Fig. 8

The C-terminus of PHYB is a signaling-output module for PIF3 degradation. a In vitro co-immunoprecipitation experiments showing that the D1040V mutation disrupts the interaction between the C-terminal module of PHYB (BC) and PIF3. HA-tagged PIFs (HA-PIF3) and Myc-tagged C-terminal domain of PHYB or PHYB18, Myc-BC or Myc-BC18, were co-translated in vitro. HA-PIF3 was pulled down by anti-HA affinity matrix. The input and bound HA-PIF3, Myc-BC, and Myc-BC18 were detected by immunoblots using either anti-HA or anti-Myc antibodies. The estimated bound fractions of Myc-BC and Myc-BC18 are shown below the anti-Myc blots. b GST pull-down assays using GST-PIF3 to pull down PHYB-HA and PHYB18-HA in apoprotein, Pr, or Pfr forms. The upper panels are immunoblots using anti-HA antibodies showing bound and input fractions of PHYB-HA or PHYB18-HA. The lower panels are Coomassie Blue-stained gels showing immobilized GST and GST-PIF3. The estimated bound fractions of PHYB-HA or PHYB18-HA for each pulldown assays are shown below the blots. G, GST; P, GST-PIF3. The asterisk indicates nonspecific bands. c Images of 4-day-old Ler, phyB-5, PBG, PBG-R110Q, PBG-G111D, PBG-R352K lines grown in 10 μmol m⁻² s⁻¹ R light. d Box and whisker plots of hypocotyl length measurements of the respective seedlings shown in c. Samples with different letters indicate statistically significant differences in hypocotyl length (ANOVA, Tukey’s HSD, P < 0.05, n > 40). e Immunoblots showing the levels of PHYB and PIF3 in 4-day-old Ler, phyB-5, PBG, PBG-R110Q, PBG-G111D, and PBG-R352K lines grown in 10 μmol m⁻² s⁻¹ R light. Dark-grown pifq samples were used as negative controls for the PIF3 immunoblots. RPN6 was used as a loading control. The relative levels of PHYB and PIF3 were normalized against the corresponding levels of RPN6 and are shown below the blots. Asterisks indicate nonspecific bands. f Model for the structure-function relationship of Arabidopsis PHYB. The N-terminal photosensory module of PHYB is responsible for light sensing through the bilin chromophore in the GAF domain and subsequent conformational changes of the tongue (green loop). Photoactivation of the N-terminal module induces an Pfr-specific interaction between the knot lasso and PIF3, this interaction contributes to light signaling by repressing the transcriptional activity of PIF3. The GAF domain provides a dimerization interface within the N-terminal module. The C-terminal output module of PHYB interacts directly with PIF3 to mediate its degradation. The PRD is responsible for mediating the nuclear accumulation of PHYB and the entire C-terminal module is required for photobody localization. The entire HKRD provides another dimerization domain. The HKRD communicates with the PRD to facilitate PHYB nuclear accumulation. Dimerization of the HKRD is required for both nuclear and photobody localization of PHYB. The domain structure of PHYB is modified from Burgie and Vierstra⁷