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Almost all plant species interact with one or more symbioses somewhere within their distribution range. Bringing together plant trait data and growth responses to symbioses spanning 552 plant species, we provide for the first time on a large scale (597 studies) a quantitative synthesis on plant performance differences between eight major types of symbiosis, including mycorrhizas, N-fixing bacteria, fungal endophytes and ant-plant interactions. Frequency distributions of plant growth responses varied considerably between different types of symbiosis, in terms of both mean effect and 'risk', defined here as percentage of experiments reporting a negative effect of symbiosis on plants. Contrary to expectation, plant traits were poor predictors of growth response across and within all eight symbiotic associations. Our analysis showed no systematic additive effect when a host plant engaged in two functionally different symbioses. This synthesis suggests that plant species' ecological strategies have little effect in determining the influence of a symbiosis on host plant growth. Reliable quantification of differences in plant performance across symbioses will prove valuable for developing general hypotheses on how species become engaged in mutualisms without a guarantee of net returns., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

The family Leguminosae comprises approximately 20,000 species that mostly form symbioses with arbuscular mycorrhizal fungi (AMF) and nitrogen-fixing bacteria (NFB). This study is aimed at investigating and confirming the dependence on nodulation and biological nitrogen fixation in the specie Piptadenia gonoacantha (Mart.) Macbr., which belongs to the Piptadenia group. Two consecutive experiments were performed in a greenhouse. The experiments were fully randomized with six replicates and a factorial scheme. For the treatments, the two AMF species and three NFB strains were combined to nodulate P. gonoacantha in addition to the control treatments. The results indicate this species' capacity for nodulation without the AMF; however, the AMF+NFB combinations yielded a considerable gain in P. gonoacantha shoot weight compared with the treatments that only included inoculating with bacteria or AMF. The results also confirm that the treatment effects among the AMF+NFB combinations produced different shoot dry weight/root dry weight ratios. We conclude that AMF is not necessary for nodulation and that this dependence improves species development because plant growth increases upon co-inoculation., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

BackgroundMercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Horizontal gene transfer has potential to confer immediate de novo adaptations to stress. We used comparative genomics of high quality de novo assemblies to identify structural differences in the genomes of nitrogen-fixing rhizobia that were isolated from a mercury (Hg) mine site that show high variation in their tolerance to Hg.ResultsOur analyses identified multiple structurally conserved merA homologs in the genomes of Sinorhizobium medicae and Rhizobium leguminosarum but only the strains that possessed a Mer operon exhibited 10-fold increased tolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while in bacteroids showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant. Transfer of a plasmid containing the Mer operon from the most tolerant strain to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the Mer operon is able to confer hyper tolerance to Hg.ConclusionsMer operons have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.

The Lotus japonicus population carrying new Lotus retrotransposon 1 (LORE1) insertions represents a valuable biological resource for genetic research. New insertions were generated by activation of the endogenous retroelement LORE1a in the germline of the G329-3 plant line and arranged in a 2-D system for reverse genetics. LORE1 mutants identified in this collection contributes substantially to characterize candidate genes involved in symbiotic association of L. japonicus with its cognate symbiont, the nitrogen-fixing bacteria Mesorhizobium loti that infects root nodules intracellularly. In this study we aimed to identify novel players in the poorly explored intercellular infection induced by Agrobacterium pusense IRBG74 sp. For this purpose, a forward screen of > 200,000 LORE1 seedlings, obtained from bulk propagation of G329-3 plants, inoculated with IRBG74 was performed. Plants with perturbed nodulation were scored and the offspring were further tested on plates to confirm the symbiotic phenotype. A total of 110 Lotus mutants with impaired nodulation after inoculation with IRBG74 were obtained. A comparative analysis of nodulation kinetics in a subset of 20 mutants showed that most of the lines were predominantly affected in nodulation by IRBG74. Interestingly, additional defects in the main root growth were observed in some mutant lines. Sequencing of LORE1 flanking regions in 47 mutants revealed that 92 Lotus genes were disrupted by novel LORE1 insertions in these lines. In the IM-S34 mutant, one of the insertions was located in the 5'UTR of the LotjaGi5g1v0179800 gene, which encodes the AUTOPHAGY9 protein. Additional mutant alleles, named atg9-2 and atg9-3, were obtained in the reverse genetic collection. Nodule formation was significantly reduced in these mutant alleles after M. loti and IRBG74 inoculation, confirming the effectiveness of the mutant screening. This study describes an effective forward genetic approach to obtain novel mutants in Lotus with a phenotype of interest and to identify the causative gene(s). [ABSTRACT FROM AUTHOR]

Nodules are formed as a result of symbiosis with nitrogen-fixing bacteria on the roots of legume plants. Symbiotic nodule development shares common developmental processes with lateral roots, including cell proliferation of the pericycle and cortical cell layers leading to primordium formation, and both these organs are assumed to be evolutionarily related. The progression of their development is realized with the help of phytohormones, in particular auxin and cytokinin. Due to common evolutionary origin, the hormonal pathway and response may have similar functionality in cell fate specification during lateral root and nodule development. It is known that auxin and cytokinin both positively regulate nodule primordia development. However during lateral root development, auxin is a key positive regulator, while cytokinin suppresses lateral root development. Therefore, a comparative review of auxin and cytokinin action in the development of these two organs to elucidate how their identity is established should be of special interest. This review discusses the involvement of auxin and cytokinin in the development of symbiotic nodules and lateral roots, with a special focus on the similarity and differences of their action during lateral root and nodule developmental processes. The spatio-temporal peculiarities of auxin and cytokinin biosynthesis, transport and response during lateral root and symbiotic nodule formation are reviewed, which should help to clarify how the identity of these two root lateral organs depends on auxin-cytokinin interplay. [ABSTRACT FROM AUTHOR]

Nitrogen-fixing cyanobacteria from the order Nostocales are able to establish symbiotic relationships with diverse plant species. They are promiscuous symbionts, as the same strain of cyanobacterium is able to form symbiotic biological nitrogen-fixing relationships with different plants species. This review will focus on the different types of cyanobacterial–plant associations, both endophytic and epiphytic, and provide insights from a structural viewpoint, as well as our current understanding of the mechanisms involved in the symbiotic crosstalk. In all these symbioses, the benefit for the plant is clear; it obtains from the cyanobacterium fixed nitrogen and other bioactive compounds, such as phytohormones, polysaccharides, siderophores, or vitamins, leading to enhanced plant growth and productivity. Additionally, there is increasing use of different cyanobacterial species as bio-inoculants for biological nitrogen fixation to improve soil fertility and crop production, thus providing an eco-friendly, alternative, and sustainable approach to reduce the over-reliance on synthetic chemical fertilizers. [ABSTRACT FROM AUTHOR]

Plant root architecture is developmentally plastic in response to fluctuating nutrient levels in the soil. Part of this developmental plasticity is the formation of dedicated root cells and organs to host mutualistic symbionts. Structures like nitrogen‐fixing nodules serve as alternative nutrient acquisition strategies during starvation conditions. Some root systems can also form myconodules—globular root structures that can host mycorrhizal fungi. The myconodule association is different from the wide‐spread arbuscular mycorrhization. This range of symbiotic associations provides different degrees of compartmentalisation, from the cellular to organ scale, which allows the plant host to regulate the entry and extent of symbiotic interactions. In this review, we discuss the degrees of symbiont compartmentalisation by the plant host as a developmental strategy and speculate how spatial confinement mitigates risks associated with root symbiosis. Summary statement: In this review, we aim to synthesize a picture of how plants manage microorganism associations through a common theme of compartmentalising symbiotic microbes into specific cell‐types or organs. We discuss a range of examples of symbiosis, including 'myconodules'—nodule‐like organs that host endosymbiotic fungi and, in some species, are co‐colonized with nitrogen‐fixing bacteria. The context of growth and safety trade‐offs associated with microbe engagement may help explain both extant and extinct types of endosymbiotic nodules. [ABSTRACT FROM AUTHOR]

