Yeast One-Hybrid Screening and Confirmation

Yeast one-hybrid screening was performed using the Matchmaker Gold Yeast One-Hybrid Library Screening System (Clontech, Mountain View, CA, United States). The promoter of EjMYB8 was isolated using the GenomeWalker universal kit (Clontech) then inserted into the pAbAi vector. Primers used for genome walking and pAbAi construction are listed in Supplementary Tables S1, S2, respectively. The recombinant EjMYB8-pAbAi vector was linearized and transformed into a Y1HGold yeast strain. After testing for autoactivation, 100 ng/ml Aureobasidin A (AbA) was used for library screening (Supplementary Figure S1). The Y1HGold[EjMYB8/AbAi] yeast strain was transfected together with the cDNA library, which was previously constructed by Zhang et al. (2016). After 5 days of growth at 30°C, every individual yeast colony was picked for PCR analysis and then sequenced by Huajin Company (Shanghai, China). The obtained cDNA fragments were used for an in silico search for annotations using the Basic Local Alignment Search Tool.¹ Five putative transcription factors were selected for further confirmation (listed in Supplementary Table S3). Primers designed for full-length CDS isolation and construction of pGADT7 vectors were listed in Supplementary Tables S4, S5, respectively. The obtained sequences were uploaded to the NCBI database (GenBank numbers are given in Supplementary Table S6).

To confirm true positive interactions, we separately transformed pGADT7 vectors with the genes listed in Supplementary Table S5 into the Y1HGold [EjMYB8/pAbAi] yeast strain. Empty pGADT7 plasmids were transformed as the negative control. Transformed yeast was grown on SD/-Leu medium containing 100 ng/ml of AbA.