A New Classification of Lysin Motif Receptor-Like Kinases in Lotus japonicus.

Lysin motif receptor-like kinases (LysM-RLKs) are a plant-specific receptor protein family that sense components from soil microorganisms, regulating innate immunity and symbiosis. Every plant species possesses multiple LysM-RLKs in order to interact with a variety of soil microorganisms; however, most receptors have not been characterized yet. Therefore, we tried to identify LysM-RLKs from diverse plant species and proposed a new classification to indicate their evolution and characteristics, as well as to predict new functions. In this study, we have attempted to explore and update LysM-RLKs in Lotus japonicus using the latest genome sequencing and divided 20 LysM-RLKs into 11 clades based on homolog identity and phylogenetic analysis. We further identified 193 LysM-RLKs from 16 Spermatophyta species including L. japonicus and divided these receptors into 14 clades and one out-group special receptor based on the classification of L. japonicus LysM-RLKs. All plant species not only have clade I receptors such as Nod factor or chitin receptors but also have clade III receptors where most of the receptors are uncharacterized. We also identified dicotyledon- and monocotyledon-specific clades and predicted evolutionary trends in LysM-RLKs. In addition, we found a strong correlation between plant species that did not possess clade II receptors and those that lost symbiosis with arbuscular mycorrhizal fungi. A clade II receptor in L. japonicus Lys8 was predicted to express during arbuscular mycorrhizal symbiosis. Our proposed new inventory classification suggests the evolutionary pattern of LysM-RLKs and might help in elucidating novel receptor functions in various plant species.

Role of Nod factor receptors and its allies involved in nitrogen fixation.

MAIN CONCLUSION: Lysin motif (LysM)-receptor-like kinase (RLK) and leucine-rich repeat (LRR)-RLK mediated signaling play important roles in the development and regulation of root nodule symbiosis in legumes. The availability of water and nutrients in the soil is a major limiting factor affecting crop productivity. Plants of the Leguminosae family form a symbiotic association with nitrogen-fixing Gram-negative soil bacteria, rhizobia for nitrogen fixation. This symbiotic relationship between legumes and rhizobia depends on the signal exchange between them. Plant receptor-like kinases (RLKs) containing lysin motif (LysM) and/or leucine-rich repeat (LRR) play an important role in the perception of chemical signals from rhizobia for initiation and establishment of root nodule symbiosis (RNS) that results in nitrogen fixation. This review highlights the diverse aspects of LysM-RLK and LRR receptors including their specificity, functions, interacting partners, regulation, and associated signaling in RNS. The activation of LysM-RLKs and LRR-RLKs is important for ensuring the successful interaction between legume roots and rhizobia. The intracellular regions of the receptors enable additional layers of signaling that help in the transduction of signals intracellularly. Additionally, symbiosis receptor-like kinase (SYMRK) containing the LRR motif acts as a co-receptor with Nod factors receptors (LysM-RLK). Cleavage of the malectin-like domain from the SYMRK ectodomain is a mechanism for controlling SYMRK stability. Overall, this review has discussed different aspects of legume receptors that are critical to the perception of signals from rhizobia and their subsequent role in creating the mutualistic relationship necessary for nitrogen fixation. Additionally, it has been discussed how crucial it is to extrapolate the knowledge gained from model legumes to crop legumes such as chickpea and common bean to better understand the mechanism underlying nodule formation in crop legumes. Future directions have also been proposed in this regard.

Genome-Wide Identification of the Soybean LysM-RLK Family Genes and Its Nitrogen Response.

Lysin-Motif receptor-like kinase (LysM-RLK) proteins are widely distributed in plants and serve a critical role in defending against pathogens and establishing symbiotic relationships. However, there is a lack of comprehensive identification and analysis of LysM-RLK family members in the soybean genome. In this study, we discovered and named 27 LysM-RLK genes in soybean. The majority of LysM-RLKs were highly conserved in Arabidopsis and soybean, while certain members of subclades III, VI, and VII are unique to soybean. The promoters of these LysM-RLKs contain specific cis-elements associated with plant development and responses to environmental factors. Notably, all LysM-RLK gene promoters feature nodule specificity elements, while 51.86% of them also possess NBS sites (NIN/NLP binding site). The expression profiles revealed that genes from subclade V in soybean roots were regulated by both rhizobia and nitrogen treatment. The expression levels of subclade V genes were then validated by real-time quantitative PCR, and it was observed that the level of GmLYK4a and GmLYK4c in roots was inhibited by rhizobia but induced via varying concentrations of nitrate. Consequently, our findings provide a comprehensive understanding of the soybean LysM-RLK gene family and emphasize the role of subclade V in coupling soybean symbiotic nitrogen fixation and nitrogen response.

