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.     

Receptor quality control in the endoplasmic reticulum for plant innate immunity

Pattern recognition receptors in eukaryotes initiate defence responses on detection of microbe‐associated molecular patterns shared by many microbe species. The Leu‐rich repeat receptor‐like kinases FLS2 and EFR recognize the bacterial epitopes flg22 and elf18, derived from flagellin and elongation factor‐Tu, respectively. We describe Arabidopsis ‘priority in sweet life’ (psl) mutants that show de‐repressed anthocyanin accumulation in the presence of elf18. EFR accumulation and signalling, but not of FLS2, are impaired in psl1, psl2, and stt3a plants. PSL1 and PSL2, respectively, encode calreticulin3 (CRT3) and UDP‐glucose:glycoprotein glycosyltransferase that act in concert with STT3A‐containing oligosaccharyltransferase complex in an N‐glycosylation pathway in the endoplasmic reticulum. However, EFR‐signalling function is impaired in weak psl1 alleles despite its normal accumulation, thereby uncoupling EFR abundance control from quality control. Furthermore, salicylic acid‐induced, but EFR‐independent defence is weakened in psl2 and stt3a plants, indicating the existence of another client protein than EFR for this immune response. Our findings suggest a critical and selective function of N‐glycosylation for different layers of plant immunity, likely through quality control of membrane‐localized regulators.     

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.     

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.     

Pattern Recognition Receptors Require N-Glycosylation to Mediate Plant Immunity

N-Glycans attached to the ectodomains of plasma membrane pattern recognition receptors constitute likely initial contact sites between plant cells and invading pathogens. To assess the role of N-glycans in receptor-mediated immune responses, we investigated the functionality of Arabidopsis receptor kinases EFR and FLS2, sensing bacterial translation elongation factor Tu (elf18) and flagellin (flg22), respectively, in N-glycosylation mutants. As revealed by binding and responses to elf18 or flg22, both receptors tolerated immature N-glycans induced by mutations in various Golgi modification steps. EFR was specifically impaired by loss-of-function mutations in STT3A, a subunit of the endoplasmic reticulum resident oligosaccharyltransferase complex. FLS2 tolerated mild underglycosylation occurring in stt3a but was sensitive to severe underglycosylation induced by tunicamycin treatment. EFR accumulation was significantly reduced when synthesized without N-glycans but to lesser extent when underglycosylated in stt3a or mutated in single amino acid positions. Interestingly, EFR^N143Q lacking a single conserved N-glycosylation site from the EFR ectodomain accumulated to reduced levels and lost the ability to bind its ligand and to mediate elf18-elicited oxidative burst. However, EFR-YFP protein localization and peptide:N-glycosidase F digestion assays support that both EFR produced in stt3a and EFR^N143Q in wild type cells correctly targeted to the plasma membrane via the Golgi apparatus. These results indicate that a single N-glycan plays a critical role for receptor abundance and ligand recognition during plant-pathogen interactions at the cell surface.     

FLS2-BAK1 Extracellular Domain Interaction Sites Required for Defense Signaling Activation

