Adaptation of Rhizobium leguminosarum sv. trifolii strains to low temperature stress in both free-living stage and during symbiosis with clover.

Legume-rhizobial symbiosis plays an important role in agriculture and ecological restoration. This process occurs within special new structures, called nodules, formed mainly on legume roots. Soil bacteria, commonly known as rhizobia, fix atmospheric dinitrogen, converting it into a form that can be assimilated by plants. Various environmental factors, including a low temperature, have an impact on the symbiotic efficiency. Nevertheless, the effect of temperature on the phenotypic and symbiotic traits of rhizobia has not been determined in detail to date. Therefore, in this study, the influence of temperature on different cell surface and symbiotic properties of rhizobia was estimated. In total, 31 Rhizobium leguminosarum sv. trifolii strains isolated from root nodules of red clover plants growing in the subpolar and temperate climate regions, which essentially differ in year and day temperature profiles, were chosen for this analysis. Our results showed that temperature has a significant effect on several surface properties of rhizobial cells, such as hydrophobicity, aggregation, and motility. Low temperature also stimulated EPS synthesis and biofilm formation in R. leguminosarum sv. trifolii. This extracellular polysaccharide is known to play an important protective role against different environmental stresses. The strains produced large amounts of EPS under tested temperature conditions that facilitated adherence of rhizobial cells to different surfaces. The high adaptability of these strains to cold stress was also confirmed during symbiosis. Irrespective of their climatic origin, the strains proved to be highly effective in attachment to legume roots and were efficient microsymbionts of clover plants. However, some diversity in the response to low temperature stress was found among the strains. Among them, M16 and R137 proved to be highly competitive and efficient in nodule occupancy and biomass production; thus, they can be potential yield-enhancing inoculants of legumes.

CNGC15-DMI1 Gating in Nuclear Calcium Signaling: Opening New Questions and Closing Controversies.

Nuclear Ca²âº signaling is crucial for symbiotic interactions between legumes and beneficial microbes, such as rhizobia and arbuscular mycorrhizal fungi. Key to generating repetitive nuclear Ca²âº oscillations are the ion channels DMI1 and CNGC15. Despite over 20 years of research on symbiotic nuclear Ca²âº spiking, important questions remain, including the exact function of the DMI1 channel. This review highlights recent developments that have filled knowledge gaps regarding the regulation of CNGC15 and its interplay with DMI1. We also explore new insights into the evolutionary conservation of DMI1-induced symbiotic nuclear Ca²âº oscillations and the roles of CNGC15 and DMI1 beyond symbiosis, such as in nitrate signaling, and discuss new questions this raises. As we delve deeper into the regulatory mechanisms and evolutionary history of these ion channels, we move closer to fully understanding the roles of nuclear Ca²âº signaling in plant life.

GA dynamics governing nodulation revealed using GIBBERELLIN PERCEPTION SENSOR 2 in Medicago truncatula lateral organs.

During nutrient scarcity, plants can adapt their developmental strategy to maximize their chance of survival. Such plasticity in development is underpinned by hormonal regulation, which mediates the relationship between environmental cues and developmental outputs. In legumes, endosymbiosis with nitrogen fixing bacteria (rhizobia) is a key adaptation for supplying the plant with nitrogen in the form of ammonium. Rhizobia are housed in lateral root-derived organs termed nodules that maintain an environment conducive to Nitrogenase in these bacteria. Several phytohormones are important for regulating the formation of nodules, with both positive and negative roles proposed for gibberellin (GA). In this study, we determine the cellular location and function of bioactive GA during nodule organogenesis using a genetically-encoded second generation GA biosensor, GIBBERELLIN PERCEPTION SENSOR 2 in Medicago truncatula. We find endogenous bioactive GA accumulates locally at the site of nodule primordia, increasing dramatically in the cortical cell layers, persisting through cell divisions and maintaining accumulation in the mature nodule meristem. We show, through mis-expression of GA catabolic enzymes that suppress GA accumulation, that GA acts as a positive regulator of nodule growth and development. Furthermore, increasing or decreasing GA through perturbation of biosynthesis gene expression can increase or decrease the size of nodules, respectively. This is unique from lateral root formation, a developmental program that shares common organogenesis regulators. We link GA to a wider gene regulatory program by showing that nodule-identity genes induce and sustain GA accumulation necessary for proper nodule formation.

Root nodules of red alder (Alnus rubra) and sitka alder (Alnus viridis ssp. sinuata) are inhabited by taxonomically diverse cultivable microbial endophytes.

