Volume 21, Issue 5, Pages 825-834 (November 2011) Dual Role of BKI1 and 14-3-3 s in Brassinosteroid Signaling to Link Receptor with Transcription Factors  Haijiao Wang, Cangjin Yang, Chi Zhang, Niyan Wang, Dihong Lu, Jie Wang, Shanshan Zhang, Zhi-Xin Wang, Hong Ma, Xuelu Wang  Developmental Cell  Volume 21, Issue 5, Pages 825-834 (November 2011) DOI: 10.1016/j.devcel.2011.08.018 Copyright © 2011 Elsevier Inc. Terms and Conditions

Developmental Cell 2011 21, 825-834DOI: (10.1016/j.devcel.2011.08.018) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 The Amino and Carboxyl Regions of BKI1 Have Distinct Functions (A and B) The phenotype of plants overexpressing the empty vector (V), the full-length BKI1-YFP (FL), the BKI1-N-YFP (N), and the BKI1-C-YFP (C) in the Col-0 (A) and bri1-301 (B) backgrounds. The lower panels show the corresponding protein levels. Ponceau-S Stained Rubisco large subunit serves as a loading control. (C–F) The expression levels of some BR-responsive genes in various BKI1-related transgenic plants, Col-0, and bri1-301. CPD (C), DWF4 (D), and Saur-AC1 (E) are in the Col-0 background. The seedlings were treated with 1.0 μM eBL for 1 hr. (F) The expression levels of CPD, DWF4, and Saur-AC1 in the bri1-301 background. All error bars indicate standard deviation (SD). Asterisks indicate a statistically significant difference from the wild-type (p < 0.05); pounds indicate a significant difference from the eBL treated control (p < 0.05). See also Figure S1 and Table S1. Developmental Cell 2011 21, 825-834DOI: (10.1016/j.devcel.2011.08.018) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Cytosolical Localization of the Carboxyl Termini of BKI1 Enhances BR Signaling (A–I) The subcellular localization of BKI1-YFP (A–C), BKI1-N-YFP (D–F), and BKI1-C-YFP (G–I) before and after eBL treatment in Col-0 background. Scale bars, 20 μm. Seedlings were grown on 1/2 Murashige & Skoog (MS) medium for 4 days. (A), (D), and (G) were untreated; (B), (E), and (H) were treated with 1 μM eBL for 5 min; and (C), (F), and (I) were treated with 1 μM eBL for 10 min. See also Figure S2. (J) The phenotype of plants expressing BKI1-C fused to a nuclear localization signal (NLS) or a nonfunctional NLS (nls) in bri1-301 background. The T1 seedlings were grown under dim light for 14 days. (K) The accumulation of BES1 in the pooled larger plants and the pooled bri1-301-like plants was detected by western blotting with anti-BES1 antibody. Ponceau-S Stained Rubisco large subunit serves as a loading control. Developmental Cell 2011 21, 825-834DOI: (10.1016/j.devcel.2011.08.018) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 The S270 and S274 Sites of BKI1 Play Essential Roles in BKI1 Dissociation from the Plasma Membrane (A) The phosphorylation status of BKI1-FLAG on Serine residues in seedlings with or without eBL treatment. Immunoprecipitation (IP) with anti-FLAG agarose; immunoblotting with anti-pSer or anti-FLAG antibody. The seedlings were grown on 1/2 MS medium containing 1 μM BRZ220 and were treated with 5 μM eBL or mock (DMSO) for 1 hr. (B) The subcellular localization of BKI1S270/274A-GFP, BKI1S270A-YFP, and BKI1S274A-YFP proteins in Col-0 before and after 1 μM eBL treatment. The time of each treatment is indicated. Scale bars, 10 μm. (C) The phenotype of plants overexpressing BKI1S270/274A-GFP, BKI1S270A-YFP or BKI1S274A-YFP in Col-0 background. Their protein levels were detected by western blotting with anti-GFP antibody. (D) The BES1 accumulation status in the BKI1-YFP and BKI1S270/274A-GFP transgenic plants. Immunoblotting with anti-BES1 antibody shows levels of the phosphorylated BES1 (p-BES1) and the dephosphorylated BES1. The 14-old-day seedlings were treated with 1 μM eBL (+) or DMSO (−) for 1 hr. In (C) and (D), Ponceau-S Stained Rubisco large subunit serves as a loading control. (E) Phosphorylation assay of wild-type and mutated BKI1 by BRI1 kinase. An equal amount of recombinant proteins, as indicated by the Coomassie Brilliant Blue (CBB) staining (bottom), was incubated with [γ-32P] ATP and separated by SDS-PAGE, followed by autoradiography (top). See also Figure S3 and Table S2. Developmental Cell 2011 21, 825-834DOI: (10.1016/j.devcel.2011.08.018) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 eBL Treatment Enhances the Interaction of BKI1 with 14-3-3κ In Vitro and In Vivo (A) The interaction of BKI1 with 14-3-3λ and 14-3-3κ was detected by semi-in vivo pull-down assay. 