Legumes are pivotal for sustainable agriculture, yet their productivity is hindered by soilborne pathogens such as Aphanomyces euteiches . This study introduces Aeschynomene evenia as a novel model to investigate legume immunity and its interplay with Nod factor-independent symbiosis (NFIS). Inoculation of A. evenia accessions with A. euteiches strains revealed a range of quantitative resistance levels. Phenotypic and cytological analyses showed a compatible interaction, including root browning, intracellular colonisation, and oospore production; along with partial resistance traits such as phenolic accumulation at the endodermal barrier. Transcriptomic analyses at 1 and 3 dpi identified 3,403 differentially expressed genes (DEGs), with upregulated genes associated with pathogen perception, immune signalling and specialised metabolite biosynthesis. Kinase-mediated signalling, ROS homeostasis, and WRKY transcription factors were among the most enriched functional categories. Comparative transcriptomics with Medicago truncatula confirmed conserved immune responses, while A. evenia displayed broader transcriptional repression. Integration with symbiotic transcriptomic data revealed overlapping gene signatures, including AeRLCK2 and AeCRK , previously found to be NFIS-specific, as putative dual-function kinases. Aerlck2 and Aecrk mutants exhibited enhanced resistance to A. euteiches , supporting their roles in immune regulation. This work positions A. evenia - A. euteiches as a valuable system to dissect legume quantitative resistance and its intersection with symbiosis. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-20-CE20-0017-01, ANR 10 LABX 41, ANR-10-INBS-04 Ministère de l’Enseignement Supérieur et de la Recherche, https://ror.org/03sjk9a61

SummaryMedicago truncatulaNod Factor Perception (MtNFP) is a lysin-domain Receptor-Like Kinase (LysM-RLK) that plays a key role in the Rhizobium-legume symbiosis, and is involved in plant immunity. MtNFP also has an inactive kinase domain, suggesting that the protein is involved in different receptor complexes. Using the MtNFP pseudo-kinase domain as a bait in a Yeast two Hybrid screen, we identifiedM. truncatulaSUPPRESSOR OF BIR1 (MtSOBIR1) as a new interactor of MtNFP. We showed that an interaction between the two RLKs can occurin plantaand that the kinase domain of MtSOBIR1 is active and can transphosphorylate the pseudo-kinase domain of MtNFP. Like in other plants, our data suggest a positive role of MtSOBIR1 in immunity;MtSOBIR1could functionally complement anAtsobir1mutant for defence activation, and aMtsobir1mutant was defective in pathogen-induced defence gene expression. We also showed thatMtSOBIR1has a symbiotic role withMtsobir1mutants showing a strong symbiotic phenotype in a plant genotype- and rhizobial strain-specific manner. The symbiotic role was apparent both at an early stage of rhizobial infection and in nodules. Together, these data suggest that, like MtNFP, MtSOBIR1 has a dual role, and can control immunity in both pathogenic and beneficial situations, with positive or negative roles, respectively.

Medicago truncatula Nod Factor Perception (MtNFP) plays a role in both the Rhizobium-Legume (RL) symbiosis and plant immunity, and evidence suggests that the immune-related function of MtNFP is relevant for symbiosis. To better understand these roles of MtNFP, we sought to identify new interacting partners. We screened a yeast-2-hybrid cDNA library from Aphanomyces euteiches infected and noninfected M. truncatula roots. The M. truncatula leucine-rich repeat (LRR) receptor-like kinase SUPPRESSOR OF BIR1 (MtSOBIR1) was identified as an interactor of MtNFP and was characterised for kinase activity, and potential roles in symbiosis and plant immunity. We showed that the kinase domain of MtSOBIR1 is active and can transphosphorylate the pseudo-kinase domain of MtNFP. MtSOBIR1 could functionally complement Atsobir1 and Nbsobir1/sobir1-like mutants for defence activation, and Mtsobir1 mutants were defective in immune responses to A. euteiches . For symbiosis, we showed that Mtsobir1 mutant plants had both a strong, early infection defect and defects in the defence suppression in nodules, and both effects were plant genotype- and rhizobial strain-specific. This work highlights a conserved function for MtSOBIR1 in activating defence responses to pathogen attack, and potentially novel symbiotic functions of downregulating defence in association with the control of symbiotic specificity.

