Nod Factor Receptor Research Articles

Heritability, signal perception and autoregulation of root nodulation in chickpea

Chickpea (Cicer arietinum L.) establishes symbiotic interactions with Mesorhizobium to develop root nodules where nitrogen fixation occurs. This symbiotic relationship can fix atmospheric nitrogen (N2) up to 140 kg N/ha that contribute nearly 80% nitrogen requirement of the crop. Global researchers had revealed the existence of natural variations in chickpea germplasm for nodulation traits with high heritability. Surprisingly, the contribution of environmental variation is too low for Biological Nitrogen Fixation (BNF) traits and high broad-sense heritability (>60) was observed for early nodulation, late nodule senescence and high nodule number traits. Correlation studies indicated a strong positive correlation between nodule number at flowering stage with total nodule weight and plant biomass and seed protein content. Nod Factor receptors in chickpea (CaNFR1 and CaNFR5) are characterized recently that forms phylogenetically distinct group along with M. truncatula, P. sativum, and L. japonicus. Critical role of cytokinin signalling through members of two component system (TCS) in nodulation was investigated in chickpea. The chickpea ortholog CaHK19 was the master spigot of cytokinin perception in chickpea. The co-expression pattern of CaHKs and CaNIN clearly indicated a link between cytokinin perception and downstream expression of CaNIN in chickpea as earlier established in Medicago. Genes involved in AON pathway are partially revealed in chickpea. CaRND1, CaRDN2, and CaRDN3 (C. arietinum Root-Determined Nodulation) function as receptors for signals produced from the roots. Revealing the molecular basis of root nodule organogenesis and their regulatory mechanisms along with identification of potential genetic stock will help on breeding or engineering chickpea genotypes with high symbiotic efficiency, extended nitrogen fixation and high symbiotic efficiency make grain legumes as nitrogen fixing factories to fertilize the soil in a sustainable way.

SymRK regulates G-protein signaling during nodulation in soybean (Glycine max) by modifying RGS phosphorylation and activity.

Molecular inter-species dialogue between leguminous plants and nitrogen-fixing rhizobia results in the development of symbiotic root nodules. This is initiated by several nodulation-related receptors present on the surface of root hair epidermal cells. We have shown previously that specific subunits of heterotrimeric G proteins and their regulatory RGS (regulator of G-protein signaling) proteins act as molecular links between the receptors and downstream components during nodule formation in soybeans. Nod factor receptor 1 (NFR1) interacts with and phosphorylates RGS proteins to regulate the G-protein cycle. Symbiosis receptor-like kinases (SymRK) phosphorylate Gα to make it inactive and unavailable for Gβγ. We now show that like NFR1, SymRK also interacts with the RGS proteins to phosphorylate them. Phosphorylated RGS has higher GTP accelerating activity, which favors conversion of active Gα to its inactive form. Phosphorylation of RGS proteins is physiologically relevant, as overexpression of a phospho-mimic version of RGS protein enhances nodule formation in soybean. These results reveal an intricate fine-tuning of the G-protein signaling during nodulation, where a negative regulator (Gα) is effectively deactivated by RGS due to the concerted efforts of several receptor proteins to ensure adequate nodulation.

RinRK1 enhances NF receptors accumulation in nanodomain-like structures at root-hair tip

Legume-rhizobia root-nodule symbioses involve the recognition of rhizobial Nod factor (NF) signals by NF receptors, triggering both nodule organogenesis and rhizobial infection. RinRK1 is induced by NF signaling and is essential for infection thread (IT) formation in Lotus japonicus. However, the precise mechanism underlying this process remains unknown. Here, we show that RinRK1 interacts with the extracellular domains of NF receptors (NFR1 and NFR5) to promote their accumulation at root hair tips in response to rhizobia or NFs. Furthermore, Flotillin 1 (Flot1), a nanodomain-organizing protein, associates with the kinase domains of NFR1, NFR5 and RinRK1. RinRK1 promotes the interactions between Flot1 and NF receptors and both RinRK1 and Flot1 are necessary for the accumulation of NF receptors at root hair tips upon NF stimulation. Our study shows that RinRK1 and Flot1 play a crucial role in NF receptor complex assembly within localized plasma membrane signaling centers to promote symbiotic infection.

