Legume Lotus Japonicus Research Articles

Lotus resistance against Ralstonia is enhanced by Mesorhizobium and does not impair mutualism.

Legumes establish nitrogen-fixing symbioses with rhizobia. On the contrary, they can be attacked concomitantly by pathogens, raising the question of potential trade-offs between mutualism and immunity. In order to study such trade-offs, we used a tripartite system involving the model legume Lotus japonicus, its rhizobial symbiont Mesorhizobium loti and the soilborne pathogen Ralstonia solanacearum. We investigated the impact of mutualism on plant defense and the reciprocal influence of plant defense on mutualism. We found that Lotus age-related resistance against Ralstonia was improved by the interaction with rhizobia especially when nodulation is triggered. Conversely, age-related resistance did not compromise nodule organogenesis or functioning under pathogen attack. Proteomic characterization indicates that this resistance is associated with distinct proteome modifications in roots and nodules. This resistance questions the concept of interference between efficient defense reactions and mutualistic interactions and is of great interest for agricultural purposes as it not only restricts pathogen colonization, but would also preserve nitrogen fixation and yield.

A mitochondrial metalloprotease FtsH4 is required for symbiotic nitrogen fixation in Lotus japonicus nodules

Symbiotic nitrogen fixation is a highly coordinated process involving legume plants and nitrogen-fixing bacteria known as rhizobia. In this study, we investigated a novel Fix− mutant of the model legume Lotus japonicus that develops root nodules with endosymbiotic rhizobia but fails in nitrogen fixation. Map-based cloning identified the causal gene encoding the filamentation temperature-sensitive H (FtsH) protein, designated as LjFtsH4. The LjFtsH4 gene was expressed in all plant organs without increased levels during nodulation. Subcellular localization revealed that LjFtsH4, fused with a fluorescent protein, localized in mitochondria. The Ljftsh4 mutant nodules showed signs of premature senescence, including symbiosome membrane collapse and bacteroid disintegration. Additionally, nodule cells of Ljftsh4 mutant displayed mitochondria with indistinct crista structures. Grafting and complementation tests confirmed that the Fix− phenotype was determined by the root genotype, and that protease activity of LjFtsH4 was essential for nodule nitrogen fixation. Furthermore, the ATP content in Ljftsh4 mutant roots and nodules was lower than in the wild-type, suggesting reduced mitochondrial function. These findings underscore the critical role of LjFtsH4 in effective symbiotic nitrogen fixation in root nodules.

A fungal endophyte increases plant resilience to low nutrient availabilities: a case of Fe acquisition in legumes.

Microbial inocula are considered a promising and effective alternative solution to the use of chemical fertilizers to support plant growth and productivity since they play a key role in the availability and uptake of nutrients. Here, the effect of a beneficial of a fungal root endophyte, Fusarium solani strain K (FsK), on nutrient acquisition efficiency of the legume Lotus japonicus was studied, and putative mode-of-action of the endophyte at a molecular level was determined. Plant colonization with the endophyte resulted in increased shoot and root fresh weight under Fe deficiency compared to control nutrient conditions. Plant inoculation with FsK was associated with a significant increase in macro- and micronutrient concentration in leaves at an early stage of endophyte inoculation and a replenishment of Fe content under prolonged iron starvation. The mechanistic basis of the plant growth promotion capabilities of the endophyte is exerted at the transcriptional level since we recorded changes in the expression levels of genes related to iron uptake in FsK-colonized plants under stress conditions compared to uninoculated plants. In addition, the observed changes in the ethylene biosynthesis-related genes suggest a possible implication of ethylene in the mode of action used by FsK to enhance plant response to nutrient stress conditions. Finally, we demonstrated that the endophyte possesses a reductive high-affinity Fe uptake system and identified a ferric reductase that was induced in planta under Fe deficiency conditions, indicating that this fungal Fe homeostasis mechanism may result in a benefit in nutrient acquisition for the plant as well.

