Yeast Two-Hybrid (Y2H) Screening/Assay

Yeast two-hybrid screening/assay was performed using the Matchmaker^TM Gold yeast two-hybrid system (Clontech, Cat. No. 630489) following the User Manual. Briefly, to screen the interacting proteins, the coding sequence of MaICE1 was cloned into pGBKT7 vector to fuse with the DNA-binding domain (DBD) as bait, and transformed into yeast strain Gold Y2H by the lithium acetate method. The cDNA library (2.0 × 10⁹ cfu/ml) was generated by TAKARA BIOTECHNOLOGY (DALIAN) CO., LTD using poly (A)⁺ mRNAs extracted from banana fruit that were stored under cold stress, fusing to pGADT7 with activation domain (AD) and was transformed into Gold Y2H carrying the MaICE1 bait. The transformed cells (approximately 6.0 × 10⁶ cfu) were placed on DDO medium (minimal media double dropouts, SD medium with -Leu/-Trp), and positive clones among the transformants were identified by scoring growth on QDO medium (minimal media quadruple dropouts, SD medium with -Leu/-Trp/-Ade/-His). Plasmids of positive clones was extracted from the yeast cells using a TIANprep yeast plasmid DNA kit (Tiangen) and then transformed into Escherichia coli for sequencing.

To confirm the MaSINA1-MaICE1 interaction, the coding sequences of MaSINA1 and MaICE1 were inserted into pGBKT7 or pGADT7 vector as bait and prey, respectively, and were co-transformed into Gold Y2H. Yeast cells were grown on DDO medium for 3 days, then transformed colonies were plated onto QDO medium, as well as QDO media containing 4 mg mL-1 X-α-Gal (α-Gal) for blue color development, to verify the possible interaction between MaSINA1 and MaICE1 according to their growth status and the activity of α-galactosidase. Primers used for Y2H assay are listed in Supplementary Table 1.