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.

Biological nitrogen (N2) fixation between legumes and rhizobia is the result of a symbiosis characterized by the formation of a new organ, the nodule, in which the plants house and feed the bacteria. Oxygen (O2) concentration inside the nodule is very low (of the order of a few tens of nanomolar). The nitrogenase responsible for the reduction of atmospheric N2 to ammonia is irreversibly inhibited by traces of O2, while O2 is required for the overall process of N2 fixation which consumes high amounts of energy and reducing power. How is this paradox solved? The setting up of a physical and chemical O2 diffusion barrier, including the synthesis of numerous leghemoglobins, a class of hemoproteins with a very high O2 absorption capacity, was evidenced. However, why are so many leghemoglobin genes present when they appear to be mainly expressed in the same nodule zone? Furthermore, why do the bacterial symbionts contain multiple O2 sensors despite the existence of a very low O2 tension inside the nodule? On the other hand, reactive oxygen species (ROS), which play important metabolic and signaling roles in symbiosis, appear to act as Janus molecules, exhibiting opposite effects throughout symbiosis establishment and nodule life. We aim to provide possible answers to the questions asked and to highlight the dual roles of ROS in nodule development, functioning, and senescence.

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.

The establishment of the Legume-Rhizobia symbiosis is generally dependent on the production of rhizobial lipochitooligosaccharidic Nod factors (NFs) and their perception by plant Lysin Motif Receptor-Like Kinases (LysM-RLKs). In this study, we characterized a cluster of LysM-RLK genes implicated in strain-specific recognition in two highly divergent and widely-studied Medicago truncatula genotypes, A17 and R108. We then used reverse genetic approaches and biochemical analyses to study the function of selected genes in the clusters and the ability of their encoded proteins to bind NFs. Our study has revealed that the LYK cluster exhibits a high degree of variability among M. truncatula genotypes, which in A17 and R108 includes recent recombination events within the cluster and a transposon insertion in A17. The essential role of LYK3 in nodulation in A17 is not conserved in R108 despite similar sequences and good nodulation expression profiles. Although, LYK2, LYK5 and LYK5bis are not essential for nodulation of the two genotypes, some evidence points to accessory roles in nodulation, but not through high-affinity NF binding. This work shows that recent evolution in the LYK cluster provides a source of variation for nodulation, and potential robustness of signaling through genetic redundancy.

Rhizobial lipochitooligosaccharidic Nod factors (NFs), specified by nod genes, are the primary determinants of host specificity in the legume-Rhizobia symbiosis. We examined the nodulation ability of Medicago truncatula cv Jemalong A17 and M. truncatula ssp. tricycla R108 with the Sinorhizobium meliloti nodF/nodL mutant, which produces modified NFs. We then applied genetic and functional approaches to study the genetic basis and mechanism of nodulation of R108 by this mutant. We show that the nodF/nodL mutant can nodulate R108 but not A17. Using genomics and reverse genetics, we identified a newly evolved, chimeric LysM receptor-like kinase gene in R108, LYK2bis, which is responsible for the phenotype and can allow A17 to gain nodulation with the nodF/nodL mutant. We found that LYK2bis is involved in nodulation by mutants producing nonO-acetylated NFs and interacts with the key receptor protein NFP. Many, but not all, natural S. meliloti and S. medicae strains tested require LYK2bis for efficient nodulation of R108. Our findings reveal that a newly evolved gene in R108, LYK2bis, extends nodulation specificity to mutants producing nonO-acetylated NFs and is important for nodulation by many natural Sinorhizobia. Evolution of this gene may present an adaptive advantage to allow nodulation by a greater variety of strains.

The model legume Medicago truncatula possesses a single outward Shaker K+ channel, while Arabidopsis thaliana possesses two channels of this type, named SKOR and GORK, the former having been shown to play a major role in K+ secretion into the xylem sap in the root vasculature and the latter to mediate the efflux of K+ across the guard cell membrane upon stomatal closure. Here we show that the expression pattern of the single M. truncatula outward Shaker channel, which has been named MtGORK, includes the root vasculature, guard cells and root hairs. As shown by patch-clamp experiments on root hair protoplasts, besides the Shaker-type slowly-activating outwardly-rectifying K+ conductance encoded by MtGORK , a second K+-permeable conductance, displaying fast activation and weak rectification, can be expressed by M. truncatula . A KO mutation resulting in absence of MtGORK activity is shown to weakly reduce K+ translocation to shoots, and only in plants engaged in rhizobial symbiosis, but to strongly affect the control of stomatal aperture and transpitational water loss. In legumes, the early electrical signaling pathway triggered by Nod Factor perception is known to comprise a short transient depolarization of the root hair plasma membrane. In absence of MtGORK functional expression, while the rate of the membrane repolarization is shown to be decreased by about 3 times, this defect is without any consequence on infection thread development and nodule production, indicating that the plant capacity to engage rhizobial symbiosis does not require integrity of the early electrical signaling events.

