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Three bacterial strains, 1AS14IT, 1AS12I and 6AS6, isolated from root nodules of Acacia saligna, were characterized using a polyphasic approach. Phylogenetic analysis based on rrs sequences placed all three strains within the Rhizobium leguminosarum complex. Further phylogeny, based on 1 756 bp sequences of four concatenated housekeeping genes (recA, atpD, glnII and gyrB), revealed their distinction from known rhizobia species of the R. leguminosarum complex (Rlc), forming a distinct clade. The closest related species, identified as Rhizobium laguerreae, with a sequence identity of 96.4% based on concatenated recA-atpD-glnII-gyrB sequences. The type strain, 1AS14IT, showed average nucleotide identity (ANI) values of 94.9, 94.3 and 94.1% and DNA-DNA hybridization values of 56.1, 57.4 and 60.0% with the type strains of closest known species: R. laguerreae, Rhizobium acaciae and 'Rhizobium indicum', respectively. Phylogenomic analyses using 81 up-to-date bacteria core genes and the Type (Strain) Genome Server pipeline further supported the uniqueness of strains 1AS14IT, 1AS12I and 6AS6. The relatedness of the novel strains to NCBI unclassified Rhizobium sp. (396 genomes) and metagenome-derived genomes showed ANI values from 76.7 to 94.8% with a species-level cut-off of 96%, suggesting that strains 1AS14I, 1AS12I and 6AS6 are a distinct lineage. Additionally, differentiation of strains 1AS14IT, 1AS12I and 6AS6 from their closest phylogenetic neighbours was achieved using phenotypic, physiological and fatty acid content analyses. Based on the genomic, phenotypic and biochemical data, we propose the establishment of a novel rhizobial species, Rhizobium aouanii sp. nov., with strain 1AS14IT designated as the type strain (=DSM 113914T=LMG 33206T). This study contributes to the understanding of microbial diversity in nitrogen-fixing symbioses, specifically within Acacia saligna ecosystems in Tunisia.

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Root exudates are known signaling agents that influence legume root nodulation, but the molecular mechanisms for nonflavonoid molecules remain largely unexplored. The number of soybean root nodules during the initial growth phase shows substantial discrepancies at distinct developmental junctures. Using a combination of metabolomics analyses on root exudates and nodulation experiments, we identify a pivotal role for certain root exudates during the rapid growth phase in promoting nodulation. Phenoxyacetic acid (POA) was found to activate the expression of GmGA2ox10 and thereby facilitate rhizobial infection and the formation of infection threads. Furthermore, POA exerts regulatory control on the miR172c-NNC1 module to foster nodule primordia development and consequently increase nodule numbers. These findings collectively highlight the important role of POA in enhancing nodulation during the accelerated growth phase of soybeans.

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Soybean plants form symbiotic nitrogen-fixing nodules with specific rhizobia bacteria. The root hair is the initial infection site for the symbiotic process before the nodules. Since roots and nodules grow in soil and are hard to perceive, little knowledge is available on the process of soybean root hair deformation and nodule development over time. In this study, adaptive microrhizotrons were used to observe root hairs and to investigate detailed root hair deformation and nodule formation subjected to different rhizobia densities. The result showed that the root hair curling angle increased with the increase of rhizobia density. The largest curling angle reached 268° on the 8th day after inoculation. Root hairs were not always straight, even in the uninfected group with a relatively small angle (<45°). The nodule is an organ developed after root hair curling. It was inoculated from curling root hairs and swelled in the root axis on the 15th day after inoculation, with the color changing from light (15th day) to a little dark brown (35th day). There was an error between observing the diameter and the real diameter; thus, a diameter over 1 mm was converted to the real diameter according to the relationship between the perceived diameter and the real diameter. The diameter of the nodule reached 5 mm on the 45th day. Nodule number and curling number were strongly related to rhizobia density with a correlation coefficient of determination of 0.92 and 0.93, respectively. Thus, root hair curling development could be quantified, and nodule number could be estimated through derived formulation.