The Bax inhibitor GmBI-1α interacts with a Nod factor receptor and plays a dual role in the legume-rhizobia symbiosis.

The gene networks surrounding Nod factor receptors that govern the symbiotic process between legumes and rhizobia remain largely unexplored. Here, we identify 13 novel GmNFR1α-associated proteins by yeast two-hybrid screening, and describe a potential interacting protein, GmBI-1α. GmBI-1α had the highest positive correlation with GmNFR1α in a co-expression network analysis, and its expression at the mRNA level in roots was enhanced by rhizobial infection. Moreover, GmBI-1α-GmNFR1α interaction was shown to occur in vitro and in vivo. The GmBI-1α protein was localized to multiple subcellular locations, including the endoplasmic reticulum and plasma membrane. Overexpression of GmBI-1α increased the nodule number in transgenic hairy roots or transgenic soybean, whereas down-regulation of GmBI-1α transcripts by RNA interference reduced the nodule number. In addition, the nodules in GmBI-1α-overexpressing plants became smaller in size and infected area with reduced nitrogenase activity. In GmBI-1α-overexpressing transgenic soybean, the elevated GmBI-1α also promoted plant growth and suppressed the expression of defense signaling-related genes. Infection thread analysis of GmBI-1α-overexpressing plants showed that GmBI-1α promoted rhizobial infection. Collectively, our findings support a GmNFR1α-associated protein in the Nod factor signaling pathway and shed new light on the regulatory mechanism of GmNFR1α in rhizobial symbiosis.

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

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.

A novel secreted protein, NISP1, is phosphorylated by soybean Nodulation Receptor Kinase to promote nodule symbiosis.

Nodulation Receptor Kinase (NORK) functions as a co-receptor of Nod factor receptors to mediate rhizobial symbiosis in legumes, but its direct phosphorylation substrates that positively mediate root nodulation remain to be fully identified. Here, we identified a GmNORK-Interacting Small Protein (GmNISP1) that functions as a phosphorylation target of GmNORK to promote soybean nodulation. GmNORKα directly interacted with and phosphorylated GmNISP1. Transcription of GmNISP1 was strongly induced after rhizobial infection in soybean roots and nodules. GmNISP1 encodes a peptide containing 90 amino acids with a "DY" consensus motif at its N-terminus. GmNISP1 protein was detected to be present in the apoplastic space. Phosphorylation of GmNISP1 by GmNORKα could enhance its secretion into the apoplast. Pretreatment with either purified GmNISP1 or phosphorylation-mimic GmNISP112D on the roots could significantly increase nodule numbers compared with the treatment with phosphorylation-inactive GmNISP112A . The data suggested a model that soybean GmNORK phosphorylates GmNISP1 to promote its secretion into the apoplast, which might function as a potential peptide hormone to promote root nodulation.

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.

Arbuscular mycorrhizal fungi induce lateral root development in angiosperms via a conserved set of MAMP receptors

Root systems regulate their branching patterns in response to environmental stimuli. Lateral root development in both monocotyledons and dicotyledons is enhanced in response to inoculation with arbuscular mycorrhizal (AM) fungi, which has been interpreted as a developmental response to specific, symbiosis-activating chitinaceous signals. Here, we report that generic instead of symbiosis-specific, chitin-derived molecules trigger lateral root formation. We demonstrate that this developmental response requires the well-known microbe-associated molecular pattern (MAMP) receptor, Chitin Elicitor Receptor Kinase 1 (CERK1), in rice, Medicago truncatula, and Lotus japonicus, as well as the non-host of AM fungi, Arabidopsis thaliana, lending further support for a broadly conserved signal transduction mechanism across angiosperms. Using rice mutants impaired in strigolactone biosynthesis and signaling, we show that strigolactone signaling is necessary to regulate this developmental response. Rice CERK1 operates together with either Chitin Elicitor Binding Protein (CEBiP) or Nod Factor Receptor 5 (NFR5) in immunity and symbiosis signaling, respectively; for the lateral root response, however, all three LysM receptors are required. Our work, therefore, reveals an overlooked but a conserved role of LysM receptors integrating MAMP perception with developmental responses in plants, an ability that might influence the interaction between roots and the rhizosphere biota.

