Rapid heteromerization and phosphorylation of ligand-activated plant transmembrane receptors and their associated kinase BAK1

Abstract

In plants leucine-rich repeat receptor kinases (LRR-RKs) located at the plasma membrane play a pivotal role in the perception of extracellular signals. For two of these LRR-RKs, the brassinosteroid receptor BRI1 and the flagellin receptor FLS2, interaction with the LRR receptor-like kinase BAK1 (BRI1-associated receptor kinase 1) was shown to be required for signal transduction. Here we report that FLS2.BAK1 heteromerization occurs almost instantaneously after perception of the ligand, the flagellin-derived peptide flg22. Flg22 can induce formation of a stable FLS2.BAK1 complex in microsomal membrane preparations in vitro, and the kinase inhibitor K-252a does not prevent complex formation. A kinase dead version of BAK1 associates with FLS2 in a flg22-dependent manner but does not restore responsiveness to flg22 in cells of bak1 plants, demonstrating that kinase activity of BAK1 is essential for FLS2 signaling. Furthermore, using in vivo phospholabeling, we are able to detect de novo phosphorylation of both FLS2 and BAK1 within 15 s of stimulation with flg22. Similarly, brassinolide induces BAK1 phosphorylation within seconds. Other triggers of plant defense, such as bacterial EF-Tu and the endogenous AtPep1 likewise induce rapid formation of heterocomplexes consisting of de novo phosphorylated BAK1 and proteins representing the ligand-specific binding receptors EF-Tu receptor and Pep1 receptor 1, respectively. Thus, we propose that several LRR-RKs form tight complexes with BAK1 almost instantaneously after ligand binding and that the subsequent phosphorylation events are key initial steps in signal transduction.

FIGURE 1.

Heteromerization of BAK1 and FLS2 occurs within seconds after flg22 treatment and can be stimulated in vitro. A, Arabidopsis cells were stimulated or not with flg22, and the sample tube was frozen either immediately (<1 s before freezing) or a few seconds after treatment. The solubilized membrane proteins were immunoprecipitated (IP) with anti-BAK1 antibodies and analyzed by Western blot (WB) with anti-FLS2 (upper panel) and anti-BAK1 antibodies (lower panel). B, microsomes were isolated at 4 °C from Arabidopsis cells treated with flg22 (in vivo) or from untreated cells. Microsomes from untreated cells were supplemented with 10 μm flg22, the inactive flg22-Δ2, or the EFR ligand elf18 and incubated at room temperature for 10 min (in vitro). C, microsomes from nonelicited cells were pretreated (+NP40) or not (control) with 1% Nonidet P-40 for 10 min on ice, before the addition of flg22. D, effect of temperature on complex formation. The microsomes were treated as described for B on ice (4 °C) or at room temperature (RT).

FIGURE 2.

The FLS2·BAK1 complex is phosphorylated within seconds after flg22 perception, clearly before the onset of plant responses. A, Arabidopsis cells were stimulated with flg22 at time 0 and incubated with [³³P]phosphate for 1 min at the time points indicated (gray bars). Labeling was stopped by freezing at the end of the phosphate pulse. Control samples (without flg22) were incubated with [³³P]phosphate only. B, analysis of solubilized membrane proteins after IP with anti-FLS2 (left panels) or anti-BAK1 antibodies (right panels). The proteins were separated by SDS-PAGE, blotted, and analyzed by autoradiography (upper panel) and Western analysis (WB, lower panels). The invariably phosphorylated protein of ∼120 kDa in the IPs with anti-FLS2 corresponds to the cross-reacting mitochondrial protein Q9FIC2 (At5g09840) (8). C, time course of de novo phosphorylation of FLS2 and BAK1 in comparison with the onset of medium alkalinization induced by flg22. Three independent repetitions of the experiment as in B were quantified for incorporation of [³³P]phosphate into FLS2 and BAK1. The data represent the averages ± S.E. (n = 3) of radioactivity in the immunoprecipitates. CNT, counts integrated from bands on the blots. Phosphorylation of the co-immunoprecipitating partner proteins shows similar kinetics (as shown in B).