Summary: Symbiotic nitrogen fixation carried out by the interaction between legumes and rhizobia is the main source of nitrogen in natural ecosystems and in sustainable agriculture. For the symbiosis to be viable, nutrient exchange between the partners is essential. Transition metals are among the nutrients delivered to the nitrogen‐fixing bacteria within the legume root nodule cells. These elements are used as cofactors for many of the enzymes controlling nodule development and function, including nitrogenase, the only known enzyme able to convert N2 into NH3. In this review, we discuss the current knowledge on how iron, zinc, copper, and molybdenum reach the nodules, how they are delivered to nodule cells, and how they are transferred to nitrogen‐fixing bacteria within. [ABSTRACT FROM AUTHOR]

Simple Summary: Acacia longifolia is an invasive plant highly dispersed in the Mediterranean region. Being a legume, the association with nitrogen-fixing bacteria (i.e., rhizobia) allows access to readily fixed nitrogen, facilitating its growth, development, and dispersal. The extent to which this relation is restricted to bacteria was investigated in this study. We aimed to (i) characterize the microbial community inside root nodules, i.e., structures where nitrogen fixation occurs, and (ii) understand how these partners change following a fire event. In fact, fire potentiates A. longifolia invasion, so it is of remarkable interest to understand the role of nodulation in dispersal. We found that root nodules are a guesthouse for microorganisms and, while some are common between unburnt and burnt sites, others shift, particularly considering fungal community. Acacia longifolia is one of the most aggressive invaders worldwide whose invasion is potentiated after a fire, a common perturbation in Mediterranean climates. As a legume, this species establishes symbioses with nitrogen-fixing bacteria inside root nodules; however, the overall microbial diversity is still unclear. In this study, we addressed root nodules' structure and biodiversity through histology and Next-Generation Sequencing, targeting 16S and 25S-28S rDNA genes for bacteria and fungi, respectively. We wanted to evaluate the effect of fire in root nodules from 1-year-old saplings, by comparing unburnt and burnt sites. We found that although having the same general structure, after a fire event, nodules had a higher number of infected cells and greater starch accumulation. Starch accumulated in uninfected cells can be a possible carbon source for the microbiota. Regarding diversity, Bradyrhizobium was dominant in both sites (ca. 77%), suggesting it is the preferential partner, followed by Tardiphaga (ca. 9%), a non-rhizobial Alphaproteobacteria, and Synechococcus, a cyanobacteria (ca. 5%). However, at the burnt site, additional N-fixing bacteria were included in the top 10 genera, highlighting the importance of this process. Major differences were found in the mycobiome, which was diverse in both sites and included genera mostly described as plant endophytes. Coniochaeta was dominant in nodules from the burnt site (69%), suggesting its role as a facilitator of symbiotic associations. We highlight the presence of a large bacterial and fungal community in nodules, suggesting nodulation is not restricted to nitrogen fixation. Thus, this microbiome can be involved in facilitating A. longifolia invasive success. [ABSTRACT FROM AUTHOR]

Carnivorous pitcher plants are uniquely adapted to nitrogen limitation, using pitfall traps to acquire nutrients from insect prey. Pitcher plants in the genus Sarracenia may also use nitrogen fixed by bacteria inhabiting the aquatic microcosms of their pitchers. Here, we investigated whether species of a convergently evolved pitcher plant genus, Nepenthes, might also use bacterial nitrogen fixation as an alternative strategy for nitrogen capture. First, we constructed predicted metagenomes of pitcher organisms from three species of Singaporean Nepenthes using 16S rRNA sequence data and correlated predicted nifH abundances with metadata. Second, we used gene-specific primers to amplify and quantify the presence or absence of nifH directly from 102 environmental samples and identified potential diazotrophs with significant differential abundance in samples that also had positive nifH PCR tests. Third, we analyzed nifH in eight shotgun metagenomes from four additional Bornean Nepenthes species. Finally, we conducted an acetylene reduction assay using greenhouse-grown Nepenthes pitcher fluids to confirm nitrogen fixation is indeed possible within the pitcher habitat. Results show active acetylene reduction can occur in Nepenthes pitcher fluid. Variation in nifH from wild samples correlates with Nepenthes host species identity and pitcher fluid acidity. Nitrogen-fixing bacteria are associated with more neutral fluid pH, while endogenous Nepenthes digestive enzymes are most active at low fluid pH. We hypothesize Nepenthes species experience a trade-off in nitrogen acquisition; when fluids are acidic, nitrogen is primarily acquired via plant enzymatic degradation of insects, but when fluids are neutral, Nepenthes plants take up more nitrogen via bacterial nitrogen fixation. [ABSTRACT FROM AUTHOR]

In the boreal forests, feather mosses such as Hylocomium splendens and Pleurozium schreberi are colonized by cyanobacteria, which provide large amounts of nitrogen to forest ecosystems through nitrogen fixation. Although these feather mosses are also ubiquitous in subalpine forests of East Asia, little is known regarding their associated cyanobacteria and their ability to fix nitrogen. In this study, we investigated (1) whether cyanobacteria co-exist and fix nitrogen in the two species of feather mosses that cover the ground surface in a subalpine forest of Mt. Fuji, (2) whether cyanobacteria belonging to a common cluster with boreal forests are found in feather mosses in Mt. Fuji, and (3) whether moss-associated nitrogen fixation rates differed among moss growing substrates, canopy openness, and moss nitrogen concentrations in the same forest area. Our results showed that cyanobacteria colonized feather mosses in the subalpine forests of Mt. Fuji and acetylene reduction rates as an index of nitrogen fixation tended to be higher in H. splendens than in P. schreberi. Based on analysis of the nifH gene, 43 bacterial operational taxonomic units (OTUs) were identified, 28 of which represented cyanobacteria. Among the five clusters of cyanobacteria classified based on their nifH gene and identified in northern Europe, four (Nostoc cluster I, Nostoc cluster II, Stigonema cluster, and nifH2 cluster) were also found at Mt. Fuji. The acetylene reduction rate differed depending on the moss growing substrate and the total nitrogen concentration of moss shoots, and a strong negative correlation was observed with the total nitrogen concentration. [ABSTRACT FROM AUTHOR]

Understanding the composition and activation of multicomponent receptor complexes is a challenge in biology. To address this, we developed a synthetic approach based on nanobodies to drive assembly and activation of cell surface receptors and apply the concept by manipulating receptors that govern plant symbiosis with nitrogen-fixing bacteria. We show that the Lotus japonicus Nod factor receptors NFR1 and NFR5 constitute the core receptor complex initiating the cortical root nodule organogenesis program as well as the epidermal program controlling infection. We find that organogenesis signaling is mediated by the intracellular kinase domains whereas infection requires functional ectodomains. Finally, we identify evolutionarily distant barley receptors that activate root nodule organogenesis, which could enable engineering of biological nitrogen-fixation into cereals. [ABSTRACT FROM AUTHOR]

The maintenance of intracellular nitrogen-fixing bacteria causes changes in proteins' location and in gene expression that may be detrimental to the host cell fitness. We hypothesized that the nodule's high vulnerability toward salt stress might be due to alterations in mechanisms involved in the exclusion of Na+ from the host cytoplasm. Confocal and electron microscopy immunolocalization analyses of Na+/K+ exchangers in the root nodule showed the plasma membrane (MtNHX7) and endosome/tonoplast (MtNHX6) signal in non-infected cells; however, in mature infected cells the proteins were depleted from their target membranes and expelled to vacuoles. This mistargeting suggests partial loss of the exchanger's functionality in these cells. In the mature part of the nodule 7 of the 20 genes encoding ion transporters, channels, and Na+/K+ exchangers were either not expressed or substantially downregulated. In nodules from plants subjected to salt treatments, low temperature-scanning electron microscopy and X-ray microanalysis revealed the accumulation of 5–6 times more Na+ per infected cell versus non-infected one. Hence, the infected cells' inability to withstand the salt may be the integral result of preexisting defects in the localization of proteins involved in Na+ exclusion and the reduced expression of key genes of ion homeostasis, resulting in premature senescence and termination of symbiosis. [ABSTRACT FROM AUTHOR]