Genome-wide in silico identification of LysM-RLK genes in potato (Solanum tuberosum L.).

The receptor like kinases (RLKs) belong to the RLK/Pelle superfamily, one of the largest gene families in plants. RLKs play an important role in plant development, as well as in response to biotic and abiotic stresses. The lysine motif receptor like kinases (LysM-RLKs) are a subfamily of RLKs containing at least one lysine motif (LysM) that are involved in the perception of elicitors or pathogen-associated molecular patterns (PAMPs). In the present study, 77 putative RLKs genes and three receptor like proteins were identified in potato (Solanum tuberosum) genome, following a genome-wide search. The 77 potato RLK proteins are classified into two major phylogenetic groups based on their kinase domain amino acid sequence similarities. Out of 77 RLKs, 10 proteins had at least one LysM. Among them three RLP proteins were found in potato genome with either 2 or three tandem LysM but these lacked a cytoplasmic kinase domain. Expression analyses of a potato LysM-RLKs (StLysM-RLK05) was carried out by a Real time RT-PCR, following inoculation of potato leaves and immature tubers with late blight and common scab pathogens, respectively. The expression was significantly higher in resistant than in susceptible following S. scabies inoculation. The StLysM-RLK05 sequence was verified and it was polymorphic in scab susceptible cultivar. The present study provides an overview of the StLysM-RLKs gene family in potato genome. This information is helpful for future functional analysis of such an important protein family, in Solanaceae species.

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.

A novel Arabidopsis CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) mutant with enhanced pathogen-induced cell death and altered receptor processing.

Plants detect pathogens by sensing microbe-associated molecular patterns (MAMPs) through pattern recognition receptors. Pattern recognition receptor complexes also have roles in cell death control, but the underlying mechanisms are poorly understood. Here, we report isolation of cerk1-4, a novel mutant allele of the Arabidopsis chitin receptor CERK1 with enhanced defense responses. We identified cerk1-4 in a forward genetic screen with barley powdery mildew and consequently characterized it by pathogen assays, mutant crosses and analysis of defense pathways. CERK1 and CERK1-4 proteins were analyzed biochemically. The cerk1-4 mutation causes an amino acid exchange in the CERK1 ectodomain. Mutant plants maintain chitin signaling capacity but exhibit hyper-inducible salicylic acid concentrations and deregulated cell death upon pathogen challenge. In contrast to chitin signaling, the cerk1-4 phenotype does not require kinase activity and is conferred by the N-terminal part of the receptor. CERK1 undergoes ectodomain shedding, a well-known process in animal cell surface proteins. Wild-type plants contain the full-length CERK1 receptor protein as well as a soluble form of the CERK1 ectodomain, whereas cerk1-4 plants lack the N-terminal shedding product. Our work suggests that CERK1 may have a chitin-independent role in cell death control and is the first report of ectodomain shedding in plants.

New sources of Sym2A allele in the pea (Pisum sativum L.) carry the unique variant of candidate LysM-RLK gene LykX.

