Yeast assays
The yeast two-hybrid and three-hybrid assay were performed using a
Clontech kit (PT3024-1 (PR973283)). For yeast two-hybrid assays, the
bait vectors (pGBKT7 plus candidate genes) and prey vectors (pGAGT7 plus
candidate genes) were transformed to Gold and Y187 yeast strain,
respectively, then pick each one colony of them to do the mating, and
spray the mating solution on SD/-Trp/-Leu/X-α-Gal/AbA (DDO/X/A) agar
plates, the positive results were confirmed by growing them on
SD/-Ade/-His/-Trp/-Leu/X-α-Gal/AbA (QDO/X/A) agar plates.
For
yeast three-hybrid assay, COP1-pGADT7 and COL3-COL13-pBbidge constructs
were co-transformed into Gold yeast strain. Pick each colony and grow
them in SD/-Leu/-Met/-Trp and SD/-Leu/-Trp solution, respectively.
Normalized Miller Units were calculated as a ratio of α-galactosidase
activity in yeast. For all yeast assays, we used empty vector as
controls.