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

Figure 3.

Figure 3

BnMKK5–BnMPK3-mediated phosphorylation of BnWRKY33 is necessary to increase BnWRKY33 transcription. A, Relative LUC activity of BnaA03.WRKY28, BnWRKY33, and BnWRKY33DD when binding to the BnWRKY33 promoter. DD, conserved Ser residues mutated to Asp. B, Comparisons of the transcriptional activities of BnaA03.WRKY28, BnWRKY33, and BnWRKY33DD. The TF fused with the yeast GAL4 binding domain (GAL4BD) was used as an effector; GAL4-fLUC was used as a reporter and AtUbi:rLUC was used as an internal control. C, A diagram of the N-terminus of BnWRKY33 showing the positions of five conserved Ser residues according to AtWRKY33. Asterisks indicate the positions of conserved residue; marked S represents serine residue, and marked D represents aspartic acid residue. D, Interaction detection between BnWRKY33 and BnaA06.MPK3/BnaC03.MPK3 in Y2H. E, Co-IP to explore the interactions of BnWRKY33 with BnaA06.MPK3/BnaC03.MPK3 in vivo. Fusion proteins were co-expressed in N. benthamiana leaves. Total proteins were extracted (Input) and immunoprecipitated (IP) with GFP beads. F, Phosphorylation detection of BnWRKY33 by activated BnaA06.MPK3 (MPK3-A6) and BnaC03.MPK3 (MPK3-C3) in vitro. MPK3-A6 and MPK3-C3 were activated with constitutively active BnaA03.MKK5DD (MKK5DD). WRKY33-N, the N-terminus of BnWRKY33 containing the five conserved Ser residues; WRKY33AA-N, five conserved Ser residues in the BnWRKY33 N-terminus all mutated to Ala. Phosphorylation reactions were incubated in protein kinase buffer containing [γ-32P] ATP. Phosphorylation signals were detected via autoradiography (top panels). Protein inputs were assessed using Coomassie Brilliant Blue (CBB) staining (bottom panels). G, Dual-LUC transient transcriptional activity assays showing the effect of MAPK cascade on BnWRKY33-BnWRKY33 module. BnWRKY33 (W33), BnaA06.MPK3 (MPK3-A6), BnaC03.MPK3 (MPK3-C3), and BnaA03.MKK5DD (MKK5DD) were introduced into pGreenII 62-SK (SK) as effectors. The BnWRKY33 promoter (p346, –326 to –246 bp) was inserted into pGreenII-0800 as reporter. H, In vitro EMSA to explore the binding of phosphorylated BnWRKY33 (WRKY33DD) to the BnWRKY33 promoter. W33box, W1, W2, and W3 were used as probes. In (A), (B), and (G), data are shown as means ± sd (n = 3). Asterisks indicate significant differences (t test; ns, P > 0.05, *P < 0.05).