Soil salinity is one of the most important abiotic stresses affecting plant growth. In legumes, symbiotic nitrogen fixation in nodules is affected by salt stress, and salinity tolerance is variable among species. Genes from the High affinity K+ Transporter (HKT) family are known to play crucial roles in salt stress tolerance in different plant species. In legumes these transporters are still very poorly characterized.. Here we study the HKT transporter family from the model legume Medicago trunacatula , which is moderately tolerant to salinity. The genome of this species comprises five HKT genes, hereafter named MtHKT1;1 to MtHKT1;5 . Phylogenetic analysis indicated that the MtHKT polypeptides belong to HKT subfamily 1. Three members ( MtHKT1;2, MtHKT1;4 and MtHKT1;5 ) of the Medicago truncatula family were cloned and expressed in Xenopus oocytes. Their electrophysiological properties revealed a permeability 10 times higher for Na+ than for K+ and varying rectification properties. Expression analyses of the three MtHKT genes under different biotic and abiotic conditions suggested that MtHKT1;5 is the main transporter from this family in the root, the three genes sharing a decrease of expression in drought and salt stress conditions in non inoculated plants as well as plants inoculated with rhizobia. In the shoot, the three MtHKT would be present at similar levels independently on the applied stresses. Based on biomass and ion content analysis, the nodule appeared as the most sensitive organ to the applied salt and drought stresses. The level of expression of the three MtHKT genes was strongly decreased by both stresses in the nodule.

The interaction between legumes and bacteria of rhizobia type results in a beneficial symbiotic relationship characterized by the formation of new root organs, called nodules. Within these nodules the bacteria, released in plant cells, differentiate into bacteroids and fix atmospheric nitrogen through the nitrogenase activity. This mutualistic interaction has evolved sophisticated signaling networks to allow rhizobia entry, colonization, bacteroid differentiation and persistence in nodules. Nodule cysteine rich (NCR) peptides, reactive oxygen species (ROS), reactive nitrogen species (RNS), and toxin-antitoxin (TA) modules produced by the host plants or bacterial microsymbionts have a major role in the control of the symbiotic interaction. These molecules described as weapons in pathogenic interactions have evolved to participate to the intracellular bacteroid accommodation by escaping control of plant innate immunity and adapt the functioning of the nitrogen-fixation to environmental signalling cues.

Climate change is increasingly impacting the water deficit over the world. Because of drought and the high pressure of the rising human population, water is becoming a scarce and expensive commodity, especially in developing countries. The identification of crops presenting a higher acclimation to drought stress is thus an important objective in agriculture. The present investigation aimed to assess the adaptation of three Vicia faba genotypes, Aguadulce (AD), Luz d'Otonio (LO) and Reina Mora (RM) to water deficit. Multiple physiological and biochemical parameters were used to analyse the response of the three genotypes to two soil water contents (80% and 40% of field capacity). A significant lower decrease in shoot, root and nodule dry weight was observed for AD compared to LO and RM. The better growth performance of AD was correlated to higher carbon and nitrogen content than in LO and RM under water deficit. Leaf parameters such as relative water content, mass area, efficiency of photosystem II and chlorophyll and carotenoid content were significantly less affected in AD than in LO and RM. Significantly higher accumulation of proline was correlated to the higher performance of AD compared to LO and RM. Additionally, the better growth of AD genotype was related to an important mobilisation of antioxidant enzyme activities such as ascorbate peroxidase and catalase. Taken together, these results allow us to suggest that AD is a water deficit tolerant genotype compared to LO and RM. Our multiple physiological and biochemical analyses show that nitrogen content, leaf proline accumulation, reduced leaf hydrogen peroxide accumulation and leaf antioxidant enzymatic activities (ascorbate peroxidase, guaiacol peroxidase, catalase and polyphenol oxidase) are potential biological markers useful to screen for water deficit resistant Vicia faba genotypes.