Aims: Silicon (Si) uptake and accumulation improves plant resilience to environmental stresses, but most studies examining this functional role of Si have focussed on grasses (Poaceae) and neglected other important plant groups, such as legumes (Fabaceae). Legumes have evolved a symbiotic relationship with nitrogen-fixing bacteria (rhizobia) housed in root nodules. Our study determined the impacts of silicon (Si) supplementation on Medicago truncatula inoculated with Ensifer meliloti rhizobial strains that differed in their capacity for nitrogen fixation: Sm1021 ('low-efficiency') or Sm1022 ('high-efficiency'). Methods: We examined how Si and rhizobial efficacy influence nodule and plant functional traits, including their chemical aspects. These combinations were supplied with or without Si in a glasshouse experiment, where we quantified nodule flavonoids and foliar chemistry (free amino acids, soluble protein, elemental C, N and Si). Results: Si supply increased nodule number per plant, specific nodule flavonoid concentrations, contents of foliar nitrogenous compounds and foliar C, but not foliar Si. We also demonstrated that rhizobial efficacy altered the magnitude of Si effects on certain traits. For example, Si significantly promoted concentrations of foliar N and soluble protein in the plants associated with the 'low-efficiency' strain only, and this was not the case with the 'high-efficiency' one. Conclusions: Collectively, our study indicates that Si generates positive effects on M. truncatula, particularly when the association with rhizobia is relatively inefficient, and may play a more prominent role in rhizobial functionality than previously thought. [ABSTRACT FROM AUTHOR]

Acquisition of mobile genetic elements can confer novel traits to bacteria. Some integrative and conjugative elements confer upon members of Bradyrhizobium the capacity to fix nitrogen in symbiosis with legumes. These so-called symbiosis integrative conjugative elements (symICEs) can be extremely large and vary as monopartite and polypartite configurations within chromosomes of related strains. These features are predicted to impose fitness costs and have defied explanation. Here, we show that chromosome architecture is largely conserved despite diversity in genome composition, variations in locations of attachment sites recognized by integrases of symICEs, and differences in large-scale chromosomal changes that occur upon integration. Conversely, many simulated nonnative chromosome–symICE combinations are predicted to result in lethal deletions or disruptions to architecture. Findings suggest that there is compatibility between chromosomes and symICEs. We hypothesize that the size and structural flexibility of symICEs are important for generating combinations that maintain chromosome architecture across a genus of nitrogen-fixing bacteria with diverse and dynamic genomes. [ABSTRACT FROM AUTHOR]

Members of the agrobacteria-rhizobia complex (ARC) have multiple and diverse plasmids. The extent to which these plasmids are shared and the consequences of their interactions are not well understood. We extracted over 4000 plasmid sequences from 1251 genome sequences and constructed a network to reveal interactions that have shaped the evolutionary histories of oncogenic virulence plasmids. One newly discovered type of oncogenic plasmid is a mosaic with three incomplete, but complementary and partially redundant virulence loci. Some types of oncogenic plasmids recombined with accessory plasmids or acquired large regions not known to be associated with pathogenicity. We also identified two classes of partial virulence plasmids. One class is potentially capable of transforming plants, but not inciting disease symptoms. Another class is inferred to be incomplete and non-functional but can be found as coresidents of the same strain and together are predicted to confer pathogenicity. The modularity and capacity for some plasmids to be transmitted broadly allow them to diversify, convergently evolve adaptive plasmids and shape the evolution of genomes across much of the ARC. [ABSTRACT FROM AUTHOR]

Silicon (Si) uptake and deposition (silicification) in tissues is known to alleviate stresses and generally improve plant health. This is mostly studied in Si-high accumulators, such as grasses, with comparatively less known about its effects on other plant functional groups, such as legumes. There is speculation that Si may positively impact the symbiosis between legumes and the nitrogen-fixing bacteria (rhizobia) they associate with, but this is poorly understood. This study examined the effects of Si enrichment on legume species associated with rhizobia and the potential underlying mechanism of Si impacts. 2. We conducted a glasshouse experiment with lucerne Medicago sativa and barrel medic M. truncatula associated with a model rhizobial strain. Six genotypes (three per species) were either supplemented with Si (+Si) or untreated (-Si). We quantified 16 functional traits which could be classified as plant growth, physiology, elemental chemistry, nodule activity and nitrogen fixation. 3. The two legume species responded to Si distinctively. For example, Si supplementation increased shoot biomass by more than 10% in lucerne but growth was unaffected in barrel medic. Conversely, nitrogen-fixing enzyme (nitrogenase) activity was promoted by more than 85% in +Si barrel medic plants but not in lucerne. Moreover, Si supplementation of lucerne increased the concentrations of Si in leaves by more than 36% but not in root nodules. Increased foliar concentrations of Si in lucerne were positively associated with increased shoot and root biomass in Sequel and Trifecta genotypes, respectively. Conversely, Si supplementation of barrel medic increased the concentration of Si in root nodules by 29% but not that in foliar tissues. Nitrogenase activity and where silicification occurred, differed between genotypes in barrel medic; nitrogenase activity was correlated with concentrations of Si in root nodules rather than that in foliar tissues in one genotype (Sephi) but the reverse was true in another (Hannaford). 4. This study demonstrates that two closely related legume species can respond to Si in distinct ways, depending on plant genotype and symbiosis. These results present the overlooked function of Si in legume-rhizobia interactions, which could potentially enhance productivity of this important group of plants. [ABSTRACT FROM AUTHOR]

Nearly all organisms participate in multiple mutualisms, and complementarity within these complex interactions can result in synergistic fitness effects. However, it remains largely untested how multiple mutualisms impact eco‐evolutionary dynamics in interacting species. We tested how multiple microbial mutualists—N‐fixing bacteria and mycorrrhizal fungi—affected selection and heritability of traits in their shared host plant (Medicago truncatula), as well as fitness alignment between partners. Our results demonstrate for the first time that multiple mutualisms synergistically affect the selection and heritability of host traits and enhance fitness alignment between mutualists. Specifically, we found interaction with multiple microbial symbionts doubled the strength of natural selection on a plant architectural trait, resulted in 2‐ to 3‐fold higher heritability of plant reproductive success, and more than doubled fitness alignment between N‐fixing bacteria and plants. These findings show synergism generated by multiple mutualisms extends to key components of microevolutionary change, emphasising the importance of multiple mutualism effects on evolutionary trajectories. [ABSTRACT FROM AUTHOR]

Legumes are high in protein and form a valuable part of human diets due to their interaction with symbiotic nitrogen-fixing bacteria known as rhizobia. Plants house rhizobia in specialized root nodules and provide the rhizobia with carbon in return for nitrogen. However, plants usually housemultiple rhizobial strains that vary in their fixation ability, so the plant faces an investment dilemma. Plants are known to sanction strains that do not fix nitrogen, but nonfixers are rare in field settings, while intermediate fixers are common. Here, we modeled how plants should respond to an intermediate fixer that was otherwise isogenic and tested model predictions using pea plants. Intermediate fixers were only tolerated when a better strain was not available. In agreement with model predictions, nodules containing the intermediate-fixing strain were large and healthy when the only alternative was a nonfixer, but nodules of the intermediate-fixing strain were small and white when the plant was coinoculated with a more effective strain. The reduction in nodule size was preceded by a lower carbon supply to the nodule even before differences in nodule size could be observed. Sanctioned nodules had reduced rates of nitrogen fixation, and in later developmental stages, sanctioned nodules contained fewer viable bacteria than nonsanctioned nodules. This indicates that legumes can make conditional decisions, most likely by comparing a local nodule-dependent cue of nitrogen output with a global cue, giving them remarkable control over their symbiotic partners. [ABSTRACT FROM AUTHOR]

