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. 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. IntroductionOne of the central themes in cell biology is the sensing of extracellular chemical signals through cell surface receptors: How does the event of receptor-ligand interaction on the outside of the cell activate a signal transduction chain in the inside of the cell? In higher plants, the most prominent class of membrane receptors is formed by proteins with intracellular serine/threonine-type protein kinases. These receptors account for ∼2.5–4% of all proteins encoded by the genome of a plant (1.Shiu S.H. Karlowski W.M. Pan R. Tzeng Y.H. Mayer K.F. Li W.H. Plant Cell. 2004; 16: 1220-1234Crossref PubMed Scopus (812) Google Scholar). Despite their importance, there is still little experimental evidence on the molecular activation mechanisms of plant transmembrane receptor kinases. Current models are based on the precedent of animal receptor tyrosine kinases where ligand binding causes receptor tyrosine kinases to form homo- or hetero-oligomers, followed by transphosphorylation (2.Schlessinger J. Cell. 2002; 110: 669-672Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar). In the case of the epidermal growth factor receptor, these phosphorylation events occur within 60 s of receptor activation (3.Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2777) Google Scholar).The best studied plant transmembrane receptor kinase is BRI1, the receptor for the brassinosteroid growth hormones (4.Wang Z.Y. Seto H. Fujioka S. Yoshida S. Chory J. Nature. 2001; 410: 380-383Crossref PubMed Scopus (596) Google Scholar). BRI1 is one of the 224 members of LRR-RKs 2The abbreviations used are: LRR-RKleucine-rich-repeat receptor kinaseBLbrassinolideEFREF-Tu receptorPEPR1Pep1 receptor 1IPimmunoprecipitationSERKsomatic embryogenesis receptor kinase. in Arabidopsis (5.Shiu S.H. Bleecker A.B. Plant Physiol. 2003; 132: 530-543Crossref PubMed Scopus (590) Google Scholar). Upon ligand binding BRI1 interacts with a second LRR receptor-like kinase named BAK1 (6.Li J. Wen J. Lease K.A. Doke J.T. Tax F.E. Walker J.C. Cell. 2002; 110: 213-222Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar, 7.Nam K.H. Li J.M. Cell. 2002; 110: 203-212Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar). Two further well characterized plant LRR-RKs are the flagellin receptor FLS2 (flagellin sensing 2) (8.Chinchilla D. Bauer Z. Regenass M. Boller T. Felix G. Plant Cell. 2006; 18: 465-476Crossref PubMed Scopus (549) Google Scholar) and the EF-Tu receptor (EFR) (9.Kunze G. Zipfel C. Robatzek S. Niehaus K. Boller T. Felix G. Plant Cell. 2004; 16: 3496-3507Crossref PubMed Scopus (611) Google Scholar). FLS2 perceives a generally conserved 22-amino acid epitope (flg22) of bacterial flagellin (8.Chinchilla D. Bauer Z. Regenass M. Boller T. Felix G. Plant Cell. 2006; 18: 465-476Crossref PubMed Scopus (549) Google Scholar), and EFR senses the N-terminal amino acids (elf18/elf26) of bacterial elongation factor EF-Tu (10.Zipfel C. Kunze G. Chinchilla D. Caniard A. Jones J.D. Boller T. Felix G. Cell. 2006; 125: 749-760Abstract Full Text Full Text PDF PubMed Scopus (1261) Google Scholar). FLS2 and EFR are typical pattern recognition receptors involved in plant immunity and render plants more resistant to bacterial attack (10.Zipfel C. Kunze G. Chinchilla D. Caniard A. Jones J.D. Boller T. Felix G. Cell. 2006; 125: 749-760Abstract Full Text Full Text PDF PubMed Scopus (1261) Google Scholar, 11.Zipfel C. Robatzek S. Navarro L. Oakeley E.J. Jones J.D. Felix G. Boller T. Nature. 2004; 428: 764-767Crossref PubMed Scopus (1201) Google Scholar). However, pattern recognition receptors are also involved in the recognition of endogenous peptide signals, such as the wound-induced AtPep1, which is recognized by the LRR-RK PEPR1 and is suggested to be part of a positive feedback loop to amplify the plant immune response (12.Huffaker A. Pearce G. Ryan C.A. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10098-10103Crossref PubMed Scopus (374) Google Scholar, 13.Yamaguchi Y. Pearce G. Ryan C.A. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10104-10109Crossref PubMed Scopus (280) Google Scholar, 14.Ryan C.A. Huffaker A. Yamaguchi Y. Cell Microbiol. 