Cyanobacteria-bryophyte symbioses

Cyanobacteria are a large group of photosynthetic prokaryotesof enormous environmental importance, being responsible fora large proportion of global CO₂ and N₂ fixation. They formsymbiotic associations with a wide range of eukaryotic hostsincluding plants, fungi, sponges, and protists. The cyanobacterialsymbionts are often filamentous and fix N₂ in specialized cellsknown as heterocysts, enabling them to provide the host withfixed nitrogen and, in the case of non-photosynthetic hosts,with fixed carbon. The best studied cyanobacterial symbiosesare those with plants, in which the cyanobacteria can infectthe roots, stems, leaves, and, in the case of the liverwortsand hornworts, the subject of this review, the thallus. Thesymbionts are usually Nostoc spp. that gain entry to the hostby means of specialized motile filaments known as hormogonia.The host plant releases chemical signals that stimulate hormogoniaformation and, by chemoattraction, guide the hormogonia to thepoint of entry into the plant tissue. Inside the symbiotic cavity,host signals inhibit further hormogonia formation and stimulateheterocyst development and dinitrogen fixation. The cyanobiontsundergo morphological and physiological changes, including reducedgrowth rate and CO₂ fixation, and enhanced N₂ fixation, andrelease to the plant much of the dinitrogen fixed. This shortreview summarizes knowledge of the cyanobacterial symbioseswith liverworts and hornworts, with particular emphasis on theimportance of pili and gliding motility for the symbiotic competenceof hormogonia. Key words: Bryophyte, cyanobacteria, gliding motility, pili, symbiosis Received 1 August 2007; Revised 23 December 2007 Accepted 7 January 2008     

The hmp chemotaxis cluster regulates gliding in the filamentous cyanobacterium Nostoc punctiforme

Many bacteria are capable of movement over surfaces without flagella or pili; they glide. Nostoc punctiforme is a cyanobacterium that differentiates specialized gliding filaments called hormogonia, but the mechanism underlying their movement is currently unknown. Risser et al. characterize the h ormogonia m otility and p olysaccharide (hmp) locus that encodes proteins homologous to well‐studied chemotaxis systems. All but one of the genes in the locus were required for gliding motility and each protein localized as a ring near the cell junction. One protein, the CheA homologue HmpE, was capable of autophosphorylation and phosphotransfer to the CheY homologue HmpB. This study reveals the hmp locus as an important regulator of gliding and highlights N. punctiforme as a model for understanding gliding motility in a complex multicellular bacterium.     

Phylogeny of symbiotic cyanobacteria within the genus <Emphasis Type="Italic">Nostoc</Emphasis> based on 16S rDNA sequence analyses

A phylogenetic analysis of selected symbiotic Nostoc strain sequences and available database 16S rDNA sequences of both symbiotic and free-living cyanobacteria was carried out using maximum likelihood and Bayesian inference techniques. Most of the symbiotic strains fell into well separated clades. One clade consisted of a mixture of symbiotic and free-living isolates. This clade includes Nostoc sp. strain PCC 73102, the reference strain proposed for Nostoc punctiforme. A separate symbiotic clade with isolates exclusively from Gunnera species was also obtained, suggesting that not all symbiotic Nostoc species can be assigned to N. punctiforme. Moreover, isolates from Azolla filiculoides and one from Gunnera dentata were well nested within a clade comprising most of the Anabaena sequences. This result supports the affiliation of the Azolla isolates with the genus Anabaena and shows that strains within this genus can form symbioses with additional hosts. Furthermore, these symbiotic strains produced hormogonia, thereby verifying that hormogonia formation is not absent in Anabaena and cannot be used as a criterion to distinguish it from Nostoc.The GenBank accession numbers for the cyanobacterial 16S rRNA gene sequences determined in this study are AY742447-AY742454.     

Motility in cyanobacteria: polysaccharide tracks and Type IV pilus motors

Motility in cyanobacteria is useful for purposes that range from seeking out favourable light environments to establishing symbioses with plants and fungi. No known cyanobacterium is equipped with flagella, but a diverse range of species is able to ‘glide’ or ‘twitch’ across surfaces. Cyanobacteria with this capacity range from unicellular species to complex filamentous forms, including species such as Nostoc punctiforme, which can generate specialised motile filaments called hormogonia. Recent work on the model unicellular cyanobacterium Synechocystis sp. PCC 6803 has shown that its means of propulsion has much in common with the twitching motility of heterotrophs such as Pseudomonas and Myxococcus. Movement depends on Type IV pili, which are extended, adhere to the substrate and then retract to pull the cell across the surface. Previous work on filamentous cyanobacteria suggested a very different mechanism, with movement powered by the directional extrusion of polysaccharide from pores close to the cell junctions. Now a new report by Khayatan and colleagues in this issue of Molecular Microbiology suggests that the motility of Nostoc hormogonia has much more in common with Synechocystis than was previously thought. In both cases, polysaccharide secretion is important for preparing the surface, but the directional motive force comes from Type IV pili.     

