Nod Factors Research Articles

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.

Sinorhizobium meliloti succinylated high-molecular-weight succinoglycan and the Medicago truncatula LysM receptor-like kinase MtLYK10 participate independently in symbiotic infection.

SummaryThe formation of nitrogen‐fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen‐fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin‐motif receptor‐like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild‐type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild‐type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan‐defective strains was achieved by in trans rescue with a Nod factor‐deficient S. meliloti mutant. While the Nod factor‐deficient strain was always more abundant inside nodules, the succinoglycan‐deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.

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.

Sinorhizobium fredii HH103 syrM inactivation affects the expression of a large number of genes, impairs nodulation with soybean and extends the host-range to Lotus japonicus.

Sinorhizobium fredii HH103 RifR is a broad host-range rhizobial strain able to nodulate with soybean and Lotus burttii, but it is ineffective with L. japonicus. Here, we study the role of the HH103 RifR SyrM protein in the regulation of gene expression and its relevance in symbiosis with those three legumes. RNAseq analyses show that HH103 SyrM is an important transcriptional regulator not only in the presence of inducer flavonoids but also in its absence. Lack of SyrM increases Nod factors production and decreases genistein-mediated repression of exopolysaccharide production in HH103. In symbiosis, mutation of syrM partially impaired interaction with soybean but improves effectiveness with L. burttii and extends the host-rage to L. japonicus Gifu. In addition, HH103 syrM mutants enter in both Lotus species by infection threads, whereas HH103 uses the more primitive intercellular infection to enter into L. burttii roots These symbiotic phenotypes were previously observed in two other HH103 mutants affected in symbiotic regulators, nodD2 and nolR, revealing that in S. fredii HH103 numerous transcriptional regulators finely modulate symbiotic gene expression.

Affinity Labeling and Purification of Plant Chitin-Binding LysM Receptor with Chitin Octasaccharide Derivatives.

Lysin motif (LysM) is a carbohydrate-binding modules found in all kingdoms. LysM binds to N-acetylglucosamine-containing molecules such as peptidoglycan, chitin, Nod factor, and Myc factor and is found in peptidoglycan hydrolases, chitinases, and plant pathogen effectors and plant receptor/co-receptor for defense and symbiosis signaling. This chapter describes the synthesis of a nonradioactive chitin ligand, biotinylated chitin octasaccharide, (GlcNAc)8-Bio, and its application for the detection and characterization of chitin-binding LysM receptor CEBiP in the microsomal membrane fraction of rice suspension-cultured cells by affinity labeling. We also describe the purification of CEBiP from the plasma membrane of the rice cells by affinity chromatography with the synthesized (GlcNAc)8-APEA-CH-Sepharose as an affinity matrix.

Nod factors improve the nitrogen content and rhizobial diversity of faba bean and alter soil dehydrogenase, protease, and acid phosphomonoesterase activities

Nod factors improve the nitrogen content and rhizobial diversity of faba bean and alter soil dehydrogenase, protease, and acid phosphomonoesterase activities

An Exploration of Common Greenhouse Gas Emissions by the Cyanobiont of the Azolla-Nostoc Symbiosis and Clues as to Nod Factors in Cyanobacteria.

Azolla is a genus of aquatic ferns that engages in a unique symbiosis with a cyanobiont that is resistant to cultivation. Azolla spp. are earmarked as a possible candidate to mitigate greenhouse gases, in particular, carbon dioxide. That opinion is underlined here in this paper to show the broader impact of Azolla spp. on greenhouse gas mitigation by revealing the enzyme catalogue in the Nostoc cyanobiont to be a poor contributor to climate change. First, regarding carbon assimilation, it was inferred that the carboxylation activity of the Rubisco enzyme of Azolla plants is able to quench carbon dioxide on par with other C3 plants and fellow aquatic free-floating macrophytes, with the cyanobiont contributing on average ~18% of the carboxylation load. Additionally, the author demonstrates here, using bioinformatics and past literature, that the Nostoc cyanobiont of Azolla does not contain nitric oxide reductase, a key enzyme that emanates nitrous oxide. In fact, all Nostoc species, both symbiotic and nonsymbiotic, are deficient in nitric oxide reductases. Furthermore, the Azolla cyanobiont is negative for methanogenic enzymes that use coenzyme conjugates to emit methane. With the absence of nitrous oxide and methane release, and the potential ability to convert ambient nitrous oxide into nitrogen gas, it is safe to say that the Azolla cyanobiont has a myriad of features that are poor contributors to climate change, which on top of carbon dioxide quenching by the Calvin cycle in Azolla plants, makes it an efficient holistic candidate to be developed as a force for climate change mitigation, especially in irrigated urea-fed rice fields. The author also shows that Nostoc cyanobionts are theoretically capable of Nod factor synthesis, similar to Rhizobia and some Frankia species, which is a new horizon to explore in the future.