Systemic long-distance sulfur transport and its role in symbiotic root nodule protein turnover

Sulfur is an essential nutrient for all plants, but also crucial for the nitrogen fixing symbiosis between legumes and rhizobia. Sulfur limitation can hamper nodule development and functioning. Until now, it remained unclear whether sulfate uptake into nodules is local or mainly systemic via the roots, and if long-distance transport from shoots to roots and into nodules occurs. Therefore, this work investigates the systemic regulation of sulfur transportation in the model legume Lotus japonicus by applying stable isotope labeling to a split-root system. Metabolite and protein extraction together with mass spectrometry analyses were conducted to determine the plants molecular phenotype and relative isotope protein abundances. Data show that treatments of varying sulfate concentrations including the absence of sulfate on one side of a nodulated root was not affecting nodule development as long as the other side of the root system was provided with sufficient sulfate. Concentrations of shoot metabolites did not indicate a significant stress response caused by a lack of sulfur. Further, we did not observe any quantitative changes in proteins involved in biological nitrogen fixation in response to the different sulfate treatments. Relative isotope abundance of 34S confirmed a long-distance transport of sulfur from one side of the roots to the other side and into the nodules. Altogether, these results provide evidence for a systemic long-distance transport of sulfur via the upper part of the plant to the nodules suggesting a demand driven sulfur distribution for the maintenance of symbiotic N-fixation.

A micro RNA mediates shoot control of root branching

Plants extract mineral nutrients from the soil, or from interactions with mutualistic soil microbes via their root systems. Adapting root architecture to nutrient availability enables efficient resource utilization, particularly in patchy and dynamic environments. Root growth responses to soil nitrogen levels are shoot-mediated, but the identity of shoot-derived mobile signals regulating root growth responses has remained enigmatic. Here we show that a shoot-derived micro RNA, miR2111, systemically steers lateral root initiation and nitrogen responsiveness through its root target TML (TOO MUCH LOVE) in the legume Lotus japonicus, where miR2111 and TML were previously shown to regulate symbiotic infections with nitrogen fixing bacteria. Intriguingly, systemic control of lateral root initiation by miR2111 and TML/HOLT (HOMOLOGUE OF LEGUME TML) was conserved in the nonsymbiotic ruderal Arabidopsis thaliana, which follows a distinct ecological strategy. Thus, the miR2111-TML/HOLT regulon emerges as an essential, conserved factor in adaptive shoot control of root architecture in dicots.

Heme catabolism mediated by heme oxygenase in uninfected interstitial cells enables efficient symbiotic nitrogen fixation in Lotus japonicus nodules.

Legume nodules produce large quantities of heme required for the synthesis of leghemoglobin (Lb) and other hemoproteins. Despite the crucial function of Lb in nitrogen fixation and the toxicity of free heme, the mechanisms of heme homeostasis remain elusive. Biochemical, cellular, and genetic approaches were used to study the role of heme oxygenases (HOs) in heme degradation in the model legume Lotus japonicus. Heme and biliverdin were quantified and localized, HOs were characterized, and knockout LORE1 and CRISPR/Cas9 mutants for LjHO1 were generated and phenotyped. We show that LjHO1, but not the LjHO2 isoform, is responsible for heme catabolism in nodules and identify biliverdin as the invivo product of the enzyme in senescing green nodules. Spatiotemporal expression analysis revealed that LjHO1 expression and biliverdin production are restricted to the plastids of uninfected interstitial cells. The nodules of ho1 mutants showed decreased nitrogen fixation, and the development of brown, rather than green, nodules during senescence. Increased superoxide production was observed in ho1 nodules, underscoring the importance of LjHO1 in antioxidant defense. We conclude that LjHO1 plays an essential role in degradation of Lb heme, uncovering a novel function of nodule plastids and uninfected interstitial cells in nitrogen fixation.

