Interaction of OsMPK20-4 with OsMPK3 by yeast two-hybrid screening. (A) Reporter yeast strain AH109 was co-transformed with the recombinant pGBKT7 and pGADT7 encoding the listed gene constructs. Transformants were selected on SD [-leu-trp] double dropout (DDO) media and the interaction was checked on SD [-trp-leu-ade-his] quadruple dropout (QDO) media. (B) β-galactosidase assay showing the lacZ reporter gene expression by using ONPG as substrate. Liquid cultures of AH109 reporter strain transformed with the mentioned constructs were checked for β-galactosidase activity by adding ONPG and reading the absorbance at OD = 420 nm.AH109 co-transformed with SV40 large T antigen in pGADT7 vector and p53 in pGBKT7 vector served as positive control of protein interaction in all the experiments.