The Medicago truncatula PIN2 auxin transporter mediates basipetal auxin transport but is not necessary for nodulation.

The development of root nodules leads to an increased auxin response in early nodule primordia, which is mediated by changes in acropetal auxin transport in some legumes. Here, we investigated the role of root basipetal auxin transport during nodulation. Rhizobia inoculation significantly increased basipetal auxin transport in both Medicago truncatula and Lotus japonicus. In M. truncatula, this increase was dependent on functional Nod factor signalling through NFP, NIN, and NSP2, as well as ethylene signalling through SKL. To test whether increased basipetal auxin transport is required for nodulation, we examined a loss-of-function mutant of the M. truncatula PIN2 gene. The Mtpin2 mutant exhibited a reduction in basipetal auxin transport and an agravitropic phenotype. Inoculation of Mtpin2 roots with rhizobia still led to a moderate increase in basipetal auxin transport, but the mutant nodulated normally. No clear differences in auxin response were observed during nodule development. Interestingly, inoculation of wild-type roots increased lateral root numbers, whereas inoculation of Mtpin2 mutants resulted in reduced lateral root numbers compared with uninoculated roots. We conclude that the MtPIN2 auxin transporter is involved in basipetal auxin transport, that its function is not essential for nodulation, but that it plays an important role in the control of lateral root development.

The peptide-encoding MtRGF3 gene negatively regulates nodulation of Medicago truncatula

Leguminous root nodules specifically induced by rhizobium species fix nitrogen gas to gain nitrogen sources, which is important in sustainable agriculture and ecological balance. Several peptide signals are reported to be involved in regulation of legume nodule number and development. There are fifteen genes coding Root Meristem Growth Factor (RGF) peptide in Medicago truncatula, herein we find the expression of MtRGF3 is significantly induced by Sinorhizobium meliloti with production of Nod factors. The gene promoter is active in nodule primordia, young nodules and the meristem region of mature nodules. Knock-down (RNAi) roots of the gene (MtRGF3-RNAi) formed more root nodules than the empty vector control, and the nodule number decreased in MtRGF3-overexpressing (MtRGF3-OX) roots. Exogenous addition of the synthesized peptide significantly promoted primary root growth and inhibited lateral root emergence, in addition, the peptide application reduced the number of infection threads, nodule primordia and root nodules of M. truncatula. We also found that tyrosine sulfation determines the biological activity of MtRGF3 functioning in nodulation process, and MtRGF3 peptide negatively regulates nodulation in a dosage manner. These results demonstrate that the MtRGF3 peptide is a novel regulator during nodulation of Medicago trucatula.

The expression of LjROP4 is required for rhizobial infection in Lotus japonicus

Increasing evidence suggests that Rho of plant (ROP) GTPases play important roles in the rhizobium–legume symbiotic nodulation, but the molecular mechanisms of their regulation in symbiosis remain poorly understood. In this study, we showed that ROP4 in Lotus japonicus (LjROP4) is involved in the symbiotic interaction between L. japonicus and Mesorhizobium loti. Tissue expression analysis showed that LjROP4 expressed highly in the root. Histochemical staining analysis showed that after rhizobia inoculation, GUS reporter activity increased in the root vascular bundle, root tip, and lateral root primordia. During nodule development, GUS activity was detected in the cortex of nodule primordia and young nodules. In the mature nodules, GUS activity was detected only in the vascular bundle. Compared with the control, the overexpression of ROP4 and ROP4-CA produced much more pronounced root hair swelling and curling induced by M. loti. The infection event and nodule number noticeably increased, which was consistent with this promotion of root hair deformation. Moreover, RNA interference of LjROP4 produced opposite phenotypes. These data suggest that LjROP4 is required for root hair deformation during rhizobial infection. Thus, our study provides important information about root hair deformation responses induced by nod factors in the early stages of symbiotic interaction.

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 Sym2A 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 Sym2E 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 Sym2A-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 Sym2A, as has been confirmed by an allelism test with Sym2A 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.

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.

NoeM, a New Nodulation Gene Involved in the Biosynthesis of Nod Factors with an Open-Chain Oxidized Terminal Residue and in the Symbiosis with Mimosa pudica.