Main conclusion Our study demonstrated that the NAPDH oxidase gene MtRbohE is expressed in arbusculated cells and plays a role in arbuscule development. Plant NADPH oxidases, known as respiratory burst oxidase homologs (RBOH), belong to a multigenic family that plays an important role in the regulation of plant development and responses to biotic and abiotic stresses. In this study, we monitored the expression profiles of five Rboh genes ( MtRbohA, MtRbohB, MtRbohE, MtRbohG, MtRbohF ) in the roots of the model species Medicago truncatula upon colonization by arbuscular mycorrhizal fungi. A complementary cellular and molecular approach was used to monitor changes in mRNA abundance and localize transcripts in different cell types from mycorrhizal roots. Rboh transcript levels did not drastically change in total RNA extractions from whole mycorrhizal and non-mycorrhizal roots. Nevertheless, the analysis of laser microdissected cells and Agrobacterium rhizogenes -transformed roots expressing a GUS transcriptional fusion construct highlighted the MtRbohE expression in arbuscule-containing cells. Furthermore, the down regulation of MtRbohE by an RNAi approach generated an altered colonization pattern in the root cortex, when compared to control roots, with fewer arbuscules and multiple penetration attempts. Altogether our data indicate a transient up-regulation of MtRbohE expression in cortical cells colonized by arbuscules and suggest a role for MtRbohE in arbuscule accommodation within cortical cells.

Plant NADPH oxidases are the major source of reactive oxygen species (ROS) that plays key roles as both signal and stressor in several plant processes, including defense responses against pathogens. ROS accumulation in root cells during arbuscular mycorrhiza (AM) development has raised the interest in understanding how ROS-mediated defense programs are modulated during the establishment of this mutualistic interaction. We have recently analyzed the expression pattern of 5 NADPH oxidase (also called RBOH) encoding genes in Medicago truncatula, showing that only one of them (MtRbohE) is specifically upregulated in arbuscule-containing cells. In line with this result, RNAi silencing of MtRbohE generated a strong alteration in root colonization, with a significant reduction in the number of arbusculated cells. On this basis, we propose that MtRBOHE-mediated ROS production plays a crucial role in the intracellular accommodation of arbuscules.

The symbiotic interaction between legumes and nitrogen-fixing rhizobium bacteria leads to the formation of a new organ, the nodule. Early steps of the interaction are characterized by the production of bacterial Nod factors, the reorientation of root-hair tip growth, the formation of an infection thread (IT) in the root hair, and the induction of cell division in inner cortical cells of the root, leading to a nodule primordium formation. Reactive oxygen species (ROS) and nitric oxide (NO) have been detected in early steps of the interaction. ROS/NO are determinant signals to arbitrate the specificity of this mutualistic association and modifications in their content impair the development of the symbiotic association. The decrease of ROS level prevents root hair curling and ITs formation, and that of NO conducts to delayed nodule formation. In root hairs, NADPH oxidases were shown to produce ROS which could be involved in the hair tip growth process. The use of enzyme inhibitors suggests that nitrate reductase and NO synthase-like enzymes are the main route for NO production during the early steps of the interaction. Transcriptomic analyses point to the involvement of ROS and NO in the success of the infection process, the induction of early nodulin gene expression, and the repression of plant defense, thereby favoring the establishment of the symbiosis. The occurrence of an interplay between ROS and NO was further supported by the finding of both S-sulfenylated and S-nitrosylated proteins during early symbiotic interaction, linking ROS/NO production to a redox-based regulation of the symbiotic process.