2007; 9: 1902-1908Crossref PubMed Scopus (72) Google Scholar). Surprisingly, in recent work we and others found that stimulation with flg22 leads to heteromerization of FLS2 with BAK1, the same associated kinase employed by the hormone receptor BRI1 (15.Chinchilla D. Zipfel C. Robatzek S. Kemmerling B. Nürnberger T. Jones J.D. Felix G. Boller T. Nature. 2007; 448: 497-500Crossref PubMed Scopus (1230) Google Scholar, 16.Heese A. Hann D.R. Gimenez-Ibanez S. Jones A.M. He K. Li J. Schroeder J.I. Peck S.C. Rathjen J.P. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12217-12222Crossref PubMed Scopus (764) Google Scholar). Consistently, bak1 mutants show strongly reduced responses to flg22, indicating that complex formation of FLS2 with BAK1 is essential for flg22 signaling. Despite these recent advances, the biochemical function of BAK1 and the precise mechanism underlying BAK1·FLS2 interaction and activation of the flagellin receptor remain unclear. In the case of brassinosteroid perception, it was shown by means of mobility shift and mass spectrometry that BRI1 and BAK1 are transphosphorylating in vivo, resulting in amplification of brassinosteroid signaling (17.Wang X. Kota U. He K. Blackburn K. Li J. Goshe M.B. Huber S.C. Clouse S.D. Dev. Cell. 2008; 15: 220-235Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 18.Oh M.H. Wang X. Kota U. Goshe M.B. Clouse S.D. Huber S.C. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 658-663Crossref PubMed Scopus (207) Google Scholar, 19.Yun H.S. Bae Y.H. Lee Y.J. Chang S.C. Kim S.K. Li J. Nam K.H. Mol. Cells. 2009; 27: 183-190Crossref PubMed Scopus (41) Google Scholar). However, these phosphorylation events have been studied only after a considerable lag time of 20–90 min following ligand perception. In contrast, 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. This special class of non-RD kinases generally shows weak autophosphorylation activity (20.Dardick C. Ronald P. PLoS Pathog. 2006; 2: 14-28Crossref Scopus (193) Google Scholar).In previous work we reported that FLS2 associates with BAK1 within ∼2 min of treatment with the flg22 ligand, concomitantly with the early physiological responses observed in Arabidopsis cells (15.Chinchilla D. Zipfel C. Robatzek S. Kemmerling B. Nürnberger T. Jones J.D. Felix G. Boller T. Nature. 2007; 448: 497-500Crossref PubMed Scopus (1230) Google Scholar). Here we performed a more detailed kinetic analysis, showing that heteromerization of FLS2 and BAK1 occurs almost instantaneously and is independent of kinase activity. Using an in vivo labeling approach with radioactive orthophosphate, we studied the dynamics of phosphorylation events occurring within the receptor complex. 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. Thus, 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.DISCUSSIONThe aim of this study was to investigate how early signaling events such as phosphorylation of plant LRR-RKs, exemplified by FLS2 and BAK1, are temporally connected with the activation of these receptors. We used in vivo labeling with short pulses of [33P]orthophosphate, which allowed us to study de novo phosphorylation events occurring during the first seconds after ligand stimulation. This approach complements and differs from other methods like mass spectrometry or Western blot analysis detecting phospho-residues that have been successfully used for the characterization of the phosphorylation status of the BRI1-BAK1 system (17.Wang X. Kota U. He K. Blackburn K. Li J. Goshe M.B. Huber S.C. Clouse S.D. Dev. Cell. 2008; 15: 220-235Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 18.Oh M.H. Wang X. Kota U. Goshe M.B. Clouse S.D. Huber S.C. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 658-663Crossref PubMed Scopus (207) Google Scholar, 19.Yun H.S. Bae Y.H. Lee Y.J. Chang S.C. Kim S.K. Li J. Nam K.H. Mol. Cells. 