A dual legume-rhizobium transcriptome of symbiotic nodule senescence reveals coordinated plant and bacterial responses.

Senescence determines plant organ lifespan depending on aging and environmental cues. During the endosymbiotic interaction with rhizobia, legume plants develop a specific organ, the root nodule, which houses nitrogen (N)-fixing bacteria. Unlike earlier processes of the legume-rhizobium interaction (nodule formation, N fixation), mechanisms controlling nodule senescence remain poorly understood. To identify nodule senescence-associated genes, we performed a dual plant-bacteria RNA sequencing approach on Medicago truncatula-Sinorhizobium meliloti nodules having initiated senescence either naturally (aging) or following an environmental trigger (nitrate treatment or salt stress). The resulting data allowed the identification of hundreds of plant and bacterial genes differentially regulated during nodule senescence, thus providing an unprecedented comprehensive resource of new candidate genes associated with this process. Remarkably, several plant and bacterial genes related to the cell cycle and stress responses were regulated in senescent nodules, including the rhizobial RpoE2-dependent general stress response. Analysis of selected core nodule senescence plant genes allowed showing that MtNAC969 and MtS40, both homologous to leaf senescence-associated genes, negatively regulate the transition between N fixation and senescence. In contrast, overexpression of a gene involved in the biosynthesis of cytokinins, well-known negative regulators of leaf senescence, may promote the transition from N fixation to senescence in nodules.

Role of Nitric Oxide of Bacterial Origin in the Medicago truncatula-Sinorhizobium meliloti Symbiosis.

Nitric oxide (NO) is a small ubiquitous gaseous molecule that has been found in many host-pathogen interactions. NO has been shown to be part of the defense arsenal of animal cells and more recently of plant cells. To fight this molecular weapon, pathogens have evolved responses consisting of adaptation to NO or degradation of this toxic molecule. More recently, it was shown that NO could also be produced by the pathogen and contributes likewise to the success of the host cell infection. NO is also present during symbiotic interactions. Despite growing knowledge about the role of NO during friendly interactions, data on the specificity of action of NO produced by each partner are scarce, partly due to the multiplicity of NO production systems. In the nitrogen-fixing symbiosis between the soil bacterium Sinorhizobium meliloti and the model legume Medicago truncatula, NO has been detected at all steps of the interaction, where it displays various roles. Both partners contribute to NO production inside the legume root nodules where nitrogen fixation occurs. The study focuses on the role of bacterial NO in this interaction. We used a genetic approach to identify bacterial NO sources in the symbiotic context and to test the phenotype in planta of bacterial mutants affected in NO production. Our results show that only denitrification is a source of bacterial NO in Medicago nodules, giving insight into the role of bacteria-derived NO at different steps of the symbiotic interaction. [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.

Rhizobia: highways to NO.

The interaction between rhizobia and their legume host plants conduces to the formation of specialized root organs called nodules where rhizobia differentiate into bacteroids which fix atmospheric nitrogen to the benefit of the plant. This beneficial symbiosis is of importance in the context of sustainable agriculture as legumes do not require the addition of nitrogen fertilizer to grow. Interestingly, nitric oxide (NO) has been detected at various steps of the rhizobium-legume symbiosis where it has been shown to play multifaceted roles. Both bacterial and plant partners are involved in NO synthesis in nodules. To better understand the role of NO, and in particular the role of bacterial NO, at all steps of rhizobia-legumes interaction, the enzymatic sources of NO have to be elucidated. In this review, we discuss different enzymatic reactions by which rhizobia may potentially produce NO. We argue that there is most probably no NO synthase activity in rhizobia, and that instead the NO2- reductase nirK, which is part of the denitrification pathway, is the main bacterial source of NO. The nitrate assimilation pathway might contribute to NO production but only when denitrification is active. The different approaches to measure NO in rhizobia are also addressed.

Tyrosine Nitration of Flagellins: a Response of Sinorhizobium meliloti to Nitrosative Stress.

