Fig 2: HSP90.1 and HSP90.2 interact with BRI1 receptor domains in vivo and in vitro. (A) Yeast two-hybrid assays using BRI1LRR and BRI1KS as baits and HSPSP90.1 or HSP90.2 as preys. Upper column, co-transformants grown on SD-Leu-Trp medium (SD-2) demonstrate that both bait and prey plasmids are present in yeast cells. Positive selection of yeast two-hybrid interactions with minimal selection on SD-Leu-Trp-His medium (SD-3). (B) Interactions between HSP90s and BRI1LRR or BRI1KS domains by BiFC assays in tobacco epidermis. Interaction was detected between HSP90.1 and BRI1LRR or BRI1KS, and between HSP90.2 and BRI1KS. Scale bars, 20 μm. (C) Co-immunoprecipitation assays of HSP90.1 with BRI1LRR and BRI1KS and HSP90.2 with BRI1KS. The co-immunoprecipitated proteins were detected by anti-c-Myc or anti-HA.

Fig 3: HSP90 is required for hypocotyl elongation. (A) Hypocotyl lengths sensitivity assays of 5-day-old etiolated Col-0, hsp90.1, and hsp90.2, seedlings grown in the presence of 60 nM HΒL or 2 μM GDA. (B) 5-day-old hsp90.1 and hsp90.2 seedlings grown in different concentrations of HBL. Left plant of each pair of seedlings shows growth under control conditions and right upon treatment with HBL. (C) Hypocotyl length sensitivity assays in 5-day-old etiolated Col-0, hsp90.1, and hsp90.2 seedlings grown on a gradient of HBL concentrations. In box plots the middle line in the box represents median, the × shows mean, the bottom line depicts the 1st quartile, while the top line describes the 3rd quartile; the vertical lines (whiskers) extend to the minimum and maximum value within the 1.5× interquartile range (distance between the 1st and the 3rd quartile); points outside of the whiskers mark outliers (values outside of the 1.5× interquartile range). Data were analyzed by one-way ANOVA followed by Tukey’s test for multiple comparison (*): p < 0.05, (**): p < 0.01, ns: non-significant (n ≥ 30). In graph (A), the significance between the Col-0 at control conditions and the other treatment, Col-0 and hsp90.1 or hsp90.2 as well as between the control conditions of hsp90 mutants and treatments are indicated. In graph (C), the significance between control conditions and treatments is presented for each genotype. (D) In vivo interactions between BRI1 and HSP90.1 or HSP90.2 were confirmed by BiFC assays in tobacco leaf epidermis Scale bars, 20 μm.

Fig 4: A model for stabilization of BRI1 and POR by DAY in different subcellular compartments.DAY binds to and stabilizes BRI1 and POR in the endomembrane system and chloroplast, respectively. The cTP sequence is sufficient to transfer DAY to the inner envelope membrane of chloroplast and the first transmembrane domain is required for DAY localization in ER, TGN/EE, and plasma membrane. DAY-mediated BR signaling and chlorophyll biosynthesis are essential for photomorphogenesis. The illustration of nuclear envelope-ER-Golgi is purchased from Biorender.

Fig 5: Impaired HSP90 function affects BRI1 protein levels at the plasma membrane of hypocotyl cells. (A) BRI1:BRI1-GFP localization in hypocotyl cells of 5-d-old, etiolated wild-type seedlings growing under control conditions or after treatment with 10 μΜ GDA for 24 h or hsp90.1 mutant in resting conditions and upon the exogenous application of 100 nM HBL. The white lines mark the cells used for the quantification of the fluorescence signal intensity presented in plot profiles at the right of the images, generated by normalized values of fluorescence intensity. Propidium iodide (PI) cell wall stain was used to visualize the cell patterns in etiolated hypocotyls. Scale bars, 20 μm. (B,C) Quantification of relative plasma membrane (PM) fluorescence intensity of untreated and treated with 10 μm GDA BRI1-GFP hypocotyls (B) and hsp90.1 and hsp90.2 mutants (C), in the absence or presence of 100 nΜ HBL. PΜ fluorescence intensity was normalized to background fluorescence for each measurement (n = 30). In box plots, the middle line in the box represents median, the × shows mean, the bottom line depicts the 1st quartile, while the top line describes the 3rd quartile; the vertical lines (whiskers) extend to the minimum and maximum value within the 1.5× interquartile range (distance between the 1st and the 3rd quartile); points outside of the whiskers mark outliers (values outside of the 1.5× interquartile range). Data were analyzed with ANOVA followed by Tukey’s test, ** is significant at p ˂ 0.01, ns is non-significant. (D) Images of 5-day-old etiolated WS-2, bak1-1, and bri1-4 seedlings grown in the presence of 60 nM HΒL, 2 μM GDA and 60 nM HBL or 2 μM GDA. Scale bars, 0.5 cm. (E) Hypocotyl length sensitivity of 5-day-old etiolated WS-2, bak1-1 and bri1-4 seedlings grown under the indicated conditions. Data are presented as means ± SD (n > 30). Data were analyzed with one-way ANOVA followed by Tukey’s test, ** is significant at p ˂ 0.01, * is significant at p ˂ 0.05, ns is non-significant. (F) Western blot analysis using anti-BRI1 antibody. Proteins were extracted from 5-day-old etiolated seedlings of WS-2, bri1-4, or bak1-1 treated for 4 h with 60 nM HΒL or 2 μM GDA. CBB staining served as loading control.