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Phaseolus vulgaris is a globally important legume cash crop, which can carry out symbiotic nitrogen fixation with rhizobia. The presence of suitable rhizobia in cultivating soils is crucial for legume cropping, especially in areas beyond the plant-host native range, where soils may lack efficient symbiotic partners. We analyzed the distribution patterns and traits of native rhizobia associated with P. vulgaris in soils of Yunnan, where the common bean experienced a recent expansion. A total of 608 rhizobial isolates were tracked from soils of fifteen sampling sites using two local varieties of P. vulgaris. The isolates were discriminated into 43 genotypes as defined by IGS PCR-RFLP. Multiple locus sequence analysis based on recA, atpD and rpoB of representative strains placed them into 11 rhizobial species of Rhizobium involving Rhizobium sophorae, Rhizobium acidisoli, Rhizobium ecuadorense, Rhizobium hidalgonense, Rhizobium vallis, Rhizobium sophoriradicis, Rhizobium croatiense, Rhizobium anhuiense, Rhizobium phaseoli, Rhizobium chutanense and Rhizobium etli, and five unknown Rhizobium species; Rhizobium genosp. I~V. R. phaseoli and R. anhuiense were the dominant species (28.0% and 28.8%) most widely distributed, followed by R. croatiense (14.8%). The other rhizobial species were less numerous or site-specific. Phylogenies of nodC and nifH markers, were divided into two specific symbiovars, sv. phaseoli regardless of the species affiliation and sv. viciae associated with R. vallis. Through symbiotic effect assessment, all the tested strains nodulated both P. vulgaris varieties, often resulting with a significant greenness index (91-98%). However, about half of them exhibited better plant biomass performance, at least on one common bean variety, and two isolates (CYAH-6 and BLYH-15) showed a better symbiotic efficiency score. Representative strains revealed diverse abiotic stress tolerance to NaCl, acidity, alkalinity, temperature, drought and glyphosate. One strain efficient on both varieties and exhibiting stress abiotic tolerance (BLYH-15) belonged to R. genosp. IV sv. phaseoli, a species first found as a legume symbiont.

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Legumes are ecologically and economically important plants that contribute to nutrient cycling and agricultural sustainability, features tied to their intimate symbiosis with nitrogen-fixing rhizobia. Rhizobia vary dramatically in quality, ranging from highly growth-promoting to non-beneficial; therefore, legumes must optimize their symbiosis with rhizobia through host mechanisms that select for beneficial rhizobia and limit losses to non-beneficial strains. In this Perspective, we examine the considerable scientific progress made in decoding host control over rhizobia, empirically examining both molecular and cellular mechanisms and their effects on rhizobia symbiosis and its benefits. We consider pre-infection controls, which require the production and detection of precise molecular signals by the legume to attract and select for compatible rhizobia strains. We also discuss post-infection mechanisms that leverage the nodule-level and cell-level compartmentalization of symbionts to enable host control over rhizobia development and proliferation in planta. These layers of host control each contribute to legume fitness by directing host resources towards a narrowing subset of more-beneficial rhizobia.

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BACKGROUND: Legumes utilize a long-distance signaling feedback pathway, termed Autoregulation of Nodulation (AON), to regulate the establishment and maintenance of their symbiosis with rhizobia. Several proteins key to this pathway have been discovered, but the AON pathway is not completely understood. RESULTS: We report a new hypernodulating mutant, defective in autoregulation, with disruption of a gene, DAR (Medtr2g450550/MtrunA17_Chr2g0304631), previously unknown to play a role in AON. The dar-1 mutant produces ten-fold more nodules than wild type, similar to AON mutants with disrupted SUNN gene function. As in sunn mutants, suppression of nodulation by CLE peptides MtCLE12 and MtCLE13 is abolished in dar. Furthermore, dar-1 also shows increased root length colonization by an arbuscular mycorrhizal fungus, suggesting a role for DAR in autoregulation of mycorrhizal symbiosis (AOM). However, unlike SUNN which functions in the shoot to control nodulation, DAR functions in the root. CONCLUSIONS: DAR encodes a membrane protein that is a member of a small protein family in M. truncatula. Our results suggest that DAR could be involved in the subcellular transport of signals involved in symbiosis regulation, but it is not upregulated during symbiosis. DAR gene family members are also present in Arabidopsis, lycophytes, mosses, and microalgae, suggesting the AON and AOM may use pathway components common to other plants, even those that do not undergo either symbiosis.

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BACKGROUND: The symbiosis among plants, rhizobia, and arbuscular mycorrhizal fungi (AMF) is one of the most well-known symbiotic relationships in nature. However, it is still unclear how bilateral/tripartite symbiosis works under resource-limited conditions and the diverse genetic backgrounds of the host. RESULTS: Using a full factorial design, we manipulated mungbean accessions/subspecies, rhizobia, and AMF to test their effects on each other. Rhizobia functions as a typical facilitator by increasing plant nitrogen content, plant weight, chlorophyll content, and AMF colonization. In contrast, AMF resulted in a tradeoff in plants (reducing biomass for phosphorus acquisition) and behaved as a competitor in reducing rhizobia fitness (nodule weight). Plant genotype did not have a significant effect on AMF fitness, but different mungbean accessions had distinct rhizobia affinities. In contrast to previous studies, the positive relationship between plant and rhizobia fitness was attenuated in the presence of AMF, with wild mungbean being more responsive to the beneficial effect of rhizobia and attenuation by AMF. CONCLUSIONS: We showed that this complex tripartite relationship does not unconditionally benefit all parties. Moreover, rhizobia species and host genetic background affect the symbiotic relationship significantly. This study provides a new opportunity to re-evaluate the relationships between legume plants and their symbiotic partners.

