Yeast three-hybrid (Y3H) assay
CsEGL3-CsJAZ6/pBridge and CsAN1/pGAD constructs were generated for yeast three-hybrid analyses. The pBridge vector in the Y3H system utilizes an inducible Met25 promoter, while CsJAZ6 was subcloned into the pBridge vector and driven by the Met25 promoter. Co-transforming the constructs into AH109 yeast cells and selected on SD/-Leu/-Trp plates. Then colonies were moved to selective dropout liquid medium (SD/-Met/-Leu/-Trp/-His) with different concentrations of Met, and the β-galactosidase activity associated with CsAN1-CsEGL3 binding activities was quantified using the O-nitrophenyl-β-D-galactopyranoside (ONPG) liquid assay according to the manufacturer’s protocol (Clontech).