Marine nitrogen-fixing microorganisms are an important source of fixed nitrogen in oceanic ecosystems. The colonial cyanobacterium Trichodesmium and diatom symbionts were thought to be the primary contributors to oceanic N2 fixation until the discovery of the unusual uncultivated symbiotic cyanobacterium UCYN-A (Candidatus Atelocyanobacterium thalassa). UCYN-A has atypical metabolic characteristics lacking the oxygen-evolving photosystem II, the tricarboxylic acid cycle, the carbon-fixation enzyme RuBisCo and de novo biosynthetic pathways for a number of amino acids and nucleotides. Therefore, it is obligately symbiotic with its single-celled haptophyte algal host. UCYN-A receives fixed carbon from its host and returns fixed nitrogen, but further insights into this symbiosis are precluded by both UCYN-A and its host being uncultured. In order to investigate how this syntrophy is coordinated, we reconstructed bottom-up genome-scale metabolic models of UCYN-A and its algal partner to explore possible trophic scenarios, focusing on nitrogen fixation and biomass synthesis. Since both partners are uncultivated and only the genome sequence of UCYN-A is available, we used the phylogenetically related Chrysochromulina tobin as a proxy for the host. Through the use of flux balance analysis (FBA), we determined the minimal set of metabolites and biochemical functions that must be shared between the two organisms to ensure viability and growth. We quantitatively investigated the metabolic characteristics that facilitate daytime N2 fixation in UCYN-A and possible oxygen-scavenging mechanisms needed to create an anaerobic environment to allow nitrogenase to function. This is the first application of an FBA framework to examine the tight metabolic coupling between uncultivated microbes in marine symbiotic communities and provides a roadmap for future efforts focusing on such specialized systems. Author summary: Reduction of dinitrogen gas to biologically useful forms via nitrogen fixation is a key component of the biogeochemical cycle. In the marine environment, the cyanobacteria UCYN-A (Candidatus Atelocyanobacterium thalassa) has been found to be a primary contributor to biological nitrogen fixation at a global scale. UCYN-A exhibits a highly streamlined genome which lacks genes coding for essential cyanobacterial processes such as the energy-generating TCA cycle, oxygen-producing photosystem II, the carbon-fixing RuBisCo and de novo production pathways for numerous amino acids and nucleotides. Thus, it exists in a symbiosis with unicellular planktonic algae where it exchanges fixed nitrogen for fixed carbon with its host. However, both UCYN-A and its symbiotic partner remain uncultured under laboratory conditions. This necessitates implementing a computational approach to glean insights into UCYN-A's unique physiology and metabolic processes governing the symbiotic association. To this end, we develop a constraints-based framework that infers all possible trophic scenarios consistent with the observed data. Possible mechanisms employed by UCYN-A to accommodate diazotrophy with daytime carbon fixation by the host (i.e., two mutually incompatible processes) are also elucidated. We envision that the developed framework using UCYN-A and its algal host will be used as a roadmap and motivate the study of similarly unique microbial systems in the future. [ABSTRACT FROM AUTHOR]

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SUMMARY: Legumes and nitrogen‐fixing rhizobial bacteria establish root nodule symbiosis, which is orchestrated by several plant hormones. Exogenous addition of biologically active gibberellic acid (GA) is known to inhibit root nodule symbiosis. However, the precise role of GA has not been elucidated because of the trace amounts of these hormones in plants and the multiple functions of GAs. Here, we found that GA signaling acts as a key regulator in a long‐distance negative‐feedback system of root nodule symbiosis called autoregulation of nodulation (AON). GA biosynthesis is activated during nodule formation in and around the nodule vascular bundles, and bioactive GAs accumulate in the nodule. In addition, GA signaling induces expression of the symbiotic transcription factor NODULE INCEPTION (NIN) via a cis‐acting region on the NIN promoter. Mutants with deletions of this cis‐acting region have increased susceptibility to rhizobial infection and reduced GA‐induced CLE‐RS1 and CLE‐RS2 expression, suggesting that the inhibitory effect of GAs occurs through AON. This is supported by the GA‐insensitive phenotypes of an AON‐defective mutant of HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1) and a reciprocal grafting experiment. Thus, endogenous GAs induce NIN expression via its GA‐responsive cis‐acting region, and subsequently the GA‐induced NIN activates the AON system to regulate nodule formation. [ABSTRACT FROM AUTHOR]

Priority effects occur when the order of species arrival affects the final community structure. Mutualists often interact with multiple partners in different orders, but if or how priority effects alter interaction outcomes is an open question. In the field, we paired the legume Medicago lupulina with two nodulating strains of Ensifer bacteria that vary in nitrogen-fixing ability. We inoculated plants with strains in different orders and measured interaction outcomes. The first strain to arrive primarily determined plant performance and final relative abundances of rhizobia on roots. Plants that received effective microbes first and ineffective microbes second grew larger than plants inoculated with the same microbes in the opposite order. Our results show that mutualism outcomes can be influenced not just by partner identity, but by the interaction order. Furthermore, hosts receiving high-quality mutualists early can better tolerate low-quality symbionts later, indicating that priority effects may help explain the persistence of ineffective symbionts. [ABSTRACT FROM AUTHOR]

Background: A wide variety of bacterial adaptative responses to environmental conditions are mediated by signal transduction pathways. Two-component signal transduction systems are one of the predominant means used by bacteria to sense the signals of the host plant and adjust their interaction behaviour. A total of seven open reading frames have been identified as putative two-component response regulators in the gram-negative nitrogen-fixing bacteria Azorhizobium caulinodans ORS571. However, the biological functions of these response regulators in the symbiotic interactions between A. caulinodans ORS571 and the host plant Sesbania rostrata have not been elucidated to date. Results: In this study, we identified and investigated a two-component response regulator, AcfR, with a phosphorylatable N-terminal REC (receiver) domain and a C-terminal HTH (helix-turn-helix) LuxR DNA-binding domain in A. caulinodans ORS571. Phylogenetic analysis showed that AcfR possessed close evolutionary relationships with NarL/FixJ family regulators. In addition, six histidine kinases containing HATPase_c and HisKA domains were predicted to interact with AcfR. Furthermore, the biological function of AcfR in free-living and symbiotic conditions was elucidated by comparing the wild-type strain and the ΔacfR mutant strain. In the free-living state, the cell motility behaviour and exopolysaccharide production of the ΔacfR mutant were significantly reduced compared to those of the wild-type strain. In the symbiotic state, the ΔacfR mutant showed a competitive nodule defect on the stems and roots of the host plant, suggesting that AcfR can provide A. caulinodans with an effective competitive ability for symbiotic nodulation. Conclusions: Our results showed that AcfR, as a response regulator, regulates numerous phenotypes of A. caulinodans under the free-living conditions and in symbiosis with the host plant. The results of this study help to elucidate the involvement of a REC + HTH_LuxR two-component response regulator in the Rhizobium-host plant interaction. [ABSTRACT FROM AUTHOR]

