Figure 7.

MYB30 Inhibits the Interaction between PIF4/PIF5 and phyB.

(A) Yeast three-hybrid assays showing that MYB30 inhibits the interaction between PIF5 and Pfr-phyB in yeast cells. AD-PIF5, phyB-BD, and MYC-MYB30 were individually expressed in the yeast strain Y190 as indicated. The β-galactosidase activities were measured by liquid culture assays using ONPG as the substrate. Error bars represent sd of three independent yeast cultures. Different letters represent statistical significances determined by an ANOVA with Tukey’s post hoc test (P < 0.05; Supplemental Data Set 3).

(B) and (C) Pull-down assays showing that MYB30 inhibits the interactions of PIF5 (B) and PIF4 (C) with phyB. Total proteins extracted from 4-d-old R light–grown 35S_pro:phyB-GFP seedlings were the bait, and His-PIF5/His-PIF4 fusion proteins were the prey. Equivalent amounts of phyB-GFP protein extract and His-PIF5/His-PIF4 fusion proteins were added, respectively, as indicated, and increasing amounts of MBP-His-MYB30 were added before anti-GFP IP assays were performed. The pulled-down proteins were analyzed by immunoblotting with antibodies against phyB (top panels), PIF4 or PIF5 (middle panels), and MBP (bottom panels). Numbers below the immunoblots indicate the relative intensities of His-PIF5 (B) and His-PIF4 (C) bands normalized to those of phyB-GFP, and the ratio was set to 100 for the first band.