The Arabidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens

Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) constitutes an important layer of innate immunity in plants. The leucine-rich repeat (LRR) receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN SENSING2 (FLS2) are the PRRs for the peptide PAMPs elf18 and flg22, which are derived from bacterial EF-Tu and flagellin, respectively. Using coimmunoprecipitation and mass spectrometry analyses, we demonstrated that EFR and FLS2 undergo ligand-induced heteromerization in planta with several LRR receptor-like kinases that belong to the SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, including BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1/SERK3 (BAK1/SERK3) and BAK1-LIKE1/SERK4 (BKK1/SERK4). Using a novel bak1 allele that does not exhibit pleiotropic defects in brassinosteroid and cell death responses, we determined that BAK1 and BKK1 cooperate genetically to achieve full signaling capability in response to elf18 and flg22 and to the damage-associated molecular pattern AtPep1. Furthermore, we demonstrated that BAK1 and BKK1 contribute to disease resistance against the hemibiotrophic bacterium Pseudomonas syringae and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Our work reveals that the establishment of PAMP-triggered immunity (PTI) relies on the rapid ligand-induced recruitment of multiple SERKs within PRR complexes and provides insight into the early PTI signaling events underlying this important layer of plant innate immunity.     

Danger peptide receptor signaling in plants ensures basal immunity upon pathogen‐induced depletion of BAK1

Pathogens infect a host by suppressing defense responses induced upon recognition of microbe‐associated molecular patterns (MAMPs). Despite this suppression, MAMP receptors mediate basal resistance to limit host susceptibility, via a process that is poorly understood. The Arabidopsis leucine‐rich repeat (LRR) receptor kinase BAK1 associates and functions with different cell surface LRR receptors for a wide range of ligands, including MAMPs. We report that BAK1 depletion is linked to defense activation through the endogenous PROPEP peptides (Pep epitopes) and their LRR receptor kinases PEPR1/PEPR2, despite critical defects in MAMP signaling. In bak1‐knockout plants, PEPR elicitation results in extensive cell death and the prioritization of salicylate‐based defenses over jasmonate‐based defenses, in addition to elevated proligand and receptor accumulation. BAK1 disruption stimulates the release of PROPEP3, produced in response to Pep application and during pathogen challenge, and renders PEPRs necessary for basal resistance. These findings are biologically relevant, since specific BAK1 depletion coincides with PEPR‐dependent resistance to the fungal pathogen Colletotrichum higginsianum. Thus, the PEPR pathway ensures basal resistance when MAMP‐triggered defenses are compromised by BAK1 depletion.     

Mutations in FLS2 Ser-938 Dissect Signaling Activation in FLS2-Mediated Arabidopsis Immunity

FLAGELLIN-SENSING 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing FLS2_S938A, while the acidic phosphomimic mutants FLS2_S938D and FLS2_S938E conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2_S938A, demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2_S938A and FLS2_S938D mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic Arabidopsis thaliana plants expressing FLS2_S938A or FLS2_D997A (a kinase catalytic site mutant), but was normally induced in FLS2_S938D plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.     

Phytophthora infestans RXLR-WY Effector AVR3a Associates with Dynamin-Related Protein 2 Required for Endocytosis of the Plant Pattern Recognition Receptor FLS2

Pathogens utilize effectors to suppress basal plant defense known as PTI (Pathogen-associated molecular pattern-triggered immunity). However, our knowledge of PTI suppression by filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previous work revealed that the co-receptor BAK1/SERK3 contributes to basal immunity against the potato pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the cell death induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. The P. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediated cell death by binding the plant E3 ligase CMPG1. In contrast, AVR3a^KI-Y147del, a deletion mutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and does not suppress INF1-mediated cell death. Here, we studied the extent to which AVR3a and its variants perturb additional BAK1/SERK3-dependent PTI responses in N. benthamiana using the elicitor/receptor pair flg22/FLS2 as a model. We found that all tested variants of AVR3a suppress defense responses triggered by flg22 and reduce internalization of activated FLS2. Moreover, we discovered that AVR3a associates with the Dynamin-Related Protein 2 (DRP2), a plant GTPase implicated in receptor-mediated endocytosis. Interestingly, silencing of DRP2 impaired ligand-induced FLS2 internalization but did not affect internalization of the growth receptor BRI1. Our results suggest that AVR3a associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis. We conclude that AVR3a is a multifunctional effector that can suppress BAK1/SERK3-mediated immunity through at least two different pathways.     

Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1

Plants rely heavily on receptor-like kinases (RLKs) for perception and integration of external and internal stimuli. The Arabidopsis regulatory leucine-rich repeat RLK (LRR-RLK) BAK1 is involved in steroid hormone responses, innate immunity, and cell death control. Here, we describe the differential regulation of three different BAK1-dependent signaling pathways by a novel allele of BAK1, bak1-5. Innate immune signaling mediated by the BAK1-dependent RKs FLS2 and EFR is severely compromised in bak1-5 mutant plants. However, bak1-5 mutants are not impaired in BR signaling or cell death control. We also show that, in contrast to the RD kinase BRI1, the non-RD kinases FLS2 and EFR have very low kinase activity, and we show that neither was able to trans-phosphorylate BAK1 in vitro. Furthermore, kinase activity for all partners is completely dispensable for the ligand-induced heteromerization of FLS2 or EFR with BAK1 in planta, revealing another pathway specific mechanistic difference. The specific suppression of FLS2- and EFR-dependent signaling in bak1-5 is not due to a differential interaction of BAK1-5 with the respective ligand-binding RK but requires BAK1-5 kinase activity. Overall our results demonstrate a phosphorylation-dependent differential control of plant growth, innate immunity, and cell death by the regulatory RLK BAK1, which may reveal key differences in the molecular mechanisms underlying the regulation of ligand-binding RD and non-RD RKs.     

BAK1 is not a target of the Pseudomonas syringae effector AvrPto

Plant cell surface-localized receptor kinases such as FLS2, EFR, and CERK1 play a crucial role in detecting invading pathogenic bacteria. Upon stimulation by bacterium-derived ligands, FLS2 and EFR interact with BAK1, a receptor-like kinase, to activate immune responses. A number of Pseudomonas syringae effector proteins are known to block immune responses mediated by these receptors. Previous reports suggested that both FLS2 and BAK1 could be targeted by the P. syringae effector AvrPto to inhibit plant defenses. Here, we provide new evidence further supporting that FLS2 but not BAK1 is targeted by AvrPto in plants. The AvrPto-FLS2 interaction prevented the phosphorylation of BIK1, a downstream component of the FLS2 pathway.     

Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE proteins serve brassinosteroid-dependent and -independent signaling pathways

The Arabidopsis (Arabidopsis thaliana) SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) genes belong to a small family of five plant receptor kinases that are involved in at least five different signaling pathways. One member of this family, BRASSINOSTEROID INSENSITIVE1 (BRI1)-ASSOCIATED KINASE1 (BAK1), also known as SERK3, is the coreceptor of the brassinolide (BR)-perceiving receptor BRI1, a function that is BR dependent and partially redundant with SERK1. BAK1 (SERK3) alone controls plant innate immunity, is also the coreceptor of the flagellin receptor FLS2, and, together with SERK4, can mediate cell death control, all three in a BR-independent fashion. SERK1 and SERK2 are essential for male microsporogenesis, again independent from BR. SERK5 does not appear to have any function under the conditions tested. Here, we show that the different SERK members are only redundant in pairs, whereas higher order mutant combinations only show additive phenotypes. Surprisingly, SERK members that are redundant within one are not redundant in another pathway. We also show that this evolution of functional pairs occurred by a change in protein function and not by differences in spatial expression. We propose that, in plants, closely related receptor kinases have a minimal homo- or heterodimeric configuration to achieve specificity.     

