Figure 8.

HUB1 and HUB2 regulated the expression of WRKY33 in response to Vd-toxins, and WRKY33 regulated the expression of AtRbohD in Arabidopsis. A, The relative expression levels of WRKY33 induced by 150 μg mL⁻¹ Vd-toxins in wild-type Columbia-0 (Col-0), the hub1-4, hub2-2, and hub1-4 hub2-2 mutants, and the HUB1/hub1-4 complementation line. Error bars indicate sd. B, Schematic diagram of the WRKY33 gene. P1 and P2 are gene body regions; the arrow indicates the transcription start site. C and D, Relative enrichments of H2Bub1 and H3K4me3 in the WRKY33 locus after treatment with 150 μg mL⁻¹ Vd-toxins in the wild type, the hub1-4, hub2-2, and hub1-4 hub2-2 mutants, and the HUB1/hub1-4 complementation line. Chromatin was extracted 12 h after the Vd-toxins treatment, and immunoprecipitated DNA was analyzed by qPCR. Data were determined as the percentages of H2Bub1/H3 and H3K4me3/H3 for each individual gene position. Relative enrichments of H2Bub1 and H3K4me3 in the AtRbohF P1 locus were used as negative controls. Error bars indicate sd. Different letters in A, C, and D represent significant differences at P < 0.05 by one-way ANOVA with Tukey’s honestly significant difference posthoc tests. E, WRKY33 regulates the expression of AtRbohD. The protoplasts of Arabidopsis harboring 35S:WRKY33 were used. The expression of AtRbohD was examined after the protoplasts were exposed to Vd-toxins for 4 h. pSuper1300 was used as the negative control. Error bars indicate sd. F, GUS activity measurement in N. benthamiana leaves after the transient expression of ProRbohD:GUS and 35S:WRKY33. pCAMBIA1391 and pSuper1300 were used as negative controls. LUC was used as an internal control. G, Schematic diagram showing the promoter structure of the AtRbohD gene. The upstream regions and part of the coding region are indicated by black wide lines and a white box, respectively. The solid arrowheads indicate the sites containing W-boxes in the AtRbohD promoter. The two fragments (P1 and P2) used for the yeast one-hybrid assay and EMSA are indicated. H, Yeast one-hybrid assay showing that WRKY33 binds to the AtRbohD promoter. I, EMSA showing WRKY33 binding to the AtRbohD promoter. Biotin-labeled fragments of the AtRbohD promoter that contained W-boxes were used as probes. Each experiment was repeated three times with similar results.