Signaling initiation by receptor-like kinases (RLKs) at the plasma membrane of plant cells often requires regulatory leucine-rich repeat (LRR) RLK proteins such as SERK or BIR proteins. The present work examined how the microbe-associated molecular pattern (MAMP) receptor FLS2 builds signaling complexes with BAK1 (SERK3). We first, using in vivo methods that validate separate findings by others, demonstrated that flg22 (flagellin epitope) ligand-initiated FLS2-BAK1 extracellular domain interactions can proceed independent of intracellular domain interactions. We then explored a candidate SERK protein interaction site in the extracellular domains (ectodomains; ECDs) of the significantly different receptors FLS2, EFR (MAMP receptors), PEPR1 (damage-associated molecular pattern (DAMP) receptor), and BRI1 (hormone receptor). Repeat conservation mapping revealed a cluster of conserved solvent-exposed residues near the C-terminus of models of the folded LRR domains. However, site-directed mutagenesis of this conserved site in FLS2 did not impair FLS2-BAK1 ECD interactions, and mutations in the analogous site of EFR caused receptor maturation defects. Hence this conserved LRR C-terminal region apparently has functions other than mediating interactions with BAK1. In vivo tests of the subsequently published FLS2-flg22-BAK1 ECD co-crystal structure were then performed to functionally evaluate some of the unexpected configurations predicted by that crystal structure. In support of the crystal structure data, FLS2-BAK1 ECD interactions were no longer detected in in vivo co-immunoprecipitation experiments after site-directed mutagenesis of the FLS2 BAK1-interaction residues S554, Q530, Q627 or N674. In contrast, in vivo FLS2-mediated signaling persisted and was only minimally reduced, suggesting residual FLS2-BAK1 interaction and the limited sensitivity of co-immunoprecipitation data relative to in vivo assays for signaling outputs. However, Arabidopsis plants expressing FLS2 with the Q530A+Q627A double mutation were impaired both in detectable interaction with BAK1 and in FLS2-mediated responses, lending overall support to current models of FLS2 structure and function.     

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.     

Ligand-dependent reduction in the membrane mobility of FLAGELLIN SENSITIVE2, an arabidopsis receptor-like kinase

Arabidopsis Flagellin sensitive2 (FLS2) is a transmembrane leucine-rich repeat receptor-like kinase, which recognizes a conserved 22 amino acid peptide (flg22) of bacterial flagellin and activates downstream defense signaling pathways resulting in enhanced resistance against plant pathogens. The underlying mechanisms for the activation of FLS2 in the cell membrane, however, are not fully understood. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that approximately 75% of the FLS2 in the plasma membrane diffuses laterally with a diffusion coefficient of 0.34 microm(2) s(-1), indicating that it moves rapidly. Further, we show that FLS2 is less mobile in the presence of flg22, suggesting its ligand-dependent confinement to microdomains or transient interaction with other less mobile membrane proteins. Using an in vivo bimolecular fluorescence complementation (BiFC) system and fluorescence resonance energy transfer (FRET), which reveals in vivo protein-protein interactions, we show that FLS2 does not homodimerize either constitutively or in the presence of flg22. Our data suggest that the reduced mobility of FLS2 after binding flg22 and its existence in monomeric form are important mechanistic features of FLS2 early signaling.     

PAMP-triggered immunity: Early events in the activation of FLAGELLIN SENSITIVE2

The Arabidopsis FLAGELLIN SENSITIVE2 (FLS2) protein is a leucine-rich repeat receptor-like kinase (LRR-RLK) that plays important roles in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). The binding of bacterial flagellin, one of the PAMPs, to the extracellular domain of FLS2 leads to activation of signaling cascades resulting in activation or repression of a specific set of genes involved in plant defense. The mechanisms at the cell membrane that lead to the activation of this signalling pathway are, however, not fully understood. Recently, we have shown that after ligand-treatment the mobility of FLS2 in the cell membrane is reduced and that the activation of FLS2 does not involve its constitutive or ligand-dependent homodimerization. Our data together with recently published reports suggest that FLS2 activation involves its association with other proteins, including BRI1-associated kinase 1 (BAK1), another LRR-RLK, and localization to less mobile areas, probably lipid rafts, in a ligand-dependent manner to initiate PTI.Key words: PTI, BiFC, flg22, FLS2, FRAP, FRET, membrane protein, RLK     

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.     

Repression of sucrose/ultraviolet B light-induced flavonoid accumulation in microbe-associated molecular pattern-triggered immunity in Arabidopsis