The tomato I gene for Fusarium wilt resistance encodes an atypical leucine‐rich repeat receptor‐like protein whose function is nevertheless dependent on SOBIR1 and SERK3/BAK1

We have identified the tomato I gene for resistance to the Fusarium wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol) and show that it encodes a membrane‐anchored leucine‐rich repeat receptor‐like protein (LRR‐RLP). Unlike most other LRR‐RLP genes involved in plant defence, the I gene is not a member of a gene cluster and contains introns in its coding sequence. The I gene encodes a loopout domain larger than those in most other LRR‐RLPs, with a distinct composition rich in serine and threonine residues. The I protein also lacks a basic cytosolic domain. Instead, this domain is rich in aromatic residues that could form a second transmembrane domain. The I protein recognises the Fol Avr1 effector protein, but, unlike many other LRR‐RLPs, recognition specificity is determined in the C‐terminal half of the protein by polymorphic amino acid residues in the LRRs just preceding the loopout domain and in the loopout domain itself. Despite these differences, we show that I/Avr1‐dependent necrosis in Nicotiana benthamiana depends on the LRR receptor‐like kinases (RLKs) SERK3/BAK1 and SOBIR1. Sequence comparisons revealed that the I protein and other LRR‐RLPs involved in plant defence all carry residues in their last LRR and C‐terminal LRR capping domain that are conserved with SERK3/BAK1‐interacting residues in the same relative positions in the LRR‐RLKs BRI1 and PSKR1. Tyrosine mutations of two of these conserved residues, Q922 and T925, abolished I/Avr1‐dependent necrosis in N. benthamiana, consistent with similar mutations in BRI1 and PSKR1 preventing their interaction with SERK3/BAK1.     

LRR conservation mapping to predict functional sites within protein leucine-rich repeat domains

Computational prediction of protein functional sites can be a critical first step for analysis of large or complex proteins. Contemporary methods often require several homologous sequences and/or a known protein structure, but these resources are not available for many proteins. Leucine-rich repeats (LRRs) are ligand interaction domains found in numerous proteins across all taxonomic kingdoms, including immune system receptors in plants and animals. We devised Repeat Conservation Mapping (RCM), a computational method that predicts functional sites of LRR domains. RCM utilizes two or more homologous sequences and a generic representation of the LRR structure to identify conserved or diversified patches of amino acids on the predicted surface of the LRR. RCM was validated using solved LRR+ligand structures from multiple taxa, identifying ligand interaction sites. RCM was then used for de novo dissection of two plant microbe-associated molecular pattern (MAMP) receptors, EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2). In vivo testing of Arabidopsis thaliana EFR and FLS2 receptors mutagenized at sites identified by RCM demonstrated previously unknown functional sites. The RCM predictions for EFR, FLS2 and a third plant LRR protein, PGIP, compared favorably to predictions from ODA (optimal docking area), Consurf, and PAML (positive selection) analyses, but RCM also made valid functional site predictions not available from these other bioinformatic approaches. RCM analyses can be conducted with any LRR-containing proteins at www.plantpath.wisc.edu/RCM, and the approach should be modifiable for use with other types of repeat protein domains.     

The Pseudomonas syringae effector HopF2 suppresses Arabidopsis immunity by targeting BAK1

Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage immune responses and modulate physiology to favor infection. The P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple microbe‐associated molecular patterns (MAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms for suppression of two branches of MAMP‐activated MAP kinase (MAPK) cascades. In addition to blocking MKK5 (MAPK kinase 5) activation in the MEKK1 (MAPK kinase kinase 1)/MEKKs–MKK4/5–MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in the MEKK1–MKK1/2–MPK4 cascade and the plasma membrane‐localized receptor‐like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane‐localized receptor‐like kinase that is involved in multiple MAMP signaling. The interaction between BAK1 and HopF2 and between two other P. syringae effectors, AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of AvrPto, AvrPtoB and HopF2, the strong growth defects or lethality associated with ectopic expression of these effectors in wild‐type Arabidopsis transgenic plants were largely alleviated in bak1 mutant plants. Thus, our results provide genetic evidence to show that BAK1 is a physiological target of AvrPto, AvrPtoB and HopF2. Identification of BAK1 as an additional target of HopF2 virulence not only explains HopF2 suppression of multiple MAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors act through multiple targets in host cells.     