Diverse roles of the GlcP glucose permease in free-living and symbiotic cyanobacteria

Certain cyanobacteria can form symbiotic associations with plants, where the symbiont supplies the plant partner with nitrogen and in return obtains sugars. We recently showed that in the symbiotic cyanobacterium Nostoc punctiforme, a glucose specific permease, GlcP, is necessary for the symbiosis to be formed. Results presented here from growth yield measurements of mutant strains with inactivated or overexpressing sugar transporters suggest that GlcP could be induced by a symbiosis specific substance. We also discuss that the transporter may have a role other than nutritional once the symbiosis is established, i.e., during infection, and more specifically in the chemotaxis of the symbiont. Phylogenetic analysis shows that the distribution of GlcP among cyanobacteria is likely influenced by horizontal gene transfer, but also that it is not correlated with symbiotic competence. Instead, regulatory patterns of the transporter in Nostoc punctiforme likely constitute symbiosis specific adaptations.     

Mutation at different sites in the Nostoc punctiforme cyaC gene, encoding the multiple-domain enzyme adenylate cyclase, results in different levels of infection of the host plant Blasia pusilla

The filamentous cyanobacterium Nostoc punctiforme forms symbioses with plants. Disruption of the catalytic domain of the N. punctiforme adenylate cyclase (CyaC) significantly increased symbiotic competence, whereas reduced infectivity was observed in a mutant with a disruption close to the N terminus of CyaC. The total cellular cyclic AMP levels were significantly reduced in both mutants.     

Multiple Roles of Soluble Sugars in the Establishment of Gunnera-Nostoc Endosymbiosis

