Saccharomyces cerevisiae

The entire ORF of EsDREB2B was amplified with specific primers (Y-f and Y-r), and the primer sequences were Y-f: 5′-GTCGACTGATGAGTGCAACTTGCATGC-3′ and Y-r: 5′-CTGCAGTCAAGACAATGAAGGATCGG-3′). The amplification product was inserted between the PstI and SalI restriction sites of the yeast expression vector pBridge, which also contained the binding domain (BD) of GAL4 (pBridge -BD-EsDREB2B). The recombinant plasmids were introduced into Saccharomyces cerevisiae (strain AH109 yeast strain carrying the His3 and LacZ reporter genes) per the manufacturer’s instructions (Clontech, USA). Yeast cells containing only GAL4 (pBridge-BD-GAL4) or the pBridge vector (pBridge-BD) were used as positive and negative controls, respectively. Successful transformants were selected by growth on SD media without His and Trp at 30°C for 3 days. Positive colonies were confirmed by yeast colony PCR. In addition, the β-galactosidase activity of the transformant colonies was examined by incubating the colonies in Z buffer with x-gal at 30°C.

Coding sequences of PPR14, PPR-SMR1, and Zm-mCSF1 were cloned into the bait (pGBKT7) and prey (pGADT7) vectors (Clontech). The primers used to make these constructs are listed in Supplementary Table S1. The various combinations of GAL4 DNA binding domain (BD) and GAL4 activation domain (AD) constructs were co-transformed into the yeast (Saccharomyces cerevisiae) strain Y2HGold (Clontech). Empty vectors were used as negative controls. The other manipulations were carried out according to the user manual (Clontech).

Full-length sequences of ShD14, ShHTL1, ShHTL3, ShHTL4, ShHTL7, and AtHTL were cloned into the pGBKT7 vector as bait (Clontech), and the coding regions of ShMAX2 and AtMAX2 were cloned into the pGADT7 vector as prey (Clontech). MAX2 was supposed to be unstable without ASK1. Therefore, AtASK1 was cloned into the N terminus of ShMAX2 and AtMAX2 with a 16-residue linker for co-expression. The bait and prey plasmids were co-transformed into the yeast (Saccharomyces cerevisiae) strain Y2HGold (Clontech) by the lithium acetate-mediated method. The presence of both plasmids was confirmed by growth on SD/-Leu/-Trp (-LT) plates. The interactions between bait and prey in the control medium (−LT) and selective medium (SD/−Leu/−Trp/−His/−Ade; −LTHA) in the absence or presence of 5 μM rac-GR24 (racemic mixtures) or 5 μM KAR₁ were detected. The plates were incubated for 5 days at 30 °C.