Yeast-based Transactivation and Trans-repression Assays

For transactivation assay, we cloned the full-length coding sequence of OsMYB7 into the vector pGBKT7 (BD Biosciences Clontech) between SalI and NotI sites, and that of OsbHLH079 into the same vector between EcoRI and NotI sites. For trans-repression assay, the activation domain of GAL4 transcription factor in the vector pGADT7 (BD Biosciences Clontech) was cloned in frame between NcoI and EcoRI sites in the vector pGBKT7 (BD Biosciences Clontech) to generate the vector rGAL4, as previously described (Mathew et al., 2016 (link)). Next, we cloned the full-length coding sequences of OsMYB7 and ONAC026 into the vector rGAL4 between SalI and NotI sites. All the resulting constructs were transformed into the Saccharomyces cerevisiae strain AH109. Subsequently, yeast β-galactosidase liquid assays were performed according to the Yeast Protocols Handbook (BD Biosciences Clontech) using chlorophenol red-β-D-galactopyranoside (CPRG; Roche, Basel, Switzerland) as substrate. Briefly, we incubated yeast extracts in 8 mM CPRG (Roche) for 30 min at 30°C in the dark and measured the absorbance of extracts at a wavelength of 574 nm using a UV/VIS spectrophotometer (PowerWave X, BioTek, Winooski, USA). Primers details are provided in Supplementary Table S4.