Nitrogen fixation is a widespread metabolic trait in certain types of microorganisms called diazotrophs. Bioavailable nitrogen is limited in various habitats on land and in the sea and, accordingly, a range of plant, animal, and single-celled eukaryotes have evolved symbioses with diverse diazotrophic bacteria, with enormous economic and ecological benefits. Until recently, all known nitrogen-fixing symbionts were heterotrophs, such as nodulating rhizobia, or photoautotrophs, such as cyanobacteria. In 2016, the first chemoautotrophic nitrogen-fixing symbionts were discovered in a common family of marine clams, the Lucinidae. Chemosynthetic nitrogen-fixing symbionts use the chemical energy stored in reduced sulfur compounds to power carbon and nitrogen fixation, making them metabolic "all-rounders" with multiple functions in the symbiosis. This distinguishes them from heterotrophic symbionts that require a source of carbon from their host, and their chemosynthetic metabolism distinguishes them from photoautotrophic symbionts that produce oxygen, a potent inhibitor of nitrogenase. In this review, we consider evolutionary aspects of this discovery, by comparing strategies that have evolved for hosting intracellular nitrogen-fixing symbionts in plants and animals. The symbiosis between lucinid clams and chemosynthetic nitrogen-fixing bacteria also has important ecological impacts, since they form a nested symbiosis with endangered marine seagrasses. Notably, nitrogen fixation by lucinid symbionts may help support seagrass health by providing a source of nitrogen in seagrass habitats. These discoveries were enabled by new techniques for understanding the activity of microbial populations in natural environments. However, an animal (or plant) host represents a diverse landscape of microbial niches due to its structural, chemical, immune, and behavioral properties. In the future, methods that resolve microbial activity at the single cell level will provide radical new insights into the regulation of nitrogen fixation in chemosynthetic symbionts, shedding new light on the evolution of nitrogen-fixing symbioses in contrasting hosts and environments. [ABSTRACT FROM AUTHOR]

In the last decade, the known biogeography of nitrogen fixation in the ocean has been expanded to colder and nitrogen‐rich coastal environments. The symbiotic nitrogen‐fixing cyanobacteria group A (UCYN‐A) has been revealed as one of the most abundant and widespread nitrogen‐fixers, and includes several sublineages that live associated with genetically distinct but closely related prymnesiophyte hosts. The UCYN‐A1 sublineage is associated with an open ocean picoplanktonic prymnesiophyte, whereas UCYN‐A2 is associated with the coastal nanoplanktonic coccolithophore Braarudosphaera bigelowii, suggesting that different sublineages may be adapted to different environments. Here, we study the diversity of nifH genes present at the Santa Cruz Municipal Wharf in the Monterey Bay (MB), California, and report for the first time the presence of multiple UCYN‐A sublineages, unexpectedly dominated by the UCYN‐A2 sublineage. Sequence and quantitative PCR data over an 8‐year time‐series (2011–2018) showed a shift toward increasing UCYN‐A2 abundances after 2013, and a marked seasonality for this sublineage which was present during summer‐fall months, coinciding with the upwelling‐relaxation period in the MB. Increased abundances corresponded to positive temperature anomalies in MB, and we discuss the possibility of a benthic life stage of the associated coccolithophore host to explain the seasonal pattern. The dominance of UCYN‐A2 in coastal waters of the MB underscores the need to further explore the habitat preference of the different sublineages in order to provide additional support for the hypothesis that UCYN‐A1 and UCYN‐A2 sublineages are different ecotypes. [ABSTRACT FROM AUTHOR]

Aims: Revealing genetic diversity in a root nodulation symbiosis under field conditions is critical to understand the formation of ecological communities of organisms associated with hosts and the nitrogen cycle in natural ecosystems. However, our knowledge of the genetic diversity of bacterial mutualists on a local scale is still poor because of the assumption that the genetic diversity of mutualistic bacteria is constrained by their hosts. Methods: We thoroughly investigated the genetic diversity of Frankia in a local forest stand. We collected root nodules from 213 Alnus hirsuta seedlings covering the spatial range of the continuous population, which means that Alnus individuals occurred in a relatively homogeneous distribution in a continuous forest. Then, a phylogenetic and diversity analysis was performed for the nifD-K IGS region, including global Frankia sequences from Alnus hosts. Results: The genetic diversity of Frankia detected even on a local scale measured as high as that shown by previous studies conducted on local and regional scales. Moreover, a genetic structure analysis revealed a spatially mosaic-like distribution of genetic variation in Frankia despite the small spatial scale. Conclusions: The genetic diversity and composition of bacterial mutualists are heterogeneous on a local scale. Our findings demonstrate that genetically different bacterial symbionts simultaneously interact with a single host population and interaction partnerships spatially vary. The standing variation could produce dynamic ecological and evolutionary outcomes in a heterogeneous forest ecosystem. [ABSTRACT FROM AUTHOR]

The mutualistic association between leguminous plants and endosymbiotic rhizobial bacteria is a paradigmatic example of a symbiosis driven by metabolic exchanges. Here, we report the reconstruction and modelling of a genome-scale metabolic network of Medicago truncatula (plant) nodulated by Sinorhizobium meliloti (bacterium). The reconstructed nodule tissue contains five spatially distinct developmental zones and encompasses the metabolism of both the plant and the bacterium. Flux balance analysis (FBA) suggests that the metabolic costs associated with symbiotic nitrogen fixation are primarily related to supporting nitrogenase activity, and increasing N2-fixation efficiency is associated with diminishing returns in terms of plant growth. Our analyses support that differentiating bacteroids have access to sugars as major carbon sources, ammonium is the main nitrogen export product of N2-fixing bacteria, and N2 fixation depends on proton transfer from the plant cytoplasm to the bacteria through acidification of the peribacteroid space. We expect that our model, called 'Virtual Nodule Environment' (ViNE), will contribute to a better understanding of the functioning of legume nodules, and may guide experimental studies and engineering of symbiotic nitrogen fixation. The association between leguminous plants and rhizobial bacteria is a paradigmatic example of a symbiosis driven by metabolic exchanges. Here, diCenzo et al. report the reconstruction and modelling of a genome-scale metabolic network of the plant Medicago truncatula nodulated by the bacterium Sinorhizobium meliloti. [ABSTRACT FROM AUTHOR]

The availability of compatible mutualistic soil microbes could influence the invasion success of non‐native plant species. Specifically, there may be spatial variation in the distribution of compatible microbes, and species‐specific variation in plant host ability to associate with available microbes. Although either or both factors could promote or limit invasion, the scale over which most studies are conducted makes it difficult to examine these two possibilities simultaneously. However, this is critical to identifying a role of soil microbes in invasion.A series of recent research projects focused on interactions between Australian Acacia and nitrogen‐fixing bacteria (rhizobia) at multiple spatial scales, from the local to the inter‐continental, has allowed us to evaluate this question. Collectively, this research reveals that nodulation, performance and rhizobial community composition are all broadly similar across spatial scales and differentially invasive species.Synthesis and applications. We argue that current research provides convincing evidence that interactions with rhizobia do not determine invasion success in Acacia, but instead highlights key knowledge gaps that remain unfilled. Importantly, the ease with which non‐native Acacia species form mutualistic associations with rhizobia, regardless of invasive status, highlights the critical need to understand the impacts of all non‐native Acacia on native soil communities. [ABSTRACT FROM AUTHOR]

Symbiotic bacteria, commonly called rhizobia, lead a saprophytic lifestyle in the soil and form nitrogen-fixing nodules on legume roots. During their lifecycle, rhizobia have to adapt to different conditions prevailing in the soils and within host plants. To survive under these conditions, rhizobia fine-tune the regulatory machinery to respond rapidly and adequately to environmental changes. Symbiotic bacteria play an essential role in the soil environment from both ecological and economical point of view, since these bacteria provide Fabaceae plants (legumes) with large amounts of accessible nitrogen as a result of symbiotic interactions (i.e., rhizobia present within the nodule reduce atmospheric dinitrogen (N2) to ammonia, which can be utilized by plants). Because of its restricted availability in the soil, nitrogen is one of the most limiting factors for plant growth. In spite of its high content in the atmosphere, plants are not able to assimilate it directly in the N2 form. During symbiosis, rhizobia infect host root and trigger the development of specific plant organ, the nodule. The aim of root nodule formation is to ensure a microaerobic environment, which is essential for proper activity of nitrogenase, i.e., a key enzyme facilitating N2 fixation. To adapt to various lifestyles and environmental stresses, rhizobia have developed several regulatory mechanisms, e.g., reversible phosphorylation. This key mechanism regulates many processes in both prokaryotic and eukaryotic cells. In microorganisms, signal transduction includes two-component systems (TCSs), which involve membrane sensor histidine kinases (HKs) and cognate DNA-binding response regulators (RRs). Furthermore, regulatory mechanisms based on phosphoenolopyruvate-dependent phosphotranspherase systems (PTSs), as well as alternative regulatory pathways controlled by Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases (STPs) play an important role in regulation of many cellular processes in both free-living bacteria and during symbiosis with the host plant (e.g., growth and cell division, envelope biogenesis, biofilm formation, response to stress conditions, and regulation of metabolism). In this review, we summarize the current knowledge of phosphorylation systems in symbiotic nitrogen-fixing bacteria, and their role in the physiology of rhizobial cells and adaptation to various environmental conditions. [ABSTRACT FROM AUTHOR]