Recognition of microbe-associated molecular patterns (MAMPs) leads to the generation of MAMP-triggered immunity (MTI), which restricts the invasion and propagation of potentially infectious microbes. It has been described that the perception of different bacterial and fungal MAMPs causes the repression of flavonoid induction upon light stress or sucrose application. However, the functional significance of this MTI-associated signaling output remains unknown. In Arabidopsis (Arabidopsis thaliana), FLAGELLIN-SENSING2 (FLS2) and EF-TU RECEPTOR act as the pattern recognition receptors for the bacterial MAMP epitopes flg22 (of flagellin) and elf18 (of elongation factor [EF]-Tu), respectively. Here, we reveal that reactive oxygen species spiking and callose deposition are dispensable for the repression of flavonoid accumulation by both pattern recognition receptors. Importantly, FLS2-triggered activation of PATHOGENESIS-RELATED (PR) genes and bacterial basal defenses are enhanced in transparent testa4 plants that are devoid of flavonoids, providing evidence for a functional contribution of flavonoid repression to MTI. Moreover, we identify nine small molecules, of which eight are structurally unrelated, that derepress flavonoid accumulation in the presence of flg22. These compounds allowed us to dissect the FLS2 pathway. Remarkably, one of the identified compounds uncouples flavonoid repression and PR gene activation from the activation of reactive oxygen species, mitogen-activated protein kinases, and callose deposition, corroborating a close link between the former two outputs. Together, our data imply a model in which MAMP-induced repression of flavonoid accumulation serves a role in removing the inherent inhibitory action of flavonoids on an MTI signaling branch.     

Interplay between calcium signalling and early signalling elements during defence responses to microbe- or damage-associated molecular patterns

While diverse microbe- or damage-associated molecular patterns (MAMPs/DAMPs) typically trigger a common set of intracellular signalling events, comparative analysis between the MAMPs flg22 and elf18 revealed MAMP-specific differences in Ca(2+) signalling, defence gene expression and MAMP-mediated growth arrest in Arabidopsis thaliana. Such MAMP-specific differences are, in part, controlled by BAK1, a kinase associated with several receptors. Whereas defence gene expression and growth inhibition mediated by flg22 were reduced in bak1 mutants, BAK1 had no or minor effects on the same responses elicited by elf18. As the residual Ca(2+) elevations induced by diverse MAMPs/DAMPs (flg22, elf18 and Pep1) were virtually identical in bak1 mutants, a differential BAK1-mediated signal amplification to attain MAMP/DAMP-specific Ca(2+) amplitudes in wild-type plants may be hypothesized. Furthermore, abrogation of reactive oxygen species (ROS) accumulation, either in the rbohD mutant or through inhibitor application, led to loss of a second Ca(2+) peak, demonstrating a feedback effect of ROS on Ca(2+) signalling. Conversely, mpk3 mutants showed a prolonged accumulation of ROS but this did not significantly impinge on the overall Ca(2+) response. Thus, fine-tuning of MAMP/DAMP responses involves interplay between diverse signalling elements functioning both up- or downstream of Ca(2+) signalling.     

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 (

Microbe-associated molecular pattern (MAMP) signatures, synergy, size and charge: influences on perception or mobility and host defence responses

Triggering of defences by microbes has mainly been investigated using single elicitors or microbe-associated molecular patterns (MAMPs), but MAMPs are released in planta as complex mixtures together with endogenous oligogalacturonan (OGA) elicitor. We investigated the early responses in Arabidopsis of calcium influx and oxidative burst induced by non-saturating concentrations of bacterial MAMPs, used singly and in combination: flagellin peptide (flg22), elongation factor peptide (elf18), peptidoglycan (PGN) and component muropeptides, lipo-oligosaccharide (LOS) and core oligosaccharides. This revealed that some MAMPs have additive (e.g. flg22 with elf18) and even synergistic (flg22 and LOS) effects, whereas others mutually interfere (flg22 with OGA). OGA suppression of flg22-induced defences was not a result of the interference with the binding of flg22 to its receptor flagellin-sensitive 2 (FLS2). MAMPs induce different calcium influx signatures, but these are concentration dependent and unlikely to explain the differential induction of defence genes [pathogenesis-related gene 1 ( PR1 ), plant defensin gene 1.2 ( PDF1.2 ) and phenylalanine ammonia lyase gene 1 ( PAL1 )] by flg22, elf18 and OGA. The peptide MAMPs are potent elicitors at subnanomolar levels, whereas PGN and LOS at high concentrations induce low and late host responses. This difference might be a result of the restricted access by plant cell walls of MAMPs to their putative cellular receptors. flg22 is restricted by ionic effects, yet rapidly permeates a cell wall matrix, whereas LOS, which forms supramolecular aggregates, is severely constrained, presumably by molecular sieving. Thus, MAMPs can interact with each other, whether directly or indirectly, and with the host wall matrix. These phenomena, which have not been considered in detail previously, are likely to influence the speed, magnitude, versatility and composition of plant defences.     