Phosphorylation of receptor-like cytoplasmic kinases by bacterial Flagellin

Molecular mechanisms that distinguish self and non-self are fundamental in innate immunity to prevent infections in plants and animals. Recognition of the conserved microbial components triggers immune responses against a broad spectrum of potential pathogens. In Arabidopsis, bacterial flagellin was perceived by a leucine-rich repeat-receptor-like kinase (LRR-RLK) FLS2. Upon flagellin perception, FLS2 forms a complex with another LRR-RLK BAK1. The intracellular signaling events downstream of FLS2/BAK1 receptor complex are still poorly understood. We recently identified a receptor-like cytoplasmic kinase (RLCK) BIK1 that associates with flagellin receptor complex to initiate plant innate immunity. BIK1 is rapidly phosphorylated upon flagellin perception in an FLS2- and BAK1-dependent manner. BAK1 directly phosphorylates BIK1 with an in vitro kinase assay. Plants have evolved a large number of RLCK genes involved in a wide range of biological processes. We provided evidence here that additional RLCKs could also be phosphorylated by flagellin and may play redundant role with BIK1 in plant innate immunity.     

Immune responses induced by oligogalacturonides are differentially affected by AvrPto and loss of BAK1/BKK1 and PEPR1/PEPR2

Plants possess an innate immune system capable of restricting invasion by most potential pathogens. At the cell surface, the recognition of microbe‐associated molecular patterns (MAMPs) and/or damage‐associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) represents the first event for the prompt mounting of an effective immune response. Pathogens have evolved effectors that block MAMP‐triggered immunity. The Pseudomonas syringae effector AvrPto abolishes immunity triggered by the peptide MAMPs flg22 and elf18, derived from the bacterial flagellin and elongation factor Tu, respectively, by inhibiting the kinase function of the corresponding receptors FLS2 and EFR, as well as their co‐receptors BAK1 and BKK1. Oligogalacturonides (OGs), a well‐known class of DAMPs, are oligomers of α‐1,4‐linked galacturonosyl residues, released on partial degradation of the plant cell wall homogalacturonan. We show here that AvrPto affects only a subset of the OG‐triggered immune responses and that, among these responses, only a subset is affected by the concomitant loss of BAK1 and BKK1. However, the antagonistic effect on auxin‐related responses is not affected by either AvrPto or the loss of BAK1/BKK1. These observations reveal an unprecedented complexity among the MAMP/DAMP response cascades. We also show that the signalling system mediated by Peps, another class of DAMPs, and their receptors PEPRs, contributes to OG‐activated immunity. We hypothesize that OGs are sensed through multiple and partially redundant perception/transduction complexes, some targeted by AvrPto, but not necessarily comprising BAK1 and BKK1.     

Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+‐associated opening of plasma membrane anion channels

The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe‐associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN‐SENSING 2 (FLS2) and EF‐Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode‐impaled mesophyll cells and root hairs of Col‐0 Arabidopsis plants revealed rapid, dose‐dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion‐selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species‐independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2‐associated receptor‐like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1‐dependent, calcium‐associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway.     

Chimeric receptors of the Arabidopsis thaliana pattern recognition receptors EFR and FLS2

