Yeast Two-Hybrid Screening of VcSnRK2.3 and MdMYB1

The Y2H experiment was performed according to Clontech’s instructions. The ORF, N-terminus, and C-terminus of VcSnRK2.3 and the ORF of MdMYB1 were inserted into pGAD and pGBD vectors. The plasmids were co-transformed into yeast strain Y2H Gold (TaKaRa, Beijing, China) by using the lithium acetate method with reference to Veries et al. For transformation, 4-5 μl of plasmid and 50 μg of denatured salmon sperm vector DNA are mixed. Then 500 μl of polyethylene glycol (PEG) lithium acetate solution is added. After incubation 20 μl DMSO is added and incubated again. The transformed yeast strains were grown on medium lacking Leucine and Tryptophan (SD-L/-T), Leucine, Tryptophan, Histidine and Adenine (SD-L/-T/-H/-A) or Leucine, Tryptophan, Histidine, Adenine and X-gal (SD-T/-L/-H/-A+X-gal) at 28°C for 3 days.