To investigate the ability of OpWRKY2 to transcriptionally activate the OpTDC gene, the 3090 bp promoter of OpTDC was analyzed and cloned into the pGreenII0800-LUC vector. The reporter constructs wt-pOpTDC::fLUC and mutant-pOpTDC::fLUC were obtained by inserting the native promoter of OpTDC and a mutated version into the pGreenII0800-LUC vector to drive expression of the firefly luciferase gene30. The Renilla luciferase gene driven by the CaMV 35 S promoter was used as an internal control. The assembled vectors were cotransformed with the helper plasmid pSoup19 into A. tumefaciens strain GV3101. The A. tumefaciens strain GV3101 containing pHB-OpWRKY2-YFP was used as the effector, and pHB-YFP was used as the negative control. Infiltration and detection were performed as previously described, with minor modifications31. The reporter strains harboring wt-pOpTDC::fLUC or mutant-pOpTDC::fLUC were mixed with effector strains harboring either pHB-OpWRKY2-YFP or pHB-YFP at a ratio of 1:1. Leaves were collected after 48 h, and Dual-LUC assays were performed using the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega, Madison, WI, USA). Three biological replicates per treatment were measured. All primers used to amplify the OpTDC promoter are listed in Table S2.

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