The root nodules of actinorhizal plants are home to nitrogen-fixing bacterial symbionts, known as Frankia, along with a small percentage of other microorganisms. These include fungal endophytes and non-Frankia bacteria. The taxonomic and functional diversity of the microbial consortia within these root nodules is not well understood. In this study, we surveyed and analyzed the cultivable, non-Frankia fungal and bacterial endophytes of root nodules from red and Sitka alder trees that grow together. We examined their taxonomic diversity, co-occurrence, differences between hosts, and potential functional roles. For the first time, we are reporting numerous fungal endophytes of alder root nodules. These include Sporothrix guttuliformis, Fontanospora sp., Cadophora melinii, an unclassified Cadophora, Ilyonectria destructans, an unclassified Gibberella, Nectria ramulariae, an unclassified Trichoderma, Mycosphaerella tassiana, an unclassified Talaromyces, Coniochaeta sp., and Sistotrema brinkmanii. We are also reporting several bacterial genera for the first time: Collimonas, Psychrobacillus, and Phyllobacterium. Additionally, we are reporting the genus Serratia for the second time, with the first report having been recently published in 2023. Pseudomonas was the most frequently isolated bacterial genus and was found to co-inhabit individual nodules with both fungi and bacteria. We found that the communities of fungal endophytes differed by host species, while the communities of bacterial endophytes did not.

Two members of a Nodule-specific Cysteine-Rich (NCR) peptide gene cluster are required for differentiation of rhizobia in Medicago truncatula nodules.

Legumes have evolved a nitrogen-fixing symbiotic interaction with rhizobia, and this association helps them to cope with the limited nitrogen conditions in soil. The compatible interaction between the host plant and rhizobia leads to the formation of root nodules, wherein internalization and transition of rhizobia into their symbiotic form, termed bacteroids, occur. Rhizobia in the nodules of the Inverted Repeat-Lacking Clade legumes, including Medicago truncatula, undergo terminal differentiation, resulting in elongated and endoreduplicated bacteroids. This transition of endocytosed rhizobia is mediated by a large gene family of host-produced nodule-specific cysteine-rich (NCR) peptides in M. truncatula. Few NCRs have been recently found to be essential for complete differentiation and persistence of bacteroids. Here, we show that a M. truncatula symbiotic mutant FN9285, defective in the complete transition of rhizobia, is deficient in a cluster of NCR genes. More specifically, we show that the loss of the duplicated genes NCR086 and NCR314 in the A17 genotype, found in a single copy in Medicago littoralis R108, is responsible for the ineffective symbiotic phenotype of FN9285. The NCR086 and NCR314 gene pair encodes the same mature peptide but their transcriptional activity varies considerably. Nevertheless, both genes can restore the effective symbiosis in FN9285 indicating that their complementation ability does not depend on the strength of their expression activity. The identification of the NCR086/NCR314 peptide, essential for complete bacteroid differentiation, has extended the list of peptides, from a gene family of several hundred members, that are essential for effective nitrogen-fixing symbiosis in M. truncatula.

Metabolic trade-offs constrain the cell size ratio in a nitrogen-fixing symbiosis.

Biological dinitrogen (N2) fixation is a key metabolic process exclusively performed by prokaryotes, some of which are symbiotic with eukaryotes. Species of the marine haptophyte algae Braarudosphaera bigelowii harbor the N2-fixing endosymbiotic cyanobacteria UCYN-A, which might be evolving organelle-like characteristics. We found that the size ratio between UCYN-A and their hosts is strikingly conserved across sublineages/species, which is consistent with the size relationships of organelles in this symbiosis and other species. Metabolic modeling showed that this size relationship maximizes the coordinated growth rate based on trade-offs between resource acquisition and exchange. Our findings show that the size relationships of N2-fixing endosymbionts and organelles in unicellular eukaryotes are constrained by predictable metabolic underpinnings and that UCYN-A is, in many regards, functioning like a hypothetical N2-fixing organelle (or nitroplast).

Fulvic Acid Promotes Legume-Rhizobium Symbiosis by Stimulating Endogenous Flavonoids Synthesis and Secretion.

