Please cite this article in press as: Halter et al., The Leucine-Rich Repeat Receptor Kinase BIR2 Is a Negative Regulator of BAK1 in Plant Immunity, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2013.11.047 Current Biology 24, 1–10, January 20, 2014 ª2014 Elsevier Ltd All rights reserved
http://dx.doi.org/10.1016/j.cub.2013.11.047
Article The Leucine-Rich Repeat Receptor Kinase BIR2 Is a Negative Regulator of BAK1 in Plant Immunity Thierry Halter,1,8 Julia Imkampe,1,8 Sara Mazzotta,1,8,9 Michael Wierzba,2 Sandra Postel,1,10 Christoph Bu¨cherl,1,3 Christian Kiefer,1,11 Mark Stahl,4 Delphine Chinchilla,5 Xiaofeng Wang,6,12 Thorsten Nu¨rnberger,1 Cyril Zipfel,3 Steven Clouse,6 Jan Willem Borst,7 Sjef Boeren,7 Sacco C. de Vries,7 Frans Tax,2 and Birgit Kemmerling1,* 1Department of Plant Biochemistry (ZMBP), Eberhard Karls University Tu¨bingen, 72076 Tu¨bingen, Germany 2Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA 3The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, UK 4Analytics (ZMBP), Eberhard Karls University Tu ¨ bingen, 72076 Tu¨bingen, Germany 5Zurich-Basel Plant Science Center, Department of Environmental Sciences, University of Basel, 4056 Basel, Switzerland 6Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695-7609, USA 7Laboratory of Biochemistry, Wageningen University, Wageningen 6703 HA, the Netherlands
Summary Background: Transmembrane leucine-rich repeat (LRR) receptors are commonly used innate immune receptors in plants and animals but can also sense endogenous signals to regulate development. BAK1 is a plant LRR-receptor-like kinase (RLK) that interacts with several ligand-binding LRR-RLKs to positively regulate their functions. BAK1 is involved in brassinosteroid-dependent growth and development, innate immunity, and cell-death control by interacting with the brassinosteroid receptor BRI1, immune receptors, such as FLS2 and EFR, and the small receptor kinase BIR1, respectively. Results: Identification of in vivo BAK1 complex partners by LC/ESI-MS/MS uncovered two novel BAK1-interacting RLKs, BIR2 and BIR3. Phosphorylation studies revealed that BIR2 is unidirectionally phosphorylated by BAK1 and that the interaction between BAK1 and BIR2 is kinase-activity dependent. Functional analyses of bir2 mutants show differential impact on BAK1-regulated processes, such as hyperresponsiveness to pathogen-associated molecular patterns (PAMP), enhanced cell death, and resistance to bacterial pathogens, but have no effect on brassinosteroid-regulated growth. BIR2 interacts constitutively with BAK1, thereby preventing interaction with the ligand-binding LRR-RLK FLS2. PAMP
8These
authors contributed equally to this work and are co-first authors address: JKI, Institute for Biological Control, 64287 Darmstadt, Germany 10Present address: Institute of Human Virology, University of Maryland, Baltimore, MD 21201, USA 11Present address: Department of Plant Physiology, Umea ˚ University, 901 87 Umea˚, Sweden 12Present address: Northwest A&F University, Yang Ling, Shaanxi 712100, China *Correspondence:
[email protected] 9Present
perception leads to BIR2 release from the BAK1 complex and enables the recruitment of BAK1 into the FLS2 complex. Conclusions: Our results provide evidence for a new regulatory mechanism for innate immune receptors with BIR2 acting as a negative regulator of PAMP-triggered immunity by limiting BAK1-receptor complex formation in the absence of ligands. Introduction The first step in plant innate immunity is the recognition of potential invaders by cell-surface receptors. A number of such pattern recognition receptors (PRRs) have been identified with most belonging to the receptor-like kinase (RLK) family [1]. The largest subgroup of this family is the leucinerich repeat RLK (LRR-RLKs) family, including flagellin sensing 2 (FLS2) and EF-Tu receptor (EFR), the PRRs for bacterial flagellin and elongation factor Tu [2–4]. The smaller LRR-RLK BAK1/SERK3, which was originally identified as an interactor of the brassinosteroid receptor BRI1 [5, 6], and its closest homolog BAK1-like 1 (BKK1/SERK4) [7], positively regulate the function of multiple ligand-binding receptors and thereby influence multiple signaling cascades [8, 9]. Following ligand binding to the receptor, the regulatory RLK BAK1 associates with the signaling complex allowing BAK1 phosphorylation; in turn, BAK1 phosphorylates the ligand-binding receptor [10–12]. This leads to the activation of the ligand-binding receptor and initiation of downstream signaling [12, 13]. In addition, BAK1 was shown to negatively regulate cell death. bak1-null mutants show enhanced cell death after infection with bacterial and fungal pathogens [14], and in double mutants of BAK1 and its closest homolog BKK1, this cell death is severely enhanced leading to seedling lethality [7]. The BAK1-interacting receptor kinase BIR1 exhibits similar cell-death phenotypes which have been shown to be partially dependent on a salicylate (SA)-dependent resistance (R)-protein pathway and on a second pathway containing suppressor of bir1, another LRR-RLK that interacts with receptor-like proteins involved in plant immunity that are lacking a cytoplasmic kinase domain [15, 16]. During innate immunity, activation of phosphorylationdependent signaling cascades by biotrophic pathogens results in the induction of immune responses, including the production of reactive oxygen species and SA; expression of defense genes, such as pathogenesis-related (PR) genes; production of antimicrobial compounds; and hypersensitive cell death [2, 17]. These responses are usually accompanied by an arrest of plant growth. In contrast, necrotrophic pathogens are restricted by activating jasmonic acid signaling, resulting in the activation of the plant defensin gene PDF1.2 [18, 19]. In defense pathways, BAK1 may interact with Botrytisinduced kinase (BIK1), a receptor-like cytoplasmic kinase [20, 21], and BONZAI (BON1), a calcium-dependent phospholipid-binding protein involved in plant growth homeostasis and disease resistance [22]. Despite the number of interactors identified so far, little is known about how PRRs are recruited to complexes, whether preformed complexes exist, and how specificity is maintained between different
Please cite this article in press as: Halter et al., The Leucine-Rich Repeat Receptor Kinase BIR2 Is a Negative Regulator of BAK1 in Plant Immunity, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2013.11.047 Current Biology Vol 24 No 2 2