Many plants interact symbiotically with arbuscular mycorrhizal fungi to enhance inorganic phosphorus uptake, and legumes also develop a nodule symbiosis with rhizobia for nitrogen acquisition. The establishment and functioning of both symbioses rely on a common plant signaling pathway activated by structurally related Myc and Nod factors. Recently, a SPARK receptor-like kinase (RLK)/receptor-like cytoplasmic kinase (RLCK) complex was shown to be essential for arbuscular mycorrhiza formation in both monocot and dicot plants. Here, we show that in Aeschynomene legumes, the RLCK component of this receptor complex has undergone a gene duplication event and mediates a unique nodule symbiosis that is independent of rhizobial Nod factors. In Aeschynomene evenia, AeRLCK2 is crucial for nodule initiation but not for arbuscular mycorrhiza symbiosis. Additionally, AeRLCK2 physically interacts with and is phosphorylated by the cysteine-rich RLK, AeCRK, which is also required for nodulation. This finding uncovers an important molecular mechanism that controls the establishment of nodulation and is associated with Nod-independent symbiosis.

Legumes are crops of considerable economic and ecological significance. They are suitable for cultivation in a variety of environments and temperatures. They are able to form a symbiotic relationship with nitrogen-fixing soil bacteria called rhizobia, contributing to the enrichment of marginal soils with this essential nutrient, and reduce artificial fertilizer use. Similarly, legumes establish a widespread mutualistic association with soil fungi, involving a reciprocal transfer of nutrients. Global warming is reshaping the interactions of plants with their environment, exerting profound effects on global agricultural systems. Plants have evolved sensing, signalling, and adaptive molecular mechanisms to respond to (a)biotic stimuli. Reactive oxygen, nitrogen, and sulfur species (RONSS) are key players in stress tolerance mechanisms, and their homeostasis, mainly accomplished by antioxidant enzymes and metabolites, is essential to allow redox signalling while preventing oxidative damage. Here, we review recent findings, highlighting metabolic pathways of RONSS and antioxidants, with emphasis on their functions in signalling and protection in response to (a)biotic constraints in legumes. Special attention is paid to the molecular crosstalk between RONSS in response to multiple stimuli and notably how redox homeostasis adjustment can increase (a)biotic stress tolerance and potentially be exploited to mitigate the negative effects of climate change.

Many plants interact symbiotically with arbuscular mycorrhizal (AM) fungi to enhance inorganic phosphorus uptake, and legumes also develop a nodule symbiosis with rhizobia for nitrogen acquisition. Establishment and functioning of both symbioses rely on a common plant signaling pathway activated by structurally related Myc- and Nod-factors. Recently, a SPARK Receptor-like-Kinase (RLK)/Receptor-like Cytoplasmic Kinase (RLCK) complex was shown to be essential for AM in both monocot and dicot plants. Here, we show that in Aeschynomene legumes the RLCK component of this receptor complex has evolved following a gene duplication event and mediates a unique nodule symbiosis that is independent of rhizobial Nod factors. In Aeschynomene evenia , AeRLCK2 is crucial for nodule initiation but not for AM. Additionally, AeRLCK2 physically interacts with and is phosphorylated by the Cysteine-rich RLK, AeCRK, also required for nodulation. This work reveals a novel evolutionary origin of this Nod-independent symbiosis from AM. ### Competing Interest Statement The authors have declared no competing interest.

