Mutualism as a constraint on invasion success for legumes and rhizobia

Because hereditary symbiont transmission is normally absent in the mutualism of legume plants and root‐nodule bacteria (rhizobia), dispersing plants may often arrive at new habitats where mutualist partners are too rare to provide full benefits. Factors governing invasion success were explored by analysing a system of two coupled pairwise competition models: a legume invader competing with a resident non‐mutualistic plant, and a rhizobial population competing with a resident population of nonsymbiotic bacteria. The non‐linear dependence of benefits on partner abundance in this mutualism creates the possibility of two alternative population size equilibria, so that a threshold density can exist for invasion. If legumes and rhizobia exceed a critical population size, both species achieve rapid population growth, while if initial densities of both species are below their respective thresholds, they remain rare and are thus vulnerable to extinction in the presence of competitors. Overall, the results indicate that legumes may often fail at colonization attempts within habitats where mutualist partners are scarce. Data on legume prevalence in island floras and rates of geographical spread by legume weeds are consistent with this inference. Predictive insights about invasiveness may emerge from comparative research on key traits identified by the model, especially the shape of the function determining the number of nodules formed at low rhizobial density.     

Recent development and new insight of diversification and symbiosis specificity of legume rhizobia: mechanism and application

Currently, symbiotic rhizobia (sl., rhizobium) refer to the soil bacteria in α- and β-Proteobacteria that can induce root and/or stem nodules on some legumes and a few of nonlegumes. In the nodules, rhizobia convert the inert dinitrogen gas (N₂) into ammonia (NH₃) and supply them as nitrogen nutrient to the host plant. In general, this symbiotic association presents specificity between rhizobial and leguminous species, and most of the rhizobia use lipochitooligosaccharides, so called Nod factor (NF), for cooperating with their host plant to initiate the formation of nodule primordium and to inhibit the plant immunity. Besides NF, effectors secreted by type III secretion system (T3SS), exopolysaccharides and many microbe-associated molecular patterns in the rhizobia also play important roles in nodulation and immunity response between rhizobia and legumes. However, the promiscuous hosts like Glycine max and Sophora flavescens can nodulate with various rhizobial species harbouring diverse symbiosis genes in different soils, meaning that the nodulation specificity/efficiency might be mainly determined by the host plants and regulated by the soil conditions in a certain cases. Based on previous studies on rhizobial application, we propose a ‘1+n−N’ model to promote the function of symbiotic nitrogen fixation (SNF) in agricultural practice, where ‘1’ refers to appreciate rhizobium; ‘+n’ means the addition of multiple trace elements and PGPR bacteria; and ‘−N’ implies the reduction of chemical nitrogen fertilizer. Finally, open questions in the SNF field are raised to future think deeply and researches.     

Biodiversity of rhizobia isolated from a wide range of forest legumes in Brazil

Tropical forests have a high diversity of plant species; are they associated with a correspondingly rich microbial flora? We addressed this question by examining the symbiotic rhizobium bacteria that nodulate a diverse pool of forest legume species in Brazil. The 44 strains studied had been isolated from 29 legume tree species representing 13 tribes including all three subfamilies of the Leguminosae, and were chosen to represent major groups from a larger sample that had previously been characterized by SDS–PAGE of total proteins. Partial 16S rRNA gene sequence was determined, corresponding to positions 44–303 in the Escherichia coli sequence. Fifteen sequences were found, including six novel ones. However, all but one of them could be assigned to a genus because they grouped closely with sequences from previously described rhizobial species. Fast-growing strains had sequences similar to Rhizobium spp., Sinorhizobium spp. or Mesorhizobium spp., while the slow-growing strains had sequences similar to Bradyrhizobium spp. One strain with an intermediate growth rate had a unique sequence which indicated that the strain might belong to the genus Azorhizobium. Although the strains showed a variety of sequences, it was surprising that these strains isolated from taxonomically very diverse host plants in previously unexplored environments were mostly very similar to strains described previously, largely from agricultural systems.     

