LysM Domain Research Articles

Characterization of an Intrinsic Disorder Domain and Functional Activity of ChiZ Membrane Protein from Mycobacterium tuberculosis

ChiZ is a membrane protein from Mycobacterium tuberculosis (MTB) that is involved in cell division. This protein has an intracellular domain that is known to be intrinsically disordered. Interestingly, this cytoplasmic domain is able to hydrolyze peptidoglican (PG) in in vitro assays. ChiZ also contains a C-terminal LysM domain for PG binding in the periplasm. ChiZ is an interesting target for structural characterization and drug development because it is involved in cell division.This work mainly focuses on the nascent structural characterization of the intrinsically disorder region of ChiZ (ChiZ1-64) using solution NMR in addition to biochemical assays for understanding its function. Biochemical assays indicate that ChiZ1-64 is able to hydrolyze PG at pH 4.0 and 7.0. As expected, solution NMR experiments of ChiZ1-64 show a typical spectrum of an unfolded protein. However, HSQC spectra at these two pHs indicate the presence of two distinct regions with different backbone dynamics and signal intensity. Sequence alignment of ChiZ homologs found in other species of Mycobacterium also shows two different regions, one of variable length (residues 1 to 37 in MTB), and another conserved region (residues 38 to 64 in MTB). Both conserved and variable regions match to the regions identified with solution NMR experiments. Removal of the variable region in ChiZ produces an active protein (ChiZ38-165) at low pH. This suggests that the conserved region is enough to hydrolyze PG. However, the variable region appears to be important for full activity at neutral pH. Additional binding assays of ChiZ1-64 show that this region is able to bind PG. This is demonstrated by the depletion of the protein from solution and the detection of protein using solid state NMR of PG and ChiZ1-64 in complex.

Negative Cooperativity and High Affinity in Chitooligosaccharide Binding by a Mycobacterium smegmatis Protein Containing LysM and Lectin Domains.

LysM domains have been recognized in bacteria and eukaryotes as carbohydrate-binding protein modules, but the mechanism of their binding to chitooligosaccharides has been underexplored. Binding of a Mycobacterium smegmatis protein containing a lectin (MSL) and one LysM domain to chitooligosaccharides has been studied using isothermal titration calorimetry and fluorescence titration that demonstrate the presence of two binding sites of nonidentical affinities per dimeric MSL-LysM molecule. The affinity of the molecule for chitooligosaccharides correlates with the length of the carbohydrate chain. Its binding to chitooligosaccharides is characterized by negative cooperativity in the interactions of the two domains. Apparently, the flexibility of the long linker that connects the LysM and MSL domains plays a facilitating role in this recognition. The LysM domain in the MSL-LysM molecule, like other bacterial domains but unlike plant LysM domains, recognizes equally well peptidoglycan fragments as well as chitin polymers. Interestingly, in the case presented here, two LysM domains are enough for binding to peptidoglycan in contrast to the three reportedly required by the LysM domains of Bacillus subtilis and Lactococcus lactis. Also, the affinity of the MSL-LysM molecule for chitooligosaccharides is higher than that of LysM-chitooligosaccharide interactions reported so far.

The LysM receptor-like kinase SlLYK10 regulates the arbuscular mycorrhizal symbiosis in tomato.

Most plants have the ability to establish a symbiosis with arbuscular mycorrhizal (AM) fungi, which allows better plant nutrition. A plant signaling pathway, called the common symbiosis signaling pathway (CSSP), is essential for the establishment of both AM and root nodule symbioses. The CSSP is activated by microbial signals. Plant receptor(s) for AM fungal signals required for the activation of the CSSP and initial fungal penetration are currently unknown. We set up conditions to use virus-induced gene silencing (VIGS) in Solanum lycopersicum to study the genes potentially involved in AM. We show that the lysin motif receptor-like kinase SlLYK10, whose orthologs in legumes are essential for nodulation, but not for AM, and SlCCaMK, a component of the CSSP, are required for penetration of the AM fungus Rhizophagus irregularis into the roots of young tomato plants. Our results support the hypothesis that the SILYK10 ancestral gene originally played a role in AM and underwent duplication and neofunctionalization for a role in nodulation in legumes. Moreover, we conclude that VIGS is an efficient method for fast screening of genes playing major roles in AM.