Lima bean (Phaseolus lunatus) is an important species of the genus Phaseolus for human consumption in tropical regions. The seeds are important source of protein for people from South America, Africa and Mexico. In addition, as a legume plant, lima bean presents the ability to perform the biological nitrogen fixation (BNF) through the symbiosis with nitrogen-fixing bacteria. The studies about diversity and efficiency of lima bean-rhizobia symbiosis have increased worldwide, mainly in Latin America. These studies have shown Bradyrhizobium and Rhizobium as the main symbionts, although Sinorhizobium, Mesorhizobium and Allorhizobium have been found associated with lima bean. Also, there is a large variation in the efficiency of N fixation by the current isolates of rhizobia and some rhizobia have presented high capability for fixing N. This review aims to explore the studies about diversity and efficiency of rhizobia in symbiosis with lima bean. [ABSTRACT FROM AUTHOR]

Major non-legume crops can form beneficial associations with nitrogen-fixing bacteria like Azospirillum brasilense. Our current understanding of the molecular aspects and signaling that occur between important crops like rice and these nitrogen-fixing bacteria is limited. In this study, we used an experimental system where the bacteria could colonize the plant roots and promote plant growth in wild type rice and symbiotic mutants (dmi3 and pollux) in rice. Our data suggest that plant growth promotion and root penetration is not dependent on these genes. We then used this colonization model to identify regulation of gene expression at two different time points during this interaction: at 1day post inoculation (dpi), we identified 1622 differentially expressed genes (DEGs) in rice roots, and at 14dpi, we identified 1995 DEGs. We performed a comprehensive data mining to classify the DEGs into the categories of transcription factors (TFs), protein kinases (PKs), and transporters (TRs). Several of these DEGs encode proteins that are involved in the flavonoid biosynthetic pathway, defense, and hormone signaling pathways. We identified genes that are involved in nitrate and sugar transport and are also implicated to play a role in other plant-microbe interactions. Overall, findings from this study will serve as an excellent resource to characterize the host genetic pathway controlling the interactions between non-legumes and beneficial bacteria which can have long-term implications towards sustainably improving agriculture. [ABSTRACT FROM AUTHOR]

Biological dinitrogen (N 2) fixation is a key metabolic process exclusively performed by prokaryotes, some of which are symbiotic with eukaryotes. Species of the marine haptophyte algae Braarudosphaera bigelowii harbor the N 2 -fixing endosymbiotic cyanobacteria UCYN-A, which might be evolving organelle-like characteristics. We found that the size ratio between UCYN-A and their hosts is strikingly conserved across sublineages/species, which is consistent with the size relationships of organelles in this symbiosis and other species. Metabolic modeling showed that this size relationship maximizes the coordinated growth rate based on trade-offs between resource acquisition and exchange. Our findings show that the size relationships of N 2 -fixing endosymbionts and organelles in unicellular eukaryotes are constrained by predictable metabolic underpinnings and that UCYN-A is, in many regards, functioning like a hypothetical N 2 -fixing organelle (or nitroplast). [Display omitted] • Strong volume covariation in the B. bigelowii /UCYN-A nitrogen-fixing symbiosis • The constant volumetric relationship reflects symbiotic resource economy constraints • Coordination of harvesting and exchange of resources maximizes symbiotic growth rate • UCYN-A is functioning like a hypothetical N 2 -fixing organelle (or nitroplast) The robust volumetric relationship between partners of a marine planktonic nitrogen-fixing symbiosis reflects predictable, balanced, and quantitative interdependencies among CO 2 fixation, respiration, and N 2 fixation that maximize synchronized growth rate. [ABSTRACT FROM AUTHOR]

A major goal in microbial ecology is to understand the factors that structure bacterial communities across space and time. For microbes that are plant symbionts, community assembly processes can lead to either a positive or negative relationship between plant size or age and soil microbe diversity. Here, we evaluated the extent to which such relationships exist within a single legume species (Acacia acuminata) and their naturally occurring symbiotic nitrogen‐fixing bacteria (rhizobia).We quantified the diversity of rhizobia that associate with A. acuminata trees of variable size spanning a large environmental gradient in southwest Australia (72 trees in 24 sites spread across ~300,000 km2), using metabarcoding. We modelled rhizobia diversity using 16S exact genetic variants, in a binomial multivariate statistical framework that controlled for climate and local soil characteristics.We identified two major phylogenetic clades of rhizobia that associate with A. acuminata. Soil sampled at the base of larger Acacia trees contained a higher richness of rhizobia genetic variants. Each major clade responds differently to environmental factors (climate and soil characteristics), but the positive association between tree size and rhizobia genetic diversity was mainly driven by responses from one of the two clades. Overall tree size explained more variation than any other factor, resulting in a ~3‐fold increase in total richness and clade diversity from the smallest to the largest trees.Synthesis. Previous studies have shown that plant host species is important in structuring microbial soil communities in the rhizosphere. Our results show that host size or age within a single plant species can also structure diversity of at least one group of soil microbes. A positive relationship between plant host size and rhizobia diversity suggests that hosts may modify the niche space of their surrounding soil (niche construction hypothesis) enabling a higher richness of microbial taxa. That different rhizobial groups responded differently to host size and other ecological factors suggests that rhizobia is not an ecologically uniform group, and that entirely neutral explanations for our results are unlikely. Host plants may be analogous to "islands," where larger plant hosts may accumulate diversity over time, through migration opportunities. Previous studies have shown that plant host species is important in structuring microbial soil communities in the rhizosphere. Our results show that host size or age within a single plant species can also structure diversity of at least one group of soil microbes. A positive relationship between plant host size and rhizobia diversity suggests that hosts may modify the niche space of their surrounding soil (niche construction hypothesis) enabling a higher richness of microbial taxa. That different rhizobial groups responded differently to host size and other ecological factors suggests that rhizobia is not an ecologically uniform group, and that entirely neutral explanations for our results are unlikely. Host plants may be analogous to "islands," where larger plant hosts may accumulate diversity over time, through migration opportunities. [ABSTRACT FROM AUTHOR]

Nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi (AMF) form symbioses with plant roots and these are established by precise regulation of symbiont accommodation within host plant cells. In model legumes such as Lotus japonicus and Medicago truncatula, rhizobia enter into roots through an intracellular invasion system that depends on the formation of a root-hair infection thread (IT). While IT-mediated intracellular rhizobia invasion is thought to be the most evolutionarily derived invasion system, some studies have indicated that a basal intercellular invasion system can replace it when some nodulation-related factors are genetically modified. In addition, intracellular rhizobia accommodation is suggested to have a similar mechanism as AMF accommodation. Nevertheless, our understanding of the underlying genetic mechanisms is incomplete. Here we identify a L. japonicus nodulation-deficient mutant, with a mutation in the LACK OF SYMBIONT ACCOMMODATION (LAN) gene, in which root-hair IT formation is strongly reduced, but intercellular rhizobial invasion eventually results in functional nodule formation. LjLAN encodes a protein that is homologous to Arabidopsis MEDIATOR 2/29/32 possibly acting as a subunit of a Mediator complex, a multiprotein complex required for gene transcription. We also show that LjLAN acts in parallel with a signaling pathway including LjCYCLOPS. In addition, the lan mutation drastically reduces the colonization levels of AMF. Taken together, our data provide a new factor that has a common role in symbiont accommodation process during root nodule and AM symbiosis. [ABSTRACT FROM AUTHOR]