The variable part of the dnaK gene as an alternative marker for phylogenetic studies of rhizobia and related alpha Proteobacteria

DnaK is the 70 kDa chaperone that prevents protein aggregation and supports the refolding of damaged proteins. Due to sequence conservation and its ubiquity this chaperone has been widely used in phylogenetic studies. In this study, we applied the less conserved part that encodes the so-called alpha-subdomain of the substrate-binding domain of DnaK for phylogenetic analysis of rhizobia and related non-symbiotic alpha-Proteobacteria. A single 330 bp DNA fragment was routinely amplified from DNA templates isolated from the species of the genera, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium, but also from some non-symbiotic alpha Proteobacteria such as Blastochloris, Chelatobacter and Chelatococcus. Phylogenetic analyses revealed high congruence between dnaK sequences and 16S rDNA trees, but they were not identical. In contrast, the partition homogeneity tests revealed that dnaK sequence data could be combined with other housekeeping genes such as recA, atpD or glnA. The dnaK trees exhibited good resolution in the cases of the genera Mesorhizobium, Sinorhizobium and Rhizobium, even better than usually shown by 16S rDNA phylogeny. The dnaK phylogeny supported the close phylogenetic relationship of Rhizobium galegae and Agrobacterium tumefaciens (R. radiobacter) C58, which together formed a separate branch within the fast-growing rhizobia, albeit closer to the genus Sinorhizobium. The Rhizobium and Sinorhizobium genera carried an insertion composed of two amino acids, which additionally supported the phylogenetic affinity of these two genera, as well as their distinctness from the Mesorhizobium genus. Consistently with the phylogeny shown by 16S-23S rDNA intergenic region sequences, the dnaK trees divided the genus Bradyrhizobium into three main lineages, corresponding to B. japonicum, B. elkanii, and photosynthetic Bradyrhizobium strains that infect Aeschynomene plants. Our results suggest that the 330 bp dnaK sequences could be used as an additional taxonomic marker for rhizobia and related species (alternatively to the 16S rRNA gene phylogeny).     

Quorum-sensing regulation in rhizobia and its role in symbiotic interactions with legumes

Legume-nodulating bacteria (rhizobia) usually produce N-acyl homoserine lactones, which regulate the induction of gene expression in a quorum-sensing (or population-density)-dependent manner. There is significant diversity in the types of quorum-sensing regulatory systems that are present in different rhizobia and no two independent isolates worked on in detail have the same complement of quorum-sensing genes. The genes regulated by quorum sensing appear to be rather diverse and many are associated with adaptive aspects of physiology that are probably important in the rhizosphere. It is evident that some aspects of rhizobial physiology related to the interaction between rhizobia and legumes are influenced by quorum sensing. However, it also appears that the legumes play an active role, both in terms of interfering with the rhizobial quorum-sensing systems and responding to the signalling molecules made by the bacteria. In this article, we review the diversity of quorum-sensing regulation in rhizobia and the potential role of legumes in influencing and responding to this signalling system.     

Hanging by a thread: invasion of legume plants by rhizobia

Nitrogen-fixing nodules on plants such as alfalfa, pea and vetch arise from the root inner cortex and grow via a persistent meristem. Thus, these nodules are defined as indeterminate. The formation of functional indeterminate nodules requires that symbiotic bacteria, collectively called rhizobia, gain access to the interior of roots and root nodules via infection threads. Recent work has begun to elucidate the important functions of the root cell cytoskeleton in infection thread formation. It has also recently become apparent that rhizobial Nod factors and rhizobial exopolysaccharides play key roles in the initiation and elongation of infection threads.     

The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation

Establishment of the Rhizobium-legume symbiosis depends on a molecular dialogue, in which rhizobial nodulation (Nod) factors act as symbiotic signals, playing a key role in the control of specificity of infection and nodule formation. Using nodulation-defective (Nod-) mutants of Medicago truncatula to study the mechanisms controlling Nod factor perception and signalling, we have previously identified five genes that control components of a Nod factor-activated signal transduction pathway. Characterisation of a new M. truncatula Nod- mutant led to the identification of the Nod Factor Perception (NFP) locus. The nfp mutant has a novel phenotype among Nod- mutants of M. truncatula, as it does not respond to Nod factors by any of the responses tested. The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild-type plants, and no root hair deformation. The nfp mutant is also deficient in Nod factor-induced calcium spiking and early nodulin gene expression. While certain genes controlling Nod factor signal transduction also control the establishment of an arbuscular mycorrhizal symbiosis, the nfp mutant shows a wild-type mycorrhizal phenotype. These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation.     

