Characterization of the molecular mechanism underlying gibberellin perception complex formation in rice

Abstract

The DELLA protein SLENDER RICE1 (SLR1) is a repressor of gibberellin (GA) signaling in rice (Oryza sativa), and most of the GA-associated responses are induced upon SLR1 degradation. It is assumed that interaction between GIBBERELLIN INSENSITIVE DWARF1 (GID1) and the N-terminal DELLA/TVHYNP motif of SLR1 triggers F-box protein GID2-mediated SLR1 degradation. We identified a semidominant dwarf mutant, Slr1-d4, which contains a mutation in the region encoding the C-terminal GRAS domain of SLR1 (SLR1(G576V)). The GA-dependent degradation of SLR1(G576V) was reduced in Slr1-d4, and compared with SLR1, SLR1(G576V) showed reduced interaction with GID1 and almost none with GID2 when tested in yeast cells. Surface plasmon resonance of GID1-SLR1 and GID1-SLR1(G576V) interactions revealed that the GRAS domain of SLR1 functions to stabilize the GID1-SLR1 interaction by reducing its dissociation rate and that the G576V substitution in SLR1 diminishes this stability. These results suggest that the stable interaction of GID1-SLR1 through the GRAS domain is essential for the recognition of SLR1 by GID2. We propose that when the DELLA/TVHYNP motif of SLR1 binds with GID1, it enables the GRAS domain of SLR1 to interact with GID1 and that the stable GID1-SLR1 complex is efficiently recognized by GID2.

Figure 1.

Gross Morphology of Rice Semidominant Dwarf Mutant F4443, Its Response to GA₃ Treatment, and Segregation Ratio of F2 Plants. (A) Gross morphology of a wild-type Nipponbare (left), F1 plant derived from a cross between F4443 and Nipponbare (center), and F4443 homozygous plant (right). Bar = 10 cm. (B) Elongation of second leaf sheath in response to GA₃ treatment. Nipponbare was used as a control. Data are means ± sd; n = 10. (C) Segregation of F2 progeny of a self-pollinated F1 plant (F4443 × Nipponbare).

Figure 2.

The Dwarf Phenotype of F4443 Is Caused by a Mutation in the SLR1 SAW Domain, Which Leads to a Reduced Interaction with GID1. (A) Schematic structure of SLR1. The protein in F4443 (Slr1-d4) contains a G576V substitution (SLR1^G576V). (B) Comparison of the region around G576 of DELLA proteins from various plant species. Rice (Kamiya et al., 2003) and Arabidopsis (Di Laurenzio et al., 1996) SCR, which belong to another GRAS protein family, are also shown. Zm, Zea mays; At, Arabidopsis thaliana; Sm, Selaginella moellendorffii; Os, Oryza sativa. (C) Gross morphology of transgenic plants at harvest. FLAG-tagged SLR^WT and FLAG-tagged SLR1^G576V were each overproduced in wild-type T65 rice. Vector, T65 transformed with proAct-FLAG/pCAMBIA control vector. Bar = 10 cm. (D) Degradation of SLR1 and SLR1^G576V protein upon GA₃ treatment in rice callus. Nipponbare and Slr1-d4 calli were incubated with 10⁻⁵ M GA₃ for the indicated times, and the crude protein extracts were subject to immunoblot analysis using an anti-SLR1 antibody. The loading control of Coomassie blue (CBB) staining is shown in the bottom panel. (E) Interaction between GID1 and SLR1^G576V with (+) or without (−) 10⁻⁴ M GA₄. Left, β-Gal activity detected in a liquid assay with yeast strain Y187 transformants (means ± sd; n = 3). Right, Growth of yeast strain AH109 transformants on –HIS plates. GID1 was used as bait, and SLR1 and its mutants were used as prey. SLR1 (E4-R125), DELLA/TVHYNP domain; ΔDELLA, SLR1 containing a deletion in the DELLA domain (from D39 to A55).

Figure 3.

Physicochemical Analysis of the Interaction between GID1 and SLR1^WT, SLR1^G576V, and SLR1 (E4-R125). (A) Various kinetic constants of GID1-SLR1, GID1-SLR1^G576V, and GID1-SLR1 (E4-R125) interactions in the presence of GA₄. Parameters were calculated by the heterogenous ligand model for three interactions or by the 1:1 binding model for GID1-SLR1^G576V and GID1-SLR1 (E4-R125) interactions. GST-fused SLR1 and SLR1 mutant proteins were used for the analysis. (B) to (D) Interactions between GID1 and SLR1 proteins in the presence of GA₄. Note that the response unit (RU) value of the GID1-SLR1^WT interaction does not return to the baseline level at the end of the reaction due to a delayed dissociation rate (B) compared with GID1-SLR1^G576V (C) and GID1-SLR1 (E4-R125) (D), as shown by arrows. (B) GID1-SLR1^WT interaction. (C) GID1-SLR1^G576V interaction. (D) GID1-SLR1 (E4-R125) interaction.

Figure 4.

GID1, GID2, and GA-Dependent Degradation of SLR1 in Yeast Cells. (A) and (B) Interaction of SLR1-GID2 (A) and GID1-SLR1 (B) in yeast cells. Interaction of BD and AD fusion proteins in yeast cells with or without 10⁻⁴ M GA₄ were scored using β-Gal activity (means ± sd; n = 3). Either GID2 or GID1 was used as bait, SLR1 was used as prey, and either GID1 or GID2 was expressed in yeast as a third clone. (C) and (D) Accumulation of AD-HA-SLR1, HA-GID2, and HA-GID1 protein in yeast cells. Crude protein extracts from yeast grown in the absence or presence of 10⁻⁴ M GA₄ were subject to immunoblot analysis and detected using the HA antibody for HA-SLR1 and HA-GID2, and anti-GID1 antibody for HA-GID1. The loading control of Coomassie blue (CBB) staining is shown in the bottom panels. [See online article for color version of this figure.]

