Gal4 yeast two hybrid system

Interactions between BIK1 and the kinase domain of PEPR1 (residues 827–1123) or RLK7 (671–977) were tested using the GAL4 yeast two-hybrid system (Clontech). In brief, the pGADT7-PEPR1_KD or pGADT7-RLK7_KD plasmid was co-transfected with pGBKT7-BIK1 into Saccharomyces cerevisiae strain AH109. The transformed yeast cells were spotted on a synthetic dropout (SD) medium (Difco Yeast Nitrogen Base) lacking tryptophan, leucine, and histidine (SD-Y⁻-L⁻-H⁻) but supplementing with 3 mM 3-amino-1,2,4-triazole (3-AT, Sigma) to detect the His reporter activity. Transformants were also detected on the basis of lacZ reporter activity with 50 µg/mL X-gal dissolved in 25 mM phosphate buffer.

The interactions between the ICK/KRP proteins and different maize cyclins and CDKs were analysed using the GAL4 yeast two-hybrid system (Clontech). The CDS of the ZmICK genes were cloned into pMD19-T and digested with NdeI and SalI, whereas ZmCDKA1, ZmCDKB, ZmCycD1, and ZmCycD2 were digested with NdeI and BamHI. The primers used are listed in Table S3. The purified gene fragments were cloned into vectors containing the yeast two-hybrid DNA-binding domain (pGBKT7, BD) and the activation domain (pGADT7, AD). All the resulting plasmids were confirmed by sequencing. Different combinations of the BD and AD constructs were introduced into AH109 using a lithium acetate transformation method. The positive and negative controls used pGADT7-T co-transformed with pGBKT7-53 or pGBKT7-lam, respectively. The transformants were first incubated on synthetic dextrose (SD) media lacking tryptophan (Trp) and leucine (Leu) for 3 days. Then, a single clone was grown in 1 ml of SD/-Trp/-Leu for 12 hours, and 6 μl of the culture was grown on synthetic dextrose (SD) medium lacking tryptophan (Trp), leucine (Leu), adenine (Ade), and histidine (His) supplemented with 30 mM 3-AT for 3 days. Finally, 10 μM 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-gal) was smeared onto the plate to detect α-galactosidase activity.

To validate the predicted protein–protein interaction between two of our candidate TFs, AP2 and WRKY16, we used the GAL4 yeast two-hybrid system (Clontech). AP2 and WRKY16 were cloned into pGADT7-GW (Addgene Plasmid #61702) and pGBKT7-GW (Addgene Plasmid #61703) plasmids (Lu et al., 2010 (link)). Empty pGADT7 (with the GAL4 activation domain, AD) and pGBKT7 (with the GAL4 DNA-binding domain, BD) plasmids were used as negative controls. We use the yeast (Saccharomyces cerevisiae) strain AH109 in the MATCHMAKER GAL4 Two-Hybrid System (Clontech), in which HIS3, ADE2, MEL1, and LacZ are under the control of GAL4 TF. The AD and BD plasmids were co-transformed to yeast AH109 competent cells following Clontech’s user manual instructions of the polyethylene glycol/lithium acetate method and cultured in YPD Plus Medium for 90 min to promote transformation efficiency. Transformed yeast cells were assayed by culturing in SD/–Leu/–Trp medium to select for successful co-transformants and then assayed by culturing in SD/–Ade/–His/–Leu/–Trp medium with 40 μg·mL⁻¹ X-α-Gal, which provides high-stringency selection. The positive protein–protein interactions between two TFs are indicated by growth on SD/–Ade/–His/–Leu/–Trp/X-α-Gal medium plates and blue colony color.