NDR1/HIN1-Like Protein 13 Interacts with Symbiotic Receptor Kinases and Regulates Nodulation in Lotus japonicus.

Lysin-motif receptor-like kinases (LysM-RLKs) are involved in the recognition of microbe-associated molecular patterns to initiate pattern-triggered immunity (PTI). LysM-RLKs are also required for recognition of microbe-derived symbiotic signal molecules upon establishing mutualistic interactions between plants and microsymbionts. A LysM-RLK CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) plays central roles both in chitin-mediated PTI and in arbuscular mycorrhizal symbiosis, suggesting the overlap between immunity and symbiosis, at least in the signal perception and the activation of downstream signal cascades. In this study, we screened for the interacting proteins of Nod factor Receptor1 (NFR1), a CERK1 homolog in the model legume Lotus japonicus, and obtained a protein orthologous to NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 (NHL13), a protein involved in the activation of innate immunity in Arabidopsis thaliana, which we named LjNHL13a. LjNHL13a interacted with NFR1 and with the symbiosis receptor kinase SymRK. LjNHL13a also displayed positive effects in nodulation. Our results suggest that NHL13 plays a role both in plant immunity and symbiosis, possibly where they overlap. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

SymRK-dependent phosphorylation of Gα protein and its role in signaling during soybean (Glycine max) nodulation.

Heterotrimeric G proteins, comprised of Gα, Gβ and Gγ subunits, influence signaling in most eukaryotes. In metazoans, G proteins are activated by G protein-coupled receptor (GPCR)-mediated GDP to GTP exchange on Gα; however, the role(s) of GPCRs in regulating plant G-protein signaling remains equivocal. Mounting evidence suggests the involvement of receptor-like kinases (RLKs) in regulating plant G-protein signaling, but their mechanistic details remain scarce. We have previously shown that during Glycine max (soybean) nodulation, the nod factor receptor 1 (NFR1) interacts with G-protein components and indirectly affects signaling. We explored the direct regulation of G-protein signaling by RLKs using protein-protein interactions, receptor-mediated in vitro phosphorylations and the effects of such phosphorylations on soybean nodule formation. Results presented in this study demonstrate a direct, phosphorylation-based regulation of Gα by symbiosis receptor kinase (SymRK). SymRKs interact with and phosphorylate Gα at multiple residues in vitro, including two in its active site, which abolishes GTP binding. Additionally, phospho-mimetic Gα fails to interact with Gβγ, potentially allowing for constitutive signaling by the freed Gβγ. These results uncover an unusual mechanism of G-protein cycle regulation in plants where the receptor-mediated phosphorylation of Gα not only affects its activity but also influences the availability of its signaling partners, thereby exerting a two-pronged check on signaling.

Phosphorylation of MtRopGEF2 by LYK3 mediates MtROP activity to regulate rhizobial infection in Medicago truncatula.

The formation of nitrogen-fixing no dules on legume roots requires the coordination of infection by rhizobia at the root epidermis with the initiation of cell divisions in the root cortex. During infection, rhizobia attach to the tip of elongating root hairs which then curl to entrap the rhizobia. However, the mechanism of root hair deformation and curling in response to symbiotic signals is still elusive. Here, we found that small GTPases (MtRac1/MtROP9 and its homologs) are required for root hair development and rhizobial infection in Medicago truncatula. Our results show that the Nod factor receptor LYK3 phosphorylates the guanine nucleotide exchange factor MtRopGEF2 at S73 which is critical for the polar growth of root hairs. In turn, phosphorylated MtRopGEF2 can activate MtRac1. Activated MtRac1 was found to localize at the tips of root hairs and to strongly interact with LYK3 and NFP. Taken together, our results support the hypothesis that MtRac1, LYK3, and NFP form a polarly localized receptor complex that regulates root hair deformation during rhizobial infection.