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Symbiotic nodules comprise two classes, indeterminate and determinate, defined by the presence/absence of apical meristem and developmental zonation. Why meristem and zonation are absent from determinate nodules remains unclear. Here, we define cell types in developing soybean nodules, highlighting the undifferentiated infection zones and differentiated nitrogen-fixation zones. Auxin governs infection zone maintenance. GRETCHEN HAGEN 3 (GH3) enzymes deactivate auxin by conjugation and promote cell differentiation. gh3 mutants increased undifferentiated cells and enlarged infection zones. The central symbiosis-transcription factor NIN2a activates GH3.1 to reduce auxin levels and facilitates cell differentiation. High auxin promotes NIN2a protein accumulation and enhances signaling, further deactivating auxin and depleting infection zones. Our findings shed light on the NIN2a-GH3-auxin module that drives soybean nodule cell differentiation. This study challenges our understanding of determinate nodule development and proposes that the regulation of nodule zonation offers valuable insights into broader mechanisms of cell differentiation across plant species.

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The species Rhizobium indigoferae and Sinorhizobium kummerowiae were isolated from legume nodules and the 16S rRNA sequences of their respective type strains, CCBAU 71042T and CCBAU 71714T, were highly divergent from those of the other species of the genera Rhizobium and Sinorhizobium, respectively. However, the 16S rRNA gene sequences obtained for strains CCBAU 71042T and CCBAU 71714T several years after description, were different from the original ones, showing 100 % similarity to the type strains of Rhizobium leguminosarum and Sinorhizobium meliloti, respectively. Phylogenetic analyses of two housekeeping genes, recA and atpD, confirmed the high phylogenetic closeness of strains CCBAU 71042T and CCBAU 71714T to the respective type strains of R. leguminosarum and S. meliloti. In the present work, we compared the genomes of the type strains of R. indigoferae and S. kummerowiae available in several culture collections with those of the respective type strains of R. leguminosarum and S. meliloti, some of them obtained in this study. The calculated average nucleotide identity-blast and digital DNA-DNA hybridization values in both cases were higher than those recommended for species differentiation, supporting the proposal for the reclassification of the type strains of R. indigoferae and S. kummerowiae into the species R. leguminosarum and S. meliloti, respectively.

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Legumes acquire nitrogen-fixing ability by forming root nodules. Transferring this capability to more crops could reduce our reliance on nitrogen fertilizers, thereby decreasing environmental pollution and agricultural production costs. Nodule organogenesis is complex, and a comprehensive transcriptomic atlas is crucial for understanding the underlying molecular events. Here, we utilized spatial transcriptomics to investigate the development of nodules in the model legume, Lotus japonicus. Our investigation has identified the developmental trajectories of two critical regions within the nodule: the infection zone and peripheral tissues. We reveal the underlying biological processes and provide gene sets to achieve symbiosis and material exchange, two essential aspects of nodulation. Among the candidate regulatory genes, we illustrate that LjNLP3, a transcription factor belonging to the NIN-LIKE PROTEIN family, orchestrates the transition of nodules from the differentiation to maturation. In summary, our research advances our understanding of nodule organogenesis and provides valuable data for developing symbiotic nitrogen-fixing crops.

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Host range specificity is a prominent feature of the legume-rhizobial symbiosis. Sinorhizobium meliloti and Sinorhizobium medicae are two closely related species that engage in root nodule symbiosis with legume plants of the Medicago genus, but certain Medicago species exhibit selectivity in their interactions with the two rhizobial species. We have identified a Medicago receptor-like kinase, which can discriminate between the two bacterial species, acting as a genetic barrier against infection by most S. medicae strains. Activation of this receptor-mediated nodulation restriction requires a bacterial gene that encodes a glycine-rich octapeptide repeat protein with distinct variants capable of distinguishing S. medicae from S. meliloti. This study sheds light on the coevolution of host plants and rhizobia, shaping symbiotic selectivity in their respective ecological niches.

