4.8. Yeast One-Hybrid (Y1H) Assay

The cDNA library was constructed in our previous work [37]. Y1H library screening was performed according to Lin et al. [39]. The Y1H assay was conducted in the yeast Y187 strain using the MATCHMAKER One Hybrid System (Clontech, CA, USA), following the manufacturer’s instructions. The TaCPOT3D promoter was inserted into the pHIS2 reporter vector to obtain pHIS2-TaCPOT3Dpro. The pHIS2-TaCPOT3Dpro and pGADT7-TaWRKY22 plasmids were co-transformed into Y187, and the culture was spread on a synthetically defined double drop-out medium lacking tryptophan (Trp) and leucine (Leu). The transformant was tested on a medium lacking leucine (Leu), tryptophan (Trp), and histidine (His) supplemented with 90 mM 3-amino-1,2,4-triazole (3-AT) for 3 days.