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Rhizobia interact with leguminous plants in the soil to form nitrogen fixing nodules in which rhizobia and plant cells coexist. Although there are emerging studies on rhizobium-associated nitrogen fixation in cereals, the legume-rhizobium interaction is more well-studied and usually serves as the model to study rhizobium-mediated nitrogen fixation in plants. Rhizobia play a crucial role in the nitrogen cycle in many ecosystems. However, rhizobia are highly sensitive to variations in soil conditions and physicochemical properties (i.e. moisture, temperature, salinity, pH, and oxygen availability). Such variations directly caused by global climate change are challenging the adaptive capabilities of rhizobia in both natural and agricultural environments. Although a few studies have identified rhizobial genes that confer adaptation to different environmental conditions, the genetic basis of rhizobial stress tolerance remains poorly understood. In this review, we highlight the importance of improving the survival of rhizobia in soil to enhance their symbiosis with plants, which can increase crop yields and facilitate the establishment of sustainable agricultural systems. To achieve this goal, we summarize the key challenges imposed by global climate change on rhizobium-plant symbiosis and collate current knowledge of stress tolerance-related genes and pathways in rhizobia. And finally, we present the latest genetic engineering approaches, such as synthetic biology, implemented to improve the adaptability of rhizobia to changing environmental conditions., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Three strains of Gram-negative bacterium, Rhizobium, were developed by gamma (γ)-irradiation random mutagenesis. The developed strains were evaluated for their augmented features for symbiotic association, nitrogen fixation, and crop yield of three leguminous plants-chickpea, field-pea, and lentil-in agricultural fields of the northern Indian state of Haryana. Crops treated with developed mutants exhibited significant improvement in plant features and the yield of crops when compared to the control-uninoculated crops and crops grown with indigenous or commercial crop-specific strains of Rhizobium. This improvement was attributed to generated mutants, MbPrRz1 (on chickpea), MbPrRz2 (on lentil), and MbPrRz3 (on field-pea). Additionally, the cocultured symbiotic response of MbPrRz1 and MbPrRz2 mutants was found to be more pronounced on all three crops. The statistical analysis using Pearson's correlation coefficients revealed that nodulation and plant biomass were the most related parameters of crop yield. Among the effectiveness of developed mutants, MbPrRz1 yielded the best results for all three tested crops. Moreover, the developed mutants enhanced macro- and micronutrients of the experimental fields when compared with fields harboring the indigenous rhizobial community. These developed mutants were further genetically characterized, predominantly expressing nitrogen fixation marker, nifH, and appeared to belong to Mesorhizobium ciceri (MbPrRz1) and Rhizobium leguminosarum (both MbPrRz2 and MbPrRz3). In summary, this study highlights the potential of developed Rhizobium mutants as effective biofertilizers for sustainable agriculture, showcasing their ability to enhance symbiotic relationships, crop yield, and soil fertility., (© 2024 The Author(s). Journal of Basic Microbiology published by Wiley‐VCH GmbH.)

The regulation of legume-rhizobia symbiosis by microorganisms has obtained considerable interest in recent research, particularly in the common rhizobacteria Bacillus. However, few studies have provided detailed explanations regarding the regulatory mechanisms involved. Here, we investigated the effects of Bacillus (Bac.B) on Bradyrhizobium-soybean (Glycine max) symbiosis and elucidated the underlying ecological mechanisms. We found that two Bradyrhizobium strains (i.e. Bra.Q2 and Bra.D) isolated from nodules significantly promoted nitrogen (N) efficiency of soybean via facilitating nodule formation, thereby enhanced plant growth and yield. However, the intrusion of Bac.B caused a reverse shift in the synergistic efficiency of N 2 fixation in the soybean-Bradyrhizobium symbiosis. Biofilm formation and naringenin may be importantin suppression of Bra.Q2 growth regulated by Bac.B. In addition, transcriptome and microbiome analyses revealed that Bra.Q2 and Bac.B might interact to regulateN transport and assimilation, thus influence the bacterial composition related to plant N nutrition in nodules. Also, the metabolisms of secondary metabolites and hormones associated with plant-microbe interaction and growth regulation were modulated by Bra.Q2 and Bac.B coinoculation. Collectively, we demonstrate that Bacillus negatively affects Bradyrhizobium-soybean symbiosis and modulate microbial interactions in the nodule. Our findings highlight a novel Bacillus-based regulation to improve N efficiency and sustainable agricultural development., (© 2024 John Wiley & Sons Ltd.)

The emergence of commensalism and mutualism often derives from ancestral parasitism. However, in the case of rhizobium-legume interactions, bacterial strains displaying both pathogenic and nodulation features on a single host have not been described yet. Here, we isolated such a bacterium from Medicago nodules. On the same plant genotypes, the T4 strain can induce ineffective nodules in a highly competitive way and behave as a harsh parasite triggering plant death. The T4 strain presents this dual ability on multiple legume species of the Inverted Repeat-Lacking Clade, the output of the interaction relying on the developmental stage of the plant. Genomic and phenotypic clustering analysis show that T4 belongs to the nonsymbiotic Ensifer adhaerens group and clusters together with T173, another strain harboring this dual ability. In this work, we identify a bacterial clade that includes rhizobial strains displaying both pathogenic and nodulating abilities on a single legume host., (© 2024. The Author(s).)