Nitrogen-fixing symbiosis between photosynthetic bacteria and legumes

Rhizobia having photosynthetic systems form nitrogen-fixing nodules on the stem and/or root of some species of the legumes Aeschynomene and Lotononis. This review is focused on the recent knowledge about the physiology, genetics and role of the photosystem in these bacteria. Photosynthetic electron transport seems to involve reaction centers, soluble cytochrome c2 and cytochrome bc1. Anaerobically, the electron transport system becomes over-reduced. The photosynthesis genes have been partially characterized; their organization is classical but their regulation is unusual as it is activated by far-red light via a bacteriophytochrome. This original mechanism of regulation seems well adapted to promote photosynthesis during stem symbiosis. Photosynthesis plays a major role in the efficiency of stem nodulation. It is also observed that infrared light stimulates nitrogen fixation in nodules containing photosynthetic bacteroids, suggesting that photosynthesis may additionally provides energy for nitrogen fixation, allowing for more efficient plant growth. Other aspects of these bacteria are discussed, in particular their taxonomic position and nodulation ability, the role of carotenoids and the potential for application of photosynthetic rhizobia in rice culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nod factors and a diffusible factor from arbuscular mycorrhizal fungi stimulate lateral root formation in Medicago truncatula via the DMI1/DMI2 signalling pathway

Legumes form two different types of intracellular root symbioses, with fungi and bacteria, resulting in arbuscular mycorrhiza and nitrogen-fixing nodules, respectively. Rhizobial signalling molecules, called Nod factors, play a key role in establishing the rhizobium-legume association and genes have been identified in Medicago truncatula that control a Nod factor signalling pathway leading to nodulation. Three of these genes, the so-called DMI1, DMI2 and DMI3 genes, are also required for formation of mycorrhiza, indicating that the symbiotic pathways activated by both the bacterial and the fungal symbionts share common steps. To analyse possible cross-talk between these pathways we have studied the effect of treatment with Nod factors on mycorrhization in M. truncatula. We show that Nod factors increase mycorrhizal colonization and stimulate lateral root formation. The stimulation of lateral root formation by Nod factors requires both the same structural features of Nod factors and the same plant genes (NFP, DMI1, DMI2, DMI3 and NSP1) that are required for other Nod factor-induced symbiotic responses such as early nodulin gene induction and cortical cell division. A diffusible factor from arbuscular mycorrhizal fungi was also found to stimulate lateral root formation, while three root pathogens did not have the same effect. Lateral root formation induced by fungal signal(s) was found to require the DMI1 and DMI2 genes, but not DMI3. The idea that this diffusible fungal factor might correspond to a previously hypothesized mycorrhizal signal, the 'Myc factor', is discussed.     

Crystal structures of NodS N-methyltransferase from Bradyrhizobium japonicum in ligand-free form and as SAH complex

NodS is an S-adenosyl-l-methionine (SAM)-dependent N-methyltransferase that is involved in the biosynthesis of Nod factor (NF) in rhizobia, which are bacterial symbionts of legume plants. NF is a modified chitooligosaccharide (COS) signal molecule that is recognized by the legume host, where it initiates symbiotic processes leading to atmospheric nitrogen fixation. We report the crystal structure of recombinant NodS protein from Bradyrhizobium japonicum, which infects lupine and serradella legumes. Two crystal forms—ligand-free NodS and NodS in complex with S-adenosyl-l-homocysteine, which is a by-product of the methylation reaction—were obtained, and their structures were refined to resolutions of 2.43 Å and 1.85 Å, respectively. Although the overall fold (consisting of a seven-stranded β-sheet flanked by layers of helices) is similar to those of other SAM-dependent methyltransferases, NodS has specific features reflecting the unique character of its oligosaccharide substrate. In particular, the N-terminal helix and its connecting loop get ordered upon SAM binding, thereby closing the methyl donor cavity and shaping a long surface canyon that is clearly the binding site for the acceptor molecule. Comparison of the two structural forms of NodS suggests that there are also other conformational changes taking place upon the binding of the donor substrate. As an enzyme that methylates a COS substrate, NodS is the first example among all SAM-dependent methyltransferases to have its three-dimensional structure elucidated. Gaining insight about how NodS binds its donor and acceptor substrates helps to better understand the mechanism of NodS activity and the basis of its functional difference in various rhizobia.     

