD in cultured rice cells. A GUS construct was used as a negative control. Firefly LUC activity was normalized against that of Renilla LUC. Values of LUC activity are shown relative to those of GAL4-W53 + GUS (n = 4); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. Statistically different data groups are indicated using different letters (p<0.01 by One-way ANOVA with Tukey post hoc test). C, Enhanced transactivation of a phosphorylation-mimic mutant of OsWRKY53 in cultured rice cells. Transient reporter gene assay was performed as in B. Values of LUC activity are shown relative to those of GAL4-W53 (n = 4); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. Statistically different data groups are indicated using different letters (p<0.05 by One-way ANOVA with Tukey post hoc test). D, Expression analysis of GAL4DB and GAL4DB-OsWRKY53 variants in cultured rice cells. qRT-PCR analysis was performed using total RNA isolated from rice cells after particle bombardment with effector, reporter and reference plasmids. Values indicate relative mRNA levels normalized to the expression of the RLUC gene (n = 4); bars indicate the standard error of the mean. W53, W53SA and W53SD indicate the native OsWRKY53, an OsWRKY53 mutants whose Ser residues in the SP cluster were substituted to Ala, and an OsWRKY53 mutant that mimics the phosphorylated form, respectively.