Rhizobial exopolysaccharides (EPS) may provide stabilization of membranes against external factors, as well as improved surface adhesion, but their role in interaction with legume and non-legume plants is still far from understanding. In this work, the transcriptional regulator RosR of Rhizobium ruizarguesonis, which regulates the synthesis of EPS, was overproduced in a pHC60 plasmid and expressed in the RCAM 1026 strain. This resulted in an improved production of EPS by this recombinant strain. Comparative analysis of the inoculation of pea Pisum sativum plants with R. ruizarguesonis pHC60-rosR and strain carrying the empty plasmid revealed an essential increase in the number of nodules, root length and biomass in plants inoculated with this EPS-overproducing strain. It demonstrates that the enhanced EPS synthesis by rhizobia may stimulate plant root colonization and subsequent nodule formation in pea plants. The influence of enhanced EPS synthesis in rhizobia on colonizing activity was also estimated in non-legume plant tomato Solanum lycopersicum. Our findings shown the increased colonization of the root surface and stimulation of the shoot biomass of inoculated plants. Inoculation of pea and tomato with EPS-overproducing rhizobial strain essentially increased plant resistance to phytopathogenic fungi Fusarium culmorum and F. oxysporum in both legume and non-legume plants, demonstrating a significant biocontrol effect of this recombinant strain. Furthermore, we have identified the PsLYK10 gene that encodes a putative EPS receptor in P. sativum, although no significant effect of PsLYK10 overexpression on nodulation in legume (pea P. sativum) and colonization of roots of non-legume plants by rhizobia was found compared to enhanced production of EPS by rhizobia., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Legume plants establish an endosymbiosis with nitrogen-fixing rhizobia bacteria, which are taken up from the environment anew by each host generation. This requires a dedicated genetic program on the host side to control microbe invasion, involving coordinated reprogramming of host cells to create infection structures that facilitate inward movement of the symbiont. Infection initiates in the epidermis, with different legumes utilizing distinct strategies for crossing this cell layer, either between cells (intercellular infection) or transcellularly (infection thread infection). Recent discoveries on the plant side using fluorescent-based imaging approaches have illuminated the spatiotemporal dynamics of infection, underscoring the importance of investigating this process at the dynamic single-cell level. Extending fluorescence-based live-dynamic approaches to the bacterial partner opens the exciting prospect of learning how individual rhizobia reprogram from rhizospheric to a host-confined state during early root infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Chitin, an N-acetyl-D-glucosamine polymer, has multiple functions in living organisms, including the induction of disease resistance and growth promotion in plants. In addition, chitin oligosaccharides (COs) are used as the backbone of the signaling molecule Nod factor secreted by soil bacteria rhizobia to establish a mutual symbiosis with leguminous plants. Nod factor perception triggers host plant responses for rhizobial symbiosis. In this study, the effects of chitins on rhizobial symbiosis were examined in the leguminous plants Lotus japonicus and soybean. Chitin nanofiber (CNF), retained with polymeric structures, and COs elicited calcium spiking in L. japonicus roots expressing a nuclear-localized cameleon reporter. Shoot growth and symbiotic nitrogen fixation were significantly increased by CNF but not COs in L.japonicus and soybean. However, treatments with chitin and cellulose nanofiber, structurally similar polymers to CNF, did not affect shoot growth and nitrogen fixation in L.japonicus. Transcriptome analysis also supported the specific effects of CNF on rhizobial symbiosis in L.japonicus. Although chitins comprise the same monosaccharides and nanofibers share similar physical properties, only CNF can promote rhizobial nitrogen fixation in leguminous plants. Taking the advantages on physical properties, CNF could be a promising material for improving legume yield by enhancing rhizobial symbiosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rhizobium inoculation has been widely applied to alleviate heavy metal (HM) stress in legumes grown in contaminated soils, but it has generated inconsistent results with regard to HM accumulation in plant tissues. Here, we conducted a meta-analysis to assess the performance of Rhizobium inoculation for regulating HM in legumes and reveal the general influencing factors and processes. The meta-analysis showed that Rhizobium inoculation in legumes primarily increased the total HM uptake by stimulating plant biomass growth rather than HM phytoavailability. Inoculation had no significant effect on the average shoot HM concentration (p > 0.05); however, it significantly increased root HM uptake by 61 % and root HM concentration by 7 % (p < 0.05), indicating safe agricultural production while facilitating HM phytostabilisation. Inoculation decreased shoot HM concentrations and increased root HM uptake in Vicia, Medicago and Glycine, whereas it increased shoot HM concentrations in Sulla, Cicer and Vigna. The effects of inoculation on shoot biomass were suppressed by nitrogen fertiliser and native microorganisms, and the effect on shoot HM concentration was enhanced by high soil pH, organic matter content, and phosphorous content. Inoculation-boosted shoot nutrient concentration was positively correlated with increased shoot biomass, whereas the changes in pH and organic matter content were insufficient to significantly affect accumulation outcomes. Nitrogen content changes in the soil were positively correlated with changes in root HM concentration and uptake, whereas nitrogen translocation changes in the tissues were positively correlated with changes in HM translocation. Phosphorus solubilisation could improve HM phytoavailability at the expense of slight biomass promotion. These results suggest that the diverse growth-promoting characteristics of Rhizobia influence the trade-off between biomass-HM phytoavailability and HM translocation, impacting HM accumulation outcomes. Our findings can assist in optimising the utilisation of legume-Rhizobium systems in HM-contaminated soils., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

Competing Interests: The author(s) declare no conflict of interest.

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., (© 2024. The Author(s).)

Root nodule symbiosis (RNS) between legumes and rhizobia is a major source of nitrogen in agricultural systems. Effective symbiosis requires precise regulation of plant defense responses. The role of the defense hormone jasmonic acid (JA) in the immune response has been extensively studied. Current research shows that JA can play either a positive or negative regulatory role in RNS depending on its concentration, but the molecular mechanisms remain to be elucidated. In this study, we found that inoculation with the rhizobia Sm1021 induces the JA pathway in Medicago truncatula, and blocking the JA pathway significantly reduces the number of infection threads. Mutations in the MtMYC2 gene, which encodes a JA signaling master transcription factor, significantly inhibited rhizobia infection, terminal differentiation, and symbiotic cell formation. Combining RNA sequencing and chromatin immunoprecipitation sequencing, we discovered that MtMYC2 regulates the expression of nodule-specific MtDNF2, MtNAD1, and MtSymCRK to suppress host defense, while it activates MtDNF1 expression to regulate the maturation of MtNCRs, which in turn promotes bacteroid formation. More importantly, MtMYC2 participates in symbiotic signal transduction by promoting the expression of MtIPD3. Notably, the MtMYC2-MtIPD3 transcriptional regulatory module is specifically present in legumes, and the Mtmyc2 mutants are susceptible to the infection by the pathogen Rhizoctonia solani. Collectively, these findings reveal the molecular mechanisms of how the JA pathway regulates RNS, broadening our understanding of the roles of JA in plant-microbe interactions., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

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 nif H 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.

Symbiotic nitrogen (N) fixation (SNF) by legumes and their rhizobial partners is one of the most important sources of bioavailable N to terrestrial ecosystems. While most work on the regulation of SNF has focussed on abiotic drivers such as light, water and soil nutrients, the diversity of rhizobia with which individual legume partners may play an important but under-recognized role in regulating N inputs from SNF. By experimentally manipulating the diversity of rhizobia available to legumes, we demonstrate that rhizobial diversity can increase average SNF rates by more than 90%, and that high rhizobial diversity can induce increased SNF even under conditions of high soil N fertilization. However, the effects of rhizobial diversity, the conditions under which diversity effects were the strongest, and the likely mechanisms driving these diversity effects differed between the two legume species we assessed. These results provide evidence that biodiversity-ecosystem function relationships can occur at the scales of an individual plant and that the effects of rhizobial diversity may be as important as long-established abiotic factors, such as N availability, in driving terrestrial N inputs via SNF.