Crosstalk between jasmonic acid, ethylene and Nod factor signaling allows integration of diverse inputs for regulation of nodulation

Plant hormones interact at many different levels to form a network of signaling pathways connected by antagonistic and synergistic interactions. Ethylene and jasmonic acid both act to regulate the plant's responsiveness to a common set of biotic stimuli. In addition ethylene has been shown to negatively regulate the plant's response to the rhizobial bacterial signal, Nod factor. This regulation occurs at an early step in the Nod factor signal transduction pathway, at or above Nod factor-induced calcium spiking. Here we show that jasmonic acid also inhibits the plant's responses to rhizobial bacteria, with direct effects on Nod factor-induced calcium spiking. However, unlike ethylene, jasmonic acid not only inhibits spiking but also suppresses the frequency of calcium oscillations when applied at lower concentrations. This effect of jasmonic acid is amplified in the ethylene-insensitive mutant skl, indicating an antagonistic interaction between these two hormones for regulation of Nod factor signaling. The rapidity of the effects of ethylene and jasmonic acid on Nod factor signaling suggests direct crosstalk between these three signal transduction pathways. This work provides a model by which crosstalk between signaling pathways can rapidly integrate environmental, developmental and biotic stimuli to coordinate diverse plant responses.     

Biology of a "babysitting" symbiosis in brittle stars: analysis of the interactions between Ophiomastix venosa and Ophiocoma scolopendrina

Abstract. "Babysitting" symbioses between brittle star species involve juveniles of one species and adults of another. During this phenomenon, reported from many localities in the Indo-Pacific Ocean, juveniles are attached to the disk or lie in the bursa of the adults. The symbiosis between members of Ophiomastix venosa and their host, Ophiocoma scolopendrina , was investigated on the Great Barrier Reef of Toliara (Madagascar) during a 14-month period. The population of O. scolopendrina only occurs on rocky spurs that frequently emerge at low tide, while the population of O. venosa lies in adjacent surge channels that are always immersed. Only juveniles of O. venosa associated with adults of O. scolopendrina may occur on the rocky spurs. Analyses conducted on the populations of the two species showed that (1) the symbiosis is facultative and that symbiotic juveniles migrate into the channels when they reach a disc diameter of 6 mm, and (2) recruitment in the channels occurs in April. Host choice experiments, Y-tube experiments, and experiments assessing the resistance of the ophiuroids to air-drying were conducted in the laboratory. The experiments clearly demonstrated that (1) symbiotic juveniles of O. venosa specifically recognize adults of O. scolopendrina , while free juveniles of the same size do not, and (2) juveniles of O. venosa would not survive air-drying conditions similar to those observed on the spurs at low tides if they were not in symbiosis with adults of O. scolopendrina .     

Positive interactions between nitrogen-fixing legumes and four different neighbouring species in a biodiversity experiment

The importance of facilitative processes due to the presence of nitrogen-fixing legumes in temperate grasslands is a contentious issue in biodiversity experiments. Despite a multitude of studies of fertilization effects of legumes on associated nonfixers in agricultural systems, we know little about the dynamics in more diverse systems. We hypothesised that the identity of target plant species (phytometers) and the diversity of neighbouring plant species would affect the magnitude of such positive species interactions. We therefore sampled aboveground tissues of phytometers planted into all plots of a grassland biodiversity–ecosystem functioning experiment and analysed their N concentrations, δ¹⁵N values and biomasses. The four phytometer species (Festuca pratensis, Plantago lanceolata, Knautia arvensis and Trifolium pratensis) each belonged to one of the four plant functional groups used in the experiment and allowed the effects of diversity on N dynamics in individual species to be assessed. We found significantly lower δ¹⁵N values and higher N concentrations and N contents (amount of N per plant) in phytometer species growing with legumes, indicating a facilitative role for legumes in these grassland ecosystems. Our data suggest that the main driving force behind these facilitative interactions in plots containing legumes was reduced competition for soil nitrate (“nitrate sparing”), with apparent N transfer playing a secondary role. Interestingly, species richness (and to a lesser extent functional group number) significantly decreased δ¹⁵N values, N concentrations and N content irrespective of any legume effect. Possible mechanisms behind this effect, such as increased N mineralisation and nitrate uptake in more diverse plots, now need further investigation. The magnitude of the positive interactions depended on the identity of the phytometer species. Evidence for increased N uptake in communities containing legumes was found in all three nonlegume phytometer species, with a subsequent strong increase in biomass in the grass F. pratensis across all diversity levels, and a lesser biomass gain in P. lanceolata and K. arvensis. In contrast, the legume phytometer species T. pratense was negatively affected when other legumes were present in their host communities across all diversity levels.     