Exploring nod factor receptors activation process in chickpea by bridging modelling, docking and molecular dynamics simulations

Plasma membrane-bound receptor proteins play crucial roles in the perception and further transmission of regulatory signals to modulate numerous developmental and metabolic events. Precise functioning and fine-tuning of Nod factor receptor (NFR) mediated signalling is a critical requirement for root nodule symbiosis. Here, we have identified, cloned and phylogenetically characterized chickpea NFR1 and NFR5, which are showing significant homology with other legume NFR receptors. Homology modelling and molecular dynamics simulations highlight the molecular structure of ligand binding ectodomains [EDs] and cytosolic kinase domains [KDs] of NFRs in chickpea. Our detailed structural analysis also revealed that both NFR1 and NFR5 share resemblance as well as dissimilarity in sequence, structure and substrate-binding pocket. Further, molecular docking simulations provide us adequate insights into the active site of receptors where the Nod factor (NF) binds. The outcome of this work sheds light on the binding specificity of NFs towards NFRs and thus may significantly contribute to the design of new strategies in improving root-nodule symbiosis towards meeting the agricultural demands.

Nod factor receptor complex phosphorylates GmGEF2 to stimulate ROP signaling during nodulation

The establishment of the symbiotic interaction between rhizobia and legumes involves the Nod factor signaling pathway. Nod factor recognition occurs through two plant receptors, NFR1 and NFR5. However, the signal transduction mechanisms downstream of NFR1-NFR5-mediated Nod factor perception remain largely unknown. Here, we report that a small guanosine triphosphatase (GTPase), GmROP9, and a guanine nucleotide exchange factor, GmGEF2, are involved in the soybean-rhizobium symbiosis. We show that GmNFR1α phosphorylates GmGEF2a at its N-terminal S86, which stimulates guanosine diphosphate (GDP)-to-GTP exchange to activate GmROP9 and that the active form of GmROP9 can associate with both GmNFR1α and GmNFR5α. We further show that a scaffold protein, GmRACK1, interacts with active GmROP9 and contributes to root nodule symbiosis. Collectively, our results highlight the symbiotic role of GmROP9-GmRACK1 and support the hypothesis that rhizobial signals promote the formation of a protein complex comprising GmNFR1, GmNFR5, GmROP9, and GmRACK1 for symbiotic signal transduction in soybean.

Distinct signaling routes mediate intercellular and intracellular rhizobial infection in Lotus japonicus.

Rhizobial infection of legume roots during the development of nitrogen-fixing root nodules can occur intracellularly, through plant-derived infection threads traversing cells, or intercellularly, via bacterial entry between epidermal plant cells. Although it is estimated that around 25% of all legume genera are intercellularly infected, the pathways and mechanisms supporting this process have remained virtually unexplored due to a lack of genetically amenable legumes that exhibit this form of infection. In this study, we report that the model legume Lotus japonicus is infected intercellularly by the IRBG74 strain, recently proposed to belong to the Agrobacterium clade of the Rhizobiaceae. We demonstrate that the resources available for L. japonicus enable insight into the genetic requirements and fine-tuning of the pathway governing intercellular infection in this species. Inoculation of L. japonicus mutants shows that Ethylene-responsive factor required for nodulation 1 (Ern1) and Leu-rich Repeat Receptor-Like Kinase (RinRK1) are dispensable for intercellular infection in contrast to intracellular infection. Other symbiotic genes, including nod factor receptor 5 (NFR5), symbiosis receptor-like kinase (SymRK), Ca2+/calmodulin dependent kinase (CCaMK), exopolysaccharide receptor 3 (Epr3), Cyclops, nodule inception (Nin), nodulation signaling pathway 1 (Nsp1), nodulation signaling pathway 2 (Nsp2), cystathionine-β-synthase (Cbs), and Vapyrin are equally important for both entry modes. Comparative RNAseq analysis of roots inoculated with IRBG74 revealed a distinctive transcriptome response compared with intracellular colonization. In particular, several cytokinin-related genes were differentially regulated. Corroborating this observation, cyp735A and ipt4 cytokinin biosynthesis mutants were significantly affected in their nodulation with IRBG74, whereas lhk1 cytokinin receptor mutants formed no nodules. These results indicate a differential requirement for cytokinin signaling during intercellular rhizobial entry and highlight distinct modalities of inter- and intracellular infection mechanisms in L. japonicus.

