Matchmaker gold yeast two hybrid library screening system

Y2H analysis was performed using the Matchmaker Gold Yeast Two‐Hybrid Library Screening System (Clontech). The ORF of bHLH genes were amplified by PCR and inserted into pGBKT7. MYB genes were cloned into pGADT7 vector. All fusion constructs were introduced into yeast strain AH109 using the LiAc‐PEG method as described in the manual from the manufacturer (Clontech). The transformants were selected on SD/‐Leu/‐Trp medium. The positive colonies were transferred to SD/‐Leu/‐Trp/‐His/‐Ade medium containing X‐α‐Gal (5‐bromo‐4‐chloro‐3‐indolyl‐α‐d‐galactopyranoside) to test for interactions between pairs of proteins. Photographs were taken after 3–5 days of growth on the medium at 28 °C.

The Y2H screening was conducted using the LRR or CC domain of MG1 as the bait following the manufacturer’s instructions (Matchmaker Gold Yeast Two-Hybrid Library Screening System, Clontech, Dalian, China). A root cDNA library of non-inoculated rice cultivar ZH11 generated with Make Your Own ‘Mate & Plate’ Library System (Clontech, Dalian, China) was used for screening. Positive protein–protein interactions were selected by growing yeast colonies on synthetic defined (SD) medium lacking leucine, tryptophan, and histidine (SD–L–T–H, TDO). Combinations of AD/SV40 with BD/p53 or BD/Lam served as positive or negative controls, respectively. To test MG1 self-interaction and to confirm the protein–protein interactions, different combinations of MG1 fragments and their interacting candidates, as indicated in the figures, were co-transformed into yeast strain AH109. Colonies grown on SD–L–T (DDO) medium were cultured, diluted, and plated on TDO medium. Plates were kept at 30 °C for 3 d before being photographed.