Fulvic acid (FA) promotes symbiosis between legumes and rhizobia. To elucidate from the aspect of symbiosis, the effects of root irrigation of water-soluble humic materials (WSHM) or foliar spraying of its highly active component, FA, on soybean root exudates and on rhizosphere microorganisms were investigated. As a result, WSHM/FA treatments significantly altered root exudate metabolite composition, and isoflavonoids were identified as key contributors in both treatments compared to the control. Increased expression of genes related to the isoflavonoid biosynthesis were validated by RT-qPCR in both treatments, which notably elevated the synthesis of symbiotic signals genistein, daidzin, coumestrol, and biochanin A. Moreover, the WSHM/FA treatments induced a change in rhizosphere microbial community, coupled with an increase in the relative abundance of rhizobia. Our findings showed that WSHM/FA promotes symbiosis by stimulating the endogenous flavonoid synthesis and leads to rhizobia accumulation in the rhizosphere. This study provides new insights into mechanisms underlying the FA-mediated promotion of symbiosis.

VapC10 toxin of the legume symbiont Sinorhizobium meliloti targets tRNASer and controls intracellular lifestyle.

The soil bacterium Sinorhizobium meliloti can establish a nitrogen-fixing symbiosis with the model legume Medicago truncatula. The rhizobia induce the formation of a specialized root organ called nodule, where they differentiate into bacteroids and reduce atmospheric nitrogen into ammonia. Little is known on the mechanisms involved in nodule senescence onset and in bacteroid survival inside the infected plant cells. Although toxin-antitoxin (TA) systems have been shown to promote intracellular survival within host cells in human pathogenic bacteria, their role in symbiotic bacteria was rarely investigated. S. meliloti encodes several TA systems, mainly of the VapBC family. Here we present the functional characterization, through a multidisciplinary approach, of the VapBC10 TA system of S. meliloti. Following a mapping by overexpression of an RNase in Escherichia coli (MORE) RNA-seq analysis, we demonstrated that the VapC10 toxin is an RNase that cleaves the anticodon loop of two tRNASer. Thereafter, a bioinformatics approach was used to predict VapC10 targets in bacteroids. This analysis suggests that toxin activation triggers a specific proteome reprogramming that could limit nitrogen fixation capability and viability of bacteroids. Accordingly, a vapC10 mutant induces a delayed senescence in nodules, associated to an enhanced bacteroid survival. VapBC10 TA system could contribute to S. meliloti adaptation to symbiotic lifestyle, in response to plant nitrogen status.

Surface Plasmon Resonance as a Tool to Elucidate the Molecular Determinants of Key Transcriptional Regulators Controlling Rhizobial Lifestyles.

Bacteria must be provided with a battery of tools integrated into regulatory networks, in order to respond and, consequently, adapt their physiology to changing environments. Within these networks, transcription factors finely orchestrate the expression of genes in response to a variety of signals, by recognizing specific DNA sequences at their promoter regions. Rhizobia are host-interacting soil bacteria that face severe changes to adapt their physiology from free-living conditions to the nitrogen-fixing endosymbiotic state inside root nodules associated with leguminous plants. One of these cues is the low partial pressure of oxygen within root nodules.Surface plasmon resonance (SPR) constitutes a technique that allows to measure molecular interactions dynamics at real time by detecting changes in the refractive index of a surface. Here, we implemented the SPR methodology to analyze the discriminatory determinants of transcription factors for specific interaction with their target genes. We focused on FixK2, a CRP/FNR-type protein with a central role in the complex oxygen-responsive regulatory network in the soybean endosymbiont Bradyrhizobium diazoefficiens. Our study unveiled relevant residues for protein-DNA interaction as well as allowed us to monitor kinetics and stability protein-DNA complex. We believe that this approach can be employed for the characterization of other relevant transcription factors which can assist to the better understanding of the adaptation of bacteria with agronomic or human interest to their different modes of life.

The Medicago truncatula nodule-specific cysteine-rich peptides, NCR343 and NCR-new35 are required for the maintenance of rhizobia in nitrogen-fixing nodules.

In the nodules of IRLC legumes, including Medicago truncatula, nitrogen-fixing rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialized for nitrogen fixation. This irreversible transition of rhizobia is mediated by host produced nodule-specific cysteine-rich (NCR) peptides, of which c. 700 are encoded in the M. truncatula genome but only few of them have been proved to be essential for nitrogen fixation. We carried out the characterization of the nodulation phenotype of three ineffective nitrogen-fixing M. truncatula mutants using confocal and electron microscopy, monitored the expression of defence and senescence-related marker genes, and analysed the bacteroid differentiation with flow cytometry. Genetic mapping combined with microarray- or transcriptome-based cloning was used to identify the impaired genes. Mtsym19 and Mtsym20 mutants are defective in the same peptide NCR-new35 and the lack of NCR343 is responsible for the ineffective symbiosis of NF-FN9363. We found that the expression of NCR-new35 is significantly lower and limited to the transition zone of the nodule compared with other crucial NCRs. The fluorescent protein-tagged version of NCR343 and NCR-new35 localized to the symbiotic compartment. Our discovery added two additional members to the group of NCR genes essential for nitrogen-fixing symbiosis in M. truncatula.

