Fig. 5: CsWRKY22 directly binds to and activates the promoter of CsLOB1.
a The transcription level of CsLOB1 in CsWRKY22-overexpressing plants (OE) and the wild-type (WT) control. b Reporter and effector vectors constructed for dual LUC assays. The CsLOB1 promoter was inserted ahead of the LUC gene to form a reporter construct (L1), and the empty vector was used as a control (C1). CaMV35S promoter-driven CsWRKY22 (W22) was used as the effector, and the empty vector was used as the control (C2). LUC firefly luciferase, REN Renilla luciferase, P35S CaMV35S promoter, T35S CaMV35S terminator. c Transient expression assay of promoter activity in tobacco leaves, shown as ratios of LUC to REN; d Schematic diagrams of CsLOB1 promoter fragments used for the Y1H assay. The black dots represent the original W-boxes. The red dots represent mutated W-boxes; e Y1H assays of CsWRKY22 and CsLOB1 promoter fragments; f EMSAs of the specific binding of CsWRKY22 to the W-box sequence in the CsLOB1 promoter. Elements shown in red are mutated W-boxes. The GST-CsWRKY22 protein (GST-W22) was incubated with a probe labeled with biotin, accompanied by or without native competitor DNA. +, presence; −, absence; g Schematic representation of the deletion of the S2-5 mutant line reported by Peng et al.7; h Expression of CsWRKY22 in the WT and S2-5 mutant lines; i, j Expression levels of CsWRKY22 and CsLOB1 in the WT and S2-5 lines with or without the transient introduction of 3×FLAG-CsWRKY22 (W22). All the data are shown as the means ± SDs (n = 3). Asterisks indicate significant differences between transgenic plants and the WT control by Student’s t-test (**P < 0.01). Different letters above the bars represent significant differences based on Duncan’s multiple range test (P < 0.05)