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Rhizobium leguminosarum aspartate aminotransferase (AatA) mutants show drastically reduced symbiotic nitrogen fixation in legume nodules. Whilst AatA reversibly transaminates the two major amino-donor compounds aspartate and glutamate, the reason for the lack of N2 fixation in the mutant has remained unclear. During our investigations into the role of AatA, we found that it catalyses an additional transamination reaction between aspartate and pyruvate, forming alanine. This secondary reaction runs at around 60 % of the canonical aspartate transaminase reaction rate and connects alanine biosynthesis to glutamate via aspartate. This may explain the lack of any glutamate-pyruvate transaminase activity in R. leguminosarum, which is common in eukaryotic and many prokaryotic genomes. However, the aspartate-to-pyruvate transaminase reaction is not needed for N2 fixation in legume nodules. Consequently, we show that aspartate degradation is required for N2 fixation, rather than biosynthetic transamination to form an amino acid. Hence, the enzyme aspartase, which catalyses the breakdown of aspartate to fumarate and ammonia, suppressed an AatA mutant and restored N2 fixation in pea nodules.

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Vicia villosa (VV) and Vicia sativa (VS) are legume forages highly valued for their excellent nitrogen fixation. However, no research has addressed the mechanisms underlying their differences in nitrogen fixation. This study employed physiological, cytological, and comparative transcriptomic approaches to elucidate the disparities in nitrogen fixation between them. Our results showed that the total amount of nitrogen fixed was 60.45% greater in VV than in VS, and the comprehensive nitrogen response performance was 94.19% greater, while the nitrogen fixation efficiency was the same. The infection zone and differentiated bacteroid proportion in mature VV root nodules were 33.76% and 19.35% greater, respectively, than those in VS. The size of the VV genome was 15.16% larger than that of the VS genome, consistent with its greater biomass. A significant enrichment of the flavonoid biosynthetic pathway was found only for VV-specific genes, among which chalcone-flavonone isomerase, caffeoyl-CoA-O-methyltransferase and stilbene synthase were extremely highly expressed. The VV-specific genes also exhibited significant enrichment in symbiotic nodulation; genes related to nodule-specific cysteine-rich peptides (NCRs) comprised 61.11% of the highly expressed genes. qRT‒PCR demonstrated that greater enrichment and expression of the dominant NCR (Unigene0004451) were associated with greater nodule bacteroid differentiation and greater nitrogen fixation in VV. Our findings suggest that the greater total nitrogen fixation of VV was attributed to its larger biomass, leading to a greater nitrogen demand and enhanced fixation physiology. This process is likely achieved by the synergistic effects of high bacteroid differentiation along with high expression of flavonoid and NCR genes.

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Host plants benefit from legume root nodule symbiosis with nitrogen-fixing bacteria under nitrogen-limiting conditions. In this interaction, the hosts must regulate nodule numbers and distribution patterns to control the degree of symbiosis and maintain root growth functions. The host response to symbiotic bacteria occurs discontinuously but repeatedly at the region behind the tip of the growing roots. Here, live-imaging and transcriptome analyses revealed oscillating host gene expression with approximately 6-hour intervals upon bacterial inoculation. Cytokinin response also exhibited a similar oscillation pattern. Cytokinin signaling is crucial to maintaining the periodicity, as observed in cytokinin receptor mutants displaying altered infection foci distribution. This periodic regulation influences the size of the root region responsive to bacteria, as well as the nodulation process progression.

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A comprehensive polyphasic taxonomic investigation integrating taxongenomic criteria was conducted on strain IRAMC:0171T isolated from the root nodules of Retama raetam in Tunisia. This Gram-stain-negative and aerobic bacterium thrived within a temperature range of 5-45 °C, optimal at 28 °C, and tolerated salt concentrations from 0-6 % NaCl, with an optimal range of 0-3 %. It displayed pH tolerance from pH 4 to 10, thriving best at pH 6.8-7.5. Chemotaxonomically, strain IRAMC:0171T was characterized by diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, and phosphatidylethanolamine as polar lipids. Its predominant fatty acid composition was C18 : 1 ω7c (61.2 %), and the primary ubiquinone was Q10 (97 %). Analysis of the 16S rRNA gene of strain IRAMC:0171T showed 99.08 % similarity to Mesorhizobium waimense ICMP 19557T, Mesorhizobium amorphae ACCC 19665T, and Mesorhizobium huakuii IAM 14158. However, digital DNA-DNA hybridization and average nucleotide identity analyses revealed values ranging from 21.1 to 25.2 % and 77.05 to 82.24 %, respectively, signifying significant deviation from established species demarcation thresholds. Phylogenetic studies, encompassing 16S rRNA, whole-genome-based tree reconstruction, and core protein analysis, positioned strain IRAMC:0171T closest to Mesorhizobium terrae KCTC 72278T and 'Mesorhizobium hungaricum' UASWS1009T, forming together a distinct branch within the genus Mesorhizobium. In consideration of this comprehensive data, we propose strain IRAMC:0171T (=DSM 112841T=CECT 30767T) as the type strain of a new species named Mesorhizobium retamae sp. nov.

