Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice

Abstract

Brassinosteroids (BR) are essential growth hormones found throughout the plant kingdom. BR bind to the receptor kinase BRI1 on the cell surface to activate a signal transduction pathway that regulates nuclear gene expression and plant growth. To understand the downstream BR signaling mechanism in rice, we studied the function of OsBZR1 using reverse genetic approaches and identified OsBZR1-interacting proteins. Suppressing OsBZR1 expression by RNAi resulted in dwarfism, erect leaves, reduced BR sensitivity, and altered BR-responsive gene expression in transgenic rice plants, demonstrating an essential role of OsBZR1 in BR responses in rice. Moreover, a yeast two-hybrid screen identified 14-3-3 proteins as OsBZR1-interacting proteins. Mutation of a putative 14-3-3-binding site of OsBZR1 abolished its interaction with the 14-3-3 proteins in yeast and in vivo. Such mutant OsBZR1 proteins suppressed the phenotypes of the Arabidopsis bri1-5 mutant and showed an increased nuclear distribution compared with the wild-type protein, suggesting that 14-3-3 proteins directly inhibit OsBZR1 function at least in part by reducing its nuclear localization. These results demonstrate a conserved function of OsBZR1 and an important role of 14-3-3 proteins in brassinosteroid signal transduction in rice.

Fig. 1.

Suppressing OsBZR1 expression reduces BR responses in rice. (A) RT-PCR analysis of the expression of OsBZR1, OsBZR2, OsBZR3, and OsBZR4 in the wild-type and two OsBZR1 RNAi transgenic lines (R-1 and R-10). (B and C) The culm length (B) and lamina joint angles (C) of flag leaf and the top second and third leaves of OsBZR1 RNAi transgenic lines (R-1 and R-10), OsBRI1 mutant d61–2, and wild type. Data are average of measurements of six plants. (Error bars indicate SEM.) (D) Leaf morphology of OsBZR1 RNAi transgenic plant. (E) Effect of 24-epiBL on the lamina inclination of the wild-type and OsBZR1 RNAi transgenic plants R-1 and R-10. (Error bars indicate SEM.) (F) RT-PCR analysis of D2, D11, and BRD1 expression in the wild-type and transgenic rice with or without 24-epiBL treatment.

Fig. 2.

OsBZR1 interacts with 14-3-3 proteins through a conserved 14-3-3 protein-binding sequence. (A) Sequences of the putative 14-3-3 binding site in OsBZR1 and its mutagenized version, S156G. (B) Interaction between OsBZR1 and GF14c in yeast two-hybrid assays. Three clones of yeast containing each combination of bait (BD) and prey (AD) vectors were grown on complete medium (+Ade) or Ade drop-out selection medium (−Ade). AD-T7, pGAD-T7 empty vector was used as a negative control. (C) Bimolecular fluorescence complementation assays of the in vivo protein interaction. Leaf epidermal cells of N. benthamiana were cotransformated with nYFP–OsBZR1 and cCFP–GF14c, nYFP–OsBZR1S156G and cCFP–GF14c, or nYFP–OsBZR1 and cCFP. PI, propidium iodide fluorescence; Merge, overlay of the YFP and PI images. (D) The bri1-5 mutant transformed with OsBZR1–GFP (MOB3–18) or OsBZR1S156G–GFP (MOB5–57) were immunoprecipitated by using anti-GFP antibodies, and the blot was probed with a 14-3-3 antibody. (E) BR treatment reduces the interaction between OsBZR1 and GF14c. Tobacco leaves cotransformed with the nYFP–OsBZR1, and cCFP–GF14c constructs were treated with mock solution (−BL) or 10 μM 24-epiBL (+BL). YFP signals were analyzed by using confocal microscopy. (Scale bar, 50 μm.)

Fig. 3.

Overexpression of OsBZR1S156G, but not wild-type OsBZR1, suppresses the bri1-5 mutant phenotypes. (A) The bri1-5 plants transformed with 35S:OsBZR1–GFP (the MOB3 lines) or 35S:OsBZR1S156G–GFP (the MOB5 lines) were grown under long day conditions for 30 days. (B) RNA expression of OsBZR1–GFP and OsBZR1S156G–GFP analyzed by RT-PCR. Expression of the EF gene was used as a loading control. (C and D) Seedlings grown in the dark on half-strength MS (C) or on 2 μM BRZ (D) for 6 days. Graphs show average hypocotyl lengths measured from at least 20 seedlings. (Error bars indicate SEM.) (E) RT-PCR analysis of CPD, DWF4, and SAUR-Ac1 expression in bri1-5 and transgenic plants. Transcript of the EF gene was analyzed as a control for equal loading.

Fig. 4.

Nuclear localization of OsBZR1 is reduced by 14-3-3 protein binding and is induced by BR treatment. (A) Localization of OsBZR1–GFP and OsBZR1S156G–GFP fusion proteins in tobacco leaf epidermal cells. (Scale bars, 50 μm.) (B) Localization of OsBZR1–GFP and OsBZR1S156G–GFP fusion proteins in hypocotyl cells of transgenic Arabidopsis plants was analyzed with a spinning disk confocal microscope. (Scale bars, 10 μm.) (C) Subcellular localization of OsBZR1-GFP in transgenic Arabidopsis seedlings grown on MS or MS plus 2 μM BRZ media. B Right and C Right show the average nuclear/cytoplasmic ratio measured from seven cells. (Scale bars, 10 μm; error bars indicate SEM.) (D) OsBZR1–GFP transgenic Arabidopsis seedlings were analyzed with spinning disk confocal microscope in liquid MS medium every 15 min for 1 h. Then, 1 μM BL (arrow) was added, and images were acquired at the times shown. Average nuclear/cytoplasmic ratios of GFP signal were calculated from multiple cells. (Error bars indicate SEM.)