The application of CRISPR/Cas9 in hairy roots to explore the functions of AhNFR1 and AhNFR5 genes during peanut nodulation

BackgroundPeanut is an important legume crop growing worldwide. With the published allotetraploid genomes, further functional studies of the genes in peanut are very critical for crop improvement. CRISPR/Cas9 system is emerging as a robust tool for gene functional study and crop improvement, which haven’t been extensively utilized in peanut yet. Peanut plant forms root nodules to fix nitrogen through a symbiotic relationship with rhizobia. In model legumes, the response of plants to rhizobia is initiated by Nod factor receptors (NFRs). However, information about the function of NFRs in peanut is still limited. In this study, we applied the CRISPR/Cas9 tool in peanut hairy root transformation system to explore the function of NFR genes.ResultsWe firstly identified four AhNFR1 genes and two AhNFR5 genes in cultivated peanut (Tifrunner). The gene expression analysis showed that the two AhNFR1 and two AhNFR5 genes had high expression levels in nodulating (Nod+) line E5 compared with non-nodulating (Nod-) line E4 during the process of nodule formation, suggesting their roles in peanut nodulation. To further explore their functions in peanut nodulation, we applied CRISPR technology to create knock-out mutants of AhNFR1 and AhNFR5 genes using hairy root transformation system. The sequencing of these genes in transgenic hairy roots showed that the selected AhNFR1 and AhNFR5 genes were successfully edited by the CRISPR system, demonstrating its efficacy for targeted mutation in allotetraploid peanut. The mutants with editing in the two AhNFR5 genes showed Nod- phenotype, whereas mutants with editing in the two selected AhNFR1 genes could still form nodules after rhizobia inoculation.ConclusionsThis study showed that CRISPR-Cas9 could be used in peanut hairy root transformation system for peanut functional genomic studies, specifically on the gene function in roots. By using CRISPR-Cas9 targeting peanut AhNFR genes in hairy root transformation system, we validated the function of AhNFR5 genes in nodule formation in peanut.

Duplication of Symbiotic Lysin Motif Receptors Predates the Evolution of Nitrogen-Fixing Nodule Symbiosis.

Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2 These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.

Expression of the Legume-Specific Nod Factor Receptor Proteins Alters Developmental and Immune Responses in Rice

Legumes form symbiosis with rhizobia, which fix nitrogen for the benefit of host plant in return for carbon resources. Development of this unique symbiosis in legumes is triggered by rhizobia-secreted nodulation (Nod) factors (NFs). NFs, upon perception, activate Nod signaling cascade, leading to reprogramming of host cell (root) developmental networks to pave way for accommodating rhizobial symbionts. A long-cherished goal of legume-rhizobia symbiosis research is to extend this symbiotic nitrogen-fixing capacity to cereal plants such as rice. As a part of achieving this ultimate goal, in this work, initially we expressed legume-specific Nod factor receptor protein (NFRP) genes, MtNFP, MtLYK3, and LjLNP, in rice and assessed their impact on NF perception and consequently triggered biological responses in roots. RNA-seq analysis revealed that roots of both control and NFRP-expressing plants perceive NFs, but NFs elicited contrasting impacts on gene expression patterns in roots of these plants. In contrast to suppressive role of NFs on expression of several genes involved in innate immune response in roots of control plants, in NFRP-expressing plants, NFs triggered massive upregulation of a vast array of genes associated with signaling, defense response, and secondary metabolism networks in roots. Expression of NFRPs in rice also conferred root hairs the ability to respond to NFs in terms of exhibiting deformations, albeit at low levels. Together, results of the study demonstrated that rice plants have inherent ability to perceive NFs, but the expression of legume NFRPs rendered rice roots hypersensitive to NFs.