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(A) Coimmunoprecipitation of BAK1-FLAG with BIR2-YFP transiently expressed in N. benthamiana leaves. Total proteins (input) were subjected to immunoprecipitation (IP) BAK1 @FLAG WB with anti-YFP antibodies followed by immunoblot analysis with anti-FLAG antibodies to @YFP WB BIR2 detect BAK1 and anti-YFP antibodies to detect BIR2. @FLAG WB BAK1 (B) Yeast two-hybrid assays with the kinase domains of BAK1 and BIR2 were performed. Three consecutive dilutions on selection medium -LW -HALW B D 1/10 1/100 1/1000 1/10 1/100 1/1000 lacking the amino acids HLW and adenine are shown; growth on medium lacking LW assures BAK1/BIR2 proper growth of transformed yeast. (C) Bimolecular fluorescence complementation eV/BIR2 analyses were done with BAK1 fused to the N terminus of YFP and BIR2 fused to the C termiNeg. control nus of YFP in N. benthamiana. As controls, BAK1 2.45 ± 0.03 2.1 ± 0.16 and BIR2 constructs were expressed in comPos. control bination with the respective YFP parts alone. BAK1-CFP BAK1-CFP/BIR2-YFP Fluorescence was visualized by epifluorescence microscopy. (D) FRET imaged by FLIM in Arabidopsis protoplasts transiently expressing BAK1-CFP or BIR2-YFP for 16 hr. Mean fluorescence lifetime values (t) in nanoseconds 6 SE and lifetime distribution are presented as pseudocolor images according to the scale. See also Figures S1–S3 and S11, Table S1, and Document S2. @YFP WB
signaling pathways. In particular, it remains unknown how PRR activation is controlled in the absence of microbial infection. To screen for BAK1-interacting proteins involved in plant defense and cell-death control, we purified in vivo BAK1 complexes by coimmunoprecipitation. Here, we describe the identification and characterization of BIR2, a novel LRR-RLK that constitutively interacts with BAK1 and differentially influences BAK1-regulated defense signaling pathways. Through a novel mechanism affecting BAK1 receptor complex formation, BIR2 negatively regulates plant innate immunity. Results Identification of BAK1-Interacting Proteins BAK1 was immunoprecipitated from Arabidopsis plants overexpressing BAK1-GFP. Proteins were extracted after infection with the necrotrophic ascomycete Alternaria (A.) brassicicola or mock treatment. Total immunoprecipitated proteins were trypsin-digested and analyzed by tandem liquid chromatography/electron spray ionization-tandem mass spectrometry (LC/ESI-MS/MS). Results obtained after MS identification of immunoprecipitated peptides are shown in Document S2 available online. Two closely related LRR-RLKs were detected in BAK1 immunoprecipitates that we named BAK1-interacting receptor-like kinase BIR2 and BIR3. Both proteins interact constitutively with BAK1 independent of the A. brassicicola infection (Document S2). A related subfamily member, BIR1, was described previously as an interactor of BAK1 [15] but was not detected in our BAK1 immunoprecipitates. As a proof of the quality of the approach, we identified additional proteins known to interact with BAK1 or BAK1-interacting receptors (Document S2). Characteristics of the BIR Family: Expression, Localization, Homologies, and Structure BIR proteins are predicted to contain a signal peptide, five LRRs, a transmembrane domain, and a cytoplasmic kinase domain and are therefore very similar in structure to BAK1
(Figures S1A and S1B). BIR2 and BIR3 form the LRR subgroup Xa along with BIR1 [15] and the close relative of BIR3, At1g69990, which we named BIR4 (Figure S1C). Microarray data show that BIR2 mRNA levels increase after infection with nonpathogenic bacteria or pathogen-associated molecular patterns (PAMP) treatment and are thus reminiscent of the expression pattern of the BAK1 gene [14]. Like BAK1, BIR2 localizes to the plasma membrane in transiently transformed Arabidopsis protoplasts and in stably transformed Arabidopsis plants (Figure S2). Interaction of BAK1 with BIR Family Proteins In directed coimmunoprecipitations of transiently expressed proteins in Nicotiana benthamiana, all four BIR family members show an association with BAK1 (Figures 1A and S3A). In yeast two-hybrid assays, the kinase domains of BIR2, BIR3, and BIR4 interact with the BAK1 kinase domain, whereas the kinase domain of BIR1 does not show interaction in this assay (Figures 1B and S3B). Fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging (FLIM) measurements revealed that BIR proteins are in the close vicinity of BAK1 in cotransformed Arabidopsis protoplasts with FRET efficiencies varying from 5.7% for BIR1, 13.8% for BIR2, 15.4% for BIR3, and 10.9% for BIR4 (Figures 1C and S3C). Bimolecular fluorescence complementation with split yellow fluorescent protein (YFP) tags fused to BAK1 and the BIR2 protein confirmed the in vivo interaction (Figure 1D). Thus, four independent assays confirmed interaction of BIR2 and BAK1. BIR2 Is an Atypical Kinase but a Substrate of BAK1 In BIR2, several residues that are described to be essential for kinase activity are not conserved [23], including the glycinerich loop and the RD and DFG motifs (Figure S4), suggesting that BIR2 is an atypical kinase. In in vitro kinase assays, strong autophosphorylation of the recombinantly expressed BAK1 kinase domain (KD) was detected, whereas BIR2 autophosphorylation activity was undetectable (Figure 2A). Incubation of BAK1 together with BIR2 resulted in a newly phosphorylated band corresponding to the BIR2 protein (Figure 2A), indicating
Please cite this article in press as: Halter et al., The Leucine-Rich Repeat Receptor Kinase BIR2 Is a Negative Regulator of BAK1 in Plant Immunity, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2013.11.047 BIR2 Negatively Regulates BAK1 during Immunity 3
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Figure 2. BIR2 Is a Substrate of BAK1 and BIR2BAK1 Interaction Is Kinase-Activity-Dependent (A) In vitro kinase assay with recombinantly expressed kinase domains of BAK1 wild-type (WT) or kinase-dead (kd) mutant K317E fused to His6 and BIR2 fused to GST (ev, empty vector control). The upper panel shows the autoradiogram, the lower Coomassie stained gel showing expressed proteins marked with asterisks. (B) Yeast two-hybrid assays with kinase domains of BIR2 and BAK1 wild-type or with the indicated mutations. Growth on selection medium indicating interaction is shown in four consecutive dilutions. Mutants with no detectable kinase activity according to [12] are indicated by an asterisk. (C) A. thaliana seedlings of the indicated genotypes were treated for 5 min with 1 mM flg22 (+) or H20 (2). Immunoprecipitation (IP) was performed with anti (a)-BAK1 antibody. Coimmunoprecipitated BIR2 was detected with anti-BIR2 antibody (WB: a-BIR2) and precipitation of BAK1 detected with anti-BAK1 antibody (western blot [WB]: a-BAK1). Western blot analysis with anti-BIR2 and -BAK1 antibodies of protein extracts before IP show protein input. Coomassie brilliant blue (CBB) staining of the membrane shows protein loading. Representative results of three independent experiments are shown. See also Figures S4 and S11 and Table S1.