Molecular Assessment of Metabolome Alterations in Lotus japonicus Roots Induced by Arbuscular Mycorrhiza

AbstractAn arbuscular mycorrhiza symbiosis, which is established by 80% of all vascular plants and Glomeromycota fungi, can boost the growth, stress tolerance, and yield of agricultural crop plants by increasing the mineral supply of the host plant [1]. Mineral nutrients absorbed by the fungus are exchanged for photoassimilates from the plant via highly branched fungal structures called arbuscules which are formed within root cortex cells [2]. The process of arbuscule formation and degradation is highly dynamic and accompanied by considerable cell reprogramming [3], but there is only limited knowledge on the influence of arbuscular mycorrhiza on the host metabolome.We investigated alterations of the host plant's root metabolome induced by arbuscular mycorrhizal symbiosis using the model legume Lotus japonicus. Methanol extracts of mycorrhizal (myc) and non‐mycorrhizal roots (mock) were analyzed employing ultra‐highperformance liquid chromatography–electrospray ionization–time‐of‐flight–ion mobility–mass spectrometry (UPLC–ESI–ToF–IM–MS) in an untargeted metabolomics approach. Up‐regulated marker metabolites were identified via principal components analysis (PCA) and orthogonal partial least squares–discriminant analysis (OPLS–DA) depicted as S‐plots, and their structures elucidated by co‐chromatography with authentic standards or by isolation from L. japonicus roots using preparative HPLC and one‐ and two‐dimensional nuclear magnetic resonance spectroscopy (NMR) experiments. We isolated three previously unknown mycorrhizal marker polyphenols from L. japonicus roots, one being a coumaronochromone, one pterocarp‐6a‐ene, and one aryl benzofuran carboxaldehyde. Additionally, we identified three more coumaronochromones, three flavonoids, three isoflavonoids, and one coumestan in mycorrhized L. japonicus.

Genome-wide identification of acyl-CoA binding proteins and possible functional prediction in legumes.

Acyl-CoA-binding proteins (ACBPs), members of a vital housekeeping protein family, are present in various animal and plant species. They are divided into four classes: small ACBPs (class I), ankyrin-repeat ACBPs (class II), large ACBPs (class III), and kelch-ACBPs (class IV). Plant ACBPs play a pivotal role in intracellular transport, protection, and pool formation of acyl-CoA esters, promoting plant development and stress response. Even though legume crops are important for vegetable oils, proteins, vegetables and green manure, legume ACBPs are not well investigated. To comprehensively explore the functions of ACBPs in nine legumes (Lotus japonicus, Medicago truncatula, Glycine max, Vigna angularis, Vigna radiata, Phaseolus vulgaris, Arachis hypogaea, Arachis duranensis, and Arachis ipaensis), we conducted genome-wide identification of the ACBP gene family. Our evolutionary analyses included phylogenetics, gene structure, the conserved motif, chromosomal distribution and homology, subcellular localization, cis-elements, and interacting proteins. The results revealed that ACBP Orthologs of nine legumes had a high identity in gene structure and conserved motif. However, subcellular localization, cis-acting elements, and interaction protein analyses revealed potentially different functions from previously reported. The predicted results were also partially verified in Arachis hypogaea. We believe that our findings will help researchers understand the roles of ACBPs in legumes and encourage them to conduct additional research.

Interplay between rhizobial nodulation and arbuscular mycorrhizal fungal colonization inLotus japonicusroots

This study aims to identify main factors that influence the tripartite association of legumes with arbuscular mycorrhiza fungi (AMF) and nitrogen-fixing rhizobia. Concurrent inoculations with Mesorhizobium loti and four AMF strains were performed on the model legume Lotus japonicus. Nodulation was significantly enhanced by all AMF strains, under normal conditions, and by specific AMF strains under heat-stress conditions. The impact of rhizobia on mycorrhizal colonization was AMF strain dependent. Co-inoculation trials, where either AMF or rhizobia were restricted outside the root, showed that the symbiotic phenotypes are not influenced by microbial interactions at the pre-symbiotic stage. External application of nutrients showed that P enhances nodulation, while N application does not enhance mycorrhizal colonization. Nodulation and mycorhization affect one another during advanced stages of the symbiosis. AMF strains may enhance nodulation under both normal and high environmental temperatures. Rhizobium-AMF compatibility is critical, as rhizobium may positively affect specific AMF strains, an effect that does not derive from increased N uptake.

FER and LecRK show haplotype-dependent cold-responsiveness and mediate freezing tolerance in Lotus japonicus.