Control of the pattern‐recognition receptor EFR by an ER protein complex in plant immunity

In plant innate immunity, the surface‐exposed leucine‐rich repeat receptor kinases EFR and FLS2 mediate recognition of the bacterial pathogen‐associated molecular patterns EF‐Tu and flagellin, respectively. We identified the Arabidopsis stromal‐derived factor‐2 (SDF2) as being required for EFR function, and to a lesser extent FLS2 function. SDF2 resides in an endoplasmic reticulum (ER) protein complex with the Hsp40 ERdj3B and the Hsp70 BiP, which are components of the ER‐quality control (ER‐QC). Loss of SDF2 results in ER retention and degradation of EFR. The differential requirement for ER‐QC components by EFR and FLS2 could be linked to N‐glycosylation mediated by STT3a, a catalytic subunit of the oligosaccharyltransferase complex involved in co‐translational N‐glycosylation. Our results show that the plasma membrane EFR requires the ER complex SDF2–ERdj3B–BiP for its proper accumulation, and provide a demonstration of a physiological requirement for ER‐QC in transmembrane receptor function in plants. They also provide an unexpected differential requirement for ER‐QC and N‐glycosylation components by two closely related receptors.     

Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense Responses

Plant plasma membrane localized pattern recognition receptors (PRRs) detect extracellular pathogen-associated molecules. PRRs such as Arabidopsis EFR and rice XA21 are taxonomically restricted and are absent from most plant genomes. Here we show that rice plants expressing EFR or the chimeric receptor EFR::XA21, containing the EFR ectodomain and the XA21 intracellular domain, sense both Escherichia coli- and Xanthomonas oryzae pv. oryzae (Xoo)-derived elf18 peptides at sub-nanomolar concentrations. Treatment of EFR and EFR::XA21 rice leaf tissue with elf18 leads to MAP kinase activation, reactive oxygen production and defense gene expression. Although expression of EFR does not lead to robust enhanced resistance to fully virulent Xoo isolates, it does lead to quantitatively enhanced resistance to weakly virulent Xoo isolates. EFR interacts with OsSERK2 and the XA21 binding protein 24 (XB24), two key components of the rice XA21-mediated immune response. Rice-EFR plants silenced for OsSERK2, or overexpressing rice XB24 are compromised in elf18-induced reactive oxygen production and defense gene expression indicating that these proteins are also important for EFR-mediated signaling in transgenic rice. Taken together, our results demonstrate the potential feasibility of enhancing disease resistance in rice and possibly other monocotyledonous crop species by expression of dicotyledonous PRRs. Our results also suggest that Arabidopsis EFR utilizes at least a subset of the known endogenous rice XA21 signaling components.     

A pair of G-type lectin receptor-like kinases modulates nlp20-mediated immune responses by coupling to the RLP23 receptor complex