2009; 27: 183-190Crossref PubMed Scopus (41) Google Scholar). In this study we show that phosphorylation of the FLS2·BAK1 complex is concomitant to the heteromerization process and is an extremely quick and specific event because it appears within 15 s of stimulation with flg22. In accordance with a role in receptor-mediated signaling, these phosphorylations clearly precede all of the responses and intracellular signaling events known so far (40.Boller T. Felix G. Annu. Rev. Plant Biol. 2009; 60: 379-406Crossref PubMed Scopus (2096) Google Scholar).Phosphorylation of BAK1 and phosphorylation of FLS2 seem to be tightly connected because both events exhibit a dose dependence for the flg22 ligand that correlates closely with the saturation curve for binding of flg22 to FLS2 (22.Bauer Z. Gómez-Gómez L. Boller T. Felix G. J. Biol. Chem. 2001; 276: 45669-45676Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), supporting the model that each flg22 molecule that binds to a FLS2 receptor site triggers formation and phosphorylation of a FLS2·BAK1 complex. Whether these complexes represent heterodimers or higher oligomerization states with a different stoichiometry of FLS2 and BAK1 remains to be studied.The protein kinase activity itself seems not required for complex formation. First, treatment with a general inhibitor of protein kinases inhibits de novo phosphorylation of BAK1 and FLS2, but it does not inhibit their ligand-induced heteromerization. Second, a kinase dead version of BAK1 can still associate with FLS2, although activation of downstream signaling is blocked. Similarly, the heteromerization process and the triggering of stable de novo phosphorylation of FLS2 and BAK1 does not depend on reduced phosphatase activity. Thus, formation of a complex with BAK1 is solely triggered by the interaction of FLS2 with its ligand, and phosphorylation is a consequence rather than a prerequisite for stable complex formation.These results contrast a previous study on the complex formation between BAK1 and BRI1 where the kinase activity of BRI1 but not that of BAK1 was found essential for heteromerization (17.Wang X. Kota U. He K. Blackburn K. Li J. Goshe M.B. Huber S.C. Clouse S.D. Dev. Cell. 2008; 15: 220-235Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). These mechanistic differences in the two BAK1-dependent systems may reflect a structural difference because FLS2 belongs to the class of non-RD kinases, whereas BRI1 is a RD kinase (20.Dardick C. Ronald P. PLoS Pathog. 2006; 2: 14-28Crossref Scopus (193) Google Scholar). Thus, it would be of great interest to investigate to what extent the FLS2 kinase activity is involved in signal transduction. Interestingly, it been that a FLS2 in Arabidopsis to protein kinase activation flg22 treatment T. G. J. L. Boller T. J. Nature. 2002; PubMed Scopus Google Scholar). However, in this case the FLS2·BAK1 complex formation was not because the and phosphorylation between BRI1 with BAK1 was not min after the BRI1 kinase activity may be for rather than formation of the we that BAK1 phosphorylated in a time in response to perception as after flg22 although a signal representing the BRI1 protein was not Thus, we the that BRI1 is phosphorylated in a time with its interaction BAK1, which the of BRI1 phosphorylation at such early time However, the of phosphorylated BRI1 may also be to of the complex during or to the of this protein in Arabidopsis the of cell used in the signal for BRI1 may be the the function of BAK1 seems not to the of the FLS2 and BRI1 signaling in BAK1 have been reported to exhibit reduced responses to various such as the bacterial or the (15.Chinchilla D. Zipfel C. Robatzek S. Kemmerling B. Nürnberger T. Jones J.D. Felix G. Boller T. Nature. 2007; 448: 497-500Crossref PubMed Scopus (1230) Google Scholar, 16.Heese A. Hann D.R. Gimenez-Ibanez S. Jones A.M. He K. Li J. Schroeder J.I. Peck S.C. Rathjen J.P. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12217-12222Crossref PubMed Scopus (764) Google Scholar, L. He P. Li J. A. Peck S.C. Nürnberger T. J. Cell 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). the BAK1 was reported to interact with the PEPR1 protein in a S. C. S. A. A. T. Kemmerling B. Nürnberger T. J. Cell Biol. 2009; PubMed Scopus Google Scholar). we biochemical evidence that BAK1 is phosphorylated in Arabidopsis cells after the perception of various like flg22, and phosphorylated in to the ligand binding LRR-RKs with BAK1 in response to the that BAK1 phosphorylation was not after of Arabidopsis cells with like is in with indicating that BAK1 is not required for signaling L. He P. Li J. A. Peck S.C. Nürnberger T. J. Cell 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, all of the proteins reported to interact with BAK1 FLS2, and to the LRR receptor kinase Thus, the study that and rapid de novo phosphorylation of BAK1 are general activation mechanisms to LRR receptor kinases. analysis with other to investigate the of BAK1 and new on BAK1 as a common signaling how these LRR-RKs complexes the same signaling is to that receptors are in complexes with BAK1 in the of of have been reported in case of the epidermal growth factor receptor in animal cells T. H. J. Mol. Biol. 2001; PubMed Scopus Google Scholar). complex formation between BAK1 and FLS2 was found to be quick to the kinetic of interaction in a time However, using we not able to of the FLS2 and BAK1 although was in from a analysis, which been employed for receptor kinase K. J. S.C. Dev. 2002; 16: PubMed Scopus Google Scholar, Plant Cell Physiol. 