Root hairs are involved in water and nutrient uptake, and thereby in plant autotrophy. In legumes, they also play a crucial role in establishment of rhizobial symbiosis. To obtain a holistic view of Medicago truncatula genes expressed in root hairs and of their regulation during the first hours of the engagement in rhizobial symbiotic interaction, a high throughput RNA sequencing on isolated root hairs from roots challenged or not with lipochitooligosaccharides Nod factors (NF) for 4 or 20 h was carried out. This provided a repertoire of genes displaying expression in root hairs, responding or not to NF, and specific or not to legumes. In analyzing the transcriptome dataset, special attention was paid to pumps, transporters, or channels active at the plasma membrane, to other proteins likely to play a role in nutrient ion uptake, NF electrical and calcium signaling, control of the redox status or the dynamic reprogramming of root hair transcriptome induced by NF treatment, and to the identification of papilionoid legume-specific genes expressed in root hairs. About 10% of the root hair expressed genes were significantly up- or down-regulated by NF treatment, suggesting their involvement in remodeling plant functions to allow establishment of the symbiotic relationship. For instance, NF-induced changes in expression of genes encoding plasma membrane transport systems or disease response proteins indicate that root hairs reduce their involvement in nutrient ion absorption and adapt their immune system in order to engage in the symbiotic interaction. It also appears that the redox status of root hair cells is tuned in response to NF perception. In addition, 1176 genes that could be considered as "papilionoid legume-specific" were identified in the M. truncatula root hair transcriptome, from which 141 were found to possess an ortholog in every of the six legume genomes that we considered, suggesting their involvement in essential functions specific to legumes. This transcriptome provides a valuable resource to investigate root hair biology in legumes and the roles that these cells play in rhizobial symbiosis establishment. These results could also contribute to the long-term objective of transferring this symbiotic capacity to non-legume plants.

Reactive oxygen species (ROS), particularly hydrogen peroxide (H(2)O(2)), play an important role in signalling in various cellular processes. The involvement of H(2)O(2) in the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction raises questions about its effect on gene expression. A transcriptome analysis was performed on inoculated roots of M. truncatula in which ROS production was inhibited with diphenylene iodonium (DPI). In total, 301 genes potentially regulated by ROS content were identified 2 d after inoculation. These genes included MtSpk1, which encodes a putative protein kinase and is induced by exogenous H(2)O(2) treatment. MtSpk1 gene expression was also induced by nodulation factor treatment. MtSpk1 transcription was observed in infected root hair cells, nodule primordia and the infection zone of mature nodules. Analysis with a fluorescent protein probe specific for H(2)O(2) showed that MtSpk1 expression and H(2)O(2) were similarly distributed in the nodule infection zone. Finally, the establishment of symbiosis was impaired by MtSpk1 downregulation with an artificial micro-RNA. Several genes regulated by H(2)O(2) during the establishment of rhizobial symbiosis were identified. The involvement of MtSpk1 in the establishment of the symbiosis is proposed.

Arbuscular mycorrhizal (AM) symbiosis is stimulated by phosphorus (P) limitation and contributes to P and nitrogen (N) acquisition. However, the effects of combined P and N limitation on AM formation are largely unknown. Medicago truncatula plants were cultivated in the presence or absence of Rhizophagus irregularis (formerly Glomus intraradices) in P-limited (LP), N-limited (LN) or combined P- and N-limited (LPN) conditions, and compared with plants grown in sufficient P and N. The highest AM formation was observed in LPN, linked to systemic signaling by the plant nutrient status. Plant free phosphate concentrations were higher in LPN than in LP, as a result of cross-talk between P and N. Transcriptome analyses suggest that LPN induces the activation of NADPH oxidases in roots, concomitant with an altered profile of plant defense genes and a coordinate increase in the expression of genes involved in the methylerythritol phosphate and isoprenoid-derived pathways, including strigolactone synthesis genes. Taken together, these results suggest that low P and N fertilization systemically induces a physiological state of plants favorable for AM symbiosis despite their higher P status. Our findings highlight the importance of the plant nutrient status in controlling plant-fungus interaction.

Leguminous biological nitrogen fixation (BNF) is very sensitive to environmental fluctuations. It is still contentious how BNF is regulated under stress conditions. The local or systemic control of BNF and the role played by reactive oxygen species (ROS) in such regulation have still not been elucidated completely. Cadmium, which belongs to the so-called heavy metals, is one of the most toxic substances released into the environment. The mechanisms involved in Cd toxicity are still not completely understood but the overproduction of ROS is one of its characteristic symptoms. In this work, we used a split-root system approach to study nodule BNF and the antioxidant machinery's response to the application of a mild Cd treatment on one side of a nodulated Medicago truncatula root system. Cd induced the majority of nodule antioxidants without generating any oxidative damage. Cd treatment also provoked BNF inhibition exclusively in nodules directly exposed to Cd, without provoking any effect on plant shoot biomass or chlorophyll content. The overall data suggest that the decline in BNF was not due to a generalized breakdown of the plant but to control exerted through leghaemoglobin/oxygen availability, affecting nitrogenase function.