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., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Plants coexist with a diverse array of microorganisms, predominantly bacteria and fungi, in both natural and agricultural environments. While some microorganisms positively influence plant development and yield, others can cause harm to the host, leading to significant adverse impacts on the environment and the economy. Plant growth-promoting microorganisms (PGPM), including plant growth-promoting bacteria, arbuscular mycorrhizal fungus (AMF), and rhizobia, have been found to increase plant biomass production by synthesizing hormones, fixing nitrogen, and solubilizing phosphate and potassium. Numerous studies have contributed to unraveling the complex process of plant-microbe interactions in recent decades. In light of the increasing global challenges such as population growth, climate change, and resource scarcity, it has become imperative to explore the potential of plant-bacteria-fungi crosstalk in promoting sustainability. This review aims to bridge existing knowledge gaps, providing a roadmap for future research in this dynamic field by synthesizing current knowledge and identifying emerging trends., Competing Interests: Declaration of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

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., (© 2024. Springer Nature Limited.)

Biological nitrogen fixation is the main source of nitrogen in ecosystems. The diversity of soil rhizobia and their effects on soybeans need further research. In this study, we collected soybean rhizosphere samples from eight sites in the black soil soybean planting area in Northeast China. A total of 94 strains of bacteria were isolated and identified using the 16S rRNA and symbiotic genes ( nodC, nifH ) analysis, of which 70 strains were identified as rhizobia belonging to the genus Bradyrhizobium . To further validate the application effects of rhizobia, we selec-ted seven representative indigenous rhizobia based on the results of phylogenetic analysis, and conducted laboratory experiments to determine their nodulation and the impacts on soybeans. The results showed that, compared to the control without rhizobial inoculation, all the seven indigenous rhizobia exhibited good promoting and nodulation abilities. Among them, strains H7-L22 and H34-L6 performed the best, with the former significantly increasing plant height by 25.7% and the latter increasing root nodule dry weight by 20.9% to 67.1% compared to other indi-genous rhizobia treatments. We tested these two efficient rhizobia strains as soybean rhizobial inoculants in field experiments. The promoting effect of mixed rhizobial inoculants was significantly better than single ones. Compared to the control without inoculation, soybean yield increased by 8.4% with the strain H7-L22 treatment and by 17.9% with the mixed inoculant treatment. Additionally, there was a significant increase in the number of four-seed pods in soybeans. In conclusion, the application of rhizobial inoculants can significantly increase soybean yield, thereby reducing dependence on nitrogen fertilizer during soybean production, improving soil health, and promoting green development in agriculture in the black soil region of Northeast China.

Background: Rhizobial inoculation in combination with fungicidal seed treatment is an effective solution for improving soybean resistance to modern climate changes due to the maximum implementation of the plant's stress-protective antioxidant properties and their nitrogen-fixing potential, which will contribute to the preservation of the environment., Methods: Model ecosystems at different stages of legume-rhizobial symbiosis formation, created by treatment before sowing soybean seeds with a fungicide (fludioxonil, 25 g/L) and inoculation with an active strain of Bradyrhizobium japonicum (titer 109 cells per mL), were subjected to microbiological, biochemical, and physiological testing methods in controlled and field conditions., Results: Seed treatment with fungicide and rhizobia showed different patterns in the dynamics of key antioxidant enzymes in soybean nodules under drought conditions. Superoxide dismutase activity increased by 32.7% under moderate stress, while catalase increased by 90.6% under long-term stress. An increase in the antioxidant enzyme activity induced the regulation of lipoperoxidation processes during drought and after the restoration of irrigation. Regeneration after stress was evident in soybean plants with a combination of fungicide seed treatment and rhizobial inoculant, where enzyme levels and lipoperoxidation processes returned to control plant levels. Applying seed treatment with fungicide and Rhizobium led to the preservation of the symbiotic apparatus functioning in drought conditions. As proof of this, molecular nitrogen fixation by nodules has a higher efficiency of 25.6% compared to soybeans without fungicide treatment. In the field, fungicidal treatment of seeds in a complex with rhizobia inoculant induced prolongation of the symbiotic apparatus functioning in the reproductive period of soybean ontogenesis. This positively affected the nitrogen-fixing activity of soybeans during the pod formation stage by more than 71.7%, as well as increasing soybean yield by 12.7% in the field., Conclusions: The application of Rhizobium inoculant and fungicide to seeds contributed to the development of antioxidant protection of soybean plants during droughts due to the activation of key enzymatic complexes and regulation of lipoperoxidation processes, which have a positive effect on nitrogen fixation and productivity of soybeans. This is a necessary element in soybean agrotechnologies to improve plant adaptation and resilience in the context of modern climate change., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Symbiosis between Glycine max and Bradyrhizobium diazoefficiens were used as a model system to investigate whether biohydrogen utilization promotes the transformation of the tetrachlorobiphenyl PCB77. Both a H 2 uptake-positive (Hup + ) strain (wild type) and a Hup - strain (a hupL deletion mutant) were inoculated into soybean nodules. Compared with Hup - nodules, Hup + nodules increased dechlorination significantly by 61.1 % and reduced the accumulation of PCB77 in nodules by 37.7 % (p < 0.05). After exposure to nickel, an enhancer of uptake hydrogenase, dechlorination increased significantly by 2.2-fold, and the accumulation of PCB77 in nodules decreased by 54.4 % (p < 0.05). Furthermore, the tetrachlorobiphenyl transformation in the soybean root nodules was mainly testified to be mediated by nitrate reductase (encoded by the gene NR) for tetrachlorobiphenyl dechlorination and biphenyl-2,3-diol 1,2-dioxygenase (bphC) for biphenyl degradation. This study demonstrates for the first time that biohydrogen utilization has a beneficial effect on tetrachlorobiphenyl biotransformation in a legume-rhizobium symbiosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Globally, agricultural productivity is facing a serious problem due to soil salinity which often causes osmotic, ionic, and redox imbalances in plants. Applying halotolerant rhizobacterial inoculants having multifarious growth-regulating traits is thought to be an effective and advantageous approach to overcome salinity stress. Here, salt-tolerant (tolerating 300 mM NaCl), exopolysaccharide (EPS) producing Rhizobium azibense SR-26 (accession no. MG063740) was assessed for salt alleviation potential by inoculating Phaseolus vulgaris (L.) plants raised under varying NaCl regimes. The metabolically active cells of strain SR-26 produced a significant amount of phytohormones (indole-3-acetic acid, gibberellic acid, and cytokinin), ACC deaminase, ammonia, and siderophore under salt stress. Increasing NaCl concentration variably affected the EPS produced by SR-26. The P-solubilization activity of the SR-26 strain was positively impacted by NaCl, as demonstrated by OD shift in NaCl-treated/untreated NBRIP medium. The detrimental effect of NaCl on plants was lowered by inoculation of halotolerant strain SR-26. Following soil inoculation, R. azibense significantly (p ≤ 0.05) enhanced seed germination (10%), root (19%) shoot (23%) biomass, leaf area (18%), total chlorophyll (21%), and carotenoid content (32%) of P. vulgaris raised in soil added with 40 mM NaCl concentration. Furthermore, strain SR-26 modulated the relative leaf water content (RLWC), proline, total soluble protein (TSP), and sugar (TSS) of salt-exposed plants. Moreover, R. azibense inoculation lowered the concentrations of oxidative stress biomarkers; MDA (29%), H 2 O 2 content (24%), electrolyte leakage (31%), membrane stability (36%) and Na + ion uptake (28%) when applied to 40 mM NaCl-treated plants. Further, R. azibense increases the salt tolerance mechanism of P. vulgaris by upregulating the antioxidant defensive responses. Summarily, it is reasonable to propose that EPS-synthesizing halotolerant R. azibense SR-26 should be applied as the most cost-effective option for increasing the yields of legume crops specifically P. vulgaris in salinity-challenged soil systems., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Legume-rhizobium symbiotic nitrogen fixation is a highly energy-consuming process. Recent studies demonstrate that nodule-specific energy sensors play important roles in modulating nodule nitrogen fixation capacity. This opens a new field in the energy regulation of symbiotic nitrogen fixation that can provide insights into designing leguminous crops with efficient nitrogen fixation., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Legumes house nitrogen-fixing endosymbiotic rhizobia in specialised polyploid cells within root nodules. This results in a mutualistic relationship whereby the plant host receives fixed nitrogen from the bacteria in exchange for dicarboxylic acids. This plant-microbe interaction requires the regulation of multiple metabolic and physiological processes in both the host and symbiont in order to achieve highly efficient symbiosis. Recent studies have showed that the success of symbiosis is influenced by the circadian clock of the plant host. Medicago and soybean plants with altered clock mechanisms showed compromised nodulation and reduced plant growth. Furthermore, transcriptomic analyses revealed that multiple genes with key roles in recruitment of rhizobia to plant roots, infection and nodule development were under circadian control, suggesting that appropriate timing of expression of these genes may be important for nodulation. There is also evidence for rhythmic gene expression of key nitrogen fixation genes in the rhizobium symbiont, and temporal coordination between nitrogen fixation in the bacterial symbiont and nitrogen assimilation in the plant host may be important for successful symbiosis. Understanding of how circadian regulation impacts on nodule establishment and function will identify key plant-rhizobial connections and regulators that could be targeted to increase the efficiency of this relationship., (© 2024 The Author(s).)