Lysin motif receptor-like kinases (LysM-RLKs) are involved in the perception of chitooligosaccharides (COs) and related lipochitooligosaccharides (LCOs) in plants. Expansion and divergence of the gene family during evolution have led to various roles in symbiosis and defense. By studying proteins of the LYR-IA subclass of LysM-RLKs of the Poaceae, we show here that they are high-affinity LCO-binding proteins with a lower affinity for COs, consistent with a role in LCO perception to establish arbuscular mycorrhiza (AM). In Papilionoid legumes, whole-genome duplication has resulted in two LYR-IA paralogs, MtLYR1 and MtNFP in Medicago truncatula, with MtNFP playing an essential role in root nodule symbiosis with nitrogen-fixing rhizobia. We show that MtLYR1 has retained the ancestral LCO-binding characteristic and is dispensable for AM. Domain swapping between the three LysMs of MtNFP and MtLYR1 and mutagenesis in MtLYR1 suggest that the MtLYR1 LCO-binding site is on the second LysM and that divergence in MtNFP led to better nodulation, but surprisingly with decreased LCO binding. These results suggest that divergence of the LCO-binding site has been important for the evolution of a role of MtNFP in nodulation with rhizobia.

On the cover: A highly simplified species for genome engineering would facilitate rational design of a synthetic plant.Eric Lam and

N-acetylglucosamine containing compounds acting as pathogenic or symbiotic signals are perceived by plant-specific Lysin Motif Receptor-Like Kinases (LysM-RLKs). The molecular mechanisms of this perception are not fully understood, notably those of lipo-chitooligosaccharides (LCOs) produced during root endosymbioses with nitrogen-fixing bacteria or arbuscular mycorrhizal fungi. In Medicago truncatula, we previously identified the LysM-RLK LYR3 (MtLYR3) as a specific LCO-binding protein. We also showed that the absence of LCO binding to LYR3 of the non-mycorrhizal Lupinus angustifolius, (LanLYR3), was related to LysM3, which differs from that of MtLYR3 by several amino acids and, particularly, by a critical tyrosine residue absent in LanLYR3. Here, we aimed to define the LCO binding site of MtLYR3 by using molecular modelling and simulation approaches, combined with site-directed mutagenesis and LCO binding experiments. 3D models of MtLYR3 and LanLYR3 ectodomains were built, and homology modelling and molecular dynamics (MD) simulations were performed. Molecular docking and MD simulation on the LysM3 identified potential key residues for LCO binding. We highlighted by steered MD simulations that in addition to the critical tyrosine, two other residues were important for LCO binding in MtLYR3. Substitution of these residues in LanLYR3-LysM3 by those of MtLYR3-LysM3 allowed the recovery of high-affinity LCO binding in experimental radioligand-binding assays. An analysis of selective constraints revealed that the critical tyrosine has experienced positive selection pressure and is absent in some LYR3 proteins. These findings now pave the way to uncover the functional significance of this specific evolutionary pattern.

Lipo-chitooligosaccharides (LCOs) were originally found as symbiotic signals called Nod Factors (Nod-LCOs) controlling the nodulation of legumes by rhizobia. More recently, LCOs were also found in symbiotic fungi and, more surprisingly, very widely in the kingdom Fungi, including in saprophytic and pathogenic fungi. The LCO-V(C18:1, fucosylated/methyl fucosylated), hereafter called Fung-LCOs, are the LCO structures most commonly found in fungi. This raises the question of how legume plants such as Medicago truncatula can discriminate between Nod-LCOs and Fung-LCOs. To address this question, we performed a genome-wide association study on 173 natural accessions of M. truncatula, using a root branching phenotype and a newly developed local score approach. Both Nod-LCOs and Fung-LCOs stimulated root branching in most accessions, but the root responses to these two types of LCO molecules were not correlated. In addition, the heritability of the root response was higher for Nod-LCOs than for Fung-LCOs. We identified 123 loci for Nod-LCO and 71 for Fung-LCO responses, of which only one was common. This suggests that Nod-LCOs and Fung-LCOs both control root branching but use different molecular mechanisms. The tighter genetic constraint of the root response to Fung-LCOs possibly reflects the ancestral origin of the biological activity of these molecules.

