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. 2011 May 17;108(20):8503-7.
doi: 10.1073/pnas.1103556108. Epub 2011 Apr 4.

Extracellular leucine-rich repeats as a platform for receptor/coreceptor complex formation

Yvon Jaillais  1 , Youssef Belkhadir, Emilia Balsemão-Pires, Jeffery L Dangl, Joanne Chory
Affiliations

Extracellular leucine-rich repeats as a platform for receptor/coreceptor complex formation

Yvon Jaillais et al. Proc Natl Acad Sci U S A. .

Abstract

Receptor kinases with leucine-rich repeat (LRR) extracellular domains form the largest family of receptors in plants. In the few cases for which there is mechanistic information, ligand binding in the extracellular domain often triggers the recruitment of a LRR-coreceptor kinase. The current model proposes that this recruitment is mediated by their respective kinase domains. Here, we show that the extracellular LRR domain of BRI1-ASSOCIATED KINASE1 (BAK1), a coreceptor involved in the disparate processes of cell surface steroid signaling and immunity in plants, is critical for its association with specific ligand-binding LRR-containing receptors. The LRRs of BAK1 thus serve as a platform for the molecular assembly of signal-competent receptors. We propose that this mechanism represents a paradigm for LRR receptor activation in plants.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Gain-of-function phenotype of bak1elg allele for the brassinosteroid signaling pathway. (A) Schematic representation of BAK1 with its extracellular LRR domain in red and intracellular kinase domain in blue. TM: transmembrane segment. The position of the elg (D122N) mutation in BAK1 (LRR3) is indicated. (B) BAK1prom:BAK1::CITRINE expression complements the bak1-3 hypocotyl growth defect. BAK1prom:bak1elg::CITRINE expression in bak1-3 leads to an elongated hypocotyl phenotype in the dark that is BRI1-dependent. Note that BAK1prom:bak1elg::CITRINE-expressing hypocotyls still elongate when BR ligand is partially depleted by 1 μM brassinazole, BRZ. Hypocotyl length is in mm ± SD (n = 25), NT/T is the ratio of nontreated (NT) over BRZ-treated (T) hypocotyl length. (C) Pictures of rosette stage transgenic homozygous Arabidopsis (T3) expressing BAK1prom:BAK1::CITRINE or BAK1prom:bak1elg::CITRINE under the control of BAK1 promoter in the bak1-3 background. The phenotypes associated with the overexpression of BRI1 (on the right, for comparison), narrow leaf blades, elongated and twisting petioles were recapitulated by driving the expression of the bak1elg::CITRINE variant. Mean value of rosette radius is indicated in mm ± SD (n = 25). (D) BAK1::CITRINE accumulates to a similar extent as bak1elg::CITRINE. Microsomal protein extracts were prepared from wild-type Col-0, BAK1prom:BAK1::CITRINE in bak1-3 and BAK1prom:bak1elg::CITRINE in bak1-3 plants. These extracts were subjected to an anti-GFP protein immunoblot analysis to detect the accumulation of the CITRINE-tagged proteins. Equal loading was ensured by protein quantification before loading and by Ponceau red staining of the membrane postprotein transfer. (E) BES1 phosphorylation in BAK1::CITRINE/bak1-3, BAK1-bak1elg::CITRINE/bak1-3 and OxBRI1 lines. P-BES is phosphorylated BES1. Equal loading was ensured by protein quantification before loading and by the signal intensity of a nonspecific band.
Fig. 2.
Fig. 2.
bak1elg has impaired flagellin response. (A) Oxidative burst triggered by 100 nM flg22 in wild-type Col-0 (blue), fls2 (red), bak1-3 (green), BAK1prom:BAK1::CITRINE in bak1-3 (purple), and BAK1prom:bak1elg::CITRINE in bak1-3 (orange) leaf discs measured in relative light units (RLU). Result are mean ± SD (n = 24). (B) Average fresh-weight ratio of 14-d-old seedlings grown for 7 d in either water or water plus 1 μM flg22. The bar graph represents the average fresh-weight ratio from wild-type Col-0, bak1-3 mutant, BAK1prom:BAK1::CITRINE in bak1-3, and BAK1prom:bak1elg::CITRINE in bak1-3. Means and SDs were calculated from 48 seedlings (six random pools of eight seedlings). (C) Callose deposits stained with aniline blue from leaves of wild type Col-0, bak1-3, BAK1prom:BAK1::CITRINE in bak1-3 and BAK1prom:bak1elg::CITRINE in bak1-3 treated with 1 μM flg22. The number of leaves showing the displayed features over the total in a given genotype is indicated in parentheses. (D) Growth of Pseudomonas syringae pv. tomato (Pto DC3000) was measured on the genetic backgrounds indicated at bottom. Leaves from 4-wk-old plants were infiltrated with a bacterial inoculum of 105 cfu·mL−1 in the presence (orange) or absence (red) of 1 μM flg22 peptide. The number of bacteria per square centimeter of leaf was plotted on a log10 scale. Error bars represent two times the SE among four internal replicate samples from one of three experiments.
Fig. 3.
Fig. 3.
A mutation in the extracellular LRR domain of BAK1 modifies its interaction with BRI1 and FLS2. (A) Transgenic Arabidopsis plants expressing either BAK1prom:BAK1::CHERRY or BAK1prom:bak1elg::CHERRY alone or with BRI1prom:BRI1::CITRINE were grown with or without the BR biosynthesis inhibitor BRZ (5 μM added from sowing of seeds). Total membrane protein was immunoprecipitated (IP) with anti-GFP antibodies and subjected to immunoblot (IB) analysis, as indicated. (B) Transgenic plants expressing either BAK1prom:BAK1::6xHA or BAK1prom:bak1elg::6xHA alone or with FLS2prom:FLS2::GFP were grown on 1/2 LS media and treated 5 min before protein extraction with 10 μM flg22. Total membrane protein was immunoprecipitated (IP) with anti-GFP antibodies and subjected to immunoblot (IB) analysis as indicated.
Fig. 4.
Fig. 4.
BRI1 is activated by a double-lock mechanism. (A) Rosette leaf phenotype of wild-type Col-0, BAK1prom:BAK1::CITRINE, BAK1prom:bak1D434N::CITRINE, BAK1prom:bak1elg::CITRINE, and BAK1prom:bak1elgD434N::CITRINE. Average rosette radius in mm ± SD (n = 25). (B) Expression level of transgenic proteins. (C) Rosette leaf phenotype of wild-type Col-0, BAK1prom:bak1elg::CITRINE, OxBKI1 and a cross between BAK1prom:bak1elg::CITRINE and OxBKI1 grown in short days. (D) Model for the formation of an active BR signaling complex. In the absence of ligand, BRI1 is maintained in an inactive state by its C-terminal tail as well as its inhibitory protein BKI1 and does not interact with BAK1 (Upper). Activation of BRI1 by BR triggers both the recruitment of BAK1 through its extracellular LRR domain as well as the BRI1-mediated phosphorylation of BKI1 inside the cell (Lower Left). This triggers dissociation of BKI1 from the plasma membrane and transphosphorylation between BRI1 and BAK1 kinase domain and leads to full activation of the receptor complex (Lower Right). BRI1 is represented as a monomer for simplicity but its isolated intracellular domain exist only as homodimers in solution (10) and 20% of the full-length receptor forms homodimers in vivo (30).
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