The legume-rhizobium symbiosis represents a unique model within the realm of plant-microbe interactions. Unlike typical cases of pathogenic invasion, the infection of rhizobia and their residence within symbiotic cells do not elicit a noticeable immune response in plants. Nevertheless, there is still much to uncover regarding the mechanisms through which plant immunity influences rhizobial symbiosis. In this study, we identify an important player in this intricate interplay: Lotus japonicus PRP1, which serves as a positive regulator of plant immunity but also exhibits the capacity to decrease rhizobial colonization and nitrogen fixation within nodules. The PRP1 gene encodes an uncharacterized protein and is named Pathogenesis-Related Protein1, owing to its orthologue in Arabidopsis thaliana, a pathogenesis-related family protein (At1g78780). The PRP1 gene displays high expression levels in nodules compared to other tissues. We observed an increase in rhizobium infection in the L. japonicus prp1 mutants, whereas PRP1-overexpressing plants exhibited a reduction in rhizobium infection compared to control plants. Intriguingly, L. japonicus prp1 mutants produced nodules with a pinker colour compared to wild-type controls, accompanied by elevated levels of leghaemoglobin and an increased proportion of infected cells within the prp1 nodules. The transcription factor Nodule Inception (NIN) can directly bind to the PRP1 promoter, activating PRP1 gene expression. Furthermore, we found that PRP1 is a positive mediator of innate immunity in plants. In summary, our study provides clear evidence of the intricate relationship between plant immunity and symbiosis. PRP1, acting as a positive regulator of plant immunity, simultaneously exerts suppressive effects on rhizobial infection and colonization within nodules., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

All non-Mimosoid nodulated genera in the legume subfamily Caesalpinioideae confine their rhizobial symbionts within cell wall-bound 'fixation threads' (FTs). The exception is the large genus Chamaecrista in which shrubs and subshrubs house their rhizobial bacteroids more intimately within symbiosomes, whereas large trees have FTs. This study aimed to unravel the evolutionary relationships between Chamaecrista growth habit, habitat, nodule bacteroid type, and rhizobial genotype. The growth habit, bacteroid anatomy, and rhizobial symbionts of 30 nodulated Chamaecrista species native to different biomes in the Brazilian state of Bahia, a major centre of diversity for the genus, was plotted onto an ITS-trnL-F-derived phylogeny of Chamaecrista. The bacteroids from most of the Chamaecrista species examined were enclosed in symbiosomes (SYM-type nodules), but those in arborescent species in the section Apoucouita, at the base of the genus, were enclosed in cell wall material containing homogalacturonan (HG) and cellulose (FT-type nodules). Most symbionts were Bradyrhizobium genotypes grouped according to the growth habits of their hosts, but the tree, C. eitenorum, was nodulated by Paraburkholderia. Chamaecrista has a range of growth habits that allow it to occupy several different biomes and to co-evolve with a wide range of (mainly) bradyrhizobial symbionts. FTs represent a less intimate symbiosis linked with nodulation losses, so the evolution of SYM-type nodules by most Chamaecrista species may have (i) aided the genus-wide retention of nodulation, and (ii) assisted in its rapid speciation and radiation out of the rainforest into more diverse and challenging habitats., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Certain legumes provide a special pathway for rhizobia to invade the root and develop nitrogen-fixing nodules, a process known as lateral root base (LRB) nodulation. This pathway involves intercellular infection at the junction of the lateral roots with the taproot, leading to nodule formation in the lateral root cortex. Remarkably, this LRB pathway serves as a backbone for various adaptative symbiotic processes. Here, we describe different aspects of LRB nodulation and highlight directions for future research to elucidate the mechanisms of this as yet little known but original pathway that will help in broadening our knowledge on the rhizobium-legume symbiosis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Nitrogen (N 2 ) fixation in oligotrophic surface waters is the main source of new nitrogen to the ocean 1 and has a key role in fuelling the biological carbon pump 2 . Oceanic N 2 fixation has been attributed almost exclusively to cyanobacteria, even though genes encoding nitrogenase, the enzyme that fixes N 2 into ammonia, are widespread among marine bacteria and archaea 3-5 . Little is known about these non-cyanobacterial N 2 fixers, and direct proof that they can fix nitrogen in the ocean has so far been lacking. Here we report the discovery of a non-cyanobacterial N 2 -fixing symbiont, 'Candidatus Tectiglobus diatomicola', which provides its diatom host with fixed nitrogen in return for photosynthetic carbon. The N 2 -fixing symbiont belongs to the order Rhizobiales and its association with a unicellular diatom expands the known hosts for this order beyond the well-known N 2 -fixing rhizobia-legume symbioses on land 6 . Our results show that the rhizobia-diatom symbioses can contribute as much fixed nitrogen as can cyanobacterial N 2 fixers in the tropical North Atlantic, and that they might be responsible for N 2 fixation in the vast regions of the ocean in which cyanobacteria are too rare to account for the measured rates., (© 2024. The Author(s).)