We recently described a regulatory loop, which we termed autoregulation of infection (AOI), by which Sinorhizobium meliloti, a Medicago endosymbiont, downregulates the root susceptibility to secondary infection events via ethylene. AOI is initially triggered by so-far unidentified Medicago nodule signals named signal 1 and signal 1’ whose transduction in bacteroids requires the S. meliloti outer-membrane-associated NsrA receptor protein and the cognate inner-membrane-associated adenylate cyclases, CyaK and CyaD1/D2, respectively. Here, we report on advances in signal 1 identification. Signal 1 activity is widespread as we robustly detected it in Medicago nodule extracts as well as in yeast and bacteria cell extracts. Biochemical analyses indicated a peptidic nature for signal 1 and, together with proteomic analyses, a universally conserved Medicago ribosomal protein of the uL2 family was identified as a candidate signal 1. Specifically, MtRPuL2A (MtrunA17Chr7g0247311) displays a strong signal activity that requires S. meliloti NsrA and CyaK, as endogenous signal 1. We have shown that MtRPuL2A is active in signaling only in a non-ribosomal form. A Medicago truncatula mutant in the major symbiotic transcriptional regulator MtNF-YA1 lacked most signal 1 activity, suggesting that signal 1 is under developmental control. Altogether, our results point to the MtRPuL2A ribosomal protein as the candidate for signal 1. Based on the Mtnf-ya1 mutant, we suggest a link between root infectiveness and nodule development. We discuss our findings in the context of ribosomal protein moonlighting.

Plant -specific lysin-motif receptor-like kinases (LysM-RLKs) are implicated in the perception of N-acetyl glucosamine-containing compounds, some of which are important signal molecules in plant-microbe interactions. Among these, both lipo-chitooligosaccharides (LCOs) and chitooligosaccharides (COs) are proposed as arbuscular mycorrhizal (AM) fungal symbiotic signals. COs can also activate plant defence, although there are scarce data about CO production by pathogens, especially nonfungal pathogens. We tested Medicago truncatula mutants in the LysM-RLK MtLYK9 for their abilities to interact with the AM fungus Rhizophagus irregularis and the oomycete pathogen Aphanomyces euteiches. This prompted us to analyse whether A. euteiches can produce COs. Compared with wild-type plants, Mtlyk9 mutants had fewer infection events and were less colonised by the AM fungus. By contrast, Mtlyk9 mutants were more heavily infected by A. euteiches and showed more disease symptoms. Aphanomyces euteiches was also shown to produce short COs, mainly CO II, but also CO III and CO IV, and traces of CO V, both ex planta and in planta. MtLYK9 thus has a dual role in plant immunity and the AM symbiosis, which raises questions about the functioning and the ancestral origins of such a receptor protein.

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.

Medicago truncatula was proposed, about three decades ago, as a model legume to study the Rhizobium-legume symbiosis. It has now been adopted to study a wide range of biological questions, including various developmental processes (in particular root, symbiotic nodule and seed development), symbiotic (nitrogen-fixing and arbuscular mycorrhizal endosymbioses) and pathogenic interactions, as well as responses to abiotic stress. With a number of tools and resources set up in M. truncatula for omics, genetics and reverse genetics approaches, massive amounts of data have been produced, as well as four genome sequence releases. Many of these data were generated with heterogeneous tools, notably for transcriptomics studies, and are consequently difficult to integrate. This issue is addressed by the LeGOO (for Legume Graph-Oriented Organizer) knowledge base (https://www.legoo.org), which finds the correspondence between the multiple identifiers of the same gene. Furthermore, an important goal of LeGOO is to collect and represent biological information from peer-reviewed publications, whatever the technical approaches used to obtain this information. The information is modeled in a graph-oriented database, which enables flexible representation, with currently over 200,000 relations retrieved from 298 publications. LeGOO also provides the user with mining tools, including links to the Mt5.0 genome browser and associated information (on gene functional annotation, expression, methylome, natural diversity and available insertion mutants), as well as tools to navigate through different model species. LeGOO is, therefore, an innovative database that will be useful to the Medicago and legume community to better exploit the wealth of data produced on this model species.