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that BIR2 is a substrate of BAK1. This phosphorylation process is unidirectional as incubation of BIR2 protein with a kinase-dead version of BAK1 (K317E) did not result in phosphorylation of either protein. This suggests that BIR2 exerts its function independently of its kinase activity but rather serves as a substrate for BAK1 kinase activity. Interaction of BIR2 and BAK1 Is Dependent on BAK1 Kinase Activity In a yeast two-hybrid analysis using BAK1 mutants affecting either kinase activity or phosphorylated residues [12], out of 14 different BAK1 mutant variants tested, only two did not interact with the BIR2 kinase domain—K317E and T455A. Interestingly, these two residues are the only ones in this mutant collection required for kinase activity of BAK1 [12] (Figure 2B). Furthermore, interaction of BIR2 with the hypoactive BAK1-5 mutant protein [11] is strongly reduced, indicating that kinase activity is also essential for proper interaction in vivo (Figure 2C). These data suggest that kinase activity of BAK1 is necessary for the interaction with BIR2. BIR2 Regulates PAMP-Induced Responses, but Not BR Responses Two transfer-DNA (T-DNA) insertion lines within the 50 upstream region of BIR2 were obtained from the GABIKAT (bir2-1; GK-793F12) and Wisconsin b-Pool collections (bir2-2; Figure S5A). As the bir2-1 allele still expresses residual BIR2 protein, we generated two independent artificial
microRNA (amiRNA) lines in the Col-0 background, which show strongly pos. control reduced transcript and protein levels (Figures S5B and S5E). BIR1 and BIR3 were not significantly affected by the amiRNA (Figures S5C and S5D). As BAK1 has been implicated in brassinosteroid (BR), PAMP, and cell-death control pathways, all bir2 knockdown lines were tested for these responses. Seedling growth inhibition upon treatment with elf18 (Figures 3A and S6A) or flg22 (Figure S6F) was strongly enhanced in all tested alleles. Early responses, such as reactive oxygen species (ROS) production and mitogen-activated protein kinase activation were also enhanced, as was FRK1 expression, a marker gene for PAMP-triggered immunity (PTI), and the callose deposition that typically accompanies PAMP responses (Figures 3B–3D, S6B–S6E, and S6G). These results strongly suggest that BIR2 is a negative regulator of PAMP-triggered responses. We tested bir2 mutant alleles for resistance to bacterial pathogens and detected increased resistance to the virulent bacterium Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) with up to 60-fold less growth as compared to the respective wild-type plants (Figures 4A and S7). All observed phenotypes could be fully complemented by the expression of the BIR2 gene under its native promoter (Figure S8). These data show that BIR2 has a negative regulatory role in immunity. In addition, bir2 mutants contain elevated levels of SA (Figure 4B), which are known to contribute to resistance [24]. The expression of the SA-responsive gene PR1 is enhanced in bir2 mutants, indicating hyperactivation of SA pathways (Figure 4C). For the BR pathway, BIR2 seems to be less critical than BAK1, as brassinolide (BL; a specific BR)-triggered responses were not significantly affected in all mutant lines tested (Figure S9).
Please cite this article in press as: Halter et al., The Leucine-Rich Repeat Receptor Kinase BIR2 Is a Negative Regulator of BAK1 in Plant Immunity, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2013.11.047 Current Biology Vol 24 No 2 4
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time [min] Figure 3. BIR2 Negatively Regulates PAMP Responses (A) Seedling growth inhibition triggered by elf18 in Col-0, bir2-1, and two amiRNA-BIR2 lines. Growth is presented relative to the mock-treated wild-type. Results are average 6 SE (n = 6). (B) FRK1 gene expression in Col-0, bir2-1, and amiRNA-BIR2 lines after elf18 treatment was measured by qRT-PCR analysis, normalized to EF1a expression, and plotted relative to expression in untreated Col-0. Results are means 6 SE (n = 3). (C) ROS production represented as relative light units (RLU) in Col-0, bak1-4, bir2-1, and two amiRNA-BIR2 lines on leaf discs after elicitation with 100 nM elf18. Results are mean 6 SE (n = 9). (D) Callose deposition visualized by aniline blue staining in 5-week-old leaves of Col-0, bak1-4, bir2-1, and two amiRNA-BIR2 lines infiltrated with 100 nM elf18 or mock treated. Representative pictures from six biological replicates are presented. The scale bar represents 0.2 mm. Quantification of mean intensities of stained tissue are given as means 6 SE as inserts in the pictures. Asterisks indicate significant differences compared to wild-type samples (*p < 0.05; ***p < 0.001; A, two-way ANOVA with no correlation between treatment and genotype effects; B–D, Student’s t test). All experiments were repeated at least three times with similar results. See also Figures S5, S6, and S8–S11 and Table S1.
bir2 Mutants Are More Susceptible to Necrotrophic Fungal Infection and Show Enhanced Cell-Death Responses In bak1 mutants, A. brassicicola infection causes spreading cell death that remains restricted to the infection sites in wild-type plants [14]. Symptoms of bir2 mutants after A. brassicicola infection scored significantly higher than those observed in the respective wild-type plants (Figures 5A, 5B, S7B, and S7C). Trypan blue staining revealed that fungal growth was enhanced in the mutants, and cell death spread to uninfected areas in bir2 mutants, comparable to the observations in bak1 mutants (Figure 5C). Most observed phenotypes correlate with the expression levels in bir2 amiRNA lines, although the promoter insertion line bir2-1 that expresses more residual protein shows the strongest phenotypes in the pathogen assays. These results show that balanced BIR2 expression has a positive impact on cell-death containment and, as a consequence, a positive impact on necrotrophic fungal resistance. Taken together, BIR2 differentially affects BAK1-regulated processes with a negative regulatory role in PAMP responses and a positive impact on cell-death control.
