Gal4 two hybrid system

Yeast two‐hybrid (Y2H) assays were performed using the matchmaker GAL4 two‐hybrid system (Clontech, CA, USA) according to the manufacturer's instructions. The coding sequences of SAPK10 were digested with EcoRI and XhoI and cloned into the pGADT7 vector, the coding sequences of SAPK10 were digested with EcoRI and PstI and cloned into the pGBKT7 vector, and the coding sequences of bZIP72 were digested with NdeI and EcoRI and cloned into the pGBKT7 vector, respectively. Primers used were listed in Table S1. The resulting constructs were cotransformed into the yeast strain Y2H Gold (Clontech) and selected on synthetic medium lacking leucine, tryptophan and histidine with 0.04 mg ml⁻¹ X‐α‐Gal and 100 ng ml⁻¹ Aureobasidin A applied.

A Matchmaker GAL4 Two-Hybrid System was applied in this study (Clontech, PT3247-1). pGADT7-Ste5 plasmids were transformed into AH109 separately and selected on SC-Leu plate. pGBKT7-Ste7 plasmids were transformed into Y187 separately and selected on SC-Trp plate. A Trp⁺ transformant was mated with a Leu⁺ transformant on YPD and diploid cells were selected on SC-Leu-Trp plates.
The quantitative β-galactosidase assay was performed as described in the Yeast Protocols Handbook (Clontech, PT3024-1). Diploid cells were grown in liquid culture and lysed by freeze/thaw cycles in liquid nitrogen. Activities in the cell lysate were measured by using ONPG (Solarbio, O8040-1g) as a substrate. 1 unit of β-galactosidase is defined as the amount which hydrolyzes 1 μmol of ONPG to o-nitrophenol and D-galactose per min per cell. To remove the autoactivation of pGBKT7-Ste7, the unit measured for a diploid containing a pGBKT7-Ste7 plasmid and a void pGADT7 vector was subtracted from the unit of a diploid containing the same pGBKT7-Ste7 plasmid and a pGADT7-Ste5 plasmid. Three different colonies of one diploid strain were subjected to the assay and activities were measured in duplicates.

The Matchmaker GAL-4 two-hybrid system was used as described by the manufacturer (Clontech, Clontech Laboratories, Inc., Palo Alto, CA, USA). A cDNA fragment, encoding the NH₂ terminus of mouse desmin (first 1–320 nucleotides), was inserted downstream of the GAL4-DNAbinding domain in the pGBKT7 bait vector. A yeast two-hybrid cDNA library (Clontech) derived from human heart muscle was screened for interacting proteins as described by the distributor. For interaction, positive clones were selected in a selection medium (SD/-Trp/-Leu,/-Ade/-His +X-α-galactosidase plates), as we have previously described [28 (link)].

The matchmaker GAL4 Two‐Hybrid System (Clontech) was employed to investigate PPIs. The full‐length coding sequences of ZmPKSB, ZmTKPR1‐1 and ZmTKPR1‐2 were cloned into pGADT7 and pGBKT7 vectors. Amino acid substitution vectors, including pGBKT7‐ZmTKPR1‐1‐9M, pGBKT7‐ZmTKPR1‐2‐7M and pGADT7‐ZmTKPR1‐1‐4M, were constructed using fusion PCR. Co‐transformation of a pGADT7/prey plasmid and a pGBKT7/bait plasmid was performed in yeast strain AH109 and verified on selective media according to the manufacturer's instructions. Negative control vectors, pGADT7‐T7 and pGBKT7‐lam, were used, as well as a positive control with pGADT7‐T7 and pGBKT7‐53. Subsequently, vector co‐transformation into yeast strains and yeast screening were conducted following the methods described in a previous study (An et al., 2020 (link)).

Yeast two-hybrid analysis was performed using the GAL4 Two-Hybrid System according to the manufacturer’s instructions (Matchmaker; Clontech, USA). The full-length cDNA of TaCHLI-7A and Tachli-7A were cloned into the bait vector pGBKT7 and the prey vector pGADT7, respectively. Pairs of the plasmids BD and AD were co-transformed into yeast strain AH109 according to the manufacturer’s instructions. Transformants were first selected on plates containing a double-dropout SD medium (lacking Leu and Trp) and then tested on selective SD medium (lacking Leu, Trp, His, and Ade).