Fig. 4.
WRKY33 SUMOylation regulates phosphorylation of WRKY33 without affecting its DNA-binding activity. (A) B. cinerea induced WRKY33 SUMOylation is essential for its transcriptional activity. Four-week-old plants of the different genotypes indicated were either sprayed with water as mock or spray-inoculated with B. cinerea and PAD3 expression was compared 16 h postinfection in the different genotypes. Means from two independent biological replicates are denoted as bar graphs with error bars representing SD EF1α was used for normalization. Bars with different letters were significantly different from others (two-tailed Student’s t test; P < 0.05). (B) SUMOylation is required for transcriptional activity of WRKY33 after flg22 treatment. PAD3 expression was analyzed in 12-d-old seedlings of the different genotypes after treatment with 1 µM flg22. For mock-treatment the seedlings were left in water for the same duration. Data presented are means ± SD from two biological replicates and normalized to EF1α expression. Bars with different letters were significantly different from others (two-tailed Student’s t test; P < 0.05). (C) SUMOylation does not affect WRKY33 DNA-binding activity. ChIP assay showing WRKY33 DNA-binding activity in WRKY33 and WRKY333K/R ± flg22 (1 µM) by comparing the binding to PAD3 promoter. Fold enrichment was calculated relative to the last exon of At4g26410. Data presented are means ± SD from two biological replicates. A two-tailed Student’s t test showed that promoter enrichment was significantly different for genotypes with different letters (P < 0.05). (D) SUMOylation is required for efficient WRKY33 phosphorylation. Total protein from 12-d-old seedlings of WRKY33 and WRKY333K/R transgenics ± flg22 (1 µM) was immunoprecipitated with anti-GFP (IP: αGFP) beads and subjected to Phos-tag gel analysis to detect WRKY33 phosphorylation status. They were immunoblotted with anti-GFP (IB: αGFP) antibodies (Upper) and a regular immunoblot was also done to detect total WRKY33 protein (Lower).