Yeast one-hybrid and two-hybrid assays

Yeast one-hybrid (Y1H) and two-hybrid assays were conducted as previously described⁴³. Yeast one-hybridization assays were performed using the Matchmaker Gold Yeast One-Hybrid System (Clontech). To construct transcription factor-expressing cassettes, the ORFs of CsMYB4, CsMYB7, CsMYB12, CsPIF3, and CsbZIP1 were recombined into the pGADT7 vector (Clontech, Palo Alto, USA). The cloned promoter fragments of CsMYB12, CsMYB7, CsFLS, and CsUGT78A14 were inserted into the pHIS2.1 vector. The yeast strain Y187 containing the recombinant pHIS2.1 vector was grown on –Trp–Leu (–T–L) screening medium for 3 days at 30 °C. Then, the interactions between the MYB TFs and promoter fragments were detected on medium lacking Trp, Leu and His (–T–L–H) for 3–5 days at 30 °C. Empty pGADT7 vectors were used as controls.

For yeast two-hybrid assays (Y2H), the ORFs of the CsMYB4, CsMYB7, CsbZIP1, and CsMYB12 genes were recombined into the pGBKT7 and pGADT7 vectors, respectively (Clontech, Palo Alto, USA). The recombinant plasmids were cotransformed into the yeast strain AH109 and cultured on medium lacking Trp and Leu (–T–L) for 3 days at 30 °C. For interaction screening, the yeast cells were transferred to medium lacking Trp, Leu, His and adenine (–T –L–H–A) with X-gal for 3–5 days at 30 °C. Empty pGADT7 and pGBKT7 vectors were used as controls.