2007; PubMed Scopus Google Scholar), may be of BAK1 with FLS2 may also be by a mobility of BAK1 and FLS2 in the plasma membrane because it been reported for FLS2 Plant Cell Physiol. 2007; PubMed Scopus Google Scholar). of flg22 reduced FLS2 membrane by complex formation with BAK1 and by with further of the or to membrane Plant Cell Physiol. 2007; PubMed Scopus Google Scholar, S. B. Mol. Cell 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). However, in this study we show that complex formation was successfully induced in of plant Thus, it is that the of FLS2 and BAK1 to on by on we may a model where ligand binding interaction of the extracellular of FLS2 and BAK1 and the intracellular kinase of intracellular signaling can be induced by transphosphorylation events between the BAK1 and FLS2 In the of the of animal immune receptors ligand-induced which was to a receptor complex and signaling Kim Lee Kim Lee H. Lee Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, L. Wang Y. J. D.R. 2008; PubMed Scopus Google Scholar). to the of signaling it be of to investigate bacterial and treatment to phosphorylations of the same or different in BAK1, at the early time IntroductionOne of the central themes in cell biology is the sensing of extracellular chemical signals through cell surface receptors: How does the event of receptor-ligand interaction on the outside of the cell activate a signal transduction chain in the inside of the cell? In higher plants, the most prominent class of membrane receptors is formed by proteins with intracellular serine/threonine-type protein kinases. These receptors account for ∼2.5–4% of all proteins encoded by the genome of a plant (1.Shiu S.H. Karlowski W.M. Pan R. Tzeng Y.H. Mayer K.F. Li W.H. Plant Cell. 2004; 16: 1220-1234Crossref PubMed Scopus (812) Google Scholar). Despite their importance, there is still little experimental evidence on the molecular activation mechanisms of plant transmembrane receptor kinases. Current models are based on the precedent of animal receptor tyrosine kinases where ligand binding causes receptor tyrosine kinases to form homo- or hetero-oligomers, followed by transphosphorylation (2.Schlessinger J. Cell. 2002; 110: 669-672Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar). In the case of the epidermal growth factor receptor, these phosphorylation events occur within 60 s of receptor activation (3.Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2777) Google Scholar).The best studied plant transmembrane receptor kinase is BRI1, the receptor for the brassinosteroid growth hormones (4.Wang Z.Y. Seto H. Fujioka S. Yoshida S. Chory J. Nature. 2001; 410: 380-383Crossref PubMed Scopus (596) Google Scholar). BRI1 is one of the 224 members of LRR-RKs 2The abbreviations used are: LRR-RKleucine-rich-repeat receptor kinaseBLbrassinolideEFREF-Tu receptorPEPR1Pep1 receptor 1IPimmunoprecipitationSERKsomatic embryogenesis receptor kinase. in Arabidopsis (5.Shiu S.H. Bleecker A.B. Plant Physiol. 2003; 132: 530-543Crossref PubMed Scopus (590) Google Scholar). Upon ligand binding BRI1 interacts with a second LRR receptor-like kinase named BAK1 (6.Li J. Wen J. Lease K.A. Doke J.T. Tax F.E. Walker J.C. Cell. 2002; 110: 213-222Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar, 7.Nam K.H. Li J.M. Cell. 2002; 110: 203-212Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar). Two further well characterized plant LRR-RKs are the flagellin receptor FLS2 (flagellin sensing 2) (8.Chinchilla D. Bauer Z. Regenass M. Boller T. Felix G. Plant Cell. 2006; 18: 465-476Crossref PubMed Scopus (549) Google Scholar) and the EF-Tu receptor (EFR) (9.Kunze G. Zipfel C. Robatzek S. Niehaus K. Boller T. Felix G. Plant Cell. 2004; 16: 3496-3507Crossref PubMed Scopus (611) Google Scholar). FLS2 