BIR2 Is Released from BAK1 after PAMP Treatment To explore the molecular mechanism of the negative regulatory role of BIR2 on BAK1-dependent PTI responses, we hypothesized that BIR2 may sequester BAK1 in an uninfected state and release it once micro-organisms are perceived. To test this, we immunoprecipitated BAK1 after flg22 treatment and detected coimmunoprecipitated BIR2. Significantly less BIR2 was bound to BAK1 in the presence of flg22 compared to mock-treated controls (Figures 6A and 6B). Relative quantification of immunoprecipitated BIR2 protein levels shows that at least 1/3 of BIR2 is released from BAK1 within 5 min after flg22 treatment, thereby increasing the pool of BAK1 available for binding to ligand-binding RLKs, such as FLS2. Treatment with different PAMPs or BL (Figure 6C) leads to a partial release of BIR2 from BAK1 for each treatment. After addition of a PAMP cocktail plus BL, the release of BIR2 from BAK1 was drastically enhanced (Figure 6E), indicating that BAK1 and BIR2 exist in distinct subpools that can be differentially addressed by different ligand-binding receptors after stimulation. This finding supports the hypothesis that BAK1 exists in preformed complexes with
Please cite this article in press as: Halter et al., The Leucine-Rich Repeat Receptor Kinase BIR2 Is a Negative Regulator of BAK1 in Plant Immunity, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2013.11.047 BIR2 Negatively Regulates BAK1 during Immunity 5
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Figure 4. Loss of BIR2 Leads to Enhanced SA Responses and Resistance to the Biotrophic Bacterial Pathogen Pto DC3000 (A) Wild-type Col-0, bir2-1 mutant, and two amiRNA-BIR2 lines were infiltrated with 104 cfu/ml Pto DC3000, and growth of bacteria was monitored at the indicated time points. Results represent mean 6 SE (n = 8). (B) Gas chromatography-MS quantification of SA content in 5-weekold leaves of Col-0 and bir2-1 mutants 24 hr after infiltration with 108 cfu/ml Pto DC3000 or mock treatment. Results represent mean 6 SE (n = 6). (C) PR1 transcripts measured by qRT-PCR in leaf material of 5-week-old Col-0, bir2-1, and two independent amiRNA-BIR2 lines upon infiltration with Pto DC3000 or mock treatment. Expression values were normalized to EF1a and presented as a ratio to Col-0 mock-treated samples. Bars represent mean ratios 6 SE (n = 3). All experiments were repeated at least three times with similar results. Asterisks represent significant differences from Col-0 (*p < 0.05, **p < 0.01, ***p < 0.001; Student’s t test). See also Figures S5 and S7–S11 and Table S1.
ligand-binding receptors. In the inactive state, BAK1 appears to be controlled by BIR2 until receptor stimulation leads to a release of BIR2 and recruitment of BAK1 to ligand-binding receptors.
Col-0, bir2-1, and two independent amiRNA-BIR2 lines were infected with the necrotrophic fungus A. brassicicola. (A) The disease index after 7 and 10 days is shown as mean (n = 10) of a representative of three independent experiments 6 SE. (B) Representative pictures of symptom development on infected leaves after 10 days were taken. (C) Trypan blue staining 7 days after inoculation shows necrotic tissue and fungal mycelia, representative pictures are presented (n = 6). The scale bar represents 0.2 mm. See also Figures S5 and S7–S11 and Table S1.
BIR2 Controls BAK1-FLS2 Complex Formation in a LigandDependent Manner As BIR2 negatively influences PAMP responses, we determined whether BAK1 complex formation with known ligandbinding receptors, such as FLS2, is affected by BIR2. We immunoprecipitated BAK1 from bir2 knockdown plants treated with or without flg22 and detected FLS2. Compared to the level in wild-type plants, the amount of FLS2 coimmunoprecipitated with BAK1 in bir2 knockdown plants was significantly increased, indicating that BIR2 prevents binding of BAK1 to FLS2 (Figures 7A and 7B). In the converse experiment, expression of BIR2-YFP leads to reduced PAMP responses (Figures S10A and S10B) and strongly reduced binding of FLS2 to BAK1 (Figures 7A and 7B), showing that BIR2 has a negative regulatory effect on the interaction of BAK1 with FLS2. In bir2-deficient lines, the amount of FLS2 is slightly increased in about 50% of the experiments, indicating that stress factors increase the level of prestimulation in bir2 mutants leading to enhanced PAMP responses and as a secondary effect to enhanced levels of BIR1 (Figure S5C) and FLS2 (Figures S10E and S10F). FLS2 levels are unchanged in
Please cite this article in press as: Halter et al., The Leucine-Rich Repeat Receptor Kinase BIR2 Is a Negative Regulator of BAK1 in Plant Immunity, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2013.11.047 Current Biology Vol 24 No 2 6
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BIR2-overexpressing plants (Figures S10E and S10F) which show reduced complex formation and PAMP responses suggesting that enhanced FLS2 levels in bir2 mutants are a secondary effect of enhanced PAMP responses downstream of primary BIR2 function. The enhanced FLS2 levels are not a consequence of enhanced SA levels resulting from increased cell death, but rather a consequence of prestimulated PTI signaling, as nahGbir2 double mutants do not show any difference in FLS2 expression as compared to bir2 single mutants. Taken together, our data show that BIR2 has a negative regulatory role on PTI by affecting formation of complexes that include the pattern recognition receptor FLS2 and its coreceptor BAK1. Discussion BAK1 is a general regulator of several signaling pathways, namely BR-mediated growth responses, plant immunity, and cell-death control. BAK1 interacts with ligand-binding LRRRLKs, such as BRI1, FLS2, EFR, and PEPR1/PEPR2 (and more that are likely to be identified in the future), thereby allowing full signaling capacity of ligand-binding receptors and activation of downstream responses. Here, we demonstrated a novel mechanism of negative regulation at the receptor level by constitutive interaction of BAK1 with BIR2, preventing interaction of BAK1 with ligand-binding receptors. In coimmunoprecipitation experiments of in vivo BAK1 complexes, we found two previously uncharacterized LRR-RLKs, BIR2 and BIR3, as strong and constitutive BAK1-interacting proteins showing association at the plasma membrane. We could not detect the previously published BAK1-interacting RLK BIR1 [15], which belongs to the same small LRR-RLK subfamily. BIR1 may be missing from our data set because of its weaker interaction compared to BIR2 and BIR3, as shown in in vivo and yeast two-hybrid assays. Evolutionarily, BIR1 falls into a different clade than its three other family members
Figure 6. BIR2 Is Released from BAK1 in a flg22Dependent Manner Arabidopsis seedlings of the indicated genotypes were treated for 5 min with 1 mM flg22 (+) or H20 (2). Immunoprecipitation (IP) was performed with anti (a)-BAK1 antibody. (A) Coimmunoprecipitated BIR2 was detected with anti-BIR2 antibody (WB: a-BIR2) and precipitation of BAK1 detected with anti-BAK1 antibody (WB: a-BAK1). Western blot analysis with antiBIR2 and -BAK1 antibodies of protein extracts before IP show protein input. Coomassie brilliant blue (CBB) staining of the membrane shows protein loading. (B, D, and F) Signal intensity of coimmunoprecipitated BIR2 was quantified using ImageJ software relative to the precipitated BAK1 signal after background subtraction from western blots shown in (A), (C), and (E), respectively. Mocktreated Col-0 was set to 1. (C and E) Coimmunoprecipitation as described in (A) with (C) Col-0 plants treated with 1 mM flg22 or 1 mM pep1 for 5 min or 10 nM BL for 90 min and (E) a cocktail of all three ligands as shown in (C). See also Figures S5, S8, and S10–S12 and Table S1.