Rhizobia are bacteria which can either live as free organisms in the soil or interact with plants of the legume family with, as a result, the formation of root organs called nodules in which differentiated endosymbiotic bacteria fix atmospheric nitrogen to the plant's benefit. In both lifestyles, rhizobia are exposed to nitric oxide (NO) which can be perceived as a signaling or toxic molecule. NO can act at the transcriptional level but can also modify proteins by S-nitrosylation of cysteine or nitration of tyrosine residues. However, only a few molecular targets of NO have been described in bacteria and none of them have been characterized in rhizobia. Here, we examined tyrosine nitration of Sinorhizobium meliloti proteins induced by NO. We found three tyrosine-nitrated proteins in S. meliloti grown under free-living conditions, in response to an NO donor. Two nitroproteins were identified by mass spectrometry and correspond to flagellins A and B. We showed that one of the nitratable tyrosines is essential to flagellin function in motility.IMPORTANCE Rhizobia are found as free-living bacteria in the soil or in interaction with plants and are exposed to nitric oxide (NO) in both environments. NO is known to have many effects on animals, plants, and bacteria where only a few molecular targets of NO have been described so far. We identified flagellin A and B by mass spectrometry as tyrosine-nitrated proteins in Sinorhizobium melilotiin vivo We also showed that one of the nitratable tyrosines is essential to flagellin function in motility. The results enhanced our understanding of NO effects on rhizobia. Identification of bacterial flagellin nitration opens a new possible role of NO in plant-microbe interactions.

The Nitrate Assimilatory Pathway in Sinorhizobium meliloti: Contribution to NO Production.

The interaction between rhizobia and their legume host plants culminates in the formation of specialized root organs called nodules in which differentiated endosymbiotic bacteria (bacteroids) fix atmospheric nitrogen to the benefit of the plant. Interestingly, nitric oxide (NO) has been detected at various steps of the rhizobium-legume symbiosis where it has been shown to play multifaceted roles. It is recognized that both bacterial and plant partners of the Sinorhizobium meliloti-Medicago truncatula symbiosis are involved in NO synthesis in nodules. S. meliloti can also produce NO from nitrate when living as free cells in the soil. S. meliloti does not possess any NO synthase gene in its genome. Instead, the denitrification pathway is often described as the main driver of NO production with nitrate as substrate. This pathway includes the periplasmic nitrate reductase (Nap) which reduces nitrate into nitrite, and the nitrite reductase (Nir) which reduces nitrite into NO. However, additional genes encoding putative nitrate and nitrite reductases (called narB and nirB, respectively) have been identified in the S. meliloti genome. Here we examined the conditions where these genes are expressed, investigated their involvement in nitrate assimilation and NO synthesis in culture and their potential role in planta. We found that narB and nirB are expressed under aerobic conditions in absence of ammonium in the medium and most likely belong to the nitrate assimilatory pathway. Even though these genes are clearly expressed in the fixation zone of legume root nodule, they do not play a crucial role in symbiosis. Our results support the hypothesis that in S. meliloti, denitrification remains the main enzymatic way to produce NO while the assimilatory pathway involving NarB and NirB participates indirectly to NO synthesis by cooperating with the denitrification pathway.

A partner-switching system controls activation of mixed-linkage ß-glucan synthesis by c-di-GMP in Sinorhizobium meliloti.

Sinorhizobium meliloti synthesizes a linear mixed-linkage (1 → 3)(1 → 4)-ß-d-glucan (ML ß-glucan, MLG) in response to high levels of cyclic diguanylate (c-di-GMP). Two proteins BgsA and BgsB are required for MLG synthesis, BgsA being the glucan synthase which is activated upon c-di-GMP binding to its C-terminal domain. Here we report that the product of bgrR (SMb20447) is a diguanylate cyclase (DGC) that provides c-di-GMP for the synthesis of MLG by BgsA. bgrR is the first gene of a hexacistronic bgrRSTUWV operon, likely encoding a partner-switching regulatory network where BgrR is the final target. Using different approaches, we have determined that the products of genes bgrU (containing a putative PP2C serine phosphatase domain) and bgrW (with predicted kinase effector domain), modulate the phosphorylation status and the activity of the STAS domain protein BgrV. We propose that unphosphorylated BgrV inhibits BgrR DGC activity, perhaps through direct protein-protein interactions as established for other partner switchers. A bgrRSTUWV operon coexists with MLG structural bgsBA genes in many rhizobial genomes but is also present in some MLG non-producers, suggesting a role of this partner-switching system in other processes besides MLG biosynthesis.