A method for functional testing constitutive and ligand-induced interactions of lysin motif receptor proteins

BackgroundPlant receptors with lysin motifs (LsyM) recognize microbial signals such as fungal chitin and lipo-chitooligosaccharidic Nod factors of nitrogen-fixing rhizobia. It is generally assumed that ligand-induced dimerization of LysM receptors is an essential step in activation of intracellular kinase domains and downstream signaling. Consequently, genes required for plant defense and establishment of symbiosis are expressed. We recently found that three LysM receptor proteins (namely LYK1, LYK4 and LYK5) of Arabidopsis thaliana form a tripartite receptor complex to perceive chitin. However, constitutive and ligand-induced interactions of LysM receptors generally remain difficult to be characterized.ResultsInteractions between ectodomains of LYK1, LYK4 and LYK5 were investigated by a chimeric receptor approach using hairy roots of the legume Lotus japonicus. Synthetic receptor pairs consisting of a LYK ectodomain and the intracellular domain of a L. japonicus Nod factor receptor (NFR1 and NFR5, respectively) were tested for their capacity to activate expression of the symbiotic NIN (nodule inception) gene. The results indicated constitutive (LYK4ED–LYK4ED, LYK4ED–LYK5ED) and chitin-induced interactions (LYK1ED–LYK1ED, LYK1ED–LYK5ED) of the examined ectodomains.ConclusionWe present a method to functionally analyze constitutive and ligand-induced interactions of LysM-type proteins.

The NMN Module Conducts Nodule Number Orchestra.

Legumes control nodule number through nodulation and autoregulation of nodulation (AON) pathways. Nodule Inception (NIN) is essential for rhizobial infection and nodule organogenesis in legumes. The GmNINa-miR172c-NNC1 (NMN) module, which consists of two positive regulators, GmNINa and miR172c, and a suppressor, NNC1, integrates both pathways. GmNINa activates miR172c to downregulate NNC1, leading to nodulation, while NNC1 inhibits miR172c expression, forming a negative feedback loop. GmNINa and NNC1 interact with each other and antagonistically fine-tune GmRIC1/RIC2 expression, turning AON on and off. Conversely, activation of AON inhibits GmNINa and miR172c expression, thereby reducing their inhibitory effects on NNC1 to attenuate both nodulation signaling and AON. The NMN module functions not only as an “accelerator” of the nodulation signal to promote nodulation but also as a “brake” on the signal by activating AON to orchestrate nodule number.

A LysM Receptor Heteromer Mediates Perception of Arbuscular Mycorrhizal Symbiotic Signal in Rice

Symbiotic microorganisms improve nutrient uptake by plants. To initiate mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, plants perceive Myc factors, including lipochitooligosaccharides (LCOs) and short-chain chitooligosaccharides (CO4/CO5), secreted by AM fungi. However, the molecular mechanism of Myc factor perception remains elusive. In this study, we identified a heteromer of LysM receptor-like kinases consisting of OsMYR1/OsLYK2 and OsCERK1 that mediates the perception of AM fungi in rice. CO4 directly binds to OsMYR1, promoting the dimerization and phosphorylation of this receptor complex. Compared with control plants, Osmyr1 and Oscerk1 mutant rice plants are less sensitive to Myc factors and show decreased AM colonization. We engineered transgenic rice by expressing chimeric receptors that respectively replaced the ectodomains of OsMYR1 and OsCERK1 with those from the homologous Nod factor receptors MtNFP and MtLYK3 of Medicago truncatula. Transgenic plants displayed increased calcium oscillations in response to Nod factors compared with control rice. Our study provides significant mechanistic insights into AM symbiotic signal perception in rice. Expression of chimeric Nod/Myc receptors achieves a potentially important step toward generating cereals that host nitrogen-fixing bacteria.