NIN-at the heart of NItrogen-fixing Nodule symbiosis.

Legumes and actinorhizal plants establish symbiotic relationships with nitrogen-fixing bacteria, resulting in the formation of nodules. Nodules create an ideal environment for nitrogenase to convert atmospheric nitrogen into biological available ammonia. NODULE INCEPTION (NIN) is an indispensable transcription factor for all aspects of nodule symbiosis. Moreover, NIN is consistently lost in non-nodulating species over evolutions. Here we focus on recent advances in the signaling mechanisms of NIN during nodulation and discuss the role of NIN in the evolution of nitrogen-fixing nodule symbiosis.

Gain or Loss? Evidence for Legume Predisposition to Symbiotic Interactions with Rhizobia via Loss of Pathogen-Resistance-Related Gene Families.

Nodulation is a hallmark yet non-universal characteristic of legumes. It is unknown whether the mechanisms underlying nitrogen-fixing symbioses evolved within legumes and the broader nitrogen-fixing clade (NFC) repeatedly de novo or based on common ancestral pathways. Ten new transcriptomes representing members from the Cercidoideae and Caesalpinioideae subfamilies were supplemented with published omics data from 65 angiosperms, to investigate how gene content correlates with nodulation capacity within Fabaceae and the NFC. Orthogroup analysis categorized annotated genes into 64150 orthogroups, of which 19 were significantly differentially represented between nodulating versus non-nodulating NFC species and were most commonly absent in nodulating taxa. The distribution of six over-represented orthogroups within Viridiplantae representatives suggested that genomic evolution events causing gene family expansions, including whole-genome duplications (WGDs), were unlikely to have facilitated the development of stable symbioses within Fabaceae as a whole. Instead, an absence of representation of 13 orthogroups indicated that losses of genes involved in trichome development, defense and wounding responses were strongly associated with rhizobial symbiosis in legumes. This finding provides novel evidence of a lineage-specific predisposition for the evolution and/or stabilization of nodulation in Fabaceae, in which a loss of pathogen resistance genes may have allowed for stable mutualistic interactions with rhizobia.

Nitric Oxide Metabolic Pathway in Drought-Stressed Nodules of Faba Bean (Vicia faba L.).

Drought is an environmental stress that strongly impacts plants. It affects all stages of growth and induces profound disturbances that influence all cellular functions. Legumes can establish a symbiosis with Rhizobium-type bacteria, whose function is to fix atmospheric nitrogen in organs called nodules and to meet plant nitrogen needs. Symbiotic nitrogen fixation (SNF) is particularly sensitive to drought. We raised the hypothesis that, in drought-stressed nodules, SNF inhibition is partly correlated to hypoxia resulting from nodule structure compaction and an increased O2 diffusion barrier, and that the nodule energy regeneration involves phytoglobin-nitric oxide (Pgb-NO) respiration. To test this hypothesis, we subjected faba bean (Vicia faba L.) plants nodulated with a Rhizobium laguerreae strain to either drought or osmotic stress. We monitored the N2-fixation activity, the energy state (ATP/ADP ratio), the expression of hypoxia marker genes (alcohol dehydrogenase and alanine aminotransferase), and the functioning of the Pgb-NO respiration in the nodules. The collected data confirmed our hypothesis and showed that (1) drought-stressed nodules were subject to more intense hypoxia than control nodules and (2) NO production increased and contributed via Pgb-NO respiration to the maintenance of the energy state of drought-stressed nodules.

A variable gene family encoding nodule-specific cysteine-rich peptides in pea (Pisum sativum L.).