Nitric oxide in plants: pro- or anti-senescence.

Senescence is a regulated process of tissue degeneration that can affect any plant organ and consists of the degradation and remobilization of molecules to other growing tissues. Senescent organs display changes at the microscopic level as well as modifications to internal cellular structure and differential gene expression. A large number of factors influencing senescence have been described including age, nutrient supply, and environmental interactions. Internal factors such as phytohormones also affect the timing of leaf senescence. A link between the senescence process and the production of nitric oxide (NO) in senescing tissues has been known for many years. Remarkably, this link can be either a positive or a negative correlation depending upon the organ. NO can be both a signaling or a toxic molecule and is known to have multiple roles in plants; this review considers the duality of NO roles in the senescence process of two different plant organs, namely the leaves and root nodules.

Bacterial NHEJ: a never ending story.

Double-strand breaks (DSBs) are the most detrimental DNA damage encountered by bacterial cells. DBSs can be repaired by homologous recombination thanks to the availability of an intact DNA template or by Non-Homologous End Joining (NHEJ) when no intact template is available. Bacterial NHEJ is performed by sets of proteins of growing complexity from Bacillus subtilis and Mycobacterium tuberculosis to Streptomyces and Sinorhizobium meliloti. Here, we discuss the contribution of these models to the understanding of the bacterial NHEJ repair mechanism as well as the involvement of NHEJ partners in other DNA repair pathways. The importance of NHEJ and of its complexity is discussed in the perspective of regulation through the biological cycle of the bacteria and in response to environmental stimuli. Finally, we consider the role of NHEJ in genome evolution, notably in horizontal gene transfer.

Stress-inducible NHEJ in bacteria: function in DNA repair and acquisition of heterologous DNA.

DNA double-strand breaks (DSB) in bacteria can be repaired by non-homologous end-joining (NHEJ), a two-component system relying on Ku and LigD. While performing a genetic characterization of NHEJ in Sinorhizobium meliloti, a representative of bacterial species encoding several Ku and LigD orthologues, we found that at least two distinct functional NHEJ repair pathways co-exist: one is dependent on Ku2 and LigD2, while the other depends on Ku3, Ku4 and LigD4. Whereas Ku2 likely acts as canonical bacterial Ku homodimers, genetic evidences suggest that Ku3-Ku4 form eukaryotic-like heterodimers. Strikingly, we found that the efficiency of both NHEJ systems increases under stress conditions, including heat and nutrient starvation. We found that this stimulation results from the transcriptional up-regulation of the ku and/or ligD genes, and that some of these genes are controlled by the general stress response regulator RpoE2. Finally, we provided evidence that NHEJ not only repairs DSBs, but can also capture heterologous DNA fragments into genomic breaks. Our data therefore suggest that NHEJ could participate to horizontal gene transfer from distantly related species, bypassing the need of homology to integrate exogenous DNA. This supports the hypothesis that NHEJ contributes to evolution and adaptation of bacteria under adverse environmental conditions.

DNA double-strand break repair is involved in desiccation resistance of Sinorhizobium meliloti, but is not essential for its symbiotic interaction with Medicago truncatula.

The soil bacterium Sinorhizobium meliloti, a nitrogen-fixing symbiont of legume plants, is exposed to numerous stress conditions in nature, some of which cause the formation of harmful DNA double-strand breaks (DSBs). In particular, the reactive oxygen species (ROS) and the reactive nitrogen species (RNS) produced during symbiosis, and the desiccation occurring in dry soils, are conditions which induce DSBs. Two major systems of DSB repair are known in S. meliloti: homologous recombination (HR) and non-homologous end-joining (NHEJ). However, their role in the resistance to ROS, RNS and desiccation has never been examined in this bacterial species, and the importance of DSB repair in the symbiotic interaction has not been properly evaluated. Here, we constructed S. meliloti strains deficient in HR (by deleting the recA gene) or in NHEJ (by deleting the four ku genes) or both. Interestingly, we observed that ku and/or recA genes are involved in S. meliloti resistance to ROS and RNS. Nevertheless, an S. meliloti strain deficient in both HR and NHEJ was not altered in its ability to establish and maintain an efficient nitrogen-fixing symbiosis with Medicago truncatula, showing that rhizobial DSB repair is not essential for this process. This result suggests either that DSB formation in S. meliloti is efficiently prevented during symbiosis or that DSBs are not detrimental for symbiosis efficiency. In contrast, we found for the first time that both recA and ku genes are involved in S. meliloti resistance to desiccation, suggesting that DSB repair could be important for rhizobium persistence in the soil.