(Figure S11) [15] that were created by two recent duplication events (http://chibba.agtec.uga.edu/duplication). BIR2 and BIR3 were also identified as interactors of SERK1 in a proteomics approach [25]. BIR2 interacts constitutively with BAK1, in contrast to other BAK1-RLK interactions that require ligand binding to the receptor to stably recruit BAK1 [9, 10, 12, 26, 27] indicating a different molecular mechanism for these interactions. Kinase domains appear to be sufficient for protein-protein interaction of BAK1 and BIR2, BIR3, and BIR4, as exemplified in our data from yeast two-hybrid assays. It remains to be elucidated whether extracellular domains are also involved in BIR-BAK1 interaction, as shown for BRI1 and FLS2 interaction with BAK1 [28, 29]. In contrast to BIR1 [15], in vitro kinase assays using the recombinant BIR2 kinase domain did not reveal autophosphorylation or transphosphorylation activity to BAK1. Sequence analyses of conserved residues within the kinase domain suggest that BIR2 is not an active kinase (Figure S4). Complementation experiments with kinase-dead BIR1 mutants partially complement growth defects of bir1 mutants, indicating that kinase activity is needed for some aspects of the bir1 mutant phenotypes [15]. This suggests that BIR1 acts at least partially via a different molecular mechanism than BIR2, using its intrinsic enzymatic activity. Approximately 20% of all Arabidopsis RLKs lack conserved residues required for enzymatic activity [30]. The LRR-RLK STRUBBELIG, for instance, is impaired in similar conserved residues of its kinase domain as BIR2 (Figure S4) and does not require kinase activity to control organ shape and plant organization [31]. However, transphosphorylation assays revealed that BIR2 is a substrate for BAK1 kinase activity. Kinase-inactive versions of BAK1, such as K317E and T455A mutants, do not interact with BIR2 in yeast two-hybrid assays, and hypoactive BAK1-5 proteins show weaker interaction with BIR2 in vivo, indicating that kinase activity of BAK1 is required for interaction with BIR2. This is somewhat different for BAK1
Please cite this article in press as: Halter et al., The Leucine-Rich Repeat Receptor Kinase BIR2 Is a Negative Regulator of BAK1 in Plant Immunity, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2013.11.047 BIR2 Negatively Regulates BAK1 during Immunity 7
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Figure 7. BIR2 Has a Negative Regulatory Effect on BAK1 FLS2 Complex Formation (A) Two-week-old A. thaliana seedlings of the indicated genotypes were treated for 5 min with 1 mM flg22 (+) or H20 (2). Immunoprecipitation (IP) was performed with anti (a)-BAK1 antibody. Western blot analyses show coimmunoprecipitation of FLS2, detected with anti-FLS2 antibody (WB: a-FLS2), and precipitation of BAK1 detected with anti-BAK1 antibody (WB: a-BAK1). Western blot analysis with anti-FLS2 and -BAK1 antibodies of protein extracts before IP show protein input. Coomassie brilliant blue (CBB) staining of the membrane shows protein loading. Representative results of at least three independent experiments are shown. (B) Signal intensity of coimmunoprecipitated FLS2 was quantified using ImageJ software relative to the precipitated BAK1 signal after background subtraction. Mock-treated Col-0 was set to 1. See also Figures S5, S8, and S10–S12 and Table S1.
BRI1 interaction, as kinase activity of BRI1, but not BAK1, is required for ligand-dependent in vivo interaction of these proteins [12]. In the case of FLS2 or EFR, BAK1 kinase-inactive mutants can still interact [10, 11], and the hypoactive BAK1-5 protein interacts even more strongly with the receptors FLS2, EFR, and BRI1 [11], indicating phosphorylation-dependent mechanistic differences in the formation of different BAK1 complexes. Further studies on the phosphorylation events between BAK1 and BIR2 are needed to reveal the impact of phosphorylation on these RLKs. Functional analysis of BIR2 knockout or knockdown plants implicated BAK1-related pathways controlling BR-mediated growth, PTI, and cell-death control. BIR2 knockdown lines show no differences during etiolation, brassinazole treatment, and in BL-hypocotyl growth-induction assays, indicating that loss of BIR2 does not affect BR signaling. This might be due to redundancy of BIR2 with its family members but might
also be a consequence of the lower requirement of BAK1 for BRI1 interaction as compared to FLS2 [32] or specific integration of BIR2 into preformed receptor complexes that do not include BRI1. BIR2 interacts less strongly with the hypoactive mutant BAK1-5. The bak1-5 mutation differentiates between the different pathways and is also not impaired in BR responses whereas PAMP responses are strongly reduced, indicating that BAK1 is less required for BRI1-mediated signaling [32] or that different pathways are differentially sensitive to kinase activity changes [11]. However, in contrast to null bak1 mutants that display reduced PAMP responses [26, 27] but enhanced cell death [7, 14], BIR2 has an antagonistic effect on PAMP responses but the same impact on A. brassicicolainduced cell death. This indicates that these pathways are independently regulated and that BIR2 differentially regulates BAK1-dependent signaling pathways. Cell death is activated in both bir2 and bak1 mutants. Celldeath phenotypes were also described for bir1 mutations, and based upon genetic suppression analyses, the authors proposed that BIR1 is guarded by at least two R-protein pathways leading to an SA-dependent autoimmune cell death when loss of BIR1 is sensed [15]. A guarding model might also be true for BIR2 during which the integrity of BAK1 complexes might be sensed, and in the absence of either BAK1 or BIR2, a yet unknown guarding system of the complex is alerted, thereby activating cell death. Cell death in bir2 mutants is SA-dependent as shown for bir1 mutants. BIR1mediated cell death might include suppressor of npr1-1, constitutive (SNC1) signaling [22]. However, we observed pathogen-induced cell-death responses in bak1-1 [5] and bir2-2 Ws alleles. Yet, this ecotype does not contain a functional SNC1 allele, indicating that there must be at least one additional cell-death pathway that is activated upon disturbance of BAK1 complexes. However, enhanced PAMPinduced ROS burst is only partially affected in mutants expressing nahG (Figures S10C and S10D). This shows that SA-independent PAMP responses are enhanced in bir2 mutants and that PAMP and cell-death responses can be differentiated. Plants highly overexpressing BAK1 constitutively activate cell-death responses. Cell death can be blocked when BRI1 is simultaneously overexpressed. This indicates that a balanced ratio of BAK1 to interacting complex partners is critical for preventing inappropriate activation of BAK1dependent pathways [33]. Further analysis of cell death in RLK mutants, which share effects similar to autoimmune responses [22] and hybrid incompatibility [34, 35], will shed more light on this phenomenon. However, the cell-death reactions do not explain the enhanced PAMP responses in bir2 mutants. How BIR2 functions in PTI signaling is an open question. Because of its small extracellular domain and its constitutive interaction with BAK1, we hypothesized that BIR2 negatively regulates BAK1 function. In rice, the adenosine triphosphatase XB24 acts as a negative regulator by binding to the LRR-RLK XA21. Upon ligand binding, XB24 is released and derepression of XA21 results in the activation of defense responses [36]. Similarly, BRI1 is kept in an inactive state by interaction with BRI1 kinase inhibitor (BKI1). Upon ligand binding, BKI1 is phosphorylated, released from the plasma membrane, and allows BRI1 recruitment of BAK1 resulting in the activation of BR signaling [37, 38]. In addition, BRI1 contains an autoinhibitory C-terminal tail, which is released after ligand binding and transphosphorylation by BAK1 [39]. Therefore, multiple mechanisms function to tightly regulate receptor
