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. 2021 Mar 1;118(10):e2021351118. doi: 10.1073/pnas.2021351118

Fig. 3.

Fig. 3.

WRKY33 deSUMOylation is regulated by SPF1 and SPF2 SUMO proteases. (A) WRKY33 interacts with SPF1 and SPF2. Total protein from N. benthamiana leaves transiently expressing GFP-WRKY33 either with HA-SPF1 or HA-SPF2 was subjected to IP with anti-GFP beads (IP: αGFP) and immunoblotted with anti-HA (IB: αHA) for SCE1-HA and anti-GFP (IB: αGFP) for GFP/GFP-fusion proteins. Total protein extracts were probed with anti-HA antibody (HA-SPF1/HA-SPF2 input) for equal HA-fusion proteins. (B) WRKY33 can be SUMOylated under in vitro conditions. Recombinantly expressed GST-WRKY33 was incubated with His-SAE1, SCE1, and His-SUMO1 at 30 °C for 2 h. Besides the GST-only control, independent reactions were set up as negative controls with one component missing to ensure that all components are essential for WRKY33 SUMOylation. Reactions were stopped with 4× SDS buffer and immunoblotted with anti-GST (IB: αGST) and anti-SUMO1/2 (IB: αSUMO1) antibodies. (C) About 10 μg of SUMOylated GST-WRKY33 was incubated either with His-SPF1 or His-SPF2 for 16 h and the reactions were stopped by adding 4× dye. The samples were immunoblotted with αGST for GST-WRKY33, αHis for His-SPF1 and SPF2, and with αSUMO1 for detecting SUMOylated WRKY33. (D) WRKY33 expression is higher in spf1 spf2 mutant compared to Col-0. WRKY33 expression was analyzed in 12-d-old seedlings of the different genotypes indicated before and after treatment with 1 µM flg22. Data presented are means ± SD from two independent biological replicates and normalized to EF1α expression. A two-tailed Student’s t test showed that WRKY33 expression was significantly different in genotypes with different letters (P < 0.05). (E) spf1 spf2 mutant is more resistant to B. cinerea infection. Fully expanded leaves from 4-wk-old plants of Col-0, wrky33 k/o and spf1 spf2 were excised and drop-inoculated with 5 µL B. cinerea spore suspension. Photographs were taken 72 h postinfection and quantified in F. (F) The data represent the means ± SE of lesion sizes from 20 to 25 individual leaves per genotype. A two-tailed Student’s t test was performed among means of lesion sizes. Genotypes with different letters were significantly different from others (P < 0.05).