Gunnera plants have the unique ability to form endosymbioses with N₂-fixing cyanobacteria, primarily Nostoc. Cyanobacteria enter Gunnera through transiently active mucilage-secreting glands on stems. We took advantage of the nitrogen (N)-limitation-induced gland development in Gunnera manicata to identify factors that may enable plant tissue to attract and maintain cyanobacteria colonies. Cortical cells in stems of N-stressed Gunnera plants were found to accumulate a copious amount of starch, while starch in the neighboring mature glands was nearly undetectable. Instead, mature glands accumulated millimolar concentrations of glucose (Glc) and fructose (Fru). Successful colonization by Nostoc drastically reduced sugar accumulation in the surrounding tissue. Consistent with the abundance of Glc and Fru in the gland prior to Nostoc colonization, genes encoding key enzymes for sucrose and starch hydrolysis (e.g. cell wall invertase, α-amylase, and starch phosphorylase) were expressed at higher levels in stem segments with glands than those without. In contrast, soluble sugars were barely detectable in mucilage freshly secreted from glands. Different sugars affected Nostoc’s ability to differentiate motile hormogonia in a manner consistent with their locations. Galactose and arabinose, the predominant constituents of polysaccharides in the mucilage, had little or no inhibitory effect on hormogonia differentiation. On the other hand, soluble sugars that accumulated in gland tissue, namely sucrose, Glc, and Fru, inhibited hormogonia differentiation and enhanced vegetative growth. Results from this study suggest that, in an N-limited environment, mature Gunnera stem glands may employ different soluble sugars to attract Nostoc and, once the cyanobacteria are internalized, to maintain them in the N₂-fixing vegetative state.Nitrogen (N) is an essential element for plant growth, but availability of reduced N in the soil is often limiting. Representatives from a wide range of land plants have evolved the ability to form associations with N₂-fixing microbes (Franche et al., 2009). Associations between rhizobia and legume plants are well-characterized examples of plant-bacterial N₂-fixing symbioses. Unlike rhizobia, which generally exhibit narrow host ranges (Kistner and Parniske, 2002), N₂-fixing cyanobacteria are able to form productive associations with a broad range of plants, including bryophytes (hornworts and liverworts), ferns (Azolla), gymnosperms (cycads), and angiosperms (Gunnera; for review, see Rai et al., 2000; Adams et al., 2006). Free-living cyanobacteria within the genus Nostoc can fix N in specialized microoxic cells called heterocysts. The ability of Nostoc to fix N independent of a host environment may facilitate the formation of symbioses with a wide range of plants. Understanding the physiological conditions that enable a plant host to enter into symbiotic associations with cyanobacteria may allow us to extend the benefit of biological N fixation to crops outside the legume family.Nostoc has the ability to differentiate not only into filaments bearing heterocysts but also into transiently motile filaments, known as hormogonia, which enable the cyanobacteria to enter plants (Meeks and Elhai, 2002). Nostoc can be induced to form hormogonia by different environmental stimuli and by a hormogonia-inducing factor released from N-stressed host plants (Meeks and Elhai, 2002; Adams et al., 2006). The attraction of hormogonia to plants is much less specific than that of rhizobia. Hormogonia are attracted to root extracts from either host or nonhost plants and even to certain simple sugars, such as Ara, Glc, and Gal (Nilsson et al., 2006). After entering a plant host, hormogonia revert back to filaments with N₂-fixing heterocysts. Inside the host, further hormogonia formation is suppressed, and heterocysts appear at a frequency of about 30% to 40%, 3- to 4-times higher than that found in free-living Nostoc (Meeks and Elhai, 2002). Although free-living Nostoc species can support N₂ fixation through photosynthesis, under symbiotic conditions they rely on photosynthate from the host plant. In general, the sugars (Suc, Glc, and Fru) known to support heterotrophic growth in the dark by free-living cyanobacteria coincide with those that support nitrogenase activity in Nostoc-plant associations (Meeks and Elhai, 2002). However, the Nostoc-Gunnera association may be exceptional; only Glc and Fru have been shown to sustain nitrogenase activities (Man and Silvester, 1994; Wouters et al., 2000), although Suc anddextrin were able to keep Nostoc alive without light (Wouters et al., 2000). It is evident from cyanobacterial studies that the plant hosts have evolved the ability to regulate cyanobacterial growth and differentiation during symbiotic associations (Meeks and Elhai, 2002).However, because most studies of plant-cyanobacterial associations have focused on the cyanobacterial partner (e.g. Wang et al., 2004; Ekman et al., 2006), the mechanisms through which plant hosts attract, internalize, and maintain cyanobacteria remain to be elucidated (Adams et al., 2006).The Nostoc-Gunnera association is an ideal system with which to study plant-cyanobacteria symbioses, not only because Gunnera is the only genus of angiosperms known to form endosymbioses with N₂-fixing cyanobacteria but also because the association between the two can be readily established in the laboratory (Bergman et al., 1992; Chiu et al., 2005). Nostoc hormogonia enter Gunnera plants through specialized glands located on the stem. As the gland matures, it secretes polysaccharide-rich mucilage that attracts cyanobacteria (Nilsson et al., 2006), supports their growth on the gland surface (Towata, 1985; Chiu et al., 2005), and permits further hormogonia differentiation (Rasmussen et al., 1994). From there, hormogonia enter the gland and penetrate cells near the base of the gland in the stem (Bonnett, 1990; Bergman et al., 1992). Although each gland is only transiently capable of accepting cyanobacteria, new glands continue to form on the stem at the base of each new leaf.In contrast to the development of nodules in legumes, which requires a complex exchange of signals between the two symbiotic partners (Cooper, 2007), stem gland development in Gunnera takes place in the absence of cyanobacteria (Bonnett, 1990). N limitation, however, is a prerequisite for stem gland development (Chiu et al., 2005), as it is for nodulation (Barbulova et al., 2007). We have taken advantage of the N-deficiency-induced gland development in G. manicata to identify factors that enable Gunnera to form endosymbiosis with cyanobacteria. This study investigated changes in the carbohydrate metabolism during Gunnera gland development and discovered that tissue in the mature glands accumulated high levels of soluble sugars prior to the arrival of cyanobacteria. In agreement with this finding, several key genes encoding enzymes for starch/Suc hydrolysis were expressed at higher levels in the gland compared to the stem. Furthermore, we found that various sugars cyanobacteria may encounter as they approach Gunnera glands as opposed to those they would encounter within plant cells differentially affected Nostoc’s ability to form motile hormogonia.     