Various legume plants form root nodules in which symbiotic bacteria (rhizobia) fix atmospheric nitrogen after differentiation into a symbiotic form named bacteroids. In some legume species, bacteroid differentiation is promoted by defensin-like nodule-specific cysteine-rich (NCR) peptides. NCR peptides have best been studied in the model legume Medicago truncatula Gaertn., while in many other legumes relevant information is still fragmentary. Here, we characterize the NCR gene family in pea (Pisum sativum L.) using genomic and transcriptomic data. We found 360 genes encoding NCR peptides that are expressed in nodules. The sequences of pea NCR genes and putative peptides are highly variable and differ significantly from NCR sequences of M. truncatula. Indeed, only one pair of orthologs (PsNCR47-MtNCR312) has been identified. The NCR genes in the pea genome are located in clusters, and the expression patterns of NCR genes from one cluster tend to be similar. These data support the idea of independent evolution of NCR genes by duplication and diversification in related legume species. We also described spatiotemporal expression profiles of NCRs and identified specific transcription factor (TF) binding sites in promoters of "early" and "late" NCR genes. Further, we studied the expression of NCR genes in nodules of Fix- mutants and predicted potential regulators of NCR gene expression, one among them being the TF ERN1 involved in the early steps of nodule organogenesis. In general, this study contributes to understanding the functions of NCRs in legume nodules and contributes to understanding the diversity and potential antibiotic properties of pea nodule-specific antimicrobial molecules.

A comparative meta-proteomic pipeline for the identification of plasmodesmata proteins and regulatory conditions in diverse plant species.

BACKGROUND: A major route for cell-to-cell signalling in plants is mediated by cell wall-embedded pores termed plasmodesmata forming the symplasm. Plasmodesmata regulate the plant development and responses to the environment; however, our understanding of what factors or regulatory cues affect their structure and permeability is still limited. In this paper, a meta-analysis was carried out for the identification of conditions affecting plasmodesmata transport and for the in silico prediction of plasmodesmata proteins in species for which the plasmodesmata proteome has not been experimentally determined. RESULTS: Using the information obtained from experimental proteomes, an analysis pipeline (named plasmodesmata in silico proteome 1 or PIP1) was developed to rapidly generate candidate plasmodesmata proteomes for 22 plant species. Using the in silico proteomes to interrogate published transcriptomes, gene interaction networks were identified pointing to conditions likely affecting plasmodesmata transport capacity. High salinity, drought and osmotic stress regulate the expression of clusters enriched in genes encoding plasmodesmata proteins, including those involved in the metabolism of the cell wall polysaccharide callose. Experimental determinations showed restriction in the intercellular transport of the symplasmic reporter GFP and enhanced callose deposition in Arabidopsis roots exposed to 75-mM NaCl and 3% PEG (polyethylene glycol). Using PIP1 and transcriptome meta-analyses, candidate plasmodesmata proteins for the legume Medicago truncatula were generated, leading to the identification of Medtr1g073320, a novel receptor-like protein that localises at plasmodesmata. Expression of Medtr1g073320 affects callose deposition and the root response to infection with the soil-borne bacteria rhizobia in the presence of nitrate. CONCLUSIONS: Our study shows that combining proteomic meta-analysis and transcriptomic data can be a valuable tool for the identification of new proteins and regulatory mechanisms affecting plasmodesmata function. We have created the freely accessible pipeline PIP1 as a resource for the screening of experimental proteomes and for the in silico prediction of PD proteins in diverse plant species.

Salicylic Acid in Plant Symbioses: Beyond Plant Pathogen Interactions.

Plants form beneficial symbioses with a wide variety of microorganisms. Among these, endophytes, arbuscular mycorrhizal fungi (AMF), and nitrogen-fixing rhizobia are some of the most studied and well understood symbiotic interactions. These symbiotic microorganisms promote plant nutrition and growth. In exchange, they receive the carbon and metabolites necessary for their development and multiplication. In addition to their role in plant growth and development, these microorganisms enhance host plant tolerance to a wide range of environmental stress. Multiple studies have shown that these microorganisms modulate the phytohormone metabolism in the host plant. Among the phytohormones involved in the plant defense response against biotic environment, salicylic acid (SA) plays an important role in activating plant defense. However, in addition to being a major actor in plant defense signaling against pathogens, SA has also been shown to be involved in plant-microbe symbiotic interactions. In this review, we summarize the impact of SA on the symbiotic interactions. In addition, we give an overview of the impact of the endophytes, AMF, and rhizobacteria on SA-mediated defense response against pathogens.

Analysis of Ensifer aridi Mutants Affecting Regulation of Methionine, Trehalose, and Inositol Metabolisms Suggests a Role in Stress Adaptation and Symbiosis Development.

