Fig. 5: PhyB inhibits the dimerization of PIF6 and influences its DNA-binding and transcriptional activation activities.
From: Structural insight into PIF6-mediated red light signal transduction of plant phytochrome B
a, b LCI assays demonstrate that red light-irradiated phyB (a) or phyBY276H (b) inhibits the dimerization of PIF6αΔC bundle composed of helicesΔC in Nicotiana benthamiana leaves. Three independent experiments were performed, with similar results. PhyBQ109A and phyBY276H/Q109A are mutants hardly interacting with PIF6. c EMSA analysis shows that PIF6 can bind to the DNA probe containing the G-box (5′-CACGTG-3′) motif. PhyB-Pfr influences the DNA-binding activity of PIF6. A reported G-box-containing DNA probe “G-wt”30 is used in the EMSA assays. The DNA probe is labeled with FAM at the 5′ end. Left panel: the final DNA probe concentrations in Lanes 1‒5 are all 15 nM, and the corresponding protein concentrations are 0, 0.375, 0.75, 1.5, and 3.0 μM, respectively. Middle and right panels: EMSA analysis shows that red light-irradiated phyB-Pfr influences the DNA-binding activity of PIF6αΔC bundle composed of helicesΔC. The final concentrations of PIF6αΔC bundle composed of helicesΔC in Lane 1‒5 are all 1.5 μM, and the corresponding concentrations of phyB-Pfr are 0, 0.75, 1.5, 3.0, and 6.0 μM, respectively. d Reporter (FT promoter: Luc and SV40: Renilla), PIF6αΔC bundle composed of helicesΔC, and phyBY276H are transfected into the HeLa cells, as indicated by “‒” or “+”. The relative firefly luciferase (LUC) activities are normalized to the Renilla luciferase (REN) activity. FT, FLOWERING LOCUS T. Data are presented as means ± SD (n = 3). **P < 0.01; ***P < 0.001; ****P < 0.0001. PhyBY276H/Q109A is a mutant hardly interacting with PIF6. e A proposed model for the mechanisms of Pr→Pfr photoconversion and PIF6-mediated signal transduction of plant phyB. PhyB-Pfr forms a homodimer in a “head-to-head” manner. PIF6 stabilizes the dimerization of the PSMs and maintains the conformation of phyB-Pfr, and meanwhile, phyB-Pfr inhibits the dimerization and influences the DNA-binding and the transcriptional activation activities of PIF6. Due to the lack of electron density, the structure of C-terminal region (PAS1-PAS2-HKRD domains) in phyB-Pfr requires further investigation.