Structural Insight into Fungal Cell Wall Recognition by a CVNH Protein with a Single LysM Domain

MGG_03307 is a lectin isolated from Magnaporte oryzae, a fungus that causes devastating rice blast disease. Its function is associated with protecting M.oryzae from the host immune response in plants. To provide the structural basis of how MGG_03307 protects the fungus, crystal structures of its CVNH-LysM module were determined in the absence and presence of GlcNAc-containing cell wall chitin constituents, which can act as pathogen-associated molecular patterns. Our structures revealed that glycan binding is accompanied by a notable conformational change in the LysM domain and that GlcNAc3 and GlcNAc4 are accommodated similarly. GlcNAc5 and GlcNAc6 interact with the LysM domain in multiple conformations, as evidenced by solution nuclear magnetic resonance studies. No dimerization of MoCVNH3 via its LysM domain was observed upon binding to GlcNAc6, unlike in multiple LysM domain-containing proteins. Importantly, we define a specific consensus binding mode for the recognition of GlcNAc oligomers by single LysM domains.

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.

Inter-organismal signaling and management of the phytomicrobiome.

The organisms of the phytomicrobiome use signal compounds to regulate aspects of each other’s behavior. Legumes use signals (flavonoids) to regulate rhizobial nod gene expression during establishment of the legume-rhizobia N2-fixation symbiosis. Lipochitooligosaccharides (LCOs) produced by rhizobia act as return signals to the host plant and are recognized by specific lysine motif receptor like kinases, which triggers a signal cascade leading to nodulation of legume roots. LCOs also enhance plant growth, particularly when plants are stressed. Chitooligosaccharides activate plant immune responses, providing enhanced resistance against diseases. Co-inoculation of rhizobia with other plant growth promoting rhizobacteria (PGPR) can improve nodulation and crop growth. PGPR also alleviate plant stress by secreting signal compounds including phytohormones and antibiotics. Thuricin 17, a small bacteriocin produced by a phytomicrobiome member promotes plant growth. Lumichrome synthesized by soil rhizobacteria function as stress-sensing cues. Inter-organismal signaling can be used to manage/engineer the phytomicrobiome to enhance crop productivity, particularly in the face of stress. Stressful conditions are likely to become more frequent and more severe because of climate change.

The role of FlhF and HubP as polar landmark proteins in Shewanella putrefaciens CN-32.

Spatiotemporal regulation of cell polarity plays a role in many fundamental processes in bacteria and often relies on 'landmark' proteins which recruit the corresponding clients to their designated position. Here, we explored the localization of two multi-protein complexes, the polar flagellar motor and the chemotaxis array, in Shewanella putrefaciens CN-32. We demonstrate that polar positioning of the flagellar system, but not of the chemotaxis system, depends on the GTPase FlhF. In contrast, the chemotaxis array is recruited by a transmembrane protein which we identified as the functional ortholog of Vibrio cholerae HubP. Mediated by its periplasmic N-terminal LysM domain, SpHubP exhibits an FlhF-independent localization pattern during cell cycle similar to its Vibrio counterpart and also has a role in proper chromosome segregation. In addition, while not affecting flagellar positioning, SpHubP is crucial for normal flagellar function and is involved in type IV pili-mediated twitching motility. We hypothesize that a group of HubP/FimV homologs, characterized by a rather conserved N-terminal periplasmic section required for polar targeting and a highly variable acidic cytoplasmic part, primarily mediating recruitment of client proteins, serves as polar markers in various bacterial species with respect to different cellular functions.

Structural and functional analysis of SleL, a peptidoglycan lysin involved in germination of Bacillus spores.