Sinorhizobium meliloti Controls Nitric Oxide-Mediated Post-Translational Modification of a Medicago truncatula Nodule Protein.

Nitric oxide (NO) is involved in various plant-microbe interactions. In the symbiosis between soil bacterium Sinorhizobium meliloti and model legume Medicago truncatula, NO is required for an optimal establishment of the interaction but is also a signal for nodule senescence. Little is known about the molecular mechanisms responsible for NO effects in the legume-rhizobium interaction. Here, we investigate the contribution of the bacterial NO response to the modulation of a plant protein post-translational modification in nitrogen-fixing nodules. We made use of different bacterial mutants to finely modulate NO levels inside M. truncatula root nodules and to examine the consequence on tyrosine nitration of the plant glutamine synthetase, a protein responsible for assimilation of the ammonia released by nitrogen fixation. Our results reveal that S. meliloti possesses several proteins that limit inactivation of plant enzyme activity via NO-mediated post-translational modifications. This is the first demonstration that rhizobia can impact the course of nitrogen fixation by modulating the activity of a plant protein.

A putative bifunctional histidine kinase/phosphatase of the HWE family exerts positive and negative control on the Sinorhizobium meliloti general stress response.

The EcfG-type sigma factor RpoE2 is the regulator of the general stress response in Sinorhizobium meliloti. RpoE2 activity is negatively regulated by two NepR-type anti-sigma factors (RsiA1/A2), themselves under the control of two anti-anti-sigma factors (RsiB1/B2) belonging to the PhyR family of response regulators. The current model of RpoE2 activation suggests that in response to stress, RsiB1/B2 are activated by phosphorylation of an aspartate residue in their receiver domain. Once activated, RsiB1/B2 become able to interact with the anti-sigma factors and release RpoE2, which can then associate with the RNA polymerase to transcribe its target genes. The purpose of this work was to identify and characterize proteins involved in controlling the phosphorylation status of RsiB1/B2. Using in vivo approaches, we show that the putative histidine kinase encoded by the rsiC gene (SMc01507), located downstream from rpoE2, is able to both positively and negatively regulate the general stress response. In addition, our data suggest that the negative action of RsiC results from inhibition of RsiB1/B2 phosphorylation. From these observations, we propose that RsiC is a bifunctional histidine kinase/phosphatase responsible for RsiB1/B2 phosphorylation or dephosphorylation in the presence or absence of stress, respectively. Two proteins were previously proposed to control PhyR phosphorylation in Caulobacter crescentus and Sphingomonas sp. strain FR1. However, these proteins contain a Pfam:HisKA_2 domain of dimerization and histidine phosphotransfer, whereas S. meliloti RsiC harbors a Pfam:HWE_HK domain instead. Therefore, this is the first report of an HWE_HK-containing protein controlling the general stress response in Alphaproteobacteria.

An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing.

Rhizobium-induced root nodules are specialized organs for symbiotic nitrogen fixation. Indeterminate-type nodules are formed from an apical meristem and exhibit a spatial zonation which corresponds to successive developmental stages. To get a dynamic and integrated view of plant and bacterial gene expression associated with nodule development, we used a sensitive and comprehensive approach based upon oriented high-depth RNA sequencing coupled to laser microdissection of nodule regions. This study, focused on the association between the model legume Medicago truncatula and its symbiont Sinorhizobium meliloti, led to the production of 942 million sequencing read pairs that were unambiguously mapped on plant and bacterial genomes. Bioinformatic and statistical analyses enabled in-depth comparison, at a whole-genome level, of gene expression in specific nodule zones. Previously characterized symbiotic genes displayed the expected spatial pattern of expression, thus validating the robustness of our approach. We illustrate the use of this resource by examining gene expression associated with three essential elements of nodule development, namely meristem activity, cell differentiation and selected signaling processes related to bacterial Nod factors and redox status. We found that transcription factor genes essential for the control of the root apical meristem were also expressed in the nodule meristem, while the plant mRNAs most enriched in nodules compared with roots were mostly associated with zones comprising both plant and bacterial partners. The data, accessible on a dedicated website, represent a rich resource for microbiologists and plant biologists to address a variety of questions of both fundamental and applied interest.