Many plant species have succeeded in colonizing a wide range of diverse climates through local adaptation, but the underlying molecular genetics remain obscure. We previously found that winter survival was a direct target of selection during colonization of Japan by the perennial legume Lotus japonicus and identified associated candidate genes. Here, we show that two of these, FERONIA-receptor like kinase (LjFER) and a S-receptor-like kinase gene (LjLecRK), are required for non-acclimated freezing tolerance and show haplotype-dependent cold-responsive expression. Our work suggests that recruiting a conserved growth regulator gene, FER, and a receptor-like kinase gene, LecRK, into the set of cold-responsive genes has contributed to freezing tolerance and local climate adaptation in L. japonicus, offering functional genetic insight into perennial herb evolution.

Role of phytohormones in legumes infected intercellularly by rhizobia without infection threads formation

Rhizobia grow in the soil as free-living organisms but they also can live as nitrogen-fixing symbionts inside root nodules of legumes. Two modes of rhizobial entry into roots are a) intracellularly, through root hairs, inside infection threads, and b) intercellularly, between epidermal cells, never involving root hair or transcellular infection thread formation. Intracellular entry is the best-known infection process, especially in the model legumes Medicago truncatula and Lotus japonicus. Intercellular entry of rhizobia never involving infection thread formation is found in some of the dalbergioid and genistoid legumes, such as Aeschynomene spp. and Arachis hypogaea. This infection mechanism has been scarcely investigated and little is known about molecular players involved in this infection mechanism. Here we focus our interest in comparing the role of phytohormones between legumes infected intracellular and intercellularly. To reach this aim we selected the legumes A. hypogaea and Aeschynomene spp., and the model legumes M. truncatula and L. japonicus.

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.

Characterization of zinc uptake and translocation visualized with positron-emitting 65Zn tracer and analysis of transport-related gene expression in two Lotus japonicus accessions.

Zinc (Zn) is an essential element for humans and plants. However, Zn deficiency is widespread and 25 % of the world's population is at risk of Zn deficiency. To overcome the deficiency of Zn intake, crops with high Zn content are required. However, most crop-producing areas have Zn-deficient soils, therefore crops with excellent Zn uptake/transport characteristics (i.e. high Zn efficiency) are needed. Our objective was to identify the crucial factors responsible for high Zn efficiency in the legume Lotus japonicus. We evaluated Zn efficiency by static and real-time visualization of radioactive Zn (65Zn) uptake/transport in two L. japonicus accessions, MG-20 and B-129, that differ in Zn efficiency. The combination of visualization methods verified the dynamics of Zn accumulation and transport within the plant. We compared gene expression under a normal Zn concentration (control) and Zn deficiency to evaluate genetic factors that may determine the differential Zn efficiency of the accessions. The accession B-129 accumulated almost twice the amount of Zn as MG-20. In the static 65Zn images, 65Zn accumulated in meristematic tissues, such as root tips and the shoot apex, in both accessions. The positron-emitting tracer imaging system (PETIS), which follows the transport process in real time, revealed that 65Zn transport to the shoot was more rapid in B-129 than in MG-20. Many genes associated with Zn uptake and transport were more highly expressed in B-129 than in MG-20 under the control condition. These gene expression patterns under Zn deficiency differed from those under the control Zn condition. PETIS confirmed that the real-time transport of 65Zn to the shoot was faster in B-129 than in MG-20. The high Zn efficiency of B-129 may be due to the elevated expression of a suite of Zn uptake- and transport-related genes.

Molecular Mechanisms of Intercellular Rhizobial Infection: Novel Findings of an Ancient Process.

Establishment of the root-nodule symbiosis in legumes involves rhizobial infection of nodule primordia in the root cortex that is dependent on rhizobia crossing the root epidermal barrier. Two mechanisms have been described: either through root hair infection threads or through the intercellular passage of bacteria. Among the legume genera investigated, around 75% use root hair entry and around 25% the intercellular entry mode. Root-hair infection thread-mediated infection has been extensively studied in the model legumes Medicago truncatula and Lotus japonicus. In contrast, the molecular circuit recruited during intercellular infection, which is presumably an ancient and simpler pathway, remains poorly known. In recent years, important discoveries have been made to better understand the transcriptome response and the genetic components involved in legumes with obligate (Aeschynomene and Arachis spp.) and conditional (Lotus and Sesbania spp.) intercellular rhizobial infections. This review addresses these novel findings and briefly considers possible future research to shed light on the molecular players that orchestrate intercellular infection in legumes.