The β-rhizobium Cupriavidus taiwanensis is a nitrogen-fixing symbiont of Mimosa pudica. Nod factors produced by this species were previously found to be pentameric chitin-oligomers carrying common C18:1 or C16:0 fatty acyl chains, N-methylated and C-6 carbamoylated on the nonreducing terminal N-acetylglucosamine and sulfated on the reducing terminal residue. Here, we report that, in addition, C. taiwanensis LMG19424 produces molecules where the reducing sugar is open and oxidized. We identified a novel nodulation gene located on the symbiotic plasmid pRalta, called noeM, which is involved in this atypical Nod factor structure. noeM encodes a transmembrane protein bearing a fatty acid hydroxylase domain. This gene is expressed during symbiosis with M. pudica and requires NodD and luteolin for optimal expression. The closest noeM homologs formed a separate phylogenetic clade containing rhizobial genes only, which are located on symbiosis plasmids downstream from a nod box. Corresponding proteins, referred to as NoeM, may have specialized in symbiosis via the connection to the nodulation pathway and the spread in rhizobia. noeM was mostly found in isolates of the Mimoseae tribe, and specifically detected in all tested strains able to nodulate M. pudica. A noeM deletion mutant of C. taiwanensis was affected for the nodulation of M. pudica, confirming the role of noeM in the symbiosis with this legume.

The rhizobial type III effector ErnA confers the ability to form nodules in legumes

Several Bradyrhizobium species nodulate the leguminous plant Aeschynomene indica in a type III secretion system-dependent manner, independently of Nod factors. To date, the underlying molecular determinants involved in this symbiotic process remain unknown. To identify the rhizobial effectors involved in nodulation, we mutated 23 out of the 27 effector genes predicted in Bradyrhizobium strain ORS3257. The mutation of nopAO increased nodulation and nitrogenase activity, whereas mutation of 5 other effector genes led to various symbiotic defects. The nopM1 and nopP1 mutants induced a reduced number of nodules, some of which displayed large necrotic zones. The nopT and nopAB mutants induced uninfected nodules, and a mutant in a yet-undescribed effector gene lost the capacity for nodule formation. This effector gene, widely conserved among bradyrhizobia, was named ernA for "effector required for nodulation-A." Remarkably, expressing ernA in a strain unable to nodulate A. indica conferred nodulation ability. Upon its delivery by Pseudomonas fluorescens into plant cells, ErnA was specifically targeted to the nucleus, and a fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy approach supports the possibility that ErnA binds nucleic acids in the plant nuclei. Ectopic expression of ernA in A. indica roots activated organogenesis of root- and nodule-like structures. Collectively, this study unravels the symbiotic functions of rhizobial type III effectors playing distinct and complementary roles in suppression of host immune functions, infection, and nodule organogenesis, and suggests that ErnA triggers organ development in plants by a mechanism that remains to be elucidated.

Matching population diversity of rhizobial nodA and legume NFR5 genes in plant-microbe symbiosis.

We hypothesized that population diversities of partners in nitrogen‐fixing rhizobium–legume symbiosis can be matched for “interplaying” genes. We tested this hypothesis using data on nucleotide polymorphism of symbiotic genes encoding two components of the plant–bacteria signaling system: (a) the rhizobial nodA acyltransferase involved in the fatty acid tail decoration of the Nod factor (signaling molecule); (b) the plant NFR5 receptor required for Nod factor binding. We collected three wild‐growing legume species together with soil samples adjacent to the roots from one large 25‐year fallow: Vicia sativa, Lathyrus pratensis, and Trifolium hybridum nodulated by one of the two Rhizobium leguminosarum biovars (viciae and trifolii). For each plant species, we prepared three pools for DNA extraction and further sequencing: the plant pool (30 plant indiv.), the nodule pool (90 nodules), and the soil pool (30 samples). We observed the following statistically significant conclusions: (a) a monotonic relationship between the diversity in the plant NFR5 gene pools and the nodule rhizobial nodA gene pools; (b) higher topological similarity of the NFR5 gene tree with the nodA gene tree of the nodule pool, than with the nodA gene tree of the soil pool. Both nonsynonymous diversity and Tajima's D were increased in the nodule pools compared with the soil pools, consistent with relaxation of negative selection and/or admixture of balancing selection. We propose that the observed genetic concordance between NFR5 gene pools and nodule nodA gene pools arises from the selection of particular genotypes of the nodA gene by the host plant.

Differential tetraspanin genes expression and subcellular localization during mutualistic interactions in Phaseolus vulgaris.

