Electrophoretic mobility shift assay
The PtrWRKY75 cDNA was cloned into the PET28a vector, then the construct was inserted by heat shock into Escherichia coli strain BL21. Production of the His-PtrWRKY75 fusion protein was induced by 0.2 Mm isopropyl β-D-1-thiogalactopyranoside.
Oligonucleotide probes (PAL1 W-box: TTGACC) were synthesized by Sangon (Beijing, China) and labeled with biotin using an EMSA Probe Biotin Labeling Kit (GS008, Beyotime Biotechnology, Jiangsu, China). For the mutated probe, the single mutation site was located in the core sequence of the W-box (changed from TGAC to TAAC). The EMSA was performed using a Chemiluminescent EMSA kit (GS009, Beyotime Biotechnology, Beijing, China) in accordance with the manufacturer’s instructions. Briefly, biotin-labeled probes were incubated with the fusion proteins in binding buffer for 30 min at room temperature (20–25 °C). The reaction products were transferred to 4% polyacrylamide gel, then electrophoresed in 0.5× Tris-borate-EDTA (TBE) buffer for 60 min. All oligonucleotide sequences are listed in Table S2.