Characteristics of Hormogonia Formation by Symbiotic Nostoc spp. in Response to the Presence of Anthoceros punctatus or Its Extracellular Products

Nostocacean cyanobacteria typically produce gliding filaments termed hormogonia at a low frequency as part of their life cycle. We report here that all Nostoc spp. competent in establishing a symbiotic association with the hornwort Anthoceros punctatus formed hormogonial filaments at a high frequency in the presence of A. punctatus. The hormogonia-inducing activity was produced by A. punctatus under nitrogen-limited culture conditions. The hormogonia of the symbiotically competent Nostoc spp. were characterized as motile (gliding) filaments lacking heterocysts and with distinctly smaller cells than those of vegetative filaments; the small cells resulted from a continuation of cell division uncoupled from biomass increase. An essentially complete conversion of vegetative filaments to hormogonia occurred within 12 h of exposure of Nostoc sp. strain 7801 to A. punctatus growth-conditioned medium. Hormogonia formation was accompanied by loss of nitrogen fixation (acetylene reduction) and by decreases in photosynthetic CO₂ fixation and in vivo NH₄⁺ assimilation of 30% and approximately 40%, respectively. The rates of acetylene reduction and CO₂ fixation returned to approximately the control rates within 72 to 96 h after hormogonia induction, as the cultures of Nostoc sp. strain 7801 differentiated heterocysts and reverted to the vegetative growth state. The relationship between hormogonia formation and symbiotic competence is discussed.     

Functional characterization of the twin ZIP/SLC39 metal transporters, NpunF3111 and NpunF2202 in Nostoc punctiforme

The ZIP family of metal transporters is involved in the transport of Zn²⁺ and other metal cations from the extracellular environment and/or organelles into the cytoplasm of prokaryotes, eukaryotes and archaeotes. In the present study, we identified twin ZIP transporters, Zip11 (Npun_F3111) and Zip63 (Npun_F2202) encoded within the genome of the filamentous cyanobacterium, Nostoc punctiforme PCC73120. Sequence-based analyses and structural predictions confirmed that these cyanobacterial transporters belong to the SLC39 subfamily of metal transporters. Quantitative real-time (QRT)-PCR analyses suggested that the enzymes encoded by zip11 and zip63 have a broad allocrite range that includes zinc as well as cadmium, cobalt, copper, manganese and nickel. Inactivation of either zip11 or zip63 via insertional mutagenesis in N. punctiforme resulted in reduced expression of both genes, highlighting a possible co-regulation mechanism. Uptake experiments using ⁶⁵Zn demonstrated that both zip mutants had diminished zinc uptake capacity, with the deletion of zip11 resulting in the greatest overall reduction in ⁶⁵Zn uptake. Over-expression of Zip11 and Zip63 in an E. coli mutant strain (ZupT736::kan) restored divalent metal cation uptake, providing further evidence that these transporters are involved in Zn uptake in N. punctiforme. Our findings show the functional role of these twin metal uptake transporters in N. punctiforme, which are independently expressed in the presence of an array of metals. Both Zip11 and Zip63 are required for the maintenance of homeostatic levels of intracellular zinc N. punctiforme, although Zip11 appears to be the primary zinc transporter in this cyanobacterium, both ZIP’s may be part of a larger metal uptake system with shared regulatory elements.     

Complex quorum-sensing regulatory systems regulate bacterial growth and symbiotic nodulation in <Emphasis Type="Italic">Mesorhizobium tianshanense</Emphasis>

LuxR/LuxI-type quorum-sensing systems have been shown to be important for symbiotic interactions between a number of rhizobium species and host legumes. In this study, we found that different isolates of Mesorhizobium tianshanense, a moderately-growing Rhizobium that forms nodules on a number of types of licorice plants, produces several different N-acyl homoserine lactone-like molecules. In M. tianshanense CCBAU060A, we performed a genetic screen and identified a network of regulatory components including a set of LuxI/LuxR-family regulators as well as a MarR-family regulator that is required for quorum-sensing regulation. Furthermore, compared with the wild-type strains, quorum-sensing deficient mutants showed a reduced growth rate and were defective in nodule formation on their host plant Glycyrrhiza uralensis. These data suggest that different M. tianshanense strains may use diverse quorum-sensing systems to regulate symbiotic process. H. Cao, M. Yang, and H. Zheng contributed equally to this work.     