Arid and semi-arid areas are facing increasingly severe water deficits that are being intensified by global climate changes. Microbes associated with plants native to arid regions provide valuable benefits to plants, especially in water-stressed environments. In this study, we used 16S rDNA metabarcoding analysis to examine the bacterial communities in the bulk soil, rhizosphere and root endosphere of the plant Malva sylvestris L. in Morocco, along a gradient of precipitation. We found that the rhizosphere of M. sylvestris did not show significant differences in beta-diversity compared to bulk soil, although, it did display an increased degree of alpha-diversity. The endosphere was largely dominated by the genus Rhizobium and displayed remarkable variation between plants, which could not be attributed to any of the variables observed in this study. Overall, the effects of precipitation level were relatively weak, which may be related to the intense drought in Morocco at the time of sampling. The dominance of Rhizobium in a non-leguminous plant is particularly noteworthy and may permit the utilization of this bacterial taxon to augment drought tolerance; additionally, the absence of any notable selection of the rhizosphere of M. sylvestris suggests that it is not significatively affecting its soil environment., (© 2024 The Authors. Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

In Tunisia, Orobanche foetida Poir. is considered an important agricultural biotic constraint on faba bean (Vicia faba L.) production. An innovative control method for managing this weed in faba bean is induced resistance through inoculation by rhizobia strains. In this study, we explored the biochemical dynamics in V. faba L. minor inoculated by rhizobia in response to O. foetida parasitism. A systemic induced resistant reaction was evaluated through an assay of peroxidase (POX), polyphenol oxidase (PPO) and phenyl alanine ammonialyase (PAL) activity and phenolic compound and hydrogen peroxide (H2O2) accumulation in faba bean plants infested with O. foetida and inoculated with rhizobia. Two rhizobia strains (Mat, Bj1) and a susceptible variety of cultivar Badi were used in a co-culture Petri dish experiment. We found that Mat inoculation significantly decreased O. foetida germination and the number of tubercles on the faba bean roots by 87% and 88%, respectively. Following Bj1 inoculation, significant decreases were only observed in O. foetida germination (62%). In addition, Mat and Bj1 inoculation induced a delay in tubercle formation (two weeks) and necrosis in the attached tubercles (12.50% and 4.16%, respectively) compared to the infested control. The resistance of V. faba to O. foetida following Mat strain inoculation was mainly associated with a relatively more efficient enzymatic antioxidative response. The antioxidant enzyme activity was enhanced following Mat inoculation of the infected faba bean plant. Indeed, increases of 45%, 67% and 86% were recorded in the POX, PPO and PAL activity, respectively. Improvements of 56% and 12% were also observed in the soluble phenolic and H2O2 contents. Regarding inoculation with the Bj1 strain, significant increases were only observed in soluble phenolic and H2O2 contents and PPO activity (especially at 45 days after inoculation) compared to the infested control. These results imply that inoculation with the rhizobia strains (especially Mat) induced resistance and could bio-protect V. faba against O. foetida parasitism by inducing systemic resistance, although complete protectionwas not achieved by rhizobia inoculation. The Mat strain could be used as a potential candidate for the development of an integrated method for controlling O. foetida parasitism in faba bean., Competing Interests: No authors have a competing interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bouraoui et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

South Africa is well-known for the diversity of its legumes and their nitrogen-fixing bacterial symbionts. However, in contrast to their plant partners, remarkably few of these microbes (collectively referred to as rhizobia) from South Africa have been characterised and formally described. This is because the rules of the International Code of Nomenclature of Prokaryotes (ICNP) are at odds with South Africa's National Environmental Management: Biodiversity Act and its associated regulations. The ICNP requires that a culture of the proposed type strain for a novel bacterial species be deposited in two international culture collections and be made available upon request without restrictions, which is not possible under South Africa's current national regulations. Here, we describe seven new Mesorhizobium species obtained from root nodules of Vachellia karroo, an iconic tree legume distributed across various biomes in southern Africa. For this purpose, 18 rhizobial isolates were delineated into putative species using genealogical concordance, after which their plausibility was explored with phenotypic characters and average genome relatedness. For naming these new species, we employed the rules of the recently published Code of Nomenclature of Prokaryotes described from Sequence Data (SeqCode), which utilizes genome sequences as nomenclatural types. The work presented in this study thus provides an illustrative example of how the SeqCode allows for a standardised approach for naming cultivated organisms for which the deposition of a type strain in international culture collections is currently problematic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Symbiotic nitrogen fixation (SNF) is crucial for legumes, providing them with the nitrogen necessary for plant growth and development. Nodulation is the first step in the establishment of SNF. However, the determinant genes in soybean nodulation and the understanding of the underlying molecular mechanisms governing nodulation are still limited. Herein, we identified a phosphatase, GmPP2C61A, which was specifically induced by rhizobia inoculation. Using transgenic hairy roots harboring GmPP2C61A::GUS, we showed that GmPP2C61A was mainly induced in epidermal cells following rhizobia inoculation. Functional analysis revealed that knockdown or knock-out of GmPP2C61A significantly reduced the number of nodules, while overexpression of GmPP2C61A promoted nodule formation. Additionally, GmPP2C61A protein was mainly localized in the cytoplasm and exhibited conserved phosphatase activity in vitro. Our findings suggest that phosphatase GmPP2C61A serves as a critical regulator in soybean nodulation, highlighting its potential significance in enhancing symbiotic nitrogen fixation., (© 2024 Scandinavian Plant Physiology Society.)

Nodulation begins with the initiation of infection threads (ITs) in root hairs. Though mutual recognition and early symbiotic signaling cascades in legumes are well understood, molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis remain largely unexplored. We functionally investigated a novel pectate lyase enzyme, GmNPLa, and its transcriptional regulator GmPTF1a/b in soybean (Glycine max), where their regulatory roles in IT development and nodule formation were elucidated through investigation of gene expression patterns, bioinformatics analysis, biochemical verification of genetic interactions, and observation of phenotypic impacts in transgenic soybean plants. GmNPLa was specifically induced by rhizobium inoculation in root hairs. Manipulation of GmNPLa produced remarkable effects on IT and nodule formation. GmPTF1a/b displayed similar expression patterns as GmNPLa, and manipulation of GmPTF1a/b also severely influenced nodulation traits. LI soybeans with low nodulation phenotypes were nearly restored to HI nodulation level by complementation of GmNPLa and/or GmPTF1a. Further genetic and biochemical analysis demonstrated that GmPTF1a can bind to the E-box motif to activate transcription of GmNPLa, and thereby facilitate nodulation. Taken together, our findings potentially reveal novel mediation of cell wall gene expression involving the basic helix-loop-helix transcription factor GmPTF1a/b acts as a key early regulator of nodulation in soybean., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