Please cite this article in press as: Halter et al., The Leucine-Rich Repeat Receptor Kinase BIR2 Is a Negative Regulator of BAK1 in Plant Immunity, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2013.11.047 Current Biology Vol 24 No 2 8
complex formation and prevent unwanted induction of downstream signaling. If this model is true for BIR2 also, BIR2 should be released from the BAK1 complex after ligand perception to allow BAK1 to interact with ligand-binding receptors. BIR2 binding to BAK1 was significantly reduced after flg22 treatment, showing that BIR2 is indeed released from the complex in a ligand-dependent manner. This effect is enhanced after treatment with multiple ligands, showing that BAK1 exists in target-specific subpools, supporting the idea that BAK1 resides in the membrane in preformed complexes with BIR2 and its ligand-binding receptor partners as suggested by [40]. We could not detect direct interaction of BIR2 with FLS2 (Figure S12B). This shows that BAK1 is likely released from BIR2 prior to binding to FLS2 after activation. BAK1 binding to FLS2 is significantly reduced in BIR2-overexpressing plants and oppositely enhanced in bir2 knockdown plants indicating that BIR2 has a direct regulatory effect on BAK1receptor complex formation. In contrast to the fact that the cellular pool of BAK1 is not rate-limiting between different signaling pathways [32], regulatory BIR2 competes with FLS2 for BAK1, thereby negatively regulating FLS2-mediated signaling. It remains to be tested whether inhibition of complex formation is the only effect of BIR2 on BAK1-dependent responses. Genetic interaction studies confirmed that BAK1 is epistatic to BIR2 (Figure S12A), and therefore upstream of BAK1 action and receptor activation, excluding direct effects of BIR2 on PAMP-triggered signaling downstream of BAK1. Mechanistically, BIR2 might exist in preformed complexes together with FLS2 and BAK1. Upon ligand binding to FLS2, the affinity of BAK1 for FLS2 may increase. This is supported by BAK1 functioning as a true coreceptor binding receptorbound ligands [29, 41]. The affinity of BAK1 for the receptorligand complex might be higher than for the receptor alone allowing the recruitment of BAK1 from BIR2 to the FLS2 complex. This mechanism is also supported by mutated BAK1-5 protein where lower affinity for BIR2 correlates with higher affinity to ligand-binding receptors even in the absence of the ligand [11]. This interaction might be phosphorylationdependent as BAK1-5 has reduced enzymatic activity. A specific phosphorylation-dependent mechanism, as shown for BKI1 release from BRI1, is also conceivable, though BAK1 phosphorylation activity in general seems to be necessary for the interaction with BIR2. Conclusions We show that BIR2 is a novel type of LRR-RLK that interacts with BAK1 in a kinase-activity-dependent manner and negatively controls complex formation of BAK1 with specific ligand-binding receptors—a novel regulatory mechanism for RLKs. Cell-death reactions are independent of direct effects of BIR2 on BAK1 complex formation and likely the result of a guarding mechanism. In the absence of PAMPs, BIR2 interacts with BAK1, inhibits autoimmune cell-death responses, and keeps BAK1 under control. Only upon ligand binding to FLS2, BAK1 is released from BIR2 and recruited to the FLS2 complex to induce PAMP-triggered immune signaling.
primers listed in Table S1). Plants were grown for 5 to 6 weeks on soil in growth chambers (8 hr light, 16 hr dark; 22 C; 110 mEm22 s21) or on onehalf MS medium. Infection Procedures Pseudomonas syringae pv. tomato DC3000 infections were performed as described by Mosher et al. [43]. Alternaria brassicicola infection assays were carried out as described by Kemmerling et al. [14]. Histochemical Assays Cell death and fungal mycelium was stained with trypan blue as described in [14]. Callose was visualized as described in [43]. Seedling Growth Assays Seedling growth inhibition assays were performed as described in [43]. Oxidative Burst Measurements Leaf discs were excised from adult plants and incubated in water overnight. Water was replaced with 20 mM luminol L-012 and 10 mg/ml horseradish peroxidase, and leaf discs (n = 9) were treated with 100 nM flg22 or elf18 and analyzed with the multiplate reader Centro LB 900 (Berthold Technologies). Quantitative Real-Time PCR Transcript levels were analyzed by quantitative RT-PCR (qRT-PCR) as described in [43] with primers listed in Table S1. Hormone Measurements Salicylate and jasmonate contents were measured as described by Lenz et al. [44] Transient Expression in Nicotiana benthamiana Agrobacterium tumefaciens GV3101 containing the respective constructs were grown 36 hr at 28 C in Luria broth medium supplemented with appropriate antibiotics. Cultures were pelleted and resuspended in 10 mM MgCl2 to optical density at 600 = 1. Agrobacteria carrying different constructs were mixed 1:1 and infiltrated into 3-week-old N. benthamiana leaves. Samples were harvested 2 to 3 days after inoculation. Coimmunoprecipitations Leaves were ground in liquid nitrogen, and 1 ml extraction buffer (25 mM Tris-HCl [pH 7.5], 150 mM NaCl, Nonidet P40 1%) per g tissue powder was added. Samples were centrifuged at 4 C and 13,000 rpm for 10 min. After 33 washing with the extraction buffer, 15 ml protein A agarose beads (Roche) were incubated 1 hr with the antibodies. Supernatants containing equal amounts of protein were incubated for 4 hr at 4 C with the beads. Beads were washed three times with 50 mM Tris-HCl (pH 7.5), and 150 mM NaCl, before adding 23 loading buffer and heating at 95 C for 10 min. SDS-PAGE and Immunoblotting Proteins were separated, blotted, and incubated with antibodies as described in [11] but with 8% SDS gels and the following antibody dilutions: anti-GFP (Acris), 1:5,000; anti-FLAG (Sigma), 1:2,000; anti-c-myc (Sigma), 1:10,000; anti-BAK1, 1:2,000; anti-FLS2, 1:250; anti-BIR2, 1:4,000; antiguinea pig (Santa Cruz Biotechnology), 1:5,000; and antirabbit (Sigma), 1:50,000. Chemiluminescence was detected with the ECL western blotting system (GE Healthcare) and Kodak XJ300 films. FRET-FLIM Measurements FLIM measurements were performed as described in [45, 46]. Details are given in the Supplemental Experimental Procedures. Yeast Two-Hybrid Assays To screen for direct interactions, the Clontech matchmaker GAL4 system was used according to the manufacturer’s instructions.