A complex network of pathways coordinates nodulation and epidermal root hair infection in the symbiotic interaction between rhizobia and legume plants. Whereas nodule formation was known to be autoregulated, it was so far unclear whether a similar control is exerted on the infection process. We assessed the capacity of Medicago plants nodulated by Sinorhizobium meliloti to modulate root susceptibility to secondary bacterial infection or to purified Nod factors in split-root and volatile assays using bacterial and plant mutant combinations. Ethylene implication in this process emerged from gas production measurements, use of a chemical inhibitor of ethylene biosynthesis and of a Medicago mutant affected in ethylene signal transduction. We identified a feedback mechanism that we named AOI (for Autoregulation Of Infection) by which endosymbiotic bacteria control secondary infection thread formation by their rhizospheric peers. AOI involves activation of a cyclic adenosine 3',5'-monophosphate (cAMP) cascade in endosymbiotic bacteria, which decreases both root infectiveness and root susceptibility to bacterial Nod factors. These latter two effects are mediated by ethylene. AOI is a novel component of the complex regulatory network controlling the interaction between Sinorhizobium meliloti and its host plants that emphasizes the implication of endosymbiotic bacteria in fine-tuning the interaction.

LysM receptor-like kinases (LysM-RLKs), which are specific to plants, can control establishment of both the arbuscular mycorrhizal (AM) and the rhizobium-legume (RL) symbioses in response to signal molecules produced, respectively, by the fungal and bacterial symbiotic partners. While most studies on these proteins have been performed in legume species, there are also important findings that demonstrate the roles of LysM-RLKs in controlling symbiosis in non-legume plants. Phylogenomic studies, which have revealed the presence or absence of certain LysM-RLKs among different plant species, have provided insight into the evolutionary mechanisms underlying both the acquisition and the loss of symbiotic properties. The role of a key nodulation LysM-RLK, NFP/NFR5, in legume plants has thus probably been co-opted from an ancestral role in the AM symbiosis, and has been lost in most plant species that have lost the ability to establish the AM or the RL symbiosis. Another LysM-RLK, LYK3/NFR1, that controls the RL symbiosis probably became neo-functionalised following two rounds of gene duplication. Evidence suggests that a third LysM-RLK, LYR3/LYS12, is also implicated in perceiving microbial symbiotic signals, and this protein could have roles in symbiosis and/or plant immunity in different plant species. By focusing on these three LysM-RLKs that are widespread in plants we review their evolutionary history and what this can tell us about the evolution of both the RL and the AM symbioses.

Molecular signals released by microbes at the surface of plant roots and leaves largely determine host responses, notably by triggering either immunity or symbiosis. How these signalling pathways cross-talk upon coincident perception of pathogens and symbionts is poorly described in plants forming symbiosis. Nitrogen fixing symbiotic Rhizobia spp. and arbuscular mycorrhizal fungi produce lipo-chitooligosaccharides (LCOs) to initiate host symbiotic programmes. In Medicago truncatula roots, the perception of LCOs leads to reduced efflux of reactive oxygen species (ROS). By contrast, pathogen perception generally triggers a strong ROS burst and activates defence gene expression. Here we show that incubation of M. truncatula seedlings with culture filtrate (CF) of the legume pathogen Aphanomyces euteiches alone or simultaneously with Sinorhizobium meliloti LCOs, resulted in a strong ROS release. However, this response was completely inhibited if CF was added after pre-incubation of seedlings with LCOs. By contrast, expression of immunity-associated genes in response to CF and disease resistance to A. euteiches remained unaffected by LCO treatment of M. truncatula roots. Our findings suggest that symbiotic plants evolved ROS inhibition response to LCOs to facilitate early steps of symbiosis whilst maintaining a parallel defence mechanisms toward pathogens.