At the onset of legume-rhizobial symbiosis, the mutual recognition of partners occurs based on a complicated interaction between signal molecules and receptors. Bacterial signal molecules named Nod factors ("nodulation factors") are perceived by the plant LysM-containing receptor-like kinases (LysM-RLKs) that recognize details of its structure (i.e., unique substitutions), thus providing the conditions particular to symbiosis. In the garden pea (Pisum sativum L.), the allelic state of Sym2 gene has long been reported to regulate the symbiotic specificity: for infection to be successful, plants with the Sym2 A allele (for "Sym2 Afghan", as these genotypes originate mostly from Afghanistan) require an additional acetylation of the Nod factor which is irrelevant for genotypes with the Sym2 E allele (for "Sym2 European"). Despite being described about 90 years ago, Sym2 has not yet been cloned, though phenotypic analysis suggests it probably encodes a receptor for the Nod factor. Recently, we described a novel pea gene LykX (PsLykX) from the LysM-RLK gene family that demonstrates a perfect correlation between its allelic state and the symbiotic specificity of the Sym2 A-type. Here we report on a series of Middle-Eastern pea genotypes exhibiting the phenotype of narrow symbiotic specificity discovered in the VIR plant genetic resources gene bank (Saint-Petersburg, Russia). These genotypes are new sources of Sym2 A, as has been confirmed by an allelism test with Sym2 A pea cv. Afghanistan. Within these genotypes, LykX is present either in the allelic state characteristic for cv. Afghanistan, or in another, minor allelic state found in two genotypes from Tajikistan and Turkmenistan. Plants carrying the second allele demonstrate the same block of rhizobial infection as cv. Afghanistan when inoculated with an incompatible strain. Intriguingly, this "Tajik" allele of LykX differs from the "European" one by a single nucleotide polymorphism leading to an R75P change in the receptor part of the putative protein. Thus, our new data are in agreement with the hypothesis concerning the identity of LykX and the elusive Sym2 gene.

Nucleoporin NUP88/MOS7 is required for manifestation of phenotypes associated with the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 mutant cerk1-4.

Arabidopsis nucleoporin MOS7/NUP88 was identified in a forward-genetic screen for components that contribute to auto-immunity of the deregulated Resistance (R) gene mutant snc1, and is required for immunity to biotrophic and hemi-biotrophic pathogens. In a recent study, we showed that MOS7 is also essential to mount a full defense response against the necrotrophic fungal pathogen Botrytis cinerea, suggesting that MOS7 modulates plant defense responses to different types of pathogenic microbes. Here, we extend our analyses of MOS7-dependent plant immune responses and report the genetic requirement of MOS7 for manifestation of phenotypes associated with the CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) mutant cerk1-4.

LYR3, a high-affinity LCO-binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor.

LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Förster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves.

Cell autonomous and non-cell autonomous control of rhizobial and mycorrhizal infection in Medicago truncatula.

Legumes can form a nitrogen fixing symbiosis with soil bacteria called rhizobia (the RL symbiosis). They can also, like most plants, form symbiotic associations with arbuscular mycorrhizal (AM) fungi, which facilitate plants' phosphate nutrition. In both interactions, the symbionts are hosted inside the plant root. Nitrogen-fixing rhizobia are housed in intracellular symbiotic structures within nodules, while AM fungi form intracellular symbiotic structures, called arbuscules, within cortical root cells. These two endosymbioses present other similarities, including production by the microsymbionts of lipo-chitooligosaccharidic signals (Nod Factors and Myc-LCOs), and the involvement of common plant signaling elements. In Medicago truncatula, DMI3 encodes a calcium and calmodulin dependent protein kinase that is part of this common signaling pathway, while NFP encodes a LysM domain receptor-like kinase involved in Nod Factor perception. Using tissue specific promoters, we recently uncoupled the roles of NFP and DMI3 in the cortex and the epidermis of the root during the RL symbiosis. (1) Here, we provide additional data showing a cell autonomous tissular contribution of DMI3 in the AM symbiosis, and we comment on a non-cell autonomous cortical role of NFP during rhizobial infection.

Chitin signaling and plant disease resistance.

Chitin, a polymer of N-acetyl-D-glucosamine, is a component of the fungal cell wall and is not found in plants. Plant cells are equipped with chitin degrading enzymes to digest fungal cell walls and are capable of perceiving chitin fragments (chitooligosaccharides) released from fungal cell walls during fungal infection. Chitin recognition results in the activation of defense signaling pathways. Although chitin is a well recognized pathogen-associated molecular pattern (PAMP), little is known about the molecular mechanism of chitin signaling. Recent studies identified a number of critical components in the chitin-elicited signaling pathway including a potential receptor, MAPK cascade and transcription factor network. Interestingly, the chitin signaling pathway overlaps with the phytobacterial flagellin-and EF-Tu-elicited signaling pathways, suggesting that plant cells may perceive different PAMPs from various pathogens via specialized receptors and then utilize a conserved, common downstream pathway to mediate disease resistance. Given the fact that fungal pathogens are major problems in many agricultural systems, research on chitin signaling could have significance to sustainable agriculture and biofuel and biomaterial production.