Pt3024 1

Full length protein coding sequence of CsULT1 was cloned in yeast (Saccharomyces cerevisiae) expression vector pGBKT7 (Clontech) at NdeI-EcoRI site to express CsULT1 protein fused to GAL4 DNA-binding domain (GAL4-BD). The primers used were ULTGBKT-F and ULTGBKT-R and cycling parameters are same as described above. The resulting construct was transformed into Y187 yeast strain. The positive transformants were selected onto synthetic medium lacking tryptophan and leucine. Cells from two independent transformants were collected and assayed for β-galactosidase activity by using ortho-nitrophenyl-β-D-galactoside (ONPG) as substrate as described in clontech manual (PT3024-1).

To detect the transcriptional activity of SbSTOP1s, the bait vector pBridge expressing SbSTOP1a, SbSTOP1b, SbSTOP1c, SbSTOP1d, SbSTOP1d-NT (1–275 aa) or SbSTOP1d-CT (276–519 aa) fused to the GAL4 DNA-binding domain (BD) was used to transform the yeast strain Y2HGold. Colonies were selected on SD/-Trp-His medium (with or without 3-AT) and cultured for 3 days at 30°C. For the yeast two-hybrid assay, the prey vector pGADT7 expressing SbSTOP1b or SbSTOP1d fused to the GAL4 activation domain (AD) and the bait vector pBridge expressing SbSTOP1d-NT (1–275 aa) fused to the BD were used to co-transform the yeast strain Y2HGold (or the Y190 yeast strain for the β-galactosidase assay). Colonies were selected on SD/-Trp-Leu-His medium and cultured for 3 days at 30°C. The β-galactosidase assay was performed using chlorophenol red-β-D-galactopyranoside (CPRG) as substrate, and Miller units were calculated according to the Yeast Protocols Handbook (Clontech, PT3024-1). The experiment was conducted using three biological replicates.

Y2H assays were described previously (Jin et al., 2016). Yeast AH109 cells were co‐transformed with a specific bait and prey constructs according to the manufacturer's instructions (Clontech, CA). For Y2H assays of interactions of FaMYB10 (FaMYB10‐1 and FaMYB10‐2) with two regulators (FaWD40 and PavbHLH), FaMYB10‐1, FaMYB10‐2, FaMYB10‐1‐N (1–531 bp), FaMYB10‐1‐C (532–702 bp), FaWD40 and PavBHLH were amplified and cloned into pGADT7 vector (Clontech, CA); two regulators (FaWD40 and PavBHLH) of the flavonoid biosynthetic pathway were cloned into the pGBKT7 vector (Clontech, CA). All primers are listed in Table S6 and were acquired from Sangon Inc. (Sangon, Shanghai, China). All the constructs were confirmed by sequencing and then transformed into the yeast strain AH109 using the lithium acetate method (Gietz et al., 1995). The subsequent clones were plated onto selective medium lacking Leu and Trp (Leu, leucine; Trp, tryptophan), and finally, putative transformants were transferred onto selective medium lacking Ade, His, Leu and Trp (Ade, adenine; His, histidine). After the selected colonies had been tested, β‐galactosidase assays were undertaken following a yeast protocol (PT3024‐1; Clontech, CA). The selected colonies were transferred to plates containing 40 μg/mL X‐α‐gal (Clontech, CA), and the vector P53 plus SV40 was used to exclude false‐positive activation.