C-terminally encoded peptides (CEP) signaling peptides are drivers of systemic pathways regulating nitrogen (N) acquisition in different plants, from Arabidopsis to legumes, depending on mineral N availability (e.g. nitrate) and on the whole plant N demand. Recent studies in the Medicago truncatula model legume revealed how root-produced CEP peptides control the root competence for endosymbiosis with N fixing rhizobia soil bacteria through the activity of the Compact Root Architecture 2 (CRA2) CEP receptor in shoots. Among CEP genes, MtCEP7 was shown to be tightly linked to nodulation, and the dynamic temporal regulation of its expression reflects the plant ability to maintain a different symbiotic root competence window depending on the symbiotic efficiency of the rhizobium strain, as well as to reinitiate a new window of root competence for nodulation., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

In addition to rhizobia, many types of co-existent bacteria are found in leguminous root nodules, but their habitats are unclear. To investigate this phenomenon, we labeled Bradyrhizobium diazoefficiens USDA122 and Bradyrhizobium sp. SSBR45 with Discosoma sp. red fluorescent protein (DsRed) or enhanced green fluorescent protein (eGFP). USDA122 enhances soybean growth by forming effective root nodules, but SSBR45 does not form any nodules. Using low-magnification laser scanning confocal microscopy, we found that infected cells in the central zone of soybean nodules appeared to be occupied by USDA122. Notably, high-magnification microscopy after co-inoculation of non-fluorescent USDA122 and fluorescence-labeled SSBR45 also revealed that SSBR45 inhabits the intercellular spaces of healthy nodules. More unexpectedly, co-inoculation of eGFP-labeled USDA122 and DsRed-labeled SSBR45 (and vice versa) revealed the presence of USDA122 bacteria in both the symbiosomes of infected cells and in the apoplasts of healthy nodules. We then next inspected nodules formed after a mixed inoculation of differently-labeled USDA122, without SSBR45, and confirmed the inhabitation of the both populations of USDA122 in the intercellular spaces. In contrast, infected cells were occupied by single-labeled USDA122. We also observed Mesorhizobium loti in the intercellular spaces of active wild-type nodules of Lotus japonicus using transmission electron microscopy. Compatible intercellular rhizobia have been described during nodule formation of several legume species and in some mutants, but our evidence suggests that this type of colonization may occur much more commonly in leguminous root nodules.

Legumes represent an important source of food protein for human nutrition and animal feed. Therefore, sustainable production of legume crops is an issue of global importance. It is well-known that legume-rhizobia symbiosis allows an increase in the productivity and resilience of legume crops. The efficiency of this mutualistic association strongly depends on precise regulation of the complex interactions between plant and rhizobia. Their molecular dialogue represents a complex multi-staged process, each step of which is critically important for the overall success of the symbiosis. In particular, understanding the details of the molecular mechanisms behind the nodule formation and functioning might give access to new legume cultivars with improved crop productivity. Therefore, here we provide a comprehensive literature overview on the dynamics of the signaling network underlying the development of the legume-rhizobia symbiosis. Thereby, we pay special attention to the new findings in the field, as well as the principal directions of the current and prospective research. For this, here we comprehensively address the principal signaling events involved in the nodule inception, development, functioning, and senescence.

Grass pea (L. sativus L.) is a widely cultivated crop worldwide, forming a symbiotic relationship with nitrogen-fixing rhizobia. Glyphosate is commonly used by farmers for weed control during agricultural processes. However, the application of this chemical herbicide negatively impacts soil fertility by affecting the nitrogen-fixing rhizobia. This study aimed to assess the effects of glyphosate on rhizobia isolated from healthy and robust Grass pea plants. Specifically, Grass pea plants exhibiting vigorous growth and a healthy appearance were intentionally selected to isolate rhizobia from their root nodules. The isolated rhizobia were then characterized based on their morphological features, biochemical properties, and resistance to abiotic traits. Rhizobial isolates from grass peas exhibited Gram-negative, rod-shaped morphology, milky colony color, and variable colony sizes. Additionally, the majority displayed smooth colony surfaces on yeast extract mannitol agar medium. Based on morphological and biochemical characteristics, the isolates could be grouped under the genus Rhizobium. Optimum growth conditions for these isolates were observed at temperatures between 28 and 38 °C, pH levels ranging from 5 to 8, and salt (NaCl) concentrations of 0.5% and 1%. At a concentration of 20 mL L -1 , glyphosate inhibited 5.52-47% of the Rhizobium population. The inhibition percentage increased to 17.1-53.38% at a concentration of 40 mL L -1 . However, when exposed to a higher concentration (60 mL/L) of glyphosate, 87% of the isolates were inhibited. The number of colonies after glyphosate exposure was significantly dependent on concentration, and there were notable differences between treatments with varying glyphosate concentrations (p < 0.05). Glyphosate negatively impacted the survival of grass pea rhizobia, leading to a reduction in the Rhizobium population (CFU). However, the effect varied between Rhizobium isolated from grass pea root nodules., (© 2023. The Author(s).)

Root nodule development plays a vital role in establishing the mutualistic relationship between legumes and nitrogen-fixing rhizobia. Two primary processes are involved in nodule development: formative cell divisions in the root cortex and the subsequent differentiation of nodule cells. The first process involves the mitotic reactivation of differentiated root cortex cells to form nodule primordium after perceiving symbiotic signals. The second process enables the nascent nodule primordium cells to develop into various cell types, leading to the creation of a functional nodule capable of supporting nitrogen fixation. Thus, both division and differentiation of nodule cells are crucial for root nodule development. This review provides an overview of the most recent advancements in comprehending the cellular and molecular mechanisms underlying symbiotic nodule development in legumes., Competing Interests: Declaration of competing interest The authors declare that they do not have any competing financial interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Legumes enter into symbiotic associations with soil nitrogen-fixing rhizobia, culminating in the creation of new organs, root nodules. This complex process relies on chemical and physical interaction between legumes and rhizobia, including early signalling events informing the host legume plant of a potentially beneficial microbe and triggering the nodulation program. The great significance of this plant-microbe interaction rests upon conversion of atmospheric dinitrogen not accessible to plants into a biologically active form of ammonia available to plants. The plant cytoskeleton consists in a highly dynamic network and undergoes rapid remodelling upon sensing various developmental and environmental cues, including response to attachment, internalization, and accommodation of rhizobia in plant root and nodule cells. This dynamic nature is governed by cytoskeleton-associated proteins that modulate cytoskeletal behaviour depending on signal perception and transduction. Precisely localized cytoskeletal rearrangements are therefore essential for the uptake of rhizobia, their targeted delivery, and establishing beneficial root nodule symbiosis. This review summarizes current knowledge about rhizobia-dependent rearrangements and functions of the cytoskeleton in legume roots and nodules. General patterns and nodule type-, nodule stage-, and species-specific aspects of actin filaments and microtubules remodelling are discussed. Moreover, emerging evidence is provided about fine-tuning the root nodulation process through cytoskeleton-associated proteins. We also consider future perspectives on dynamic localization studies of the cytoskeleton during early symbiosis utilizing state of the art molecular and advanced microscopy approaches. Based on acquired detailed knowledge of the mutualistic interactions with microbes, these approaches could contribute to broader biotechnological crop improvement., Competing Interests: Declaration of Competing Interest The authors have no conflicts to declare., (Copyright © 2023. Published by Elsevier Inc.)