FLS2 and EFR are pattern recognition receptors in Arabidopsis thaliana perceiving the bacterial proteins flagellin and Elongation factor Tu (EF-Tu). Both receptors belong to the >200 membered protein family of Leucine-Rich Repeat Receptor Kinases (LRR-RKs) in Arabidopsis. FLS2 and EFR are engaged in the activation of a common intracellular signal output and they belong to the same subfamily of LRR-RKs, sharing structural features like the intracellular kinase domain and the ectodomain organized in LRRs. On the amino acid sequence level, however, they are only <50% identical even in their kinase domains. In our recently published paper¹ we demonstrated that it is possible to create chimeric receptors of EFR and FLS2 that are fully functional in ligand binding and receptor activation. Chimeric receptors consisting of the complete EFR ectodomain and the FLS2 kinase domain proved to be sensitive to elf18, the minimal peptide required for EF-Tu recognition, similar to the native EFR. In chimeric receptors where parts of the FLS2 ectodomain were swapped into the EFR LRR-domain, the receptor function was strongly affected even in cases with only small fragments exchanged. In this addendum we want to address problems and limits but also possibilities and chances of studying receptor functions using a chimeric approach.Key words: pattern recognition receptors, chimeric receptors, MAMP, flagellin perception, FLS2, EFRIn the Arabidopsis genome exist >600 genes that are predicted to encode for receptor-like kinases (RLKs).^2,3 More than 200 of them have ectodomains with LRRs. Physiological functions have been attributed only to a rather small percentage of them. Examples for known receptor-ligand pairs in A. thaliana include the well studied BRI1/Brassionlide,^4,5 AtPEPR1/Pep25,⁶ HAESA/IDA⁷ or CLV1/CLV3.⁸ While these LRR-RKs detect endogenous ligands, other members of this family function as immunoreceptors that detect ligands indicative of ‘non-self,’ such as pathogen associated molecular patterns (PAMPs). Examples of such LRR-RKs include FLS2 (Flagellin Sensing 2) and EFR (EF-Tu Receptor) from Arabidopsis and XA21 from rice.^9–11 The corresponding ligands have been identified as the flg22-epitope of bacterial flagellin for FLS2, the N-terminus of bacterial EF-Tu represented by the elf18 peptide for EFR, and the sulfated Avr21 peptide from Xanthomonas for XA21, respectively. LRR-ectodomains with related function in pathogen recognition occur also in so-called receptor-like proteins that lack the cytoplasmic kinase domains. Well studied examples include several Cf-receptor proteins which confer resistance against the fungus Cladosporium fulvum (Cf) in a gene-for-gene dependent manner. Thereby, different Cf-proteins function as recognition systems with specificity for factors determined by corresponding AvrCf products of the fungal pathogen.^12,13Receptor activation of the well studied receptor BRI1 by its ligand brassinolide involves interaction with a further receptor kinase, BAK1 (BRI1-associated receptor kinase 1).^5,14 Most interestingly, BAK1, or one of the four BAK1-related receptor kinases of the SERK protein family, also acts as a co-receptor for the ligand-dependent activation of FLS2, AtPEPR1 and EFR.^15–17 It seems that the co-receptor BAK1 plays an important role in activation of receptor kinases, serving different intracellular signaling pathways and output programs.¹⁸Up to now, little is known about the molecular details of ligand binding by the ectodomain in the apoplast and how this process leads to activation of the output signaling by the kinase moiety in the cytoplasm. The interaction with the co-receptor BAK1 suggests an activation process involving a ligand-induced intramolecular conformational change of the LRR-RK that then allows heterodimerization with the co-receptor BAK1. An initial task in elucidation of this activation process consists in defining the exact sites in the ectodomains of the receptors that interact with their corresponding ligands. So far, the clearest results for mapping ligand binding sites on LRR-receptor proteins were obtained with directed point mutations within the LRR domains as performed with the tomato receptor-like protein Cf-9,^19,20 and the Arabidopsis FLS2. There, a series of directed point mutations helped to map the LRRs 9–15 as a subdomain essential for interaction with the ligand flg22.²¹ Another interesting and promising approach consists in swaps of receptor sub-domains or exchanges of LRRs. In a remarkable, pioneering experiment this approach was used to produce chimeric receptors with the ectodomain of the brassinosteroid receptor BRI1 from Arabidopsis and the kinase domain of the immunoreceptor XA21 from rice.²² This chimera was reported to recognize the “developmental signal” brassinolide but to trigger characteristic cellular defense responses. In a recent publication²³ a domain swap between the ectodomain of the Wall Associated Kinase 1 (WAK1) and EFR was used to gain evidence for a function of the WAK1 ectodomain as a pectin receptor. Chimeric forms of the Cf receptor-like protein were used to identify subdomains carrying the specificity for the corresponding effectors from the C. fulvum pathogens.²⁴ However, as a limitation of this analysis, for none of these tomato resistance proteins a direct interaction with the corresponding effector proteins of the pathogen could be demonstrated so far.²⁵In our work, recently published in the Journal of Biochemistry,¹ we used the Arabidopsis thaliana receptors FLS2 and EFR to generate receptor chimeras. The main goal was to study the elf18 binding site in the EFR LRR-domain. In initial attempts we used EFR-constructs lacking some of the LRRs to narrow down the interaction site on the ectodomain. However, all of these truncated ectodomain versions lacking the transmembrane domain or more turned out to be unable in binding elf18 and triggering responses. In a second approach, we used the replacement of receptor parts with fragments from the structurally related receptor AtFLS2. These chimeras were tested for proper expression, localization, functionality in several plant defence related assays and affinity for the ligand elf18 in binding assays. The chimera with the complete EFR ectodomain swapped to the Kinase of FLS2 was fully functional as EF-Tu receptor. Since both receptors are known to trigger the same set of defense responses this might be not unexpected. Nevertheless, it is noteworthy that the two receptors show ∼45% sequence identity in their kinase domain, a degree of identity also shared with the kinase domains of receptors involved in other output programs, like BRI1. The 21 LRRs of EFR are sufficient for specifying full affinity for the elf18 as a ligand (

