Yeast Two-Hybrid Screening and Validation

The coding sequence of CsTFL1 was cloned into the pGBKT7 to construct the bait vector BD-CsTFL1. A normalized cucumber Mate & Plate library was constructed by Shanghai OE Biotech using equal amounts of cDNA obtained from leaf, shoot tip and flowers of the cucumber inbred line 9930. The bait BD-CsTFL1 was used to screen the Mate & Plate library according the Matchmaker Gold Yeast Two-Hybrid System User Manual (Clontech). The full-length coding sequence of the identified interacting protein was cloned into the pGADT7. The resultant pGADT7 vector and pGBKT7 or BD-CsTFL1 were co-transformed into the Y2HGOLD yeast strain and selected on SD--Ade/-His/-Leu/-Trp to verify the interaction. In addition, full-length coding sequences for CsTFL1, CsTFL1m, CsFT, CsFD, CsFDP, Cs14-3-3-3 and Cs14-3-3-5 were cloned into pGADT7 (prey) or pGBKT7 (bait) vectors, sequenced and then transformed into the yeast strain AH109. The bait and prey vectors were transformed following the instructions of Matchmaker GAL4 Two-Hybrid System 3 & Libraries (Clontech). The protein interaction assay followed the methods of Ding et al. (2015b) (link). The primer information is listed in Table S4.