Yeast Two-Hybrid Screening of Plant Transcription Factors

The Matchmaker GAL4 Two-Hybrid System 3 kit (Clontech; http://www.clontech.com) was used for Y2H assays, as described previously (Yamaji et al., 2006 (link)). The BD-fused PHYL1_OY and AD-fused SEP3 were constructed as previously reported (Maejima et al., 2014a (link)). For construction of the other AD-fused MTFs, the MTFs were amplified using the primers shown in Supplementary Table S1, and cloned into the pGADT7 vector (Clontech) digested by NdeI and EcoRI-HF (NEB), as described above. The BD-fused PHYL1_PnWB was constructed in the same manner, using the pGBKT7 vector (Clontech), digested by NdeI and EcoRI-HF.
Lithium acetate-treated yeast cells (strain AH109) were co-transformed with pairs of appropriate pGADT7 and pGBKT7 vectors. Successful co-transformants were selected on synthetically defined medium (SD) lacking tryptophan and leucine (SD/–LW). To evaluate the protein interaction, the co-transformants were cultured on three selective media: leucine/tryptophan/histidine-lacking SD (SD/–LWH), SD/–LWH containing 5 mM 3-amino-1,2,4-triazole (Sigma-Aldrich) (SD/–LWH+3AT), and leucine/tryptophan/adenine/histidine-lacking SD (SD/–LWAH).