Plant systems for recognition of pathogen-associated molecular patterns

Research of the last decade has revealed that plant immunity consists of different layers of defense that have evolved by the co-evolutional battle of plants with its pathogens. Particular light has been shed on PAMP- (pathogen-associated molecular pattern) triggered immunity (PTI) mediated by pattern recognition receptors. Striking similarities exist between the plant and animal innate immune system that point for a common optimized mechanism that has evolved independently in both kingdoms. Pattern recognition receptors (PRRs) from both kingdoms consist of leucine-rich repeat receptor complexes that allow recognition of invading pathogens at the cell surface. In plants, PRRs like FLS2 and EFR are controlled by a co-receptor SERK3/BAK1, also a leucine-rich repeat receptor that dimerizes with the PRRs to support their function. Pathogens can inject effector proteins into the plant cells to suppress the immune responses initiated after perception of PAMPs by PRRs via inhibition or degradation of the receptors. Plants have acquired the ability to recognize the presence of some of these effector proteins which leads to a quick and hypersensitive response to arrest and terminate pathogen growth.     

A Two-Hybrid-Receptor Assay Demonstrates Heteromer Formation as Switch-On for Plant Immune Receptors

Receptor kinases sense extracellular signals and trigger intracellular signaling and physiological responses. However, how does signal binding to the extracellular domain activate the cytoplasmic kinase domain? Activation of the plant immunoreceptor Flagellin sensing2 (FLS2) by its bacterial ligand flagellin or the peptide-epitope flg22 coincides with rapid complex formation with a second receptor kinase termed brassinosteroid receptor1 associated kinase1 (BAK1). Here, we show that the receptor pair of FLS2 and BAK1 is also functional when the roles of the complex partners are reversed by swapping their cytosolic domains. This reciprocal constellation prevents interference by redundant partners that can partially substitute for BAK1 and demonstrates that formation of the heteromeric complex is the molecular switch for transmembrane signaling. A similar approach with swaps between the Elongation factor-Tu receptor and BAK1 also resulted in a functional receptor/coreceptor pair, suggesting that a “two-hybrid-receptor assay” is of more general use for studying heteromeric receptor complexes.Cell surface receptors are chemical sensors, often with an exquisite specificity and sensitivity, which detect extracellular signals and initiate corresponding intracellular response programs. Many of these receptors are transmembrane proteins with an extracellular ligand-binding domain and an intracellular protein kinase domain. Higher plants, such as Arabidopsis (Arabidopsis thaliana), have several hundred genes encoding receptor like kinases (Shiu and Bleecker, 2001; Shiu and Li, 2004). How are these receptor like kinases activated by their ligands, and how do they initiate a subsequent intracellular signaling cascade? In our work, we used the leucine-rich repeat receptor kinase (LRR-RK) Flagellin sensing2 (FLS2), which specifically detects bacterial flagellin or its peptide epitope flg22 at subnanomolar concentrations (Gomez-Gomez and Boller, 2000; Gómez-Gómez et al., 2001; Chinchilla et al., 2006). FLS2 undergoes heteromeric complex formation with brassinosteroid receptor1 associated kinase1 (BAK1) within seconds after application of the flagellin-derived peptide ligand flg22 (Chinchilla et al., 2007; Heese et al., 2007; Schulze et al., 2010). Thus, BAK1 might act as a coreceptor of FLS2. However, as previously observed (Chinchilla et al., 2007; Roux et al., 2011), FLS2 is still functional in the absence of BAK1, although with a reduced efficiency. This raises the question whether the ligand-induced heteromeric complex has merely an enhancing effect or whether association with BAK1 or a functional substitute acts as the essential switch-on for transmembrane signaling of FLS2. BAK1 is one of the five members that form the somatic embryogenesis receptor kinase (SERK) family (Albrecht et al., 2008), and other members of this family might partially substitute for BAK1 (Roux et al., 2011). However, a rigorous genetic approach to delineate the role of these potential substitutes is not feasible because triple mutants (serk1 serk3 serk4) and quadruple mutants (serk1 serk2 serk3 serk4) exhibit severe general phenotypes of dwarfing or even lethality at the early embryo stage (He et al., 2007; Gou et al., 2012) that might be due to the important role of SERKs in plant developmental processes (Li et al., 2002; Nam and Li, 2002).To address the role of the heteromeric complex with BAK1 in the absence of other interfering SERKs, we took a two-hybrid-receptor approach based on the premise that the apoplastic and cytoplasmic domains of FLS2 and BAK1 function in a modular manner. A heteromeric complex might thus also form and function when the roles of FLS2 and BAK1 are reversed by reciprocal swapping of their cytoplasmic protein kinase domains (Fig. 1A, schematic view).Open in a separate windowFigure 1.Heteromeric complex formation of FLS with BAK1 switches on flagellin-dependent transmembrane signaling. A, Model for the flg22-dependent heteromeric receptor complex. Schematic representation of FLS2 (blue), BAK1 (red), and the chimeric receptor constructs Ftm-B and Btm-F. Ftm-B comprises the extracellular and the transmembrane domains of FLS2 (blue) and the cytoplasmic domain of BAK1 (red). The receptor chimera Btm-F represents the reciprocal construct with the cytoplasmic part of BAK1 replaced by that of FLS2. The cytoplasmic domain of FLS2 was C-terminally tagged with a GFP. B and C, Functional comparison of native FLS2 and BAK1 (B) with the two hybrid receptors Ftm-B and Btm-F (C). The experiments show luciferase activity in Arabidopsis fls2 bak1-4 double mutant protoplasts cotransformed with pFRK1::Luciferase as a reporter and the receptor constructs indicated. At 0 h (dashed line) protoplasts were treated with 100 nm of flg22 (black diamonds) or 100 nm of the inactive analog flg22^Atum (white circles). Light emission of the protoplasts was measured with a luminometer. Values represent averages and sds of three replicates. Data shown are representative for at least three independent repetitions of the experiments with all constructs. D and E, Dose-response relationship for flg22-dependent induction of pFRK1::Luciferase in protoplasts expressing the receptor constructs indicated. Values represent increase in luciferase activity after 5 h of treatment as percentage of the increase observed with FLS2 plus BAK1 treated with saturating doses of greater than or equal to 10 nm flg22. Comparison of (half-maximal stimulation values in D and E shows that cells coexpressing Ftm-B plus Btm-F responded at least as sensitive to flg22 as cells coexpressing FLS2 plus BAK1. A combination of Ftm-B with the kinase dead version Btm-F_KD was not functional, even when treated with 1,000 nm flg22. LU, Light units.     