Members of the plant family Leguminosae (Fabaceae) are unique in that they have evolved a symbiotic relationship with rhizobia (a group of soil bacteria that can fix atmospheric nitrogen). Rhizobia infect and form root nodules on their specific host plants before differentiating into bacteroids, the symbiotic form of rhizobia. This complex relationship involves the supply of C4-dicarboxylate and phosphate by the host plants to the microsymbionts that utilize them in the energy-intensive process of fixing atmospheric nitrogen into ammonium, which is in turn made available to the host plants as a source of nitrogen, a macronutrient for growth. Although nitrogen-fixing bacteroids are no longer growing, they are metabolically active. The symbiotic process is complex and tightly regulated by both the host plants and the bacteroids. The metabolic pathways of carbon, nitrogen, and phosphate are heavily regulated in the host plants, as they need to strike a fine balance between satisfying their own needs as well as those of the microsymbionts. A network of transporters for the various metabolites are responsible for the trafficking of these essential molecules between the two partners through the symbiosome membrane (plant-derived membrane surrounding the bacteroid), and these are in turn regulated by various transcription factors that control their expressions under different environmental conditions. Understanding this complex process of symbiotic nitrogen fixation is vital in promoting sustainable agriculture and enhancing soil fertility. [ABSTRACT FROM AUTHOR]

Legumes are plants able to engage in root nodule symbiosis with nitrogen-fixing soil bacteria, called rhizobia. This symbiotic interaction is highly specific, such that each rhizobial species interacts with only a specific group of legumes, and vice versa. The rhizobia and the host plant develop a dialogue system based on an exchange of chemical molecules. Many studies reported that the flavonoids, in combination with the rhizobial NodD transcriptional activator, stimulate the expression of the genes required in the perception and signal transduction of Nod factors and the genes of nodule formation. This review seeks to highlight the genes and molecular signals involved in legume-rhizobia symbiosis. Recent knowledge leading to bacterial access and nodule formation was focused and discussed. [ABSTRACT FROM AUTHOR]

Contents Summary 1199 I. Introduction 1199 II. Selecting beneficial symbionts: one problem, many solutions 1200 III. Control and conflict over legume nodulation 1201 IV. Control and conflict over nodule growth and senescence 1204 V. Conclusion 1204 Acknowledgements 1205 References 1205 Summary: The legume–rhizobia association is a powerful model of the limits of host control over microbes. Legumes regulate the formation of root nodules that house nitrogen‐fixing rhizobia and adjust investment into nodule development and growth. However, the range of fitness outcomes in these traits reveals intense conflicts of interest between the partners. New work that we review and synthesize here shows that legumes have evolved varied mechanisms of control over symbionts, but that host control is often subverted by rhizobia. An outcome of this conflict is that both legumes and rhizobia have evolved numerous traits that can improve their own short‐term fitness in this interaction, but little evidence exists for any net improvement in the joint trait of nitrogen fixation. [ABSTRACT FROM AUTHOR]

Abstract: Grain legumes play a significant role in global food security. They have an advantage over cereals in that they can form symbiotic associations with nitrogen‐fixing bacteria, making them self‐sufficient in terms of nitrogen acquisition. In addition to this superior agronomic trait, grain legumes have excellent nutritional properties and are thus widely used as animal feed as well as in human nutrition. Current global trends towards increased legume consumption and availability of value‐added products, as well as legume production in developing countries require the provision of improved cultivars with better productivity and adaptability. Intensive efforts are thus underway to elaborate genomic resources and gain an improved knowledge base in a number of legume crops. There is also an emerging understanding of the beneficial interactions between legume‐associated organisms, particularly rhizobia and arbuscular mycorrhizal fungi, which result in improved nodulation and nutrient acquisition. The emerging focus on legume breeding for high sustainable yields as well as improved biotic and abiotic stress tolerance traits will serve to close the current gap between grain legume production and demand. With the support from policymakers, this increase in knowledge can be readily translated into increased crop production to meet the demands of an increasing global population. [ABSTRACT FROM AUTHOR]

The symbiosis formed by nitrogen-fixing bacteria with plant hosts mainly in the legume family involves a very intimate interaction. Within the symbiotic organ (the nodule) the bacteria are fully internalized by the host cell to become an intracellular organelle surrounded by a host-derived membrane. This arrangement is probably necessary for the efficient provision of energy and the sequestration of free oxygen molecules, two conditions required for sustained nitrogen fixation. Recent advances made in model legume species, such as Medicago truncatula , are beginning to uncover the genetic components allowing rhizobia to access the host cytoplasm and establish chronic intracellular infections without overt detrimental effects. It is now known that the rhizobial compartment in M. truncatula cells, the symbiosome, retains some features of the extracellular space as the target for a redirected host protein secretory pathway. A set of vesicle trafficking proteins function specifically in symbiotic cells to ensure the faithful delivery of secretory proteins to the intracellular bacteria, or bacteroid. This system is co-opted from the more ancient association with arbuscular mycorrhizal fungi found in most land plants, highlighting the evolutionary origin of the legume–rhizobia symbiosis. In some legume lineages, this heightened capability to process secretory proteins is needed to deliver a large number of symbiosis-specific antimicrobial peptides to the bacteria. Known as NCR peptides, these molecules transform bacteroids into a state of terminal differentiation, where the microbe loses its ability to proliferate outside their host. Numbering in their hundreds, these peptides manipulate various aspects of rhizobial biology, and affect the outcome of this symbiosis in complex ways. The extreme size of the NCR peptide family seems to be the result of an evolutionary conflict between the two partners to extract maximum benefit from each other. [ABSTRACT FROM AUTHOR]

Alfalfa, usually known as the “Queen of Forages”, is the main source of vegetable protein to meat and milk production systems worldwide. This legume is extremely rich in proteins due to its highly efficient symbiotic association with nitrogen-fixing strains. In the last years, alfalfa culture has been displaced to saline environments by other important crops, including major cereals, a fact that has reduced its biomass production and symbiotic nitrogen fixation. In this short communication, we report the high forage production and nutrient quality of alfalfa under saline conditions by alfalfa transformation with the AtNHX1 Na + /H + antiporter and inoculation with the stress-resistant nitrogen-fixing strain Sinorhizobium meliloti B401. Therefore, the incorporation of transgenic traits into salt-sensitive legumes in association with the inoculation with natural stress-resistant isolates could be a robust approach to improve the productivity and quality of these important nitrogen-fixing crops. [ABSTRACT FROM AUTHOR]

Glutathione (L-γ-glutamyl-L-cysteinylglycine) (GSH), one of the key antioxidants in Sinorhizobium meliloti, is required for the development of alfalfa (Medicago sativa) nitrogen-fixing nodules. Glutathione exists as either reduced glutathione (GSH) or oxidized glutathione (GSSG), and its content is regulated by two pathways in S. meliloti. The first pathway is the de novo synthesis of glutathione from its constituent amino acids, namely, Glu, Cys, and Gly, catalyzed by γ-glutamylcysteine synthetase (GshA) and glutathione synthetase (GshB). The second pathway is the recycling of GSSG via glutathione reductase (GR). However, whether the S. meliloti GR functions similarly to GshA and GshB1 during symbiotic interactions with alfalfa remains unknown. In this study, a plasmid insertion mutation of the S. meliloti gor gene, which encodes GR, was constructed, and the mutant exhibited delayed alfalfa nodulation, with 75% reduction in nitrogen-fixing capacity. The gor mutant demonstrated increased accumulation of GSSG and a decreased GSH/GSSG ratio in cells. The mutant also showed defective growth in rich broth and minimal broth and was more sensitive to the oxidants H2O2 and sodium nitroprusside. Interestingly, the expression of gshA, gshB1, katA, and katB was induced in the mutant. These findings reveal that the recycling of glutathione is important for S. meliloti to maintain redox homeostasis and to interact symbiotically with alfalfa. [ABSTRACT FROM AUTHOR]

