Yeast one-hybrid (Y1H)assay

Total RNA was isolated from rice seedlings, as described by Almeida et al. (2017). The cDNA expression library was synthesized according to HybriZAP-2.1 XR cDNA Synthesis and Library Construction Kits (Stratagene), as described by the manufacturer. The screening was performed according to the Matchmaker, One-Hybrid System (Clontech). The reporter vectors containing bait F and bait G were introduced into yeast strain PJ69-4A, as previously reported (Assunção et al., 2010). Yeast bait strains were transformed with 1 µg of the cDNA expression library. For each reporter vector, >1 million yeast colonies were screened in CM-His supplemented with 10–40 mM 3-aminotriazole (3-AT), as previously described (Ouwerkerk and Meijer, 2001). The identified clones were re-streaked on selective medium to confirm growth. Direct PCR on the yeast colonies was performed to amplify the cDNA insert, using specific primers for the library plasmid. The sequences were used to search for homology in the rice genome.