The nodD1 Gene of Sinorhizobium fredii HH103 Restores Nodulation Capacity on Bean in a Rhizobium tropici CIAT 899 nodD1/nodD2 Mutant, but the Secondary Symbiotic Regulators nolR, nodD2 or syrM Prevent HH103 to Nodulate with This Legume.

Rhizobial NodD proteins and appropriate flavonoids induce rhizobial nodulation gene expression. In this study, we show that the nodD1 gene of Sinorhizobium fredii HH103, but not the nodD2 gene, can restore the nodulation capacity of a double nodD1/nodD2 mutant of Rhizobium tropici CIAT 899 in bean plants (Phaseolus vulgaris). S. fredii HH103 only induces pseudonodules in beans. We have also studied whether the mutation of different symbiotic regulatory genes may affect the symbiotic interaction of HH103 with beans: ttsI (the positive regulator of the symbiotic type 3 protein secretion system), and nodD2, nolR and syrM (all of them controlling the level of Nod factor production). Inactivation of either nodD2, nolR or syrM, but not that of ttsI, affected positively the symbiotic behavior of HH103 with beans, leading to the formation of colonized nodules. Acetylene reduction assays showed certain levels of nitrogenase activity that were higher in the case of the nodD2 and nolR mutants. Similar results have been previously obtained by our group with the model legume Lotus japonicus. Hence, the results obtained in the present work confirm that repression of Nod factor production, provided by either NodD2, NolR or SyrM, prevents HH103 to effectively nodulate several putative host plants.

Comparative Analysis of NADPH-Cytochrome P450 Reductases From Legumes for Heterologous Production of Triterpenoids in Transgenic Saccharomyces cerevisiae.

Triterpenoids are plant specialized metabolites with various pharmacological activities. They are widely distributed in higher plants, such as legumes. Because of their low accumulation in plants, there is a need for improving triterpenoid production. Cytochrome P450 monooxygenases (CYPs) play critical roles in the structural diversification of triterpenoids. To perform site-specific oxidations, CYPs require the electrons that are transferred by NADPH-cytochrome P450 reductase (CPR). Plants possess two main CPR classes, class I and class II. CPR classes I and II have been reported to be responsible for primary and specialized (secondary) metabolism, respectively. In this study, we first analyzed the CPR expression level of three legumes species, Medicago truncatula, Lotus japonicus, and Glycyrrhiza uralensis, showing that the expression level of CPR class I was lower and more stable, while that of CPR class II was higher in almost all the samples. We then co-expressed different combinations of CYP716As and CYP72As with different CPR classes from these three legumes in transgenic yeast. We found that CYP716As worked better with CPR-I from the same species, while CYP72As worked better with any CPR-IIs. Using engineered yeast strains, CYP88D6 paired with class II GuCPR produced the highest level of 11-oxo-β-amyrin, the important precursor of high-value metabolites glycyrrhizin. This study provides insight into co-expressing genes from legumes for heterologous production of triterpenoids in yeast.

The Lotus japonicus ROP3 Is Involved in the Establishment of the Nitrogen-Fixing Symbiosis but Not of the Arbuscular Mycorrhizal Symbiosis.

