Volume 9, Issue 9, Pages 1260-1271 (September 2016) A Small G Protein as a Novel Component of the Rice Brassinosteroid Signal Transduction  Ge Zhang, Xiaoguang Song, Hongyan Guo, Yao Wu, Xiaoying Chen, Rongxiang Fang  Molecular Plant  Volume 9, Issue 9, Pages 1260-1271 (September 2016) DOI: 10.1016/j.molp.2016.06.010 Copyright © 2016 The Author Terms and Conditions

Figure 1 Phenotypes of OsPRA2 RNAi Plants (Pi) and OsPRA2-Overexpressing Plants (Po). (A) Gross morphology of Pi plants (lines Pi-1, -2, and -3) at the reproductive stage. WT, wild-type rice plants. Scale bar, 15 cm. (B) Leaf angle of flag leaf, second leaf, and third leaf of Pi plants. Scale bar, 1 cm. (C) OsPRA2 transcript levels in Pi plants determined by qRT–PCR. The transcript levels are normalized against that of rice ACTIN1. Transcript level in WT plants was set to 1.0. Columns represent means ± SD (n = 3). (D) Gross morphology of Po plants (lines Po-1, -2, and -3) at the reproductive stage. Scale bar, 15 cm. (E) Lamina inclination of flag leaf, second leaf, and third leaf of Po plants. Scale bar, 1 cm. (F) OsPRA2 transcript levels in Po plants determined by qRT–PCR. The transcript levels are normalized against that of rice ACTIN1. Transcript level in WT plants was set to 1.0. Columns represent means ± SD (n = 3). (G) Seed size of WT, Pi-3, and Po-3 plants. Scale bar, 1 mm. (H) Internode pattern of WT and Po-3 plants. Arrowheads indicate the nodes. Scale bar, 10 cm. Molecular Plant 2016 9, 1260-1271DOI: (10.1016/j.molp.2016.06.010) Copyright © 2016 The Author Terms and Conditions

Figure 2 BR Sensitivity of Pi and Po Plants. (A) Lamina joint bending response of the wild-type (WT) and Pi-3 plants to exogenous brassinolide (BL) at 0 (−) or 10−7 M (+). (B) Dose response of lamina bending to BL in WT, Pi, and Po plants. Values are means ± SEM (n = 10). (C) Morphology of WT, Pi-3, and Po-3 seedlings grown in the dark. Arrows indicate nodes. Arrowheads indicate mesocotyls. Scale bar, 1 cm. (D) Effect of 10−7 M BL on coleoptile elongation of WT, Pi-3, and Po-3 plants. Scale bar, 1 cm. (E) Coleoptile length of WT, Pi-3, and Po-3 plants in the presence or absence of 10−7 M BL. Columns represent means ± SD (n = 10). (F) Root length of WT, Pi-3, and Po-3 plants in the presence or absence of 10−7 M BL. Columns represent means ± SD (n = 10). Molecular Plant 2016 9, 1260-1271DOI: (10.1016/j.molp.2016.06.010) Copyright © 2016 The Author Terms and Conditions

Figure 3 OsPRA2 Negatively Regulates BR Signaling in Rice. (A) Transcript levels of BR biosynthetic genes in the wild-type (WT), Pi, and Po determined by qRT–PCR. Gene transcript levels were normalized against that of rice ACTIN1. Transcript levels in WT plants were set to 1.0. Columns represent means ± SD (n = 3). (B) Abundance of phosphorylated (OsBZR1-P) and dephosphorylated (OsBZR1) forms of OsBZR1 in. Top panel: immunoblot with anti-OsBZR1 antibody (α-OsBZR1). Bottom panel: immunoblot with anti-heat shock protein antibody (α-HSP). Rice HSP was used as the internal control, indicating equal loading of total proteins. (C) Transcript levels of OsPRA2 at indicated times after treatment with exogenous BL. Columns represent mean ± SD (n = 3). Molecular Plant 2016 9, 1260-1271DOI: (10.1016/j.molp.2016.06.010) Copyright © 2016 The Author Terms and Conditions

