OsARF19 Binding to OsWOX10 Promoter

The binding of OsARF19, preferentially expressed in LRP (Yamauchi et al., 2019 (link)), to auxin response elements (AuxREs) on OsWOX10 promoter was analyzed by performing an electrophoresis mobility shift assay (EMSA). The sequence encoding the N-terminal 265 amino acids of OsARF19, including the B3 DNA-binding domain (DB), that was optimized for the codon usage of E. coli, was fused to pET32a. The OsARF19-DB was expressed in E. coli BL21(DE3), followed by purification with TALON metal affinity resin (Clontech, United States) following the manufacturer’s instructions. To prepare DNA probes, the 59 or 60-bp oligonucleotides were labeled with Cy5 fluorescence dye using Klenow fragment (TaKaRa Bio, Japan) and purified on a column (NucleoSpin^® Gel and PCR Clean-up; Macherey-Nagel, Germany) following the manufacturer’s instructions. The DNA-binding reaction was performed at 4°C for 30 min in PBS (–) (137 mM NaCl, 8.10 mM Na₂HPO₄, 2.68 mM KCl, 1.47 mM KH₂PO₄; pH 7.4) + 1 mM 2-Mercaptoethanol with 25.3 nM probe with 0, 0.5, or 1 μM recombinant OsARF19-DB, and subjected to EMSA with 5% polyacrylamide gels in 0.5 × TBE buffer at 4°C. The Cy5-labeled probes were analyzed by Typhoon FLA9000 (GE Healthcare, United States).