Main Conclusion: Rpf107 is involved in the infection process of rhizobia and the maintenance of symbiotic nitrogen fixation in black locust root nodules. The LURP-one related (LOR) protein family plays a pivotal role in mediating plant defense responses against both biotic and abiotic stresses. However, our understanding of its function in the symbiotic interaction between legumes and rhizobia remains limited. Here, Rpf107, a homolog of LOR, was identified in Robinia pseudoacacia (black locust). The subcellular localization of Rpf107 was analyzed, and its function was investigated using RNA interference (RNAi) and overexpression techniques. The subcellular localization assay revealed that Rpf107 was mainly distributed in the plasma membrane and nucleus. Rpf107 silencing prevented rhizobial infection and hampered plant growth. The number of infected cells in the nitrogen fixation zone of the Rpf107-RNAi nodules was also noticeably lower than that in the control nodules. Notably, Rpf107 silencing resulted in bacteroid degradation and the premature aging of nodules. In contrast, the overexpression of Rpf107 delayed the senescence of nodules and prolonged the nitrogen-fixing ability of nodules. These results demonstrate that Rpf107 was involved in the infection of rhizobia and the maintenance of symbiotic nitrogen fixation in black locust root nodules. The findings reveal that a member of the LOR protein family plays a role in leguminous root nodule symbiosis, which is helpful to clarify the functions of plant LOR protein family and fully understand the molecular mechanisms underlying legume-rhizobium symbiosis., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Nitrogen-fixing nodules occur in ten related taxonomic lineages interspersed with lineages of non-nodulating plant species. Nodules result from an endosymbiosis between plants and diazotrophic bacteria; rhizobia in the case of legumes and Parasponia and Frankia in the case of actinorhizal species. Nodulating plants share a conserved set of symbiosis genes, whereas related non-nodulating sister species show pseudogenization of several key nodulation-specific genes. Signalling and cellular mechanisms critical for nodulation have been co-opted from the more ancient plant-fungal arbuscular endomycorrhizal symbiosis. Studies in legumes and actinorhizal plants uncovered a key component in symbiotic signalling, the LRR-type SYMBIOSIS RECEPTOR KINASE (SYMRK). SYMRK is essential for nodulation and arbuscular endomycorrhizal symbiosis. To our surprise, however, despite its arbuscular endomycorrhizal symbiosis capacities, we observed a seemingly critical mutation in a donor splice site in the SYMRK gene of Trema orientalis, the non-nodulating sister species of Parasponia. This led us to investigate the symbiotic functioning of SYMRK in the Trema-Parasponia lineage and to address the question of to what extent a single nucleotide polymorphism in a donor splice site affects the symbiotic functioning of SYMRK., Results: We show that SYMRK is essential for nodulation and endomycorrhization in Parasponia andersonii. Subsequently, it is revealed that the 5'-intron donor splice site of SYMRK intron 12 is variable and, in most dicotyledon species, doesn't contain the canonical dinucleotide 'GT' signature but the much less common motif 'GC'. Strikingly, in T. orientalis, this motif is converted into a rare non-canonical 5'-intron donor splice site 'GA'. This SYMRK allele, however, is fully functional and spreads in the T. orientalis population of Malaysian Borneo. A further investigation into the occurrence of the non-canonical GA-AG splice sites confirmed that these are extremely rare., Conclusion: SYMRK functioning is highly conserved in legumes, actinorhizal plants, and Parasponia. The gene possesses a non-common 5'-intron GC donor splice site in intron 12, which is converted into a GA in T. orientalis accessions of Malaysian Borneo. The discovery of this functional GA-AG splice site in SYMRK highlights a gap in our understanding of splice donor sites., (© 2023. The Author(s).)

Main Conclusion: A comprehensive understanding of nitrogen signaling cascades involving heterotrimeric G-proteins and their putative receptors can assist in the production of nitrogen-efficient plants. Plants are immobile in nature, so they must endure abiotic stresses including nutrient stress. Plant development and agricultural productivity are frequently constrained by the restricted availability of nitrogen in the soil. Non-legume plants acquire nitrogen from the soil through root membrane-bound transporters. In depleted soil nitrogen conditions, legumes are naturally conditioned to fix atmospheric nitrogen with the aid of nodulation elicited by nitrogen-fixing bacteria. Moreover, apart from the symbiotic nitrogen fixation process, nitrogen uptake from the soil can also be a significant secondary source to satisfy the nitrogen requirements of legumes. Heterotrimeric G-proteins function as molecular switches to help plant cells relay diverse stimuli emanating from external stress conditions. They are comprised of Gα, Gβ and Gγ subunits, which cooperate with several downstream effectors to regulate multiple plant signaling events. In the present review, we concentrate on signaling mechanisms that regulate plant nitrogen nutrition. Our review highlights the potential of heterotrimeric G-proteins, together with their putative receptors, to assist the legume root nodule symbiosis (RNS) cascade, particularly during calcium spiking and nodulation. Additionally, the functions of heterotrimeric G-proteins in nitrogen acquisition by plant roots as well as in improving nitrogen use efficiency (NUE) have also been discussed. Future research oriented towards heterotrimeric G-proteins through genome editing tools can be a game changer in the enhancement of the nitrogen fixation process. This will foster the precise manipulation and production of plants to ensure global food security in an era of climate change by enhancing crop productivity and minimizing reliance on external inputs., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In most legumes, the rhizobial symbionts exhibit diversity across different environments. Although common bean (Phaseolus vulgaris L.) is one of the important legumes in southern Africa, there is no available information on the genetic diversity and N 2 -fixing effectiveness of its symbionts in Malkerns, Eswatini. In this study, we assessed the phylogenetic positions of rhizobial microsymbionts of common bean from Malkerns in Eswatini. The isolates obtained showed differences in morpho-physiology and N 2 -fixing efficiency. A dendrogram constructed from the ERIC-PCR banding patterns, grouped a total of 88 tested isolates into 80 ERIC-PCR types if considered at a 70% similarity cut-off point. Multilocus sequence analysis using 16S rRNA, rpoB, dnaK, gyrB, and glnII and symbiotic (nifH and nodC) gene sequences closely aligned the test isolates to the type strains of Rhizobium muluonense, R. paranaense, R. pusense, R. phaseoli and R. etli. Subjecting the isolates in this study to further description can potentially reveal novel species. Most of the isolates tested were efficient in fixing nitrogen and elicited greater stomatal conductance and photosynthetic rates in the common bean. Relative effectiveness (RE) varied from 18 to 433%, with 75 (85%) out of the 88 tested isolates being more effective than the nitrate fed control plants., (© 2023. Springer Nature Limited.)