Legumes form root mutualistic symbioses with some soil microbes promoting their growth, rhizobia, and arbuscular mycorrhizal fungi (AMF). A conserved set of plant proteins rules the transduction of symbiotic signals from rhizobia and AMF in a so-called common symbiotic signaling pathway (CSSP). Despite considerable efforts and advances over the past 20 years, there are still key elements to be discovered about the establishment of these root symbioses. Rhizobia and AMF root colonization are possible after a deep cell reorganization. In the interaction between the model legume Lotus japonicus and Mesorhizobium loti, this reorganization has been shown to be dependent on a SCAR/Wave-like signaling module, including Rho-GTPase (ROP in plants). Here, we studied the potential role of ROP3 in the nitrogen-fixing symbiosis (NFS) as well as in the arbuscular mycorrhizal symbiosis (AMS). We performed a detailed phenotypic study on the effects of the loss of a single ROP on the establishment of both root symbioses. Moreover, we evaluated the expression of key genes related to CSSP and to the rhizobial-specific pathway. Under our experimental conditions, rop3 mutant showed less nodule formation at 7- and 21-days post inoculation as well as less microcolonies and a higher frequency of epidermal infection threads. However, AMF root colonization was not affected. These results suggest a role of ROP3 as a positive regulator of infection thread formation and nodulation in L. japonicus. In addition, CSSP gene expression was neither affected in NFS nor in AMS condition in rop3 mutant. whereas the expression level of some genes belonging to the rhizobial-specific pathway, like RACK1, decreased in the NFS. In conclusion, ROP3 appears to be involved in the NFS, but is neither required for intra-radical growth of AMF nor arbuscule formation.

Molecular Characterization of Carbonic Anhydrase Genes in Lotus japonicus and Their Potential Roles in Symbiotic Nitrogen Fixation.

Carbonic anhydrase (CA) plays a vital role in photosynthetic tissues of higher plants, whereas its non-photosynthetic role in the symbiotic root nodule was rarely characterized. In this study, 13 CA genes were identified in the model legume Lotus japonicus by comparison with Arabidopsis CA genes. Using qPCR and promoter-reporter fusion methods, three previously identified nodule-enhanced CA genes (LjαCA2, LjαCA6, and LjβCA1) have been further characterized, which exhibit different spatiotemporal expression patterns during nodule development. LjαCA2 was expressed in the central infection zone of the mature nodule, including both infected and uninfected cells. LjαCA6 was restricted to the vascular bundle of the root and nodule. As for LjβCA1, it was expressed in most cell types of nodule primordia but only in peripheral cortical cells and uninfected cells of the mature nodule. Using CRISPR/Cas9 technology, the knockout of LjβCA1 or both LjαCA2 and its homolog, LjαCA1, did not result in abnormal symbiotic phenotype compared with the wild-type plants, suggesting that LjβCA1 or LjαCA1/2 are not essential for the nitrogen fixation under normal symbiotic conditions. Nevertheless, the nodule-enhanced expression patterns and the diverse distributions in different types of cells imply their potential functions during root nodule symbiosis, such as CO2 fixation, N assimilation, and pH regulation, which await further investigations.

Use of the rhizobial type III effector gene nopP to improve Agrobacterium rhizogenes-mediated transformation of Lotus japonicus

BackgroundProtocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus. The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency.ResultsStrong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL, nopM, and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1. A nodulation experiment indicated that nopP expression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti.ConclusionWe have established a novel protocol for hairy root transformation of L. japonicus. The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.

Natural variation identifies a Pxy gene controlling vascular organisation and formation of nodules and lateral roots in Lotus japonicus.

Forward and reverse genetics using the model legumes Lotus japonicus and Medicago truncatula have been instrumental in identifying the essential genes governing legume-rhizobia symbiosis. However, little information is known about the effects of intraspecific variation on symbiotic signalling. Here, we use quantitative trait locus sequencing (QTL-seq) to investigate the genetic basis of the differentiated phenotypic responses shown by the Lotus accessions Gifu and MG20 to inoculation with the Mesorhizobium loti exoU mutant that produces truncated exopolysaccharides. We identified through genetic complementation the Pxy gene as a component of this differential exoU response. Lotus Pxy encodes a leucine-rich repeat receptor-like kinase similar to Arabidopsis thaliana PXY, which regulates stem vascular development. We show that Lotus pxy insertion mutants displayed defects in root and stem vascular organisation, as well as lateral root and nodule formation. Our work links Pxy to de novo organogenesis in the root, highlights the genetic overlap between regulation of lateral root and nodule formation, and demonstrates that natural variation in Pxy affects nodulation signalling.