Isolated from desert, the nitrogen-fixing bacterium Ensifer aridi LMR001 is capable of survival under particularly harsh environmental conditions. To obtain insights in molecular mechanisms involved in stress adaptation, a recent study using RNAseq revealed that the RpoE2-mediated general stress response was activated under mild saline stress but appeared non-essential for the bacterium to thrive under stress and develop the symbiosis. Functions associated with the stress response included the metabolisms of trehalose, methionine, and inositol. To explore the roles of these metabolisms in stress adaptation and symbiosis development, and the possible regulatory mechanisms involved, mutants were generated notably in regulators and their transcriptions were studied in various mutant backgrounds. We found that mutations in regulatory genes nesR and sahR of the methionine cycle generating S-adenosylmethionine negatively impacted symbiosis, tolerance to salt, and motility in the presence of NaCl. When both regulators were mutated, an increased tolerance to detergent, oxidative, and acid stresses was found, suggesting a modification of the cell wall components which may explain these phenotypes and support a major role of the fine-tuning methylation for symbiosis and stress adaptation of the bacterium. In contrast, we also found that mutations in the predicted trehalose transport and utilization regulator ThuR and the trehalose phosphate phosphatase OtsB-encoding genes improved symbiosis and growth in liquid medium containing 0.4 M of NaCl of LMR001ΔotsB, suggesting that trehalose metabolism control and possibly trehalose-6 phosphate cellular status may be biotechnologically engineered for improved symbiosis under stress. Finally, transcriptional fusions of gfp to promoters of selected genes and expression studies in the various mutant backgrounds suggest complex regulatory interplay between inositol, methionine, and trehalose metabolic pathways.

Development of an Illumina-based analysis method to study bradyrhizobial population structure-case study on nitrogen-fixing rhizobia associating with cowpea or peanut.

Bradyrhizobia are Gram-negative soil bacteria that regroup a growing number of species. They are widespread in nature and recovered from various biomes that may be explained by a high genetic diversity in this genus. Among the numerous metabolic properties they can harbor, the nitrogen fixation resulting from the association with plants among which important crop legumes (soya bean, peanut, cowpea …) is of great interest, notably in a context of sustainable development. Metabarcoding is widely applied to study biodiversity from complex microbial communities. Here, we demonstrate that using a new species-specific and highly polymorphic 16S-23S rRNA intergenic spacer barcode, we could rapidly estimate the diversity of bradyrhizobial populations that associate with cowpea and peanut plants, two crop legumes of major interest in Senegal. Application of the method on indigenous bradyrhizobia associated with peanut and cowpea grown in soils collected in the center of the peanut basin shows that Bradyrhizobium vignae is a dominant symbiont. We also showed that the two plant species associate with distinct community profiles and that strains introduced by inoculation significantly modified the population structure with these two plants suggesting that application of elite strains as inoculants may well ensure optimized symbiotic performance. This approach may further be used to study the diversity of bradyrhizobia from contrasting agro-eco-climatic zones, to test whether the plant genotype influences the association outputs as well as to estimate the competitiveness for nodule occupancy and the fate of elite strains inoculated in the field.Key points• An amplicon sequencing approach targeting the Bradyrhizobium genus was developed.• Diversity of cowpea and peanut bradyrhizobia from cultivated soils was identified.• The method is well suited to test the competitiveness of defined Bradyrhizobium inoculants.

Pangenomics of the Symbiotic Rhizobiales. Core and Accessory Functions Across a Group Endowed with High Levels of Genomic Plasticity.

Pangenome analyses reveal major clues on evolutionary instances and critical genome core conservation. The order Rhizobiales encompasses several families with rather disparate ecological attitudes. Among them, Rhizobiaceae, Bradyrhizobiaceae, Phyllobacteriacreae and Xanthobacteriaceae, include members proficient in mutualistic symbioses with plants based on the bacterial conversion of N2 into ammonia (nitrogen-fixation). The pangenome of 12 nitrogen-fixing plant symbionts of the Rhizobiales was analyzed yielding total 37,364 loci, with a core genome constituting 700 genes. The percentage of core genes averaged 10.2% over single genomes, and between 5% to 7% were found to be plasmid-associated. The comparison between a representative reference genome and the core genome subset, showed the core genome highly enriched in genes for macromolecule metabolism, ribosomal constituents and overall translation machinery, while membrane/periplasm-associated genes, and transport domains resulted under-represented. The analysis of protein functions revealed that between 1.7% and 4.9% of core proteins could putatively have different functions.