Figure 4 Specific Interaction between OsPRA2 and OsBRI1. (A) Subcellular localization of EGFP–OsPRA2 fusion protein in rice protoplast. Scale bar, 10 μm. (B) Bimolecular fluorescence complementation assay showing interaction between OsPRA2 and OsBRI1 in plasma membrane of rice protoplasts. Scale bar, 10 μm. (C) Morphology of the wild-type (Taichung65, the background of d61-1 and d61-2, abbreviated as T65), d61-1 and d61-2 plants, and the OsPRA2 overexpression or knockdown lines in T65, and d61-1 and d61-2 mutants. Scale bar, 20 cm. (D) In vivo co-immunoprecipitation of OsPRA2 and OsBRI1. Total protein extracts from rice protoplasts transiently expressing HA-OsPRA2 (or EGFP-HA) and OsBRI1-myc were immunoprecipitated with anti-myc antibody (α-myc). Immunoblotting was performed with an anti-HA antibody (α-HA) to find the co-immunoprecipitated proteins. EGFP-HA was used as a control. (E) In vitro pulldown assay of His-OsPRA2 with the bait protein: GST–OsBRI1 or GST. Top panel: input and pulldown of His-OsPRA2. The smaller bands may represent degradation products of His-OsPRA2. Bottom panel: bait proteins after GST pulldown. Molecular Plant 2016 9, 1260-1271DOI: (10.1016/j.molp.2016.06.010) Copyright © 2016 The Author Terms and Conditions

Figure 5 OsBRI1 Kinase Activity Is Inhibited by OsPRA2. (A) Autophosphorylation activity of OsBRI1 in the presence of different amounts of OsPRA2. Purified OsBRI1 protein (2 μg) was incubated with [γ-32P]ATP and autophosphorylated. Addition of increasing amounts of OsPRA2 (2, 4, and 8 μg) led to weaker autophosphorylation activity of OsBRI1, but addition of GST protein (4 μg) did not change this activity. The upper panel shows the level of OsBRI1 phosphorylation detected by autoradiography (Autorad), and the lower panel the purified proteins detected by Coomassie brilliant blue (CBB) staining. (B) Phosphorylation activity of OsBRI1 toward substrate, OsBAK1 (M), in the presence of different amounts of OsPRA2. After incubation with [γ-32P]ATP, OsBRI1 (2 μg) showed strong autophosphorylation activity and transphosphorylation activity toward OsBAK1 (M) (4 μg), which was not autophosphorylated. Addition of increasing amounts of OsPRA2 (2, 4, and 8 μg) gradually weakened OsBRI1 transphosphorylation activity toward OsBAK1 (M), but addition of GST protein (4 μg) did not. Upper panel: the level of protein phosphorylation detected by autoradiography (Autorad). Lower panel: the purified proteins detected by CBB staining. Molecular Plant 2016 9, 1260-1271DOI: (10.1016/j.molp.2016.06.010) Copyright © 2016 The Author Terms and Conditions

Figure 6 OsPRA2 Inhibits Interaction between OsBRI1 and OsBAK1. (A and B) Luciferase complementation assay showing that OsPRA2 inhibits OsBRI1 self-interaction (A) and its interaction with OsBAK1 (B). Luminescence intensities of c and g were set to 1.0 (n = 8). **P < 0.01, Student's t-test. (C and D) Microscale thermophoresis analysis showing that OsPRA2 inhibits the OsBRI1 self-interaction (C) and the interaction between OsBRI1 and OsBAK1 (D). Binding of unlabeled OsBRI1 or OsBAK1 to fluorescently labeled OsBRI1 is quantified in PBS buffer. The concentration of unlabeled OsBRI1 and OsBAK1 was varied from 0.7 nM to 20 μM. To test the interference of OsPRA1 to these protein interactions, we added the OsPRA2 or GST protein at a constant concentration of 150 nM. The results show a strong interference of OsPRA2 on these protein interactions, whereas no interference was observed for GST protein. Molecular Plant 2016 9, 1260-1271DOI: (10.1016/j.molp.2016.06.010) Copyright © 2016 The Author Terms and Conditions