Matchmaker gal4 based two hybrid system

Yeast two-hybrid assays were performed using the Matchmaker GAL4-based two-hybrid system (Clontech, Palo Alto, CA, USA). Full-length cDNA and truncated mutants of MdHXK1, including MdHXK1^{1-245 aa} and MdHXK1^245-498aa, were inserted into the pGBT9 vector. The associated yeast two-hybrid vectors of MdbHLH3, which were inserted into vector pGAD424, are detailed in Xie et al. [32 (link)]. All of the constructs were transformed into yeast strain AH109 using a lithium acetate method. Yeast cells were cultured on minimal medium -Leu/-Trp according to the manufacturer’s instructions. Transformed colonies were plated onto minimal medium -Leu/-Trp/-His/-Ade with or without β-galactosidase to test for possible interactions.

The Matchmaker GAL4-based two-hybrid system (Clontech) was used to perform yeast two-hybrid assays. The cDNAs encoding full-length LSF2 were cloned into the pGAD424 vector (Clontech) to generate activation domain (AD) constructs. The cDNA encoding full-length MPK8 was cloned into the pGBT9 vector (Clontech) to generate binding domain (BD) constructs. All constructs and empty vector controls were transformed into yeast stain AH109 using the modified lithium acetate method. Co-transformed yeast transformants were selected on plates containing SD/-Leu/-Trp medium and screened on selective medium containing SD/-Leu/-Trp/-His with 20 mM 3-amino-1,2,4,-triazole (3-AT) to test protein interactions, and empty vectors pGBT9 and pGAD424 were used as negative controls.

Yeast two-hybrid assays were carried out using the Matchmaker GAL4-based two-hybrid system, as previously described (Clontech, Palo Alto, CA, USA). Full-length LoCOP1 and LoMYB1, LoMYB2, and LoMYB3 cDNA fragments were introduced into pGADT7 and pGBKT9 (Clontech, Palo Alto, CA, USA). All constructs were transformed into the yeast strain AH109 using the lithium acetate technique, and yeast cells were cultured on minimal medium/-Leu/-Trp according to the manufacturer guidelines (Clontech, Palo Alto, CA, USA). To screen for potential interactions, transformed colonies were plated onto minimal medium/-Leu/-Trp/-His/-Ade and stained with X-gal.

Yeast (Saccharomyces cerevisiae) two-hybrid assays were performed using the Matchmaker GAL4-based two-hybrid system according to the manufacturer’s instructions (Clontech). The coding region of LcMYB5, LcMYB5 N terminal (LcMYB5N, 1–417 bp) and LcMYB5 C terminal (LcMYB5C, 418–1002 bp) were ligated into pGADT7 vector to fuse with the activation domain (DNA-AD) (primers pairs are listed in Additional file 1: Table S2). Full length of LcMYB5 displayed auto transcriptional activation activity in yeast cells. N terminal of LcMYB5 (LcMYB5N) did not show while C terminal of LcMYB5 (LcMYB5C) showed auto transcriptional activation activity in yeast cells. Specific bait and prey constructs combinations were then co-transformed into Gold Y2H yeast strain through a lithium acetate method and then yeast cells were plated selected on SD/−Leu/−Trp medium for 3 days. The transformed colonies were then plated on SD/−Leu/−Trp/–His/−Ade medium containing appropriate amount of Aureobasidin A and X-α-Gal at 30 °C for 3 days. The interactions between LcbHLH1/3 and LcMYB5 were determined according to the growth of the yeast cells and the activity of α-galactosidase.