Brassinosteroid-independent functions of the BRI1-associated kinase BAK1/SERK3

Eukaryotes have evolved programmed cell death (PCD) mechanisms that play important roles in both, development and immunity.^1–3 We demonstrated a requirement for the Arabidopsis thaliana leucine-rich repeat receptor-like kinase (LRR-RLK), BAK1/SERK3 (BRI1-Associated receptor Kinase 1/Somatic Embryogenesis Receptor Kinase 3) in regulating the containment of microbial infection-induced necrosis. BAK1-deficient plants showed constitutive expression of defense-related genes and developed spreading cell death upon infection by necrotizing pathogens that result in enhanced susceptibility to necrotrophic pathogens. This reaction was not inducible by exposition of bak1 mutants to general stresses but appeared to be solely inducible by necrotizing pathogen infection. BAK1 is known to interact with the brassinosteroid receptor, BRI1, and thereby facilitates plant growth and development in a brassinolide (BL)-dependent manner.^4,5 Surprisingly, the cell death-related phenotype in bak1 mutants is brassinolide-independent. In this addendum we want to present recent new data on BAK1 and discuss its role as a general regulator in plant processes being as diverse as brassinosteroid signaling in development, perception of pathogen associated molecular patterns (PAMPs), and cell-death control in innate immunity.Key words: LRR-RLK, cell-death control, immunity, brassinosteroids, BAK1, SERK3, BRI1, FLS2     

Arabidopsis thaliana Pattern Recognition Receptors for Bacterial Elongation Factor Tu and Flagellin Can Be Combined to Form Functional Chimeric Receptors

