Matchmaker gal4 based yeast two hybrid system

Yeast two-hybrid assays were performed using the Matchmaker GAL4-based yeast two-hybrid system (Clontech). Various DNA fragments derived from ctn-1 were subcloned into the pGBKT7 vector in frame with the GAL4 DNA-binding domain, and the resulting constructs were transformed into the Y187 yeast strain. Various slo-1 cDNA fragments were subcloned into the PGADT7 vector in frame with the GAL4 activation domain, and the resulting constructs were transformed into the Y2H Gold strain. Mating was performed by inoculating and growing appropriate yeast colonies in YPD medium, followed by the selection of diploids that showed positive two-hybrid interactions on quadruple dropout plates (−histidine, −alanine, −leucine, and -tryptophan) containing 40 μg/ml X-α-Gal. Empty vectors served as negative controls, and constructs were tested for autoactivation.

The Matchmaker GAL4-based Yeast Two-Hybrid System (Clontech, CA, USA) was used for verifying protein–protein interactions. The full-length CDSs of NF-YB3a/3b/3c and truncated CDS of NF-YB3b/3c were amplified and linked to pGBKT7 vectors. The full-length CDSs of NF-YC1a/1b/9 and NF-YA3b were amplified and then linked into pGADT7 vectors. Saccharomyces cerevisiae strain AH109 was co-transformed with the pairs of plasmids. Yeast cells were cultured on transformation-selection (SD/−Leu−Trp) and interaction-selection (SD/−Leu-Trp−Ade−His) medium to screen for protein–protein interactions. Specific primers (Supplementary Data Table S1) were used to construct the plasmids.
For transcriptional activation assays in yeast, the full-length CDS of NF-YA3b was cloned into pGBKT7 vector, and the resulting construct was used for transformation of yeast AH109 strain. The empty vector pGBKT7 and the combination of pGBKT7-53 + pGADT7-RecT vectors were used as the negative and positive controls, respectively. Transformed yeast cells were grown on SD/−Trp, SD/−Trp−His, and SD/−Trp−His with X-α-gal media.

The Gal4‐based Matchmaker yeast two‐hybrid system (Clontech) was used to investigate the interactions of StCds1 with StNrm1 and StNrm1 with StMbp1, according to the manufacturer's instructions. The indicated full‐length cDNAs were subcloned to fuse the GAL4 activation domain (AD) in the pGADT7 vector or the GAL4 DNA‐binding domain (BD) in pGBKT7. The corresponding constructs were co‐transformed into S. cerevisiae AH109. The protocol used was described previously (Zeng et al., 2018).
Matchmaker one‐hybrid system (Clontech) was used for the yeast one‐hybrid assay to analyze the interaction between the transcription factor StMbp1 and the promoter DNA of the StPKS gene according to the manufacturer's instructions. The StPKS promoter fragment (−738 to −238) was inserted into the pAbAi vector as reporter (the promoter sequence is listed in Text S1). The reporter vectors were linearized at the BstBI site and transformed into S. cerevisiae Y1HGold strain. The full‐length cDNA of StMBP1 was cloned into the pGADT7 vector as effector and transformed into the Y1HGold strain containing the indicated reporter vectors. The yeast one‐hybrid assay was performed according to the manufacturer's protocol (Clontech). The primers used for yeast hybrid constructs are listed in Table S1.