Experimental Procedures
BIFC Assays BIR family and BAK1 coding regions were transiently expressed in N. benthamiana as fusions to the C- and N-terminal part of YFP. Fluorescence of YFP fusions was visualized by epifluorescence microscopy 16 hr after infiltration.
Plant Material and Growth Conditions T-DNA insertion mutants used are bir2-1 (GK-793F12) and bir2-2 (Wisconsin Arabidopsis Knockout Facility b-Pool; screened as described in [42] with
Kinase Activity Assays Kinase activity assays were performed as described by Schwessinger et al. [11] but incubated for 1 hr at 37 C without shaking.
Please cite this article in press as: Halter et al., The Leucine-Rich Repeat Receptor Kinase BIR2 Is a Negative Regulator of BAK1 in Plant Immunity, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2013.11.047 BIR2 Negatively Regulates BAK1 during Immunity 9
Statistical Methods Statistical significance between two groups has been checked by using Student’s t test. One-way ANOVA was performed for multiple comparisons combined with Tukey’s honest significant difference (HSD) test indicating significant differences with different letters (p < 0.01). For multivariant analysis, we applied a two-way ANOVA analysis to the original data. If statistical differences within and between the two parameters were detected and no correlation between the two parameters was calculated, we applied Tukey’s HSD test. Asterisks represent significant differences (*p < 0.05; **p < 0.01; ***p < 0.001). Supplemental Information Supplemental Information includes Supplemental Experimental Procedures, 12 figures, one table, and MS data and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2013.11.047. Acknowledgments We acknowledge funding from DFG KE1485/1, EU BRAVISSIMO, to B.K.; the Gatsby Charitable Foundation to C.Z.; and the US NSF, MCB1021363, to X.W. and S.C. Received: July 11, 2013 Revised: October 25, 2013 Accepted: November 22, 2013 Published: January 2, 2014 References 1. Postel, S., and Kemmerling, B. (2009). Plant systems for recognition of pathogen-associated molecular patterns. Semin. Cell Dev. Biol. 20, 1025–1031. 2. Boller, T., and Felix, G. (2009). A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu. Rev. Plant Biol. 60, 379–406. 3. Go´mez-Go´mez, L., and Boller, T. (2000). FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol. Cell 5, 1003–1011. 4. Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J.D., Boller, T., and Felix, G. (2006). Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125, 749–760. 5. Li, J., Wen, J., Lease, K.A., Doke, J.T., Tax, F.E., and Walker, J.C. (2002). BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell 110, 213–222. 6. Nam, K.H., and Li, J. (2002). BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell 110, 203–212. 7. He, K., Gou, X., Yuan, T., Lin, H., Asami, T., Yoshida, S., Russell, S.D., and Li, J. (2007). BAK1 and BKK1 regulate brassinosteroid-dependent growth and brassinosteroid-independent cell-death pathways. Curr. Biol. 17, 1109–1115. 8. Chinchilla, D., Shan, L., He, P., de Vries, S., and Kemmerling, B. (2009). One for all: the receptor-associated kinase BAK1. Trends Plant Sci. 14, 535–541. 9. Roux, M., Schwessinger, B., Albrecht, C., Chinchilla, D., Jones, A., Holton, N., Malinovsky, F.G., To¨r, M., de Vries, S., and Zipfel, C. (2011). The Arabidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens. Plant Cell 23, 2440–2455. 10. Schulze, B., Mentzel, T., Jehle, A.K., Mueller, K., Beeler, S., Boller, T., Felix, G., and Chinchilla, D. (2010). Rapid heteromerization and phosphorylation of ligand-activated plant transmembrane receptors and their associated kinase BAK1. J. Biol. Chem. 285, 9444–9451. 11. Schwessinger, B., Roux, M., Kadota, Y., Ntoukakis, V., Sklenar, J., Jones, A., and Zipfel, C. (2011). Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1. PLoS Genet. 7, e1002046. 12. Wang, X., Kota, U., He, K., Blackburn, K., Li, J., Goshe, M.B., Huber, S.C., and Clouse, S.D. (2008). Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling. Dev. Cell 15, 220–235.
13. Cao, Y., Aceti, D.J., Sabat, G., Song, J., Makino, S., Fox, B.G., and Bent, A.F. (2013). Mutations in FLS2 Ser-938 dissect signaling activation in FLS2-mediated Arabidopsis immunity. PLoS Pathog. 9, e1003313. 14. Kemmerling, B., Schwedt, A., Rodriguez, P., Mazzotta, S., Frank, M., Qamar, S.A., Mengiste, T., Betsuyaku, S., Parker, J.E., Mu¨ssig, C., et al. (2007). The BRI1-associated kinase 1, BAK1, has a brassinolideindependent role in plant cell-death control. Curr. Biol. 17, 1116–1122. 15. Gao, M., Wang, X., Wang, D., Xu, F., Ding, X., Zhang, Z., Bi, D., Cheng, Y.T., Chen, S., Li, X., and Zhang, Y. (2009). Regulation of cell death and innate immunity by two receptor-like kinases in Arabidopsis. Cell Host Microbe 6, 34–44. 16. Liebrand, T.W., van den Berg, G.C., Zhang, Z., Smit, P., Cordewener, J.H., America, A.H., Sklenar, J., Jones, A.M., Tameling, W.I., Robatzek, S., et al. (2013). Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection. Proc. Natl. Acad. Sci. USA 110, 10010–10015. 17. Segonzac, C., and Zipfel, C. (2011). Activation of plant pattern-recognition receptors by bacteria. Curr. Opin. Microbiol. 14, 54–61. 18. Kazan, K., and Manners, J.M. (2008). Jasmonate signaling: toward an integrated view. Plant Physiol. 146, 1459–1468. 19. Browse, J. (2009). Jasmonate passes muster: a receptor and targets for the defense hormone. Annu. Rev. Plant Biol. 60, 183–205. 20. Lu, D., Wu, S., Gao, X., Zhang, Y., Shan, L., and He, P. (2010). A receptorlike cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proc. Natl. Acad. Sci. USA 107, 496–501. 21. Zhang, J., Li, W., Xiang, T., Liu, Z., Laluk, K., Ding, X., Zou, Y., Gao, M., Zhang, X., Chen, S., et al. (2010). Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a Pseudomonas syringae effector. Cell Host Microbe 7, 290–301. 