Which role for nitric oxide in symbiotic N2-fixing nodules: toxic by-product or useful signaling/metabolic intermediate?

The interaction between legumes and rhizobia leads to the establishment of a symbiotic relationship characterized by the formation of new organs called nodules, in which bacteria have the ability to fix atmospheric nitrogen (N2) via the nitrogenase activity. Significant nitric oxide (NO) production was evidenced in the N2-fixing nodules suggesting that it may impact the symbiotic process. Indeed, NO was shown to be a potent inhibitor of nitrogenase activity and symbiotic N2 fixation. It has also been shown that NO production is increased in hypoxic nodules and this production was supposed to be linked - via a nitrate/NO respiration process - with improved capacity of the nodules to maintain their energy status under hypoxic conditions. Other data suggest that NO might be a developmental signal involved in the induction of nodule senescence. Hence, the questions were raised of the toxic effects versus signaling/metabolic functions of NO, and of the regulation of NO levels compatible with nitrogenase activity. The present review analyses the different roles of NO in functioning nodules, and discusses the role of plant and bacterial (flavo)hemoglobins in the control of NO level in nodules.

Control of NO level in rhizobium-legume root nodules: not only a plant globin story.

Nitric oxide (NO ) is a gaseous signaling molecule which plays both regulatory and defense roles in animals and plants. In the symbiosis between legumes and rhizobia, NO has been shown to be involved in bacterial infection and nodule development steps as well as in mature nodule functioning. We recently showed that an increase in NO level inside Medicago truncatula root nodules also could trigger premature nodule senescence. Here we discuss the importance of the bacterial Sinorhizobium meliloti flavohemoglobin to finely tune the NO level inside nodules and further, we demonstrate that S. meliloti possesses at least two non redundant ways to control NO and that both systems are necessary to maintain efficient nitrogen fixing activity.

Next-generation annotation of prokaryotic genomes with EuGene-P: application to Sinorhizobium meliloti 2011.

The availability of next-generation sequences of transcripts from prokaryotic organisms offers the opportunity to design a new generation of automated genome annotation tools not yet available for prokaryotes. In this work, we designed EuGene-P, the first integrative prokaryotic gene finder tool which combines a variety of high-throughput data, including oriented RNA-Seq data, directly into the prediction process. This enables the automated prediction of coding sequences (CDSs), untranslated regions, transcription start sites (TSSs) and non-coding RNA (ncRNA, sense and antisense) genes. EuGene-P was used to comprehensively and accurately annotate the genome of the nitrogen-fixing bacterium Sinorhizobium meliloti strain 2011, leading to the prediction of 6308 CDSs as well as 1876 ncRNAs. Among them, 1280 appeared as antisense to a CDS, which supports recent findings that antisense transcription activity is widespread in bacteria. Moreover, 4077 TSSs upstream of protein-coding or non-coding genes were precisely mapped providing valuable data for the study of promoter regions. By looking for RpoE2-binding sites upstream of annotated TSSs, we were able to extend the S. meliloti RpoE2 regulon by ∼3-fold. Altogether, these observations demonstrate the power of EuGene-P to produce a reliable and high-resolution automatic annotation of prokaryotic genomes.

Sinorhizobium meliloti sigma factors RpoE1 and RpoE4 are activated in stationary phase in response to sulfite.