14-3-3λ and 14-3-3κ fused with GST were expressed and purified from E. coli. Protein extracts from plants overexpressing BKI1-FLAG was used for pull-down assay. (B) The interaction between BKI1 and 14-3-3κ was tested by BiFC assay. The indicated constructs were transformed into leaves of Nicotiana benthamiana. Scale bars, 20 μm. (C) The interaction of BKI1-YFP with 14-3-3κ-FLAG was detected by Co-IP. The 14-3-3κ-FLAG was IPed with anti-FLAG agarose, and the Co-IPed BKI1-YFP was detected by immunoblotting with anti-GFP antibody. (D) Two known 14-3-3-binding sequences (mode I and mode II) are aligned against the BKI1 sequence. “X” presents any given amino acid. The phosphoserine residues are marked with bold font. (E) The interaction of BKI1S270A/274A with 14-3-3κ was detected with a semi-in vivo pull-down assay. (F) The in vivo interaction of BKI1S270A/274A with 14-3-3κ was detected by Co-IP. (G) The interaction of BKI1S270A and BKI1S274A with 14-3-3κ was detected by semi-in vivo pull-down assay. In (A), (C), and (F), the 14-day-old seedlings were treated with 5.0 μM eBL (+) or DMSO (−) for 1 hr. An equal amount of recombinant proteins were indicated by the Coomassie brilliant blue (CBB) staining (bottom) in the semi-in vivo pull-down assay. See also Figure S4. Developmental Cell 2011 21, 825-834DOI: (10.1016/j.devcel.2011.08.018) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Overexpression of 14-3-3κ Can Release the Inhibitory Effect of BKI1 on Plant Growth (A) The interaction of BKI1-C-YFP with 14-3-3κ-FLAG was detected by Co-IP. (B) The phosphorylation assay of the truncated BKI1s by BRI1 kinase. An equal amount of recombinant protein, as indicated by the Coomassie brilliant blue (CBB) staining (bottom), was incubated with [γ-32P] ATP and separated by SDS-PAGE, followed by autoradiography (top). (C) The phenotype of the 14-3-3κ-FLAG overexpression lines in Col-0 background. The protein levels of 14-3-3κ-FLAG were detected by western blotting with anti-FLAG antibody. (D–F) The phenotype of plants overexpressing 14-3-3κ-FLAG with BKI1-YFP (D), BKI1S270/274A-GFP (E), or BKI1S270A-YFP (F). The expression levels of transgenes were detected by western blotting with anti-FLAG and anti-GFP antibody. (G) The subcellular localization of BKI1-YFP or BKI1S270A-YFP in the pavement cells of Nicotiana benthamiana. See also Figure S5. Developmental Cell 2011 21, 825-834DOI: (10.1016/j.devcel.2011.08.018) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 The Phosphorylated BKI1 Competes 14-3-3κ Away from BES1 (A) The in vivo interaction of 14-3-3κ-FLAG with BES1. The protein extracts from the 14-3-3κ-FLAG line were IPed with anti-FLAG agarose, and the resulting precipitates were detected by western blotting with anti-FLAG or anti-BES1 antibody. The 14-day-old seedlings were treated with DMSO (−) or 5.0 μM eBL (+) for 1 hr. (B) The level and phosphorylation status of BES1 in cytosolic (C) or nuclear (N) fractions from the BKI1-C overexpression lines. The western blotting with anti-BES1 antibody was used to detect BES1. Histone H2B was used as a nuclear marker. (C) The hypocotyl length of dark-grown seedlings on 1/2 MS medium containing different concentrations of BRZ220 for 4 days. The “FL-High” line is the commonly used BKI1-YFP overexpression line in our study. See also Figure S6. (D) The effect of phosphorylated BKI1 on the interaction of 14-3-3κ with BES1. The protein extracts from the 14-3-3κ-FLAG plants were IPed with anti-FLAG agarose. MBP fused BKI1 and BKI1S270/274A proteins (5 μg) were preincubated with BRI1 kinase (0.5 μg) with or without 10 mM ATP for 30 min, and then they were added to the IPed 14-3-3κ-FLAG products and incubated for 2 hr. The proteins bound with anti-FLAG agarose were eluted and detected by western blotting with anti-BES1 antibody. The inputs of MBP fused proteins and 14-3-3κ were detected by western blotting with anti-MBP and anti-FLAG antibody, respectively. The input of BES1 was determined by western blotting with anti-BES1 antibody. Developmental Cell 2011 21, 825-834DOI: (10.1016/j.devcel.2011.08.018) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 A Model of the BR Signal Transduction Pathway (A) In the absence of BRs, BKI1 proximal to the plasma membrane keeps BRI1 in an inactive state through their direct interaction; BIN2 phosphorylates BES1 and BZR1 and inhibits their DNA binding activity; 14-3-3 proteins keep the phosphorylated BES1/BZR1 in the cytoplasm. (B) BR binding to BRI1 activates BRI1 kinase, which promotes BKI1 phosphorylation. The phosphorylated BKI1 dissociates from plasma membrane and BRI1, and then binds to 14-3-3 proteins in cytosol to free BES1/BZR1. The released BES1/BZR1 accumulates in the nucleus, leading to BR-responsive gene expression. Stars represent the phosphorylation state. Developmental Cell 2011 21, 825-834DOI: (10.1016/j.devcel.2011.08.018) Copyright © 2011 Elsevier Inc. Terms and Conditions