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).