Rhizobia are soil bacteria able to establish a nitrogen-fixing symbiosis with legume plants. Both in soil and in planta, rhizobia spend non-growing periods resembling the stationary phase of in vitro-cultured bacteria. The primary objective of this work was to better characterize gene regulation in this biologically relevant growth stage in Sinorhizobium meliloti. By a tap-tag/mass spectrometry approach, we identified five sigma factors co-purifying with the RNA polymerase in stationary phase: the general stress response regulator RpoE2, the heat shock sigma factor RpoH2, and three extra-cytoplasmic function sigma factors (RpoE1, RpoE3 and RpoE4) belonging to the poorly characterized ECF26 subgroup. We then showed that RpoE1 and RpoE4 i) are activated upon metabolism of sulfite-generating compounds (thiosulfate and taurine), ii) display overlapping regulatory activities, iii) govern a dedicated sulfite response by controlling expression of the sulfite dehydrogenase SorT, iv) are activated in stationary phase, likely as a result of endogenous sulfite generation during bacterial growth. We showed that SorT is required for optimal growth of S. meliloti in the presence of sulfite, suggesting that the response governed by RpoE1 and RpoE4 may be advantageous for bacteria in stationary phase either by providing a sulfite detoxification function or by contributing to energy production through sulfite respiration. This paper therefore reports the first characterization of ECF26 sigma factors, the first description of sigma factors involved in control of sulphur metabolism, and the first indication that endogenous sulfite may act as a signal for regulation of gene expression upon entry of bacteria in stationary phase.

Nitric oxide (NO): a key player in the senescence of Medicago truncatula root nodules.

Nitric oxide (NO) is a signalling and defence molecule involved in diverse plant developmental processes, as well as in the plant response to pathogens. NO has also been detected at different steps of the symbiosis between legumes and rhizobia. NO is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction, but little is known about the role of NO in mature nodules. Here, we investigate the role of NO in the late steps of symbiosis. Genetic and pharmacological approaches were conducted to modulate the NO level inside root nodules, and their effects on nitrogen fixation and root nodule senescence were monitored. An increase in endogenous NO levels led to a decrease in nitrogen fixation and early nodule senescence, characterized by cytological modifications of the nodule structure and the early expression of a specific senescence marker. By contrast, a decrease in NO levels led to a delay in nodule senescence. Together, our results strongly suggest that NO is a signal in developmental as well as stress-induced nodule senescence. In addition, this work demonstrates the pivotal role of the bacterial NO detoxification response in the prevention of early nodule senescence, and hence the maintenance of efficient symbiosis.

Nitric oxide in legume-rhizobium symbiosis.

Nitric oxide (NO) is a gaseous signaling molecule with a broad spectrum of regulatory functions in plant growth and development. NO has been found to be involved in various pathogenic or symbiotic plant-microbe interactions. During the last decade, increasing evidence of the occurrence of NO during legume-rhizobium symbioses has been reported, from early steps of plant-bacteria interaction, to the nitrogen-fixing step in mature nodules. This review focuses on recent advances on NO production and function in nitrogen-fixing symbiosis. First, the potential plant and bacterial sources of NO, including NO synthase-like, nitrate reductase or electron transfer chains of both partners, are presented. Then responses of plant and bacterial cells to the presence of NO are presented in the context of the N(2)-fixing symbiosis. Finally, the roles of NO as either a regulatory signal of development, or a toxic compound with inhibitory effects on nitrogen fixation, or an intermediate involved in energy metabolism, during symbiosis establishment and nodule functioning are discussed.

Nitric oxide is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiosis.

Nitric oxide (NO) is a gaseous molecule that participates in numerous plant signalling pathways. It is involved in plant responses to pathogens and development processes such as seed germination, flowering and stomatal closure. Using a permeable NO-specific fluorescent probe and a bacterial reporter strain expressing the lacZ gene under the control of a NO-responsive promoter, we detected NO production in the first steps, during infection threads growth, of the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction. Nitric oxide was also detected, by confocal microscopy, in nodule primordia. Depletion of NO caused by cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide), an NO scavenger, resulted in a significant delay in nodule appearance. The overexpression of a bacterial hmp gene, encoding a flavohaemoglobin able to scavenge NO, under the control of a nodule-specific promoter (pENOD20) in transgenic roots, led to the same phenotype. The NO scavenging resulting from these approaches provoked the downregulation of plant genes involved in nodule development, such as MtCRE1 and MtCCS52A. Furthermore, an Hmp-overexpressing S. meliloti mutant strain was found to be less competitive than the wild type in the nodulation process. Taken together, these results indicate that NO is required for an optimal establishment of the M. truncatula-S. meliloti symbiotic interaction.