FIGURE 3.

Complex formation and phosphorylation of FLS2 and BAK1 is only induced in the presence of the active ligand flg22. A, cells were treated simultaneously with [³³P]phosphate and flg22 or flg22-Δ2 for 1 min. Solubilized membrane proteins were immunoprecipitated (IP) with anti-FLS2 (top panels) or anti-BAK1 (bottom panels). Phosphorylation and Western blot (WB) signals were absent in IPs competed with their respective antigenic peptides (+flg22 +antigen). B, formation of the FLS2·BAK1 complex is not dependent on kinase activity. Preincubation with the kinase inhibitor K-252a (1 μm) 2 min before (−120 s) the 1-min pulse with [³³P]phosphate and flg22 prevents phosphorylation but not complex formation of FLS2 and BAK1. In contrast, K-252a given 15 s after (+15 s) flg22 treatment had no effect on phosphorylation. Controls without K-252a were treated with an equivalent amount of solvent (dimethyl sulfoxide, shown as +DMSO). autorad., autoradiography.

FIGURE 4.

A functional kinase domain of BAK1 is required for flg22-dependent response activation. A, leaf mesophyll protoplasts of bak1-4 mutant plants were co-transfected with luciferase under the flg22-responsive FRK1 promoter and either the wild type BAK1 or the kinase-deficient BAK1-KD. The samples were mock treated or treated with 100 nm flg22 (time 0), and luminescence was quantified as relative light units (RLU). The values show the means of two replicates with standard deviations bigger than ±60 relative light units (RLU) shown as error bars. B, expression of BAK1 and BAK1-KD in bak1-4 protoplasts under similar conditions as shown in A. Although the full response to flg22 was already gained after transformation of bak1-4 protoplasts with low amounts of plasmid (0.5 μg), protein accumulation was more obvious in samples transfected with higher amounts of DNA (5 μg). C, responsiveness of bak1-4 plants to flg22 was restored with wild type BAK1, but not BAK1-KD. Two T2-lines each of bak1-4 plants, transformed with either BAK1 (line-2 and line-4) or BAK1-KD (line-A and line-M) under the control of an estradiol-inducible promoter, were selected and compared with the parent bak1-4 line and with wild type Col-0 plants. Expression was induced or not in leaf pieces by treatment with 1 μm estradiol, and production of reactive oxygen species was quantified as RLU after treatment or not with 100 nm flg22. The data represent the averages ± S.D. (n = 3). D, expression of BAK1 and BAK1-KD in plants induced or not with estradiol under the conditions shown in C. E, analysis of FLS2 co-immunoprecipitation with BAK1 after 5 min of treatment with 1 μm flg22 both in BAK1 line 4 and in the BAK1-KD line A. Col-0 seedlings were used as positive control of co-IP between BAK1 and FLS2. WB, Western blot.

FIGURE 5.

Phosphorylation of BAK1 and co-immunoprecipitation of specific LRR-RKs in response to various signals. A, phosphorylation of BAK1 in response to stimulation with BL. Arabidopsis cell cultures were preincubated with brassinazole (2 μm for 6 days) and treated with BL (1 μm) or ethanol as control and were pulse-labeled with the same sampling scheme as in Fig. 2A. Solubilized membrane proteins were immunoprecipitated (IP) with anti-BAK1, separated by SDS-PAGE, blotted, and analyzed by autoradiography (autorad., upper panel) and Western analysis (WB, lower panel). B, BAK1 serves as interaction partner of several transmembrane receptors. Arabidopsis cells were treated with the peptide elicitors flg22, elf26, and AtPep1, and the fungal elicitors Pen and chitin and were simultaneously incubated with [³³P]phosphate for 5 min.