Plant cells recognize microbial patterns with the plasma-membrane-localized pattern-recognition receptors consisting mainly of receptor kinases (RKs) and receptor-like proteins (RLPs). RKs, such as bacterial flagellin receptor FLS2, and their downstream signaling components have been studied extensively. However, newly discovered regulatory components of RLP-mediated immune signaling, such as the nlp20 receptor RLP23, await identification. Unlike RKs, RLPs lack a cytoplasmic kinase domain, instead recruiting the receptor-like kinases (RLKs) BAK1 and SOBIR1. SOBIR1 specifically works as an adapter for RLP-mediated immunity. To identify new regulators of RLP-mediated signaling, we looked for SOBIR1-binding proteins (SBPs) in Arabidopsis thaliana using protein immunoprecipitation and mass spectrometry, identifying two G-type lectin RLKs, SBP1 and SBP2, that physically interacted with SOBIR1. SBP1 and SBP2 showed high sequence similarity, were tandemly repeated on chromosome 4, and also interacted with both RLP23 and BAK1. sbp1 sbp2 double mutants obtained via CRISPR-Cas9 gene editing showed severely impaired nlp20-induced reactive oxygen species burst, mitogen-activated protein kinase (MAPK) activation, and defense gene expression, but normal flg22-induced immune responses. We showed that SBP1 regulated nlp20-induced immunity in a kinase activity-independent manner. Furthermore, the nlp20-induced the RLP23–BAK1 interaction, although not the flg22-induced FLS2–BAK1 interaction, was significantly reduced in sbp1 sbp2. This study identified SBPs as new regulatory components in RLP23 receptor complex that may specifically modulate RLP23-mediated immunity by positively regulating the interaction between the RLP23 receptor and the BAK1 co-receptor.     

Probing the Arabidopsis flagellin receptor: FLS2-FLS2 association and the contributions of specific domains to signaling function

Flagellin sensing2 (FLS2) is a transmembrane receptor kinase that activates antimicrobial defense responses upon binding of bacterial flagellin or the flagellin-derived peptide flg22. We find that some Arabidopsis thaliana FLS2 is present in FLS2-FLS2 complexes before and after plant exposure to flg22. flg22 binding capability is not required for FLS2-FLS2 association. Cys pairs flank the extracellular leucine rich repeat (LRR) domain in FLS2 and many other LRR receptors, and we find that the Cys pair N-terminal to the FLS2 LRR is required for normal processing, stability, and function, possibly due to undescribed endoplasmic reticulum quality control mechanisms. By contrast, disruption of the membrane-proximal Cys pair does not block FLS2 function, instead increasing responsiveness to flg22, as indicated by a stronger oxidative burst. There was no evidence for intermolecular FLS2-FLS2 disulfide bridges. Truncated FLS2 containing only the intracellular domain associates with full-length FLS2 and exerts a dominant-negative effect on wild-type FLS2 function that is dependent on expression level but independent of the protein kinase capacity of the truncated protein. FLS2 is insensitive to disruption of multiple N-glycosylation sites, in contrast with the related receptor EF-Tu receptor that can be rendered nonfunctional by disruption of single glycosylation sites. These and additional findings more precisely define the molecular mechanisms of FLS2 receptor function.     

BAK1‐mediated phosphorylation of canonical G protein alpha during flagellin signaling in Arabidopsis

Heterotrimeric G proteins consisting of Gα, Gβ and Gγ are conserved signaling hubs in eukaryotes. Without analogs to canonical animal G protein‐coupled receptors, plant cells are thought to use RGS1 and a yet unknown mechanism to regulate the activity of Gα. Meanwhile, the exact role of canonical Gα in plant innate immunity remains controversial. Here, we report multiple immune deficiencies in the null allele of Arabidopsis Gα (GPA1) in response to bacterial flg22 elicitor, clarifying a positive regulatory role of GPA1 in flg22 signaling. We also detect overall increased phosphorylation of GPA1 but reduced phosphorylation at Thr¹⁹ upon flg22 elicitation. Interestingly, flg22 could not induce phosphorylation of GPA1^T19A and GPA1^T19D, suggesting that the dynamic Thr¹⁹ phosphorylation is required for GPA1 to respond to flg22. Moreover, flg22‐induced GPA1 phosphorylation is largely abolished in the absence of BAK1 in vivo, and BAK1 could phosphorylate GPA1 but not GPA1^T19A in vitro at the phosphorylation sites identified in vivo, suggesting BAK1 is likely the kinase for GPA1 phosphorylation in response to flg22. Furthermore, the T19A mutation could promote flg22‐induced association, rather than dissociation, between GPA1 and RGS1. Taken together, our findings shed new insights into the function and regulation of GPA1 in Arabidopsis defense signaling.