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. 2018 Mar 27;115(13):3488-3493.
doi: 10.1073/pnas.1714972115. Epub 2018 Mar 12.

Mechanistic Basis for the Activation of Plant Membrane Receptor Kinases by SERK-family Coreceptors

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Free PMC article

Mechanistic Basis for the Activation of Plant Membrane Receptor Kinases by SERK-family Coreceptors

Ulrich Hohmann et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Plant-unique membrane receptor kinases with leucine-rich repeat ectodomains (LRR-RKs) can sense small molecule, peptide, and protein ligands. Many LRR-RKs require SERK-family coreceptor kinases for high-affinity ligand binding and receptor activation. How one coreceptor can contribute to the specific binding of distinct ligands and activation of different LRR-RKs is poorly understood. Here we quantitatively analyze the contribution of SERK3 to ligand binding and activation of the brassinosteroid receptor BRI1 and the peptide hormone receptor HAESA. We show that while the isolated receptors sense their respective ligands with drastically different binding affinities, the SERK3 ectodomain binds the ligand-associated receptors with very similar binding kinetics. We identify residues in the SERK3 N-terminal capping domain, which allow for selective steroid and peptide hormone recognition. In contrast, residues in the SERK3 LRR core form a second, constitutive receptor-coreceptor interface. Genetic analyses of protein chimera between BRI1 and SERK3 define that signaling-competent complexes are formed by receptor-coreceptor heteromerization in planta. A functional BRI1-HAESA chimera suggests that the receptor activation mechanism is conserved among different LRR-RKs, and that their signaling specificity is encoded in the kinase domain of the receptor. Our work pinpoints the relative contributions of receptor, ligand, and coreceptor to the formation and activation of SERK-dependent LRR-RK signaling complexes regulating plant growth and development.

Keywords: brassinosteroid signaling; floral abscission; leucine-rich repeat domain; membrane receptor kinase; receptor activation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Two distinct SERK surface patches contribute to formation of different LRR-RK complexes. (A, Center) Structural superposition of BRI1–BL–SERK1 (PDB ID 4LSX) and HAESA–IDA–SERK1 (PDB ID 5IYX) complex structures, using the SERK1 ectodomain as reference (rmsd is ∼0.6 Å comparing 185 corresponding Cα atoms). Shown are Cα traces of the BRI1 LRR domain (blue, island domain in dark blue), the HAESA LRR domain (orange), the SERK1 ectodomain (gray) and the steroid (blue) and peptide (orange) ligands in surface representation, respectively. (A, Left) Close-up view of the BRI1–BL–SERK1 complex, highlighting the two distinct interaction surfaces and including selected interface interactions. BL is shown in bond representation (yellow). (A, Right) Details of the HAESA–IDA–SERK1 interface, with IDA shown in bonds representation (yellow). SERK residue numbering is according to Arabidopsis SERK3. (B) Grating-coupled interferometry (GCI)-derived binding kinetics for BRI1 and HAESA vs. their ligands and SERK3. Shown are sensorgrams with data in red and their respective fits in black (Materials and Methods). Table summaries of kinetic parameters are shown (ka, association rate constant; kd, dissociation rate constant; KD, dissociation constant). (C) Isothermal titration calorimetry (ITC) of the BRI1 LRR domain vs. wild-type and mutant SERK3 ectodomains and in the presence of BL. (D) ITC of the HAESA LRR domain vs. SERK3 proteins and in the presence of IDA. Table summaries for dissociation constants (KD) and binding stoichiometries (N) are shown (± fitting error; n.d., no detectable binding).
Fig. 2.
Fig. 2.
Mutations in the SERK3 ligand binding and receptor interfaces differentially impact SERK3 function and complex formation in vivo. (A) Hypocotyl growth assay in the presence and absence of the brassinosteroid biosynthesis inhibitor brassinazole (BRZ), from seedlings grown for 5 d in the dark. Serk1-1 serk3-1 mutants are BRZ hypersensitive and this phenotype can be complemented by expressing SERK3 in the mutant background (Col-0, untransformed wild type, n = 50). (B) Quantification of the data from A. The log-transformed endpoint hypocotyl length was analyzed by a mixed effects model for the ratio of the transgenic lines vs. wild type, allowing for heterogeneous variances. The ratio of the untreated and BRZ-treated hypocotyl length was calculated for wild type (rwt) and each mutant line (rm); the ratio of this ratio for wild type divided by the ratio for a given mutant line results in the ratio of ratios (RR = rwt/rm; CI, confidence interval). (C) Coimmunoprecipitation (Co-IP) of lines shown in A. SERK3:HA was immunoprecipitated from plant protein extracts using an anti-HA antibody (IP-HA) and BRI1 was detected with an anti-BRI1 antibody (14) in the IP elution.
Fig. 3.
Fig. 3.
BRI1 receptor activation requires heteromerization with a SERK coreceptor in planta. (A) Schematic view of signaling competent wild-type and chimeric BRI1–BL–SERK3 complexes. BRI1 domains are shown in blue, SERK3 domains in gray. (B) Stable transgenic lines expressing oBRI1–iSERK3:mCherry under the BRI promoter (pBRI1) and oSERK3–iBRI1:mCitrine under the SERK3 promoter (pSERK3) show a partially rescued bri1-301 growth phenotype when expressed together (Bottom Left). The expression of isolated chimera results in a dominant negative growth phenotype (Bottom Middle and Right). ∼WT corresponds to pBRI1::BRI1:mCitrine/bri1-null. (Scale bar, 1 cm.) (C) Hypocotyl growth assay in the presence and absence of BRZ (compare Fig. 2A). bri1-301 is hypersensitive to BRZ, a phenotype that is partially complemented by the expression of oBRI1–iSERK3 and oSERK3–iBRI1. Quantifications are shown, analyzed as in Fig. 2B.
Fig. 4.
Fig. 4.
The receptor’s kinase domain encodes for the signaling specificity of LRR-RK pathways. (A) Schematic overview of the oBRI1–iHAESA chimera and its envisioned signaling complex (colors as in Fig. 3A; the HAESA kinase domain is shown in orange). (B) Inflorescences of ∼9-wk-old Arabidopsis plants. Abscission of floral organs is impaired in hae hsl2 mutant plants compared with Col-0 wild type. (C) Box plots of BRI1–HAESA chimera vs. Col-0 and hae hsl2 lines. In a quantitative petal break-strength assay, the force required to remove a petal at a given position on the inflorescence is measured in gram equivalents (for each position, at least 15 independent measurements were taken). Statistical analysis is shown in SI Appendix, Table S2.

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