Arbuscular mycorrhizal fungi and rhizobia association with plants are two of the most successful plant-microbe associations that allow the assimilation of P and N by plants, respectively. These mutualistic interactions require a molecular dialogue, i.e., legume roots exude flavonoids or strigolactones which induce the Nod factors or Myc factors synthesis and secretion from the rhizobia or fungi, respectively. These Nod or Myc factors trigger several responses in the plant root, including calcium oscillations, and reactive oxygen species (ROS). Furthermore, superoxide and H2O2 have emerged as key components that regulate the transitions from proliferation to differentiation in the plant meristems. Similar to the root meristem, the nodule meristem accumulates superoxide and H2O2. Tetraspanins are transmembrane proteins that organize into tetraspanin web regions, where they recruit specific proteins into platforms required for signal transduction, membrane fusion, cell trafficking and ROS generation. Plant tetraspanins are scaffolding proteins associated with root radial patterning, biotic and abiotic stress responses, cell fate determination, and hormonal regulation and recently have been reported as a specific marker of exosomes in animal and plant cells and key players at the site of plant fungal infection. In this study, we conducted transcriptional profiling of the tetraspanin family in common bean (Phaseolus vulgaris L. var. Negro Jamapa) to determine the specific expression patterns and subcellular localization of tetraspanins during nodulation or under mycorrhizal association. Our results demonstrate that the tetraspanins are transcriptionally modulated during the mycorrhizal association, but are also expressed in the infection thread and nodule meristem development. Subcellular localization indicates that tetraspanins have a key role in vesicular trafficking, cell division, and root hair polar growth.

Atypical Receptor Kinase RINRK1 Required for Rhizobial Infection But Not Nodule Development in Lotus japonicus.

During the legume-rhizobium symbiotic interaction, rhizobial invasion of legumes is primarily mediated by a plant-made tubular invagination called an infection thread (IT). Here, we identify a gene in Lotus japonicus encoding a Leu-rich repeat receptor-like kinase (LRR-RLK), RINRK1 (Rhizobial Infection Receptor-like Kinase1), that is induced by Nod factors (NFs) and is involved in IT formation but not nodule organogenesis. A paralog, RINRK2, plays a relatively minor role in infection. RINRK1 is required for full induction of early infection genes, including Nodule Inception (NIN), encoding an essential nodulation transcription factor. RINRK1 displayed an infection-specific expression pattern, and NIN bound to the RINRK1 promoter, inducing its expression. RINRK1 was found to be an atypical kinase localized to the plasma membrane and did not require kinase activity for rhizobial infection. We propose RINRK1 is an infection-specific RLK, which may specifically coordinate output from NF signaling or perceive an unknown signal required for rhizobial infection.

The genome of Ensifer alkalisoli YIC4027 provides insights for host specificity and environmental adaptations

BackgroundEnsifer alkalisoli YIC4027, a recently characterized nitrogen-fixing bacterium of the genus Ensifer, has been isolated from root nodules of the host plant Sesbania cannabina. This plant is widely used as green manure and for soil remediation. E. alkalisoli YIC4027 can grow in saline-alkaline soils and is a narrow-host-range strain that establishes a symbiotic relationship with S. cannabina. The complete genome of this strain was sequenced to better understand the genetic basis of host specificity and adaptation to saline-alkaline soils.ResultsE. alkalisoli YIC4027 was found to possess a 6.1-Mb genome consisting of three circular replicons: one chromosome (3.7 Mb), a chromid (1.9 Mb) and a plasmid (0.46 Mb). Genome comparisons showed that strain YIC4027 is phylogenetically related to broad-host-range Ensifer fredii strains. Synteny analysis revealed a strong collinearity between chromosomes of E. alkalisoli YIC4027 and those of the E. fredii NGR234 (3.9 Mb), HH103 (4.3 Mb) and USDA257 (6.48 Mb) strains. Notable differences were found for genes required for biosynthesis of nodulation factors and protein secretion systems, suggesting a role of these genes in host-specific nodulation. In addition, the genome analysis led to the identification of YIC4027 genes that are presumably related to adaptation to saline-alkaline soils, rhizosphere colonization and nodulation competitiveness. Analysis of chemotaxis cluster genes and nodulation tests with constructed che gene mutants indicated a role of chemotaxis and flagella-mediated motility in the symbiotic association between YIC4027 and S. cannabina.ConclusionsThis study provides a basis for a better understanding of host specific nodulation and of adaptation to a saline-alkaline rhizosphere. This information offers the perspective to prepare optimal E. alkalisoli inocula for agriculture use and soil remediation.

A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation

The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5.