Figure 1. Yeast Reverse-Hybrid Screen Identifies phyBNT Missense Mutations that Result in Reduced Interaction with PIF3.

The yeast strain MaV103a, which possesses the genes LacZ and URA3 under the control of Gal4, was transformed with GAD-PIF3- and phyBNT-DBD-containing plasmids or PCR product. A) Schematic showing that in the event of normal, light-induced interaction between phyBNT-DBD and GAD-PIF3 proteins, B-galactosidase and URA3 protein are produced, resulting in cell death on media containing 5-FOA. B) Schematic showing that in the event of a mutation in phyBNT that abolishes interaction with PIF3, little URA3 protein accumulates, resulting in survival even in the presence of 5-FOA. The light blue box represents the chromophore, PCB, and the yellow star represents a missense mutation in phyBNT. C) Enzymatic Assays to test for β-galactosidase activity using ONPG as a substrate were performed for each mutant and the phyBNT wild type control exposed to 5-minute saturating pulses of either red or far red light in the presence of PCB. Red bars represent the interaction in red light and grey bars represent the interaction in far red light. β-galactosidase activity is represented in Miller Units. Technical triplicates were performed and mean values were plotted with error bars representing standard error.