WRKY46 binds to VITL1 promoter region in yeast. The 2-kb promoter region of VITL1 was indicated. The small blue boxes in the promoter represent three W-boxes (corresponding to the sequences in red). VP1-3 indicate the promoter fragments (about 50 bp) surrounding each W-box, and mVP1-3 indicate the mutated VP1-3 (with 1 bp mutation in each W-box), respectively. Yeast cells were cotransformed with a bait vector, containing a promoter fragment (VP1-3 and mVP1-3) fused to a HIS2 reporter gene, and a prey vector, containing WRKY46 fused to a GAL4 activation domain. Cells were grown in liquid media to OD600 of 0.1 (10−1) and diluted in a 10× dilution series (10−2 to 10−3). Of each dilution, 5 µL was spotted on media selecting for both plasmids (SD –Trp –Leu) and selecting for interaction (SD –Trp –Leu –His), supplemented with 25 mm
3AT to suppress background growth and test the strength of the interaction.