Figure 8.

Phosphorylation of WRKY33 Does Not Alter Its DNA Binding Ability to the W-Box cis-Element.

(A) EMSA was performed using freshly prepared recombinant WRKY33^WT or WRKY33^SA protein and ³²P-labeled W-box probe. The specificity of W-box binding activity was demonstrated by competition assay using 250-fold excess unlabeled W-box, GCC-box, or as1-box DNAs.

(B) Phosphorylation of WRKY33 does not enhance its W-box binding activity. Freshly prepared recombinant WRKY33^WT was phosphorylated using the activated MPK3 and MPK6 (equal mixture). A control reaction without MPK3/MPK6 was processed side-by-side. The W-box binding activity of the phosphorylated and unphosphorylated (from the control reaction) was determined by EMSA as in (A).