A major event in the germination of Bacillus spores concerns hydrolysis of the cortical peptidoglycan that surrounds the spore protoplast, the integrity of which is essential for maintenance of dormancy. Cortex degradation is initiated in all species of Bacillus spores by the combined activity of two semi-redundant cortex-lytic enzymes, SleB and CwlJ. A third enzyme, SleL, which has N-acetylglucosaminidase activity, cleaves peptidoglycan fragments generated by SleB and CwlJ. Here we present crystal structures of B. cereus and B. megaterium SleL at 1.6 angstroms and 1.7 angstroms, respectively. The structures were determined with a view to identifying the structural basis of differences in catalytic efficiency between the respective enzymes. The catalytic (α/β)8 -barrel cores of both enzymes are highly conserved from a structural perspective, including the spatial distribution of the catalytic residues. Both enzymes are equipped with two N-terminal peptidoglycan-binding LysM domains, which are also structurally highly conserved. However, the topological arrangement of the respective enzymes second LysM domain is markedly different, and this may account for differences in catalytic rates by impacting upon the position of the active sites with respect to their substrates. A chimeric enzyme comprising the B. megaterium SleL catalytic domain plus B. cereus SleL LysM domains displayed enzymatic activity comparable to the native B. cereus protein, exemplifying the importance of the LysM domains to SleL function. Similarly, the reciprocal construct, comprising the B. cereus SleL catalytic domain with B. megaterium SleL LysM domains, showed reduced activity compared with native B. cereus SleL.

Purification, cDNA cloning, and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense).

Chitinase-A (EaChiA), molecular mass 36 kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.

Deep Sequencing of the Medicago truncatula Root Transcriptome Reveals a Massive and Early Interaction between Nodulation Factor and Ethylene Signals.

The legume-rhizobium symbiosis is initiated through the activation of the Nodulation (Nod) factor-signaling cascade, leading to a rapid reprogramming of host cell developmental pathways. In this work, we combine transcriptome sequencing with molecular genetics and network analysis to quantify and categorize the transcriptional changes occurring in roots of Medicago truncatula from minutes to days after inoculation with Sinorhizobium medicae. To identify the nature of the inductive and regulatory cues, we employed mutants with absent or decreased Nod factor sensitivities (i.e. Nodulation factor perception and Lysine motif domain-containing receptor-like kinase3, respectively) and an ethylene (ET)-insensitive, Nod factor-hypersensitive mutant (sickle). This unique data set encompasses nine time points, allowing observation of the symbiotic regulation of diverse biological processes with high temporal resolution. Among the many outputs of the study is the early Nod factor-induced, ET-regulated expression of ET signaling and biosynthesis genes. Coupled with the observation of massive transcriptional derepression in the ET-insensitive background, these results suggest that Nod factor signaling activates ET production to attenuate its own signal. Promoter:β-glucuronidase fusions report ET biosynthesis both in root hairs responding to rhizobium as well as in meristematic tissue during nodule organogenesis and growth, indicating that ET signaling functions at multiple developmental stages during symbiosis. In addition, we identified thousands of novel candidate genes undergoing Nod factor-dependent, ET-regulated expression. We leveraged the power of this large data set to model Nod factor- and ET-regulated signaling networks using MERLIN, a regulatory network inference algorithm. These analyses predict key nodes regulating the biological process impacted by Nod factor perception. We have made these results available to the research community through a searchable online resource.

Candidate gene prediction via quantitative trait locus analysis of fruit shape index traits in apple

Fruit shape is a critical appearance quality in apple. Quantitative trait loci (QTLs) for apple fruit shape index (FSI) traits were previously mapped by our laboratory to linkage group 11 of the maternal parent in a cross population of ‘Jonathan’ × ‘Golden Delicious’ using simple sequence repeat markers. In this study, QTLs for fruit length, diameter, and FSI were identified again using a high-density single nucleotide polymorphism (SNP) genetic linkage map, and candidate genes associated with FSI were screened via whole-genome re-sequencing data for ‘Jonathan’ and ‘Golden Delicious’. Fifteen QTLs, including four for fruit length, one for fruit diameter, and ten for FSI, were identified in three sampling years. Two overlapping year-stable QTL regions related to FSI were anchored on LG 11 of ‘Jonathan’. One candidate gene (MDP0000135244) related to FSI in apple and encoding an LysM domain receptor-like kinase protein was predicted and verified in the segregated population. The nonsynonymous SNP (C11.6053728) of MDP0000135244 was present in 23 of 30 individuals with high FSI, demonstrating a close relationship between MDP0000135244 and FSI trait. These results will be useful for the application of marker-assisted selection for FSI trait in apple.

