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. 2022 Sep 21;190(4):2757–2774. doi: 10.1093/plphys/kiac439

Figure 5.

Figure 5

The BnaA09.VQ12–BnaA03.WRKY28 protein complex inhibits BnWRKY33 transcription by competitively binding to the BnWRKY33 promoter. A, Increase in lesion radius on oilseed rape leaves infected by Sclerotinia over 84 h. Lesions were measured every 3 h. B, Induced expression of BnWRKY33 in the WT, 0, 12, 24, 36, 48, and 72 h after inoculation with S. sclerotiorum. C, Relative expression levels of BnWRKY33 in BnaA03.WRKY28/BnaA09.VQ12 transgenic lines and WT plants after Sclerotinia inoculation for 24 and 48 h. Asterisks indicate significant differences compared with the WT at the corresponding time points (t test; *P < 0.05). D, EMSA showing the effect of the BnaA09.VQ12 on the binding capacity of BnaA03.WRKY28 to the BnWRKY33 promoter. Shift, indicating the binding of BnaA03.WRKY28; Super shift, indicating the binding of BnaA09.VQ12–BnaA03.WRKY28 complex. E, In vitro binding assays to compare the binding capacity of BnaA09.VQ12–BnaA03.WRKY28 complex and phosphorylated BnWRKY33 to the BnWRKY33 promoter. Equal addition of BnaA03.WRKY28 protein in Lanes 2, 3, 5, and 6; equal addition of BnWRKY33 protein in Lanes 4 and 5; equal addition of BnWRKY33DD and BnaA03.WRKY28 was mixed in Lane 5; 5:1 ratio of BnWRKY33DD:BnaA03.WRKY28 was mixed in Lane 6. Protein inputs were assessed using CBB staining (bottom panels). F and G, In vivo dual-LUC transient transcriptional activity assays to explore competitive binding of BnaA09.VQ12, BnaA03.WRKY28, and BnWRKY33 to the BnWRKY33 promoter. In (F) and (G), data are shown as means ± sd (n = 3); asterisks indicate significant differences (t test; ns, P > 0.05, *P < 0.05, **P < 0.01).