Yeast strain y2h

The full-length coding sequence of ZmERF061 was inserted into pGBKT7 vector to generate the bait plasmid (pGBKT7-ZmERF061). The coding sequence of ZmMPK6-1 was cloned into pGADT7 vector to generate prey plasmid (pGADT7-ZmMPK6-1). The prey and bait plasmids were co-transformed into the yeast strain Y₂H according to the manufacturer’s instructions (Clontech, United States). After selection on SD/-Trp/-Leu medium for 3 days at 30°C, the transformants were grown on SD/-Trp/-Leu/-His/-Ade medium containing X-α-Gal (20 μg ml^–1). Yeast cells carrying the pGBKT7-p53 and pGADT7-SV40 plasmids were used as positive controls, and yeast cells harboring the pGBKT7-Lam and pGADT7-SV40 plasmids were used as negative controls.

The yeast cloning vectors pGBKT7 and pGADT7, the control vectors pGADT7-T and pGBKT7-53, and the yeast strain Y2H used in the yeast-two hybridization assays were obtained from the Clontech company (http://www.clontech.com/). The yeast two-hybridization assays were performed according to the manufacturer's instructions. Soybean full-length CDS of GmFT2a and GmFT5a were inserted into pGBKT7 vectors to generate fused GAL4 DNA binding domains as the soybean baits. Full-length CDS of the three soybean FD-like genes containing the SAP motif (GmFDL19, GmFDL08 and GmFDL15) were cloned into pGADT7 to generate fused GAL4 DNA activation domains as the soybean preys, and the full-length CDS of Arabidopsis FD was also cloned into pGADT7 to generate a positive control. Table S4 lists the primers and restriction sites used to generate the yeast bait and prey constructs. The bait and prey plasmids were cotransformed into the yeast strain Y2H using the lithium acetate method and selected on SD medium lacking leucine (Leu) and tryptophan (Trp). After 4 days of incubation at 30°C, the yeast cells were replated on selection plates with SD medium lacking Leu, Trp, histidine (His) and adenine (Ade) but including the X-α-gal substrate for the interaction test.

To investigate the transcriptional activity of ScDREB10, the yeast two-hybrid system (Y2H) with pGBKT7 vector and Y2H yeast strain were used (Clontech). The coding sequence of ScDERB10 was fused in frame with the GAL4 DNA-binding domain (pGBKT7 vector) to produce the fusion construct of BD-ScDREB10 using the in-fusion PCR cloning system (Clontech); the full-length ORF of ScDREB10 was amplified from pMD18-T plasmids by PCR using gene-specific primers containing EcoR I and BamH I restriction sites, and the PCR product was inserted into the EcoR I/BamH I pGBKT7 vector. The construct was subsequently introduced into yeast Y2H Gold cells (Clontech). The yeast positive transformants were adjusted to an OD600 of 2.0, and the yeast cells were then 10-fold serially diluted and dropped with 2 µL on synthetic dropout (SD) medium without tryptophan (SD/−Trp), without tryptophan and histidine (SD/−Trp−His), and with SD/−Trp−His plates containing x-α-gal (5-Bromo-4-chloro-3-indolyl α-d-galactopyranoside) (SD/−Trp−His + x-α-gal). Yeast cells expressing containing the pGBKT7 empty vector or expressing GAL4 were used as the negative and positive control, respectively. The plates were incubated at 30 °C for 2 to 4 days before photographing. The primer information was listed in Table S1.