Rapid Heteromerization and Phosphorylation of Ligand-activated Plant Transmembrane Receptors and Their Associated Kinase BAK1

Tobias Mentzel,Seraina Beeler,Katharina Mueller,Delphine Chinchilla,Anna K Jehle,Thomas Boller,Birgit Schulze,Georg Felix

doi:10.1074/jbc.m109.096842

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.

Highlights

Ϳ2.5– 4% of all proteins encoded by the genome of a plant [1]
Because the ϳ75kDa signal was absent in extracts of bak1-4 mutant plants, either cross-reactivity with SERK4 and SERK5 is weak, or BAK1 is strongly predominating in these plant extracts
Using Arabidopsis cultured cells and the anti-BAK1 antibodies for co-IP experiments, we were able to detect and identify by mass spectrometry the FLS2 polypeptide in an anti-BAK1 IP performed on extracts of flg22-induced cells, but not on those of nonstimulated cells

Summary

Introduction

Ϳ2.5– 4% of all proteins encoded by the genome of a plant [1]. Despite their importance, there is still little experimental evidence on the molecular activation mechanisms of plant transmembrane receptor kinases. Bak mutants show strongly reduced responses to flg, indicating that complex formation of FLS2 with BAK1 is essential for flg signaling Despite these recent advances, the biochemical function of BAK1 and the precise mechanism underlying BAK11⁄7FLS2 interaction and activation of the flagellin receptor remain unclear. There is currently no direct evidence for phosphorylation of BAK1 and FLS2 in response to flagellin treatment This is of particular interest because FLS2 does not exhibit a conserved arginine-aspartic acid (RD) motif in the catalytic site. Thereby, we observed that rapid heteromerization with BAK1 and phosphorylation induced by ligand perception are features common to various LRR-RKs, including BRI1 and the pattern recognition receptors EFR and PEPR1. The in vivo phospholabeling approach described in this study represents a new tool to detect plant receptors that biochemically interact and rely on BAK1 as a signaling partner

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