Although most studies of mutualisms focus on a single partner at a time, host species often associate with multiple mutualist partners simultaneously. Because of potential interactions between mutualists, only studying a single type of mutualism could lead to a biased perspective of mutualism benefit and how mutualisms may scale-up to affect communities. The legume Chamaecrista fasciculata engages in a resource mutualism with nitrogen-fixing rhizobia and also forms symbiotic interactions with ants by providing nectar in exchange for defense against herbivores. Although they provide very different benefits to the plant, both mutualists receive carbon resources from the plant. As a result, these two mutualists are likely to interact, potentially competing for carbon resources or mutually benefitting each other via their positive effects on plant hosts. In a full-factorial field experiment, we explored how rhizobia and ants influence one another, C. fasciculata fitness, and the associated arthropod community. Ants reduced plant allocation to rhizobia, but ants also increased rhizobia contamination of uninoculated plants, suggesting that ants may disperse rhizobia. In turn, rhizobia increased ant abundances, with ants preferentially tending plants with rhizobia. Chamaecrista fasciculata received substantial fitness benefits from rhizobia; in contrast, associating with ants reduced fitness. Additionally, the mutualists interacted to influence the abundance of other arthropods found on the plants. Rhizobia increased arthropod abundances, likely because more nitrogen-rich leaf tissue was more attractive to arthropod herbivores, but ants negated these increases. As these results illustrate, multiple mutualists may interact, influencing each other's abundance and the abundance of other community members. [ABSTRACT FROM AUTHOR]

Strain CCMM B554, also known as FSM-MA, is a soil dwelling and nodule forming, nitrogen-fixing bacterium isolated from the nodules of the legume Medicago arborea L. in the Maamora Forest, Morocco. The strain forms effective nitrogen fixing nodules on species of the Medicago, Melilotus and Trigonella genera and is exceptional because it is a highly effective symbiotic partner of the two most widely used accessions, A17 and R108, of the model legume Medicago truncatula Gaertn. Based on 16S rRNA gene sequence, multilocus sequence and average nucleotide identity analyses, FSM-MA is identified as a new Ensifer meliloti strain. The genome is 6,70 Mbp and is comprised of the chromosome (3,64 Mbp) harboring 3574 predicted genes and two megaplasmids, pSymA (1,42 Mbp) and pSymB (1,64 Mbp) with respectively 1481 and 1595 predicted genes. The average GC content of the genome is 61.93%. The FSM-MA genome structure is highly similar and co-linear to other E. meliloti strains in the chromosome and the pSymB megaplasmid while, in contrast, it shows high variability in the pSymA plasmid. The large number of strain-specific sequences in pSymA as well as strain-specific genes on pSymB involved in the biosynthesis of the lipopolysaccharide and capsular polysaccharide surface polysaccharides may encode novel symbiotic functions explaining the high symbiotic performance of FSM-MA. [ABSTRACT FROM AUTHOR]

The development of legume nitrogen-fixing nodules is regulated by reactive oxygen species (ROS) produced by symbionts. Several regulators from Rhizobium are involved in ROS sensing. In a previous study, we found that Sinorhizobium meliloti LsrB regulates lipopolysaccharide production and is associated with H2O2 accumulation in alfalfa ( Medicago sativa) nodules. However, its underlying regulatory mechanism remains unclear. Here, we report that the cysteine residues in LsrB are required for adaptation to oxidative stress, gene expression, alfalfa nodulation and nitrogen fixation. Moreover, LsrB directly activated the transcription of lrp3 and gshA (encoding γ-glutamylcysteine synthetase, responsible for glutathione synthesis) and this regulation required the cysteine (Cys) residues in the LsrB substrate-binding domain. The Cys residues could sense oxidative stress via the formation of intermolecular disulfide bonds, generating LsrB dimers and LsrB-DNA complexes. Among the Cys residues, C238 is a positive regulatory site for the induction of downstream genes, whereas C146 and C275 play negative roles in the process. The lsrB mutants with Cys-to-Ser substitutions displayed altered phenotypes in respect to their adaptation to oxidative stress, nodulation and nitrogen fixation-related plant growth. Our findings demonstrate that S. meliloti LsrB modulates alfalfa nodule development by directly regulating downstream gene expression via a post-translational strategy. [ABSTRACT FROM AUTHOR]

Small non-coding RNAs (sRNAs) are expected to have pivotal roles in the adaptive responses underlying symbiosis of nitrogen-fixing rhizobia with legumes. Here, we provide primary insights into the function and activity mechanism of the Sinorhizobium meliloti trans-sRNA NfeR1 (Nodule Formation Efficiency RNA). Northern blot probing and transcription tracking with fluorescent promoter-reporter fusions unveiled high nfeR1 expression in response to salt stress and throughout the symbiotic interaction. The strength and differential regulation of nfeR1 transcription are conferred by a motif, which is conserved in nfeR1 promoter regions in α-proteobacteria. NfeR1 loss-of-function compromised osmoadaptation of free-living bacteria, whilst causing misregulation of salt-responsive genes related to stress adaptation, osmolytes catabolism and membrane trafficking. Nodulation tests revealed that lack of NfeR1 affected competitiveness, infectivity, nodule development and symbiotic efficiency of S. meliloti on alfalfa roots. Comparative computer predictions and a genetic reporter assay evidenced a redundant role of three identical NfeR1 unpaired anti Shine-Dalgarno motifs for targeting and downregulation of translation of multiple mRNAs from transporter genes. Our data provide genetic evidence of the hyperosmotic conditions of the endosymbiotic compartments. NfeR1-mediated gene regulation in response to this cue could contribute to coordinate nutrient uptake with the metabolic reprogramming concomitant to symbiotic transitions. [ABSTRACT FROM AUTHOR]

Bradyrhizobium comprises most tropical symbiotic nitrogen-fixing strains, but the correlation between symbiotic and core genes with host specificity is still unclear. In this study, the phylogenies of the nodY/K and nifH genes of 45 Bradyrhizobium strains isolated from legumes of economic and environmental importance in Brazil ( Arachis hypogaea , Acacia auriculiformis , Glycine max , Lespedeza striata , Lupinus albus , Stylosanthes sp. and Vigna unguiculata ) were compared to 16S rRNA gene phylogeny and genetic diversity by rep-PCR. In the 16S rRNA tree, strains were distributed into two superclades— B. japonicum and B. elkanii —with several strains being very similar within each clade. The rep-PCR analysis also revealed high intra-species diversity. Clustering of strains in the nodY/K and nifH trees was identical: 39 strains isolated from soybean grouped with Bradyrhizobium type species symbionts of soybean, whereas five others occupied isolated positions. Only one strain isolated from Stylosanthes sp. showed similar nodY/K and nifH sequences to soybean strains, and it also nodulated soybean. Twenty-one representative strains of the 16S rRNA phylogram were selected and taxonomically classified using a concatenated glnII - recA phylogeny; nodC sequences were also compared and revealed the same clusters as observed in the nodY/K and nifH phylograms. The analyses of symbiotic genes indicated that a large group of strains from the B. elkanii superclade comprised the novel symbiovar sojae, whereas for another group, including B. pachyrhizi , the symbiovar pachyrhizi could be proposed. Other potential new symbiovars were also detected. The co-evolution hypotheses is discussed and it is suggested that nodY/K analysis would be useful for investigating the symbiotic diversity of the genus Bradyrhizobium . [ABSTRACT FROM AUTHOR]