Although vacuolar phosphate transporters (VPTs) are essential for plant phosphorus adaptation, their role in Rhizobium-legume symbiosis is unclear. In this study, homologous genes of VPT1 (MtVPTs) were identified in Medicago truncatula to assess their roles in Rhizobium-legume symbiosis and phosphorus adaptation. MtVPT2 and MtVPT3 mainly positively responded to low and high phosphate, respectively. However, both mtvpt2 and mtvpt3 mutants displayed shoot phenotypes with high phosphate sensitivity and low phosphate tolerance. The root-to-shoot phosphate transfer efficiency was significantly enhanced in mtvpt3 but weakened in mtvpt2, accompanied by lower and higher root cytosolic inorganic phosphate (Pi) concentration, respectively. Low phosphate induced MtVPT2 and MtVPT3 expressions in nodules. MtVPT2 and MtVPT3 mutations markedly reduced the nodule number and nitrogenase activity under different phosphate conditions. Cytosolic Pi concentration in nodules was significantly lower in mtvpt2 and mtvpt3 than in the wildtype, especially in tissues near the base of nodules, probably due to inhibition of long-distance Pi transport and cytosolic Pi supply. Also, mtvpt2 and mtvpt3 could not maintain a stable cytosolic Pi level in the nodule fixation zone as the wildtype under low phosphate stress. These findings show that MtVPT2 and MtVPT3 modulate phosphorus adaptation and rhizobia-legume symbiosis, possibly by regulating long-distance Pi transport., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

In the Rhizobium-Legume symbiosis, the nodulation outer protein P (NopP) effector is one of the key regulators for rhizobial infection and nodule organogenesis. However, the molecular mechanism through which host legume plants sense NopP remains largely unknown. Here, we constructed an nopP deletion mutant of Mesorhizobium huakuii and found that nopP negatively regulates nodulation on Chinese milk vetch (Astragalus sinicus). Screening for NopP interacting proteins in host plants using the yeast 2-hybrid system identified NopP interacting protein 43 (AsNIP43), which encodes a G-type receptor-like kinase (LecRLK). The B-lectin domain at the N terminus of AsNIP43 was essential in mediating its interaction with NopP, which was confirmed in vitro and in vivo. Subcellular localization, co-localization, and gene expression analyses showed that AsNIP43 and NopP function tightly associated with earlier infection events. RNA interference (RNAi) knockdown of AsNIP43 expression by hairy root transformation led to decreased nodule formation. AsNIP43 plays a positive role in symbiosis, which was further verified in the model legume Medicago truncatula. Transcriptome analysis indicated that MtRLK (a homolog of AsNIP43 in M. truncatula) may function to affect defense gene expression and thus to regulate early nodulation. Taken together, we show that LecRLK AsNIP43 is a legume host target that interacts with rhizobia effector NopP is essential for rhizobial infection and nodulation., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Hydrogen sulfide (H 2 S) performs a crucial role in plant development and abiotic stress responses by interacting with other signalling molecules. However, the synergistic involvement of H 2 S and rhizobia in photosynthetic carbon (C) metabolism in soybean (Glycine max) under nitrogen (N) deficiency has been largely overlooked. Therefore, we scrutinised how H 2 S drives photosynthetic C fixation, utilisation, and accumulation in soybean-rhizobia symbiotic systems. When soybeans encountered N deficiency, organ growth, grain output, and nodule N-fixation performance were considerably improved owing to H 2 S and rhizobia. Furthermore, H 2 S collaborated with rhizobia to actively govern assimilation product generation and transport, modulating C allocation, utilisation, and accumulation. Additionally, H 2 S and rhizobia profoundly affected critical enzyme activities and coding gene expressions implicated in C fixation, transport, and metabolism. Furthermore, we observed substantial effects of H 2 S and rhizobia on primary metabolism and C-N coupled metabolic networks in essential organs via C metabolic regulation. Consequently, H 2 S synergy with rhizobia inspired complex primary metabolism and C-N coupled metabolic pathways by directing the expression of key enzymes and related coding genes involved in C metabolism, stimulating effective C fixation, transport, and distribution, and ultimately improving N fixation, growth, and grain yield in soybeans., (© 2023 John Wiley & Sons Ltd.)

Plants associate with nitrogen-fixing bacteria to secure nitrogen, which is generally the most limiting nutrient for plant growth. Endosymbiotic nitrogen-fixing associations are widespread among diverse plant lineages, ranging from microalgae to angiosperms, and are primarily one of three types: cyanobacterial, actinorhizal or rhizobial. The large overlap in the signaling pathways and infection components of arbuscular mycorrhizal, actinorhizal and rhizobial symbioses reflects their evolutionary relatedness. These beneficial associations are influenced by environmental factors and other microorganisms in the rhizosphere. In this review, we summarize the diversity of nitrogen-fixing symbioses, key signal transduction pathways and colonization mechanisms relevant to such interactions, and compare and contrast these interactions with arbuscular mycorrhizal associations from an evolutionary standpoint. Additionally, we highlight recent studies on environmental factors regulating nitrogen-fixing symbioses to provide insights into the adaptation of symbiotic plants to complex environments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Nodulation Receptor Kinase (NORK) functions as a co-receptor of Nod factor receptors to mediate rhizobial symbiosis in legumes, but its direct phosphorylation substrates that positively mediate root nodulation remain to be fully identified. Here, we identified a GmNORK-Interacting Small Protein (GmNISP1) that functions as a phosphorylation target of GmNORK to promote soybean nodulation. GmNORKα directly interacted with and phosphorylated GmNISP1. Transcription of GmNISP1 was strongly induced after rhizobial infection in soybean roots and nodules. GmNISP1 encodes a peptide containing 90 amino acids with a "DY" consensus motif at its N-terminus. GmNISP1 protein was detected to be present in the apoplastic space. Phosphorylation of GmNISP1 by GmNORKα could enhance its secretion into the apoplast. Pretreatment with either purified GmNISP1 or phosphorylation-mimic GmNISP1 12D on the roots could significantly increase nodule numbers compared with the treatment with phosphorylation-inactive GmNISP1 12A . The data suggested a model that soybean GmNORK phosphorylates GmNISP1 to promote its secretion into the apoplast, which might function as a potential peptide hormone to promote root nodulation., (© 2022 Institute of Botany, Chinese Academy of Sciences.)