4.7. Yeast One-Hybrid (Y1H) Assays

Y1H assay was carried out using the Matchmaker™ Gold Yeast One-Hybrid System (Clontech, United States). Three tandem copies of CRT/DRE were generated by oligonucleotide synthesis and cloned into the pAbAi (bait) vector (Clontech, United States). A fragment (−312 to −487) of the LlNAC2 promoter was amplified by PCR, and also cloned into the pAbAi (bait) vector to generate pAbAi-NAC-CRT/DREs plasmid (shown in Figure 4B). Full-length LlDREB1 (NCBI accession No.{"type":"entrez-nucleotide","attrs":{"text":"KJ467618","term_id":"588493661","term_text":"KJ467618"}}KJ467618) was amplified and inserted into pGADT7 (prey) vector (Clontech, United States) yielding plasmid pGADT7-LlDREB1. The bait plasmids were linearized and transformed into the yeast strain Y1HGold. Positive yeast cells were then transformed with pGADT7-LlDREB1 plasmid. The DNA–protein interaction was determined based on the growth ability of the co-transformants on SD/-Leu medium with Aureobasidin A (AbA).