The receptor kinase EFR of Arabidopsis thaliana detects the microbe-associated molecular pattern elf18, a peptide that represents the N terminus of bacterial elongation factor Tu. Here, we tested subdomains of EFR for their importance in receptor function. Transient expression of tagged versions of EFR and EFR lacking its cytoplasmic domain in leaves of Nicotiana benthamiana resulted in functional binding sites for elf18. No binding of ligand was found with the ectodomain lacking the transmembrane domain or with EFR lacking the first 5 of its 21 leucine-rich repeats (LRRs). EFR is structurally related to the receptor kinase flagellin-sensing 2 (FLS2) that detects bacterial flagellin. Chimeric receptors with subdomains of FLS2 substituting for corresponding parts of EFR were tested for functionality in ligand binding and receptor activation assays. Substituting the transmembrane domain and the cytoplasmic domain resulted in a fully functional receptor for elf18. Replacing also the outer juxtamembrane domain with that of FLS2 led to a receptor with full affinity for elf18 but with a lower efficiency in response activation. Extending the substitution to encompass also the last two of the LRRs abolished binding and receptor activation. Substitution of the N terminus by the first six LRRs from FLS2 reduced binding affinity and strongly affected receptor activation. In summary, chimeric receptors allow mapping of subdomains relevant for ligand binding and receptor activation. The results also show that modular assembly of chimeras from different receptors can be used to form functional receptors.     

Genetic evidence for an indispensable role of somatic embryogenesis receptor kinases in brassinosteroid signaling

The Arabidopsis thaliana somatic embryogenesis receptor kinases (SERKs) consist of five members, SERK1 to SERK5, of the leucine-rich repeat receptor-like kinase subfamily II (LRR-RLK II). SERK3 was named BRI1-Associated Receptor Kinase 1 (BAK1) due to its direct interaction with the brassinosteroid (BR) receptor BRI1 in vivo, while SERK4 has also been designated as BAK1-Like 1 (BKK1) for its functionally redundant role with BAK1. Here we provide genetic and biochemical evidence to demonstrate that SERKs are absolutely required for early steps in BR signaling. Overexpression of four of the five SERKs-SERK1, SERK2, SERK3/BAK1, and SERK4/BKK1-suppressed the phenotypes of an intermediate BRI1 mutant, bri1-5. Overexpression of the kinase-dead versions of these four genes in the bri1-5 background, on the other hand, resulted in typical dominant negative phenotypes, resembling those of null BRI1 mutants. We isolated and generated single, double, triple, and quadruple mutants and analyzed their phenotypes in detail. While the quadruple mutant is embryo-lethal, the serk1 bak1 bkk1 triple null mutant exhibits an extreme de-etiolated phenotype similar to a null bri1 mutant. While overexpression of BRI1 can drastically increase hypocotyl growth of wild-type plants, overexpression of BRI1 does not alter hypocotyl growth of the serk1 bak1 bkk1 triple mutant. Biochemical analysis indicated that the phosphorylation level of BRI1 in serk1 bak1 bkk1 is incapable of sensing exogenously applied BR. As a result, the unphosphorylated level of BES1 has lost its sensitivity to the BR treatment in the triple mutant, indicating that the BR signaling pathway has been completely abolished in the triple mutant. These data clearly demonstrate that SERKs are essential to the early events of BR signaling.     

Linking pattern recognition and salicylic acid responses in Arabidopsis through ACCELERATED CELL DEATH6 and receptors

The Arabidopsis membrane protein ACCELERATED CELL DEATH 6 (ACD6) and the defense signal salicylic acid (SA) are part of a positive feedback loop that regulates the levels of at least 2 pathogen-associated molecular patterns (PAMP) receptors, including FLAGELLIN SENSING 2 (FLS2) and CHITIN ELICITOR RECEPTOR (LYSM domain receptor-like kinase 1, CERK1). ACD6- and SA-mediated regulation of these receptors results in potentiation of responses to FLS2 and CERK1 ligands (e.g. flg22 and chitin, respectively). ACD6, FLS2 and CERK1 are also important for callose induction in response to an SA agonist even in the absence of PAMPs. Here, we report that another receptor, EF-Tu RECEPTOR (EFR) is also part of the ACD6/SA signaling network, similar to FLS2 and CERK1.