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Legume-rhizobia symbiosis offers a unique approach to increase leguminous crop yields. Previous studies have indicated that the number of soybean nodules are increased under elevated CO2 concentration. However, the underlying mechanism behind this phenomenon remains elusive. In this study, transcriptome analysis was applied to identify candidate genes involved in regulating soybean nodulation mediated by elevated CO2 concentration. Among the different expression genes (DEGs), we identified a gene encoding small heat shock protein (sHSP) called GmHSP23.9, which mainly expressed in soybean roots and nodules, and its expression was significantly induced by rhizobium USDA110 infection at 14 days after inoculation (DAI) under elevated CO2 conditions. We further investigated the role of GmHSP23.9 by generating transgenic composite plants carrying GmHSP23.9 overexpression (GmHSP23.9-OE), RNA interference (GmHSP23.9-RNAi), and CRISPR-Cas9 (GmHSP23.9-KO), and these modifications resulted in notable changes in nodule number and the root hairs deformation and suggesting that GmHSP23.9 function as an important positive regulator in soybean. Moreover, we found that altering the expression of GmHSP23.9 influenced the expression of genes involved in the Nod factor signaling pathway and AON signaling pathway to modulate soybean nodulation. Interestingly, we found that knocking down of GmHSP23.9 prevented the increase in the nodule number of soybean in response to elevated CO2 concentration. This research has successfully identified a crucial regulator that influences soybean nodulation under elevated CO2 level and shedding new light on the role of sHSPs in legume nodulation.

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The soybean root system is complex. In addition to being composed of various cell types, the soybean root system includes the primary root, the lateral roots, and the nodule, an organ in which mutualistic symbiosis with N-fixing rhizobia occurs. A mature soybean root nodule is characterized by a central infection zone where atmospheric nitrogen is fixed and assimilated by the symbiont, resulting from the close cooperation between the plant cell and the bacteria. To date, the transcriptome of individual cells isolated from developing soybean nodules has been established, but the transcriptomic signatures of cells from the mature soybean nodule have not yet been characterized. Using single-nucleus RNA-seq and Molecular Cartography technologies, we precisely characterized the transcriptomic signature of soybean root and mature nodule cell types and revealed the co-existence of different sub-populations of B. diazoefficiens-infected cells in the mature soybean nodule, including those actively involved in nitrogen fixation and those engaged in senescence. Mining of the single-cell-resolution nodule transcriptome atlas and the associated gene co-expression network confirmed the role of known nodulation-related genes and identified new genes that control the nodulation process. For instance, we functionally characterized the role of GmFWL3, a plasma membrane microdomain-associated protein that controls rhizobial infection. Our study reveals the unique cellular complexity of the mature soybean nodule and helps redefine the concept of cell types when considering the infection zone of the soybean nodule.

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Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness1. In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules2-8. Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops.

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The precise control of receptor levels is crucial for initiating cellular signaling transduction in response to specific ligands; however, such mechanisms regulating nodulation factor (NF) receptor (NFR)-mediated perception of NFs to establish symbiosis remain unclear. In this study, we unveil the pivotal role of the NFR-interacting RING-type E3 ligase 1 (NIRE1) in regulating NFR1/NFR5 homeostasis to optimize rhizobial infection and nodule development in Lotus japonicus. We demonstrated that NIRE1 has a dual function in this regulatory process. It associates with both NFR1 and NFR5, facilitating their degradation through K48-linked polyubiquitination before rhizobial inoculation. However, following rhizobial inoculation, NFR1 phosphorylates NIRE1 at a conserved residue, Tyr-109, inducing a functional switch in NIRE1, which enables NIRE1 to mediate K63-linked polyubiquitination, thereby stabilizing NFR1/NFR5 in infected root cells. The introduction of phospho-dead NIRE1Y109F leads to delayed nodule development, underscoring the significance of phosphorylation at Tyr-109 in orchestrating symbiotic processes. Conversely, expression of the phospho-mimic NIRE1Y109E results in the formation of spontaneous nodules in L. japonicus, further emphasizing the critical role of the phosphorylation-dependent functional switch in NIRE1. In summary, these findings uncover a fine-tuned symbiotic mechanism that a single E3 ligase could undergo a phosphorylation-dependent functional switch to dynamically and precisely regulate NF receptor protein levels.

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