A Legume Genetic Framework Controls Infection of Nodules by Symbiotic and Endophytic Bacteria

Legumes have an intrinsic capacity to accommodate both symbiotic and endophytic bacteria within root nodules. For the symbionts, a complex genetic mechanism that allows mutual recognition and plant infection has emerged from genetic studies under axenic conditions. In contrast, little is known about the mechanisms controlling the endophytic infection. Here we investigate the contribution of both the host and the symbiotic microbe to endophyte infection and development of mixed colonised nodules in Lotus japonicus. We found that infection threads initiated by Mesorhizobium loti, the natural symbiont of Lotus, can selectively guide endophytic bacteria towards nodule primordia, where competent strains multiply and colonise the nodule together with the nitrogen-fixing symbiotic partner. Further co-inoculation studies with the competent coloniser, Rhizobium mesosinicum strain KAW12, show that endophytic nodule infection depends on functional and efficient M. loti-driven Nod factor signalling. KAW12 exopolysaccharide (EPS) enabled endophyte nodule infection whilst compatible M. loti EPS restricted it. Analysis of plant mutants that control different stages of the symbiotic infection showed that both symbiont and endophyte accommodation within nodules is under host genetic control. This demonstrates that when legume plants are exposed to complex communities they selectively regulate access and accommodation of bacteria occupying this specialized environmental niche, the root nodule.     

The Sinorhizobium meliloti ntrX Gene Is Involved in Succinoglycan Production,Motility, and Symbiotic Nodulation on Alfalfa

Rhizobia establish a symbiotic relationship with their host legumes to induce the formation of nitrogen-fixing nodules. This process is regulated by many rhizobium regulators, including some two-component regulatory systems (TCSs). NtrY/NtrX, a TCS that was first identified in Azorhizobium caulinodans, is required for free-living nitrogen metabolism and symbiotic nodulation on Sesbania rostrata. However, its functions in a typical rhizobium such as Sinorhizobium meliloti remain unclear. Here we found that the S. meliloti response regulator NtrX but not the histidine kinase NtrY is involved in the regulation of exopolysaccharide production, motility, and symbiosis with alfalfa. A plasmid insertion mutant of ntrX formed mucous colonies, which overproduced succinoglycan, an exopolysaccharide, by upregulating its biosynthesis genes. This mutant also exhibited motility defects due to reduced flagella and decreased expression of flagellins and regulatory genes. The regulation is independent of the known regulatory systems of ExoR/ExoS/ChvI, EmmABC, and ExpR. Alfalfa plants inoculated with the ntrX mutant were small and displayed symptoms of nitrogen starvation. Interestingly, the deletion mutant of ntrY showed a phenotype similar to that of the parent strain. These findings demonstrate that the S. meliloti NtrX is a new regulator of succinoglycan production and motility that is not genetically coupled with NtrY.     

Production of phenolics and the emission of volatile organic compounds by perennial ryegrass (Lolium perenne L.)/Neotyphodium lolii association as a response to infection by Fusarium poae

Grasses very often form symbiotic associations with Neotyphodium/Epichloë endophytic fungi. These endophytes often allow the host grass to be protected from different pathogens. However, there is little known about the mechanisms of such endophyte influence on the host. Thus, the purpose of this research was to examine the effect of the N. lolii endophyte on the total production of phenolic compounds, VOCs emission and the resistance of three perennial ryegrass genotypes infected by pathogenic Fusarium poae. Analyses of total phenolics content were performed in control (not inoculated) and inoculated plants after 1, 2, 3, 4, 5, and 6 days (DAI) and for VOCs after 0, 3, 6 and 12 DAI. The presence of endophytes significantly reduced the disease index in two of the three genotypes relative to that in E−. Plants infected by N. lolii exhibited higher production of phenolics relative to the E− plants. The highest amounts of phenolics were observed on the second and sixth DAI. Genotype Nl22 showed the strongest effect of the endophyte on the production of phenolics, which increased by over 61%. Both the endophyte infected and non-infected plants emitted most abundantly two GLVs ((Z)-3-hexenal, (Z)-3-hexen-1-yl acetate), three terpenes (linalool, (Z)-ocimene, β-caryophyllene) and three shikimic acid pathway derivatives (benzyl acetate, indole, and methyl salicylate). The endophyte presence and the intervals of VOCs detection were a highly significant source of variation for all emitted volatiles (P < 0.001). The genotype of the perennial ryegrass significantly affected only the emission of methyl salicylate (P < 0.05) and β-caryophyllene (P < 0.05). Most of the VOCs ((Z)-3-hexen-1-yl acetate, (Z)-3-hexenal, linalool and methyl salicylate) reached their highest levels of emission on the sixth DAI, when averaged over genotypes and endophyte status. The results highlight the role of Neotyphodium spp. in the mediation of quadro-trophic interactions among plants, symbiotic endophytes, invertebrate herbivores and plant pathogenic fungi. Our results also confirm the fact that symbiotic plants can activate a defense reaction faster than non-symbiotic plants after a pathogen attack. Thus, N. lolii can be involved in the defense of perennial ryegrass against pathogens and potentially could be central to the host plants’ protection.     

