Yeast One-Hybrid Screening of cDNA Library

The yeast one-hybrid cDNA library construction and screening were performed using the Matchmaker Yeast One-Hybrid Library Screening System (Takara Bio Inc., Shiga, Japan). The Y1H pAbAi-Native bait strain was co-transformed with cDNA and pGADT7-Rec that had been linearized with SmaI (Takara Bio Inc., Shiga, Japan). This allowed for the cDNA inserts and vector to recombine in the yeast cells, placing the cDNA “prey” inserts downstream of a GAL4 activation domain. Co-transformation was undertaken using the large-scale yeast transformation method, as described in the Matchmaker Yeast One-Hybrid Library Screening System manual. To calculate the number of clones screened, 100 μL of the transformation reaction was diluted to 1/10, 1/100, 1/1000, and 1/10,000 and spread on synthetic dropout media lacking leucine (SD/-Leu). The remainder of the transformation reaction was spread onto SD/-Leu/AbA¹⁰⁰ media and incubated at 30 °C for 5 days. After 5 days, the number of clones screened was calculated as
$$clones screened=\left(\frac{cfu}{ml}on\frac{SD}{-Leu}\right)\times \left(dilution factor\right)\times (resuspension volume \left(15mls\right))$$
Colonies on the SD/-Leu/AbA¹⁰⁰ plates greater than 2 mm in diameter were re-streaked onto fresh media and grown at 30 °C for three days. Healthy colonies were analyzed by yeast colony PCR using the Matchmaker Insert Check PCR Mix 2 (Takara Bio Inc., Shiga, Japan). Products were purified using the Accuprep PCR/Gel Purification Kit (Bioneer, Corporation, Daejeon, Republic of Korea) and sequenced with a T7 primer. Vectors containing inserts of interest were rescued and transformed into DH5α E. coli via electroporation and selected by ampicillin (50 μg mL⁻¹). The construct was purified from E. coli using the Accuprep Plasmid Mini Extraction Kit (Bioneer, Corporation, Daejeon, Republic of Korea) and re-sequenced to confirm the cDNA insert sequence.
Following the identification of SbGATA22 in Screens 1 and 2, confirmation of its binding to the putative GATA transcription factor binding motifs in the SbCYP79A1 promoter region was undertaken. A mutant SbCYP79A1 (Sb01g001200) promoter region with all putative GATA transcription factor binding sites mutated from GAT into GTA (Supplementary Figure S1) was synthesized by GenScript (Piscataway, NJ, USA) and cloned into pUC57. The pAbAi-Mutant constructs were transformed into S. cerevisiae Y1H Gold (Takara Bio Inc., Shiga, Japan) and tested for autoactivation of the bait sequence as described above for the pAbAi-native construct. Y1H pAbAi-Mutant strains were then transformed with 1μg of pGADT7-Rec vector that contained the full-length SbGATA22 insert from Screen 1 (Supplementary Figure S2) and selected on SD/-Leu media using the small-scale yeast transformation method. Colonies were analyzed by yeast colony PCR using the Matchmaker Insert Check PCR Mix 2 (Takara Bio Inc., Shiga, Japan) to ensure the correct vector was present. The Y1H SbGATA22-Mutant strain was spread onto SD/-Leu/AbA¹⁰⁰ media, and growth was compared with that of the strain harboring the native promoter sequence (SbGATA22-Native) after three days at 30 °C.