perceives a generally conserved 22-amino acid epitope (flg22) of bacterial flagellin (8.Chinchilla D. Bauer Z. Regenass M. Boller T. Felix G. Plant Cell. 2006; 18: 465-476Crossref PubMed Scopus (549) Google Scholar), and EFR senses the N-terminal amino acids (elf18/elf26) of bacterial elongation factor EF-Tu (10.Zipfel C. Kunze G. Chinchilla D. Caniard A. Jones J.D. Boller T. Felix G. Cell. 2006; 125: 749-760Abstract Full Text Full Text PDF PubMed Scopus (1261) Google Scholar). FLS2 and EFR are typical pattern recognition receptors involved in plant immunity and render plants more resistant to bacterial attack (10.Zipfel C. Kunze G. Chinchilla D. Caniard A. Jones J.D. Boller T. Felix G. Cell. 2006; 125: 749-760Abstract Full Text Full Text PDF PubMed Scopus (1261) Google Scholar, 11.Zipfel C. Robatzek S. Navarro L. Oakeley E.J. Jones J.D. Felix G. Boller T. Nature. 2004; 428: 764-767Crossref PubMed Scopus (1201) Google Scholar). However, pattern recognition receptors are also involved in the recognition of endogenous peptide signals, such as the wound-induced AtPep1, which is recognized by the LRR-RK PEPR1 and is suggested to be part of a positive feedback loop to amplify the plant immune response (12.Huffaker A. Pearce G. Ryan C.A. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10098-10103Crossref PubMed Scopus (374) Google Scholar, 13.Yamaguchi Y. Pearce G. Ryan C.A. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10104-10109Crossref PubMed Scopus (280) Google Scholar, 14.Ryan C.A. Huffaker A. Yamaguchi Y. Cell Microbiol. 2007; 9: 1902-1908Crossref PubMed Scopus (72) Google Scholar). Surprisingly, in recent work we and others found that stimulation with flg22 leads to heteromerization of FLS2 with BAK1, the same associated kinase employed by the hormone receptor BRI1 (15.Chinchilla D. Zipfel C. Robatzek S. Kemmerling B. Nürnberger T. Jones J.D. Felix G. Boller T. Nature. 2007; 448: 497-500Crossref PubMed Scopus (1230) Google Scholar, 16.Heese A. Hann D.R. Gimenez-Ibanez S. Jones A.M. He K. Li J. Schroeder J.I. Peck S.C. Rathjen J.P. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12217-12222Crossref PubMed Scopus (764) Google Scholar). Consistently, bak1 mutants show strongly reduced responses to flg22, indicating that complex formation of FLS2 with BAK1 is essential for flg22 signaling. Despite these recent advances, the biochemical function of BAK1 and the precise mechanism underlying BAK1·FLS2 interaction and activation of the flagellin receptor remain unclear. In the case of brassinosteroid perception, it was shown by means of mobility shift and mass spectrometry that BRI1 and BAK1 are transphosphorylating in vivo, resulting in amplification of brassinosteroid signaling (17.Wang X. Kota U. He K. Blackburn K. Li J. Goshe M.B. Huber S.C. Clouse S.D. Dev. Cell. 2008; 15: 220-235Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 18.Oh M.H. Wang X. Kota U. Goshe M.B. Clouse S.D. Huber S.C. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 658-663Crossref PubMed Scopus (207) Google Scholar, 19.Yun H.S. Bae Y.H. Lee Y.J. Chang S.C. Kim S.K. Li J. Nam K.H. Mol. Cells. 2009; 27: 183-190Crossref PubMed Scopus (41) Google Scholar). However, these phosphorylation events have been studied only after a considerable lag time of 20–90 min following ligand perception. In contrast, 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. This special class of non-RD kinases generally shows weak autophosphorylation activity (20.Dardick C. Ronald P. PLoS Pathog. 2006; 2: 14-28Crossref Scopus (193) Google Scholar).In previous work we reported that FLS2 associates with BAK1 within ∼2 min of treatment with the flg22 ligand, concomitantly with the early physiological responses observed in Arabidopsis cells (15.Chinchilla D. Zipfel C. Robatzek S. Kemmerling B. Nürnberger T. Jones J.D. Felix G. Boller T. Nature. 2007; 448: 497-500Crossref PubMed Scopus (1230) Google Scholar). Here we performed a more detailed kinetic analysis, showing that heteromerization of FLS2 and BAK1 occurs almost instantaneously and is independent of kinase activity. Using an in vivo labeling approach with radioactive orthophosphate, we studied the dynamics of phosphorylation events occurring within the receptor complex. 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. Thus, 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