22. Wang, Z., Meng, P., Zhang, X., Ren, D., and Yang, S. (2011). BON1 interacts with the protein kinases BIR1 and BAK1 in modulation of temperature-dependent plant growth and cell death in Arabidopsis. Plant J. 67, 1081–1093. 23. Hanks, S.K., Quinn, A.M., and Hunter, T. (1988). The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 42–52. 24. Glazebrook, J. (2005). Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu. Rev. Phytopathol. 43, 205–227. 25. Smaczniak, C., Li, N., Boeren, S., America, T., van Dongen, W., Goerdayal, S.S., de Vries, S., Angenent, G.C., and Kaufmann, K. (2012). Proteomics-based identification of low-abundance signaling and regulatory protein complexes in native plant tissues. Nat. Protoc. 7, 2144–2158. 26. Chinchilla, D., Zipfel, C., Robatzek, S., Kemmerling, B., Nu¨rnberger, T., Jones, J.D.G., Felix, G., and Boller, T. (2007). A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448, 497–500. 27. Heese, A., Hann, D.R., Gimenez-Ibanez, S., Jones, A.M., He, K., Li, J., Schroeder, J.I., Peck, S.C., and Rathjen, J.P. (2007). The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc. Natl. Acad. Sci. USA 104, 12217–12222. 28. Jaillais, Y., Belkhadir, Y., Balsema˜o-Pires, E., Dangl, J.L., and Chory, J. (2011). Extracellular leucine-rich repeats as a platform for receptor/ coreceptor complex formation. Proc. Natl. Acad. Sci. USA 108, 8503– 8507. 29. Sun, Y., Li, L., Macho, A.P., Han, Z., Hu, Z., Zipfel, C., Zhou, J.M., and Chai, J. (2013). Structural basis for flg22-induced activation of the Arabidopsis FLS2-BAK1 immune complex. Science 342, 624–628. 30. Castells, E., and Casacuberta, J.M. (2007). Signalling through kinasedefective domains: the prevalence of atypical receptor-like kinases in plants. J. Exp. Bot. 58, 3503–3511. 31. Chevalier, D., Batoux, M., Fulton, L., Pfister, K., Yadav, R.K., Schellenberg, M., and Schneitz, K. (2005). STRUBBELIG defines a receptor kinase-mediated signaling pathway regulating organ development in Arabidopsis. Proc. Natl. Acad. Sci. USA 102, 9074–9079. 32. Albrecht, C., Boutrot, F., Segonzac, C., Schwessinger, B., GimenezIbanez, S., Chinchilla, D., Rathjen, J.P., de Vries, S.C., and Zipfel, C. (2012). Brassinosteroids inhibit pathogen-associated molecular pattern-triggered immune signaling independent of the receptor kinase BAK1. Proc. Natl. Acad. Sci. USA 109, 303–308.
Please cite this article in press as: Halter et al., The Leucine-Rich Repeat Receptor Kinase BIR2 Is a Negative Regulator of BAK1 in Plant Immunity, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2013.11.047 Current Biology Vol 24 No 2 10
33. Belkhadir, Y., Jaillais, Y., Epple, P., Balsema˜o-Pires, E., Dangl, J.L., and Chory, J. (2012). Brassinosteroids modulate the efficiency of plant immune responses to microbe-associated molecular patterns. Proc. Natl. Acad. Sci. USA 109, 297–302. 34. Bomblies, K., Lempe, J., Epple, P., Warthmann, N., Lanz, C., Dangl, J.L., and Weigel, D. (2007). Autoimmune response as a mechanism for a Dobzhansky-Muller-type incompatibility syndrome in plants. PLoS Biol. 5, e236. 35. Alca´zar, R., Garcı´a, A.V., Kronholm, I., de Meaux, J., Koornneef, M., Parker, J.E., and Reymond, M. (2010). Natural variation at Strubbelig Receptor Kinase 3 drives immune-triggered incompatibilities between Arabidopsis thaliana accessions. Nat. Genet. 42, 1135–1139. 36. Chen, X., Chern, M., Canlas, P.E., Ruan, D., Jiang, C., and Ronald, P.C. (2010). An ATPase promotes autophosphorylation of the pattern recognition receptor XA21 and inhibits XA21-mediated immunity. Proc. Natl. Acad. Sci. USA 107, 8029–8034. 37. Jaillais, Y., Hothorn, M., Belkhadir, Y., Dabi, T., Nimchuk, Z.L., Meyerowitz, E.M., and Chory, J. (2011). Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor. Genes Dev. 25, 232–237. 38. Wang, X., and Chory, J. (2006). Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane. Science 313, 1118–1122. 39. Wang, X., Li, X., Meisenhelder, J., Hunter, T., Yoshida, S., Asami, T., and Chory, J. (2005). Autoregulation and homodimerization are involved in the activation of the plant steroid receptor BRI1. Dev. Cell 8, 855–865. 40. Bu¨cherl, C.A., van Esse, G.W., Kruis, A., Luchtenberg, J., Westphal, A.H., Aker, J., van Hoek, A., Albrecht, C., Borst, J.W., and de Vries, S.C. (2013). Visualization of BRI1 and BAK1(SERK3) membrane receptor heterooligomers during brassinosteroid signaling. Plant Physiol. 162, 1911–1925. 41. Sun, Y., Han, Z., Tang, J., Hu, Z., Chai, C., Zhou, B., and Chai, J. (2013). Structure reveals that BAK1 as a co-receptor recognizes the BRI1bound brassinolide. Cell Res. 23, 1326–1329. 42. Sussman, M.R., Amasino, R.M., Young, J.C., Krysan, P.J., and AustinPhillips, S. (2000). The Arabidopsis knockout facility at the University of Wisconsin-Madison. Plant Physiol. 124, 1465–1467. 43. Mosher, S., Seybold, H., Rodriguez, P., Stahl, M., Davies, K.A., Dayaratne, S., Morillo, S.A., Wierzba, M., Favery, B., Keller, H., et al. (2013). The tyrosine-sulfated peptide receptors PSKR1 and PSY1R modify the immunity of Arabidopsis to biotrophic and necrotrophic pathogens in an antagonistic manner. Plant J. 73, 469–482. 44. Lenz, H.D., Haller, E., Melzer, E., Kober, K., Wurster, K., Stahl, M., Bassham, D.C., Vierstra, R.D., Parker, J.E., Bautor, J., et al. (2011). Autophagy differentially controls plant basal immunity to biotrophic and necrotrophic pathogens. Plant J. 66, 818–830. 45. Bu¨cherl, C., Aker, J., de Vries, S., and Borst, J.W. (2010). Probing protein-protein Interactions with FRET-FLIM. Methods Mol. Biol. 655, 389–399. 46. Russinova, E., Borst, J.W., Kwaaitaal, M., Can˜o-Delgado, A., Yin, Y., Chory, J., and de Vries, S.C. (2004). Heterodimerization and endocytosis of Arabidopsis brassinosteroid receptors BRI1 and AtSERK3 (BAK1). Plant Cell 16, 3216–3229.