New evidence for the symbiosis between Tuber aestivum and Picea abies

The Burgundy truffle (Tuber aestivum Vittad.), an ectomycorrhizal fungus living in association with host plants, is one of the most exclusive delicacies. The symbiosis with deciduous oak, beech, and hazel dominates our concept of truffle ecophysiology, whereas potential conifer hosts have rarely been reported. Here, we present morphological and molecular evidence of a wildlife T. aestivum symbiosis with Norway spruce (Picea abies Karst.) and an independent greenhouse inoculation experiment, to confirm our field observation in southwest Germany. A total of 27 out of 50 P. abies seedlings developed T. aestivum ectomycorrhizae with a mean mycorrhization rate of 19.6 %. These findings not only suggest P. abies to be a productive host species under suitable biogeographic conditions but also emphasize the broad ecological amplitude and great symbiotic range of T. aestivum. While challenging common knowledge, this study demonstrates a significant expansion of the species' cultivation potential to the central European regions, where P. abies forests occur on calcareous soils.     

The regulation of symbiotic N2 fixation: a conceptual model of N feedback from the ecosystem to the gene expression level

Symbiotic nitrogen (N₂) fixation in legumes may give the host plant a distinct competitive advantage; at the same time it is mainly responsible for introducing N into terrestrial ecosystems which may ultimately benefit all organisms. Depending on environmental conditions, symbiotic N₂ fixation may be tuned to the plant's N demand or specifically inhibited (a disadvantage for plants which depend mainly on symbiotic N₂ fixation), or even prevented. Thus, the ecological range for symbiotic N₂ fixation can be narrower than that of the host plants. A shortage of mineral N is the only case in which adverse environmental conditions clearly favour symbiotic N₂ fixation. Variations in number or mass of nodules or nodule morphology are persistent features, that may represent one kind of regulation of N₂ fixation. In addition, varying O₂ permeability of nodules functions as a rapid and reversible control of N₂ fixation which may compensate partially or fully for poor nodulation. The plant's demand for symbiotically fixed N is thought to play a central role in modulating both nodulation and N₂ fixation activity; an N feedback mechanism is assumed. The control of symbiotic N₂ fixation operates through a series of ecophysiological triggers which are also influenced by complex interactions between legume plants and other organisms in the ecosystem. The proportion of legume biomass and the performance of symbiotic N₂ fixation in each individual legume are the main parameters which determine the amount of symbiotically fixed N introduced into a terrestrial ecosystem. The various triggers and N feedback mechanisms from the whole ecosystem to the gene expression level which regulate symbiotic N₂ fixation in terrestrial ecosystems are reviewed and discussed in terms of a conceptual model. Although the presented model is based primarily on our knowledge about the physiology of a few leguminous crop species and of ecosystem processes in managed, perennial grassland in temperate climatic conditions, it may stimulate thinking about functional relationships between symbiotic N₂ fixation and terrestrial ecosystems at various system levels.     

Improving Soybean (Glycine max L.) N2 Fixation under Stress

Soybean is a major leguminous plant that has the ability to establish a symbiotic association with the N-fixing bacteria, Bradyrhizobium japonicum. Soils are usually subjected to stress including salinity, drought, acidity, and suboptimal root zone temperature, adversely affecting the symbiotic process between soybean and the bacteria. One of the important processes affecting the performance of soybean under stress is the inhibited exchange of symbiosis-related signaling molecules, specifically genistein, between the host legume and B. japonicum during the initiation of symbiosis. Interestingly, inoculation of B. japonicum with the signal molecule genistein can partially or completely alleviate the stress. Understanding the techniques and the precise molecular pathways, which may be influenced by the signaling molecules during the stress, can be useful to determine parameters that enhance the plant’s ability to cope with stress. For example, the use of proteomic techniques to identify proteins expressed under stress can help characterize those proteins and their involvement in stress. Biotechnological-genetic techniques, either breeding or transformation, are also among the effective methods of improving soybean’s ability to fix N₂ under stress. This can be achieved by identifying the genes, which may be expressed under stress in tolerant bacterial and plant species, and inserting them into the non-tolerant species. This article highlights some important advances in soybean N₂ fixation under different stress conditions, and reviews some of the techniques used to improve the ability of plants and bacteria to efficiently fix N₂ under stress.