Yeast assays
The yeast two-hybrid and three-hybrid assays 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 into the gold and Y187 yeast strains,
respectively, and then each colony was picked for mating, and the mating
solution was sprayed on SD/-Trp/-Leu/X-α-Gal/AbA (DDO/X/A) agar plates,
and positive results were confirmed by growing them on
SD/-Ade/-His/-Trp/-Leu/X-α-Gal/AbA (QDO/X/A) agar plates.
For
the yeast three-hybrid assay, COP1-pGADT7 and COL3-COL13-pBbidge
constructs were co-transformed into the gold yeast strain. Each colony
was picked and grown in SD/-Leu/-Met/-Trp and SD/-Leu/-Trp solutions,
respectively. Normalized Miller units were calculated as a ratio of
α-galactosidase activity in yeast. For all yeast assays, we used empty
vectors as controls.