Figure 5.

Ala Scanning Analysis of GID2 for SLR1-Interacting Activities. SLR1-interacting activity of 68 mutated GID2s and the wild-type GID2 was assessed using a Y3H assay with GID2s as baits, the full-length SLR1 as prey, and GID1 expressed as a third clone (means ± sd; n = 3). Interactions were analyzed in the absence (red bars) or presence (blue bars) of 10⁻⁴ M GA₄. Activity of wild-type GID2 is shown at the far left of the graph. The minimum region necessary for SLR1 interaction (E114-P193) is indicated within the graph. Activities of regions E114-P193 and N123-P193 are shown at the far right side of the graph. A schematic structure of GID2 is shown at the bottom.

Figure 6.

Interaction of GID2^L76A, SLR1, and GID1 in Yeast Cells. Interactions of BD and AD fusion proteins with or without 10⁻⁴ M GA₄ were scored by β-Gal activity (means ± sd; n = 3). For the Y3H assay, GID2^L76A was used as bait, SLR1 (wild-type or mutant) was used as prey, and either GID1 or gid1-1 mutant protein was expressed as a third clone. The GA-dependent interaction of SLR1 and GID1 proteins is indicated with a circle (strong interaction) or triangle (weak interaction), and “×” indicates no interaction.

Figure 7.

Ala Scanning Analysis of SLR1 for GA-Dependent GID2- and GID1-Interacting Activities. Interacting activities between 34 mutated SLR1s and GID1 or GID2^L76A are shown by blue and red bars, respectively (means ± sd; n = 3). SLR1^WT and SLR1 (E4-R125) were used as controls (far left). Activities are shown as a relative rate, with activities of wild-type SLR1 set as 1. To measure interactions between SLR1s and GID1, a Y2H assay was performed using GID1 as bait and the mutated SLR1 as prey in the presence of 10⁻⁴ M GA₄. For interactions between SLR1s and GID2, a Y3H assay was performed using GID2^L76A as bait and the mutated SLR1 as prey in the presence of GID1 and 10⁻⁴ M GA₄. The structure of the SLR1 GRAS domain is shown at the bottom of the figure, and regions important for SLR1 to interact with GID1 or GID2 are shown in rectangles at the top. SLR1 mutants, which were analyzed with SPR (Table 1), are marked with black circles.

Figure 8.

Degradation of Ala-Mutated SLR1 Proteins in Rice Calli Treated with GA₄. Wild-type T65 rice calli overproducing FLAG-tagged SLR1 mutants with changes in the domains indicated in boxes were incubated with or without 10⁻⁵ M GA₄ for 12 h, and the crude protein extracts were subject to immunoblot analysis using anti-FLAG-tag antibody. The loading control of Coomassie blue (CBB) staining is shown in the right-hand panels.

Figure 9.

Regions Necessary for Repression Activity of SLR1 Are Scattered within the GRAS Domain. (A) Gross morphology of transgenic seedlings grown under GA-deficient conditions. Seedlings were grown in the presence of 10⁻⁶ M uniconazole (an inhibitor of GA synthesis). Wild-type and mSLR1s fused with FLAG tag were overproduced in wild-type T65 rice. vec, T65 transformed with proAct-FLAG/pCAMBIA control vector. Repression activity of SLR1 and mSLR1 proteins was assessed by comparing the height of each transgenic plant to wild-type SLR1 transformants. Three-week-old seedlings, which exhibit the typical phenotype obtained for each protein tested, are shown. Red circle, level of repression activity similar to SLR1^WT; light blue, repression activity decreased but still retained; dark blue, repression activity is almost eliminated. Bar = 5 cm. (B) Results of repression activity in (A) were plotted onto the GRAS domain of SLR1. Mutation sites of loss-of-function mutants reported in DELLA proteins are also plotted. For mutants that are not derived from rice, the corresponding site in rice is plotted. Definition of colored circles is the same as in (A), except mutation sites of mutants from other studies are shown in white. cry-c, Pisum sativa DELLA protein CRY mutant (Weston et al., 2008); procera, Solanum lycopersicum DELLA protein PROCERA mutant (Bassel et al., 2008); rga2, Arabidopsis RGA mutant (Silverstone et al., 1998).

Figure 10.

GA-Dependent Interaction between GID2 and SLR1 in Vitro and in Vivo. (A) In vitro pull-down assay showing GA-dependent interaction between GST-SLR1 and T7-tagged GID1 and between GST-SLR1 and HA tagged-GID2. GST-SLR1, T7 tagged-GID1, and HA tagged-GID2 were expressed together in E. coli and purified with glutathione beads. E. coli expressing GST-SLR1 and T7-tagged GID1 was used as a control. HA-tagged GID2 was detected with anti-HA antibody. (B) BiFC analysis of in vivo interaction between GID2 and SLR1 in N. benthamiana leaf epidermis (Abe et al., 2005). BF, bright-field image; EYFP, EYFP fluorescence; DAPI, 4',6-diamidino-2-phenylindole; NY-GID2 CY-SLR1, expression of N•EYFP-GID2 and C•EYFP-SLR1 without GID1; NY-GID2 CY-SLR1+GID1, expression of N•EYFP-GID2 and C•EYFP-SLR1 with nontagged GID1. Leaves were sprayed with 10⁻⁴ M GA₄ dissolved in ethanol (+) or with ethanol alone (–) 10 min before observation of the signals. Bar = 10 μm.

Figure 11.

Molecular Model for the Formation of the GID1-SLR1-GID2 Complex. The blue circle at the center of the diagram represents the GRAS domain of SLR1. See text for details.