Phenotypic and genotypic characterization of peptidoglycan hydrolases of Lactobacillus sakei

Lactobacillus sakei, a lactic acid bacterium naturally found in fresh meat and sea products, is considered to be one of the most important bacterial species involved in meat fermentation and bio-preservation. Several enzymes of Lb. sakei species contributing to microbial safeguarding and organoleptic properties of fermented-meat were studied. However, the specific autolytic mechanisms and associated enzymes involved in Lb. sakei are not well understood. The autolytic phenotype of 22 Lb. sakei strains isolated from Tunisian meat and seafood products was evaluated under starvation conditions, at pH 6.5 and 8.5, and in the presence of different carbon sources. A higher autolytic rate was observed when cells were grown in the presence of glucose and incubated at pH 6.5. Almost all strains showed high resistance to mutanolysin, indicating a minor role of muramidases in Lb. sakei cell lysis. Using Micrococcus lysodeikticus cells as a substrate in activity gels zymogram, peptidoglycan hydrolase (PGH) patterns for all strains was characterized by two lytic bands of ∼80 (B1) and ∼70kDa (B2), except for strain BMG.167 which harbored two activity signals at a lower MW. Lytic activity was retained in high salt and in acid/basic conditions and was active toward cells of Lb. sakei, Listeria monocytogenes, Listeria ivanovii and Listeria innocua. Analysis of five putative PGH genes found in the Lb. sakei 23K model strain genome, indicated that one gene, lsa1437, could encode a PGH (N-acetylmuramoyl-L-alanine amidase) containing B1 and B2 as isoforms. According to this hypothesis, strain BMG.167 showed an allelic version of lsa1437 gene deleted of one of the five LysM domains, leading to a reduction in the MW of lytic bands and the high autolytic rate of this strain. Characterization of autolytic phenotype of Lb. sakei should expand the knowledge of their role in fermentation processes where they represent the dominant species.

Heterologous surface display on lactic acid bacteria: non-GMO alternative?

Lactic acid bacteria (LAB) are food-grade hosts for surface display with potential applications in food and therapy. Alternative approaches to surface display on LAB would avoid the use of recombinant DNA technology and genetically-modified organism (GMO)-related regulatory requirements. Non-covalent surface display of proteins can be achieved by fusing them to various cell-wall binding domains, of which the Lysine motif domain (LysM) is particularly well studied. Fusion proteins have been isolated from recombinant bacteria or from their growth medium and displayed on unmodified bacteria, enabling heterologous surface display. This was demonstrated on non-viable cells devoid of protein content, termed bacteria-like particles, and on various species of genus Lactobacillus. Of the latter, Lactobacillus salivarius ATCC 11741 was recently shown to be particularly amenable for LysM-mediated display. Possible regulatory implications of heterologous surface display are discussed, particularly those relevant for the European Union.

An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase.

LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

Improvement of LysM-mediated surface display of designed ankyrin repeat proteins (DARPins) in recombinant and nonrecombinant strains of Lactococcus lactis and Lactobacillus Species.

Safety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of Lactococcus lactis by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus Lactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control. Lactobacillus salivarius ATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides of Lb. salivarius as a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display on L. lactis was demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selecting Lactobacillus salivarius ATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display on L. lactis. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities.

Lipo-chitin oligosaccharides, plant symbiosis signalling molecules that modulate mammalian angiogenesis in vitro.

Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathways. In animals, oligo-chitins also induce innate immunity and other physiological responses but LCO recognition has not been demonstrated. Here LCO and LCO-like compounds are shown to be biologically active in mammals in a structure dependent way through the modulation of angiogenesis, a tightly-regulated process involving the induction and growth of new blood vessels from existing vessels. The testing of 24 LCO, LCO-like or oligo-chitin compounds resulted in structure-dependent effects on angiogenesis in vitro leading to promotion, or inhibition or nil effects. Like plants, the mammalian LCO biological activity depended upon the presence and type of terminal substitutions. Un-substituted oligo-chitins of similar chain lengths were unable to modulate angiogenesis indicating that mammalian cells, like plant cells, can distinguish between LCOs and un-substituted oligo-chitins. The cellular mode-of-action of the biologically active LCOs in mammals was determined. The stimulation or inhibition of endothelial cell adhesion to vitronectin or fibronectin correlated with their pro- or anti-angiogenic activity. Importantly, novel and more easily synthesised LCO-like disaccharide molecules were also biologically active and de-acetylated chitobiose was shown to be the primary structural basis of recognition. Given this, simpler chitin disaccharides derivatives based on the structure of biologically active LCOs were synthesised and purified and these showed biological activity in mammalian cells. Since important chronic disease states are linked to either insufficient or excessive angiogenesis, LCO and LCO-like molecules may have the potential to be a new, carbohydrate-based class of therapeutics for modulating angiogenesis.