Relationship between boron and calcium in the N2-fixing legume–rhizobia symbiosis

Because boron (B) and calcium (Ca²⁺) seem to have a strong effect on legume nodulation and nitrogen fixation, rhizobial symbiosis with leguminous plants, grown under varying concentrations of both nutrients, was investigated. The study of early pre‐infection events included the capacity of root exudates to induce nod genes, and the degree of adsorption of bacteria to the root surface. Both phenomena were inhibited by B deficiency, and increased by addition of Ca²⁺, resulting in an increase of the number of nodules. The infection and invasion steps were investigated by fluorescence microscopy in pea nodules harbouring a Rhizobium leguminosarum strain that constitutively expresses green fluorescent protein. High Ca²⁺ enhanced cell and tissue invasion by Rhizobium, which was highly inhibited after B deficiency. This was combined with an increased B concentration in nodules of plants grown on B‐free medium and supplemented with high Ca²⁺ concentrations, and that can be attributed to an increased B import to the nodules. Histological examination of indeterminate (pea) and determinate (bean) nodules showed an altered nodule anatomy at low B content of the tissue. The moderate increase in nodular B due to additional Ca²⁺ was not sufficient to prevent the abnormal cell wall structure and the aberrant distribution of pectin polysaccharides in B‐deficient treatments. Overall results indicate that the development of the symbiosis depends of the concentration of B and Ca²⁺, and that both nutrients are essential for nodule structure and function.     

Parasitic and mutualistic associations between a mycorrhizal fungus and soybean: The effect of phosphorus on host plant-endophyte interactions

Soybean [ Glycine max (L.) Merr. cv. Kent] plants were colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum (Thaxt. sensu Gerd.) Gerd. and Trappe in pot cultures using an inert medium and a nutrient solution. Phosphorus was provided initially as 0, 25,50, 100 or 200 mg hydroxyapatite [HAP, Ca₁₀(PO₄)₆(OH)₂] per pot. Under the low (0 mg HAP) and high (100 and 200 mg HAP) P regimes, VAM plants showed 20, 25 and 38% growth retardation, respectively, relative to non-colonized controls. At 50 mg HAP, VAM plant growth was significantly enhanced (14%). Dry weight and P content of both VAM and control plants increased with increased P availability throughout the HAP gradient. Intraradical VAM fungal biomass increased linearly with increasing P availability. Extraradical VAM fungal biomass was smaller than the intraradical component of the fungus at the lowest and highest levels of P addition in the growth medium. The ratio of extra- to intraradical mycelium, a suggested index of VAM fungal effectiveness, was greatest for the 50 mg HAP treatment, coinciding with growth enhancement of the host plant. This enhanced growth of the host at an intermediate P level was apparently a result of increased P uptake by the endophyte.     

丛枝菌根（AM）真菌-豆科植物-根瘤菌共生识别信号研究概况

<正>植物与微生物共生是自然界中普遍存在的一种生物学现象,其中高等植物和丛枝菌根(arbuscular mycorrhiza,AM)真菌共生形成的菌根、以及豆科植物和根瘤菌(rhizobia)形成的根瘤与农林牧业生产和生态系统的稳定性密切相关。豆科植物形成根瘤的同时还能与AM真菌形成菌根,最终建立三位一体