The Intimin periplasmic domain mediates dimerisation and binding to peptidoglycan.

Intimin and Invasin are prototypical inverse (Type Ve) autotransporters and important virulence factors of enteropathogenic Escherichia coli and Yersinia spp. respectively. In addition to a C-terminal extracellular domain and a β-barrel transmembrane domain, both proteins also contain a short N-terminal periplasmic domain that, in Intimin, includes a lysin motif (LysM), which is thought to mediate binding to peptidoglycan. We show that the periplasmic domain of Intimin does bind to peptidoglycan both in vitro and in vivo, but only under acidic conditions. We were able to determine a dissociation constant of 0.8 μM for this interaction, whereas the Invasin periplasmic domain, which lacks a LysM, bound only weakly in vitro and failed to bind peptidoglycan in vivo. We present the solution structure of the Intimin LysM, which has an additional α-helix conserved within inverse autotransporter LysMs but lacking in others. In contrast to previous reports, we demonstrate that the periplasmic domain of Intimin mediates dimerisation. We further show that dimerisation and peptidoglycan binding are general features of LysM-containing inverse autotransporters. Peptidoglycan binding by the periplasmic domain in the infection process may aid in resisting mechanical and chemical stress during transit through the gastrointestinal tract.

Knowing your friends and foes--plant receptor-like kinases as initiators of symbiosis or defence.

The decision between defence and symbiosis signalling in plants involves alternative and modular plasma membrane-localized receptor complexes. A critical step in their activation is ligand-induced homo- or hetero-oligomerization of leucine-rich repeat (LRR)- and/or lysin motif (LysM) receptor-like kinases (RLKs). In defence signalling, receptor complexes form upon binding of pathogen-associated molecular patterns (PAMPs), including the bacterial flagellin-derived peptide flg22, or chitin. Similar mechanisms are likely to operate during the perception of microbial symbiont-derived (lipo)-chitooligosaccharides. The structurally related chitin-oligomer ligands chitooctaose and chitotetraose trigger defence and symbiosis signalling, respectively, and their discrimination involves closely related, if not identical, LysM-RLKs. This illustrates the demand for and the challenges imposed on decision mechanisms that ensure appropriate signal initiation. Appropriate signalling critically depends on abundance and localization of RLKs at the cell surface. This is regulated by internalization, which also provides a mechanism for the removal of activated signalling RLKs. Abundance of the malectin-like domain (MLD)-LRR-RLK Symbiosis Receptor-like Kinase (SYMRK) is additionally controlled by cleavage of its modular ectodomain, which generates a truncated and rapidly degraded RLK fragment. This review explores LRR- and LysM-mediated signalling, the involvement of MLD-LRR-RLKs in symbiosis and defence, and the role of endocytosis in RLK function.

Nod factor receptors form heteromeric complexes and are essential for intracellular infection in medicago nodules.

Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. This restricted accumulation is narrower than the region of promoter activity/mRNA accumulation and might serve to prevent the induction of defense-like responses and/or to restrict the rhizobium release to precise cell layers. The distal cell layer where the receptors accumulate at the cell periphery is part of the meristem, and the proximal layer is part of the infection zone. In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. Furthermore, our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules.

The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1.

Chitin is a fungal microbe-associated molecular pattern recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1. Previous research suggested that AtCERK1 is the major chitin receptor and mediates chitin-induced signaling through homodimerization and phosphorylation. However, the reported chitin binding affinity of AtCERK1 is quite low, suggesting another receptor with high chitin binding affinity might be present. Here, we propose that AtLYK5 is the primary chitin receptor in Arabidopsis. Mutations in AtLYK5 resulted in a significant reduction in chitin response. However, AtLYK5 shares overlapping function with AtLYK4 and, therefore, Atlyk4/Atlyk5-2 double mutants show a complete loss of chitin response. AtLYK5 interacts with AtCERK1 in a chitin-dependent manner. Chitin binding to AtLYK5 is indispensable for chitin-induced AtCERK1 phosphorylation. AtLYK5 binds chitin at a much higher affinity than AtCERK1. The data suggest that AtLYK5 is the primary receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant immunity.