Rhizobien in der Pflanzenmikrobiota

Rhizobia in the plant microbiota The plant microbiota is of critical importance for plant growth and survival in soil. To explore mechanisms underlying plant‐microbiota interactions, defined commensal communities can be composed from microbiota culture collections and co‐cultivated with germ‐free plants to determine their impact on plant growth and health. The order Rhizobiales belongs to the core microbiota and includes nitrogen‐fixing bacteria that are known to engage in symbiotic interactions with legumes. Compatible host‐symbiont pairs are needed for a functional symbiosis, which involves the activation of highly specialized and interdependent signaling pathways between the two partners. Comparative genome analysis of more than 1,300 legume symbionts and rhizobial root commensals from non‐leguminous plants revealed that the most recent common ancestor of rhizobia lacked the gene repertoire needed for symbiosis and was able to colonize roots of a wide variety of plants. During evolution, key symbiosis genes were acquired multiple independent times by commensals belonging to different families of the Rhizobiales order.     

Phytoremediation of Heavy and Transition Metals Aided by Legume-Rhizobia Symbiosis

Legumes are important for nitrogen cycling in the environment and agriculture due to the ability of nitrogen fixation by rhizobia. In this review, we introduce an important and potential role of legume-rhizobia symbiosis in aiding phytoremediation of some metal contaminated soils as various legumes have been found to be the dominant plant species in metal contaminated areas. Resistant rhizobia used for phytoremediation could act on metals directly by chelation, precipitation, transformation, biosorption and accumulation. Moreover, the plant growth promoting (PGP) traits of rhizobia including nitrogen fixation, phosphorus solubilization, phytohormone synthesis, siderophore release, and production of ACC deaminase and the volatile compounds of acetoin and 2, 3-butanediol may facilitate legume growth while lessening metal toxicity. The benefits of using legumes inoculated with naturally resistant rhizobia or recombinant rhizobia with enhanced resistance, as well as co-inoculation with other plant growth promoting bacteria (PGPB) are discussed. However, the legume-rhizobia symbiosis appears to be sensitive to metals, and the effect of metal toxicity on the interaction between legumes and rhizobia is not clear. Therefore, to obtain the maximum benefits from legumes assisted by rhizobia for phytoremediation of metals, it is critical to have a good understanding of interactions between PGP traits, the symbiotic plant-rhizobia relationship and metals.     

Symbiosis specificity in the legume: rhizobial mutualism

Legume plants are able to engage in root nodule symbiosis with nitrogen-fixing soil bacteria, collectively called rhizobia. This mutualistic association is highly specific, such that each rhizobial species/strain interacts with only a specific group of legumes, and vice versa. Symbiosis specificity can occur at multiple phases of the interaction, ranging from initial bacterial attachment and infection to late nodule development associated with nitrogen fixation. Genetic control of symbiosis specificity is complex, involving fine-tuned signal communication between the symbiotic partners. Here we review our current understanding of the mechanisms used by the host and bacteria to choose their symbiotic partners, with a special focus on the role that the host immunity plays in controlling the specificity of the legume - rhizobial symbiosis.     

Multi‐symbiotic systems: functional implications of the coexistence of grass–endophyte and legume–rhizobia symbioses

The coexistence of symbionts with different functional roles in co‐occurring plants is highly probable in terrestrial ecosystems. Analyses of how plants and microbes interact above‐ and belowground in multi‐symbiotic systems are key to understand community structure and ecosystem functioning. We performed an outdoor experiment in mesocosms to investigate the consequences of the interaction of a provider belowground symbiont of legumes (nitrogen‐fixing bacteria) and a protector aerial fungal symbiont of grasses (Epichloё endophyte) on nitrogen dynamics and aboveground net primary productivity. Four plants of Trifolium repens (Trifolium, a perennial legume) either inoculated or not with Rhizobium leguminosarum, grew surrounded by 16 plants of Lolium multiflorum (Lolium, an annual grass), with either low or high levels of the endophyte Neotyphodium occultans. After five months, we quantified the number of nodules in Trifolium roots, shoot biomass of both plant species, and the contribution of atmospheric nitrogen fixation vs. soil nitrogen uptake to above ground nitrogen in each plant species. The endophyte increased grass biomass production (+ 16%), and nitrogen uptake from the soil – the main source for the grass. Further, it reduced the nodulation of neighbour Trifolium plants (?50%). Notably, due to a compensatory increase in nitrogen fixation per nodule, this reduced neither its atmospheric nitrogen fixation – the main source of nitrogen for the legume – nor its biomass production, both of which were doubled by rhizobial inoculation. In consequence, the total amount of nitrogen in aboveground biomass and aboveground productivity were greatest in mesocosms with both symbionts (i.e. high rhizobia + high endophyte). These results show that, in spite of the deleterious effect of the endophyte on the establishment of the rhizobia–legume symbiosis, the coexistence of these symbionts, leading to additive effects on nitrogen capture and aboveground productivity, can generate complementarity on the functioning of multi‐symbiotic systems.     

Laser‐ablation electrospray ionization mass spectrometry with ion mobility separation reveals metabolites in the symbiotic interactions of soybean roots and rhizobia

Technologies enabling in situ metabolic profiling of living plant systems are invaluable for understanding physiological processes and could be used for rapid phenotypic screening (e.g., to produce plants with superior biological nitrogen‐fixing ability). The symbiotic interaction between legumes and nitrogen‐fixing soil bacteria results in a specialized plant organ (i.e., root nodule) where the exchange of nutrients between host and endosymbiont occurs. Laser‐ablation electrospray ionization mass spectrometry (LAESI‐MS) is a method that can be performed under ambient conditions requiring minimal sample preparation. Here, we employed LAESI‐MS to explore the well characterized symbiosis between soybean (Glycine max L. Merr.) and its compatible symbiont, Bradyrhizobium japonicum. The utilization of ion mobility separation (IMS) improved the molecular coverage, selectivity, and identification of the detected biomolecules. Specifically, incorporation of IMS resulted in an increase of 153 differentially abundant spectral features in the nodule samples. The data presented demonstrate the advantages of using LAESI–IMS–MS for the rapid analysis of intact root nodules, uninfected root segments, and free‐living rhizobia. Untargeted pathway analysis revealed several metabolic processes within the nodule (e.g., zeatin, riboflavin, and purine synthesis). Compounds specific to the uninfected root and bacteria were also detected. Lastly, we performed depth profiling of intact nodules to reveal the location of metabolites to the cortex and inside the infected region, and lateral profiling of sectioned nodules confirmed these molecular distributions. Our results established the feasibility of LAESI–IMS–MS for the analysis and spatial mapping of plant tissues, with its specific demonstration to improve our understanding of the soybean‐rhizobial symbiosis.     

Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

The nitrogen‐fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix^– mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix^– mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix^– mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (LjAPN1), encodes a nepenthesin‐type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain‐specific Fix^– phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen‐fixing) symbiosis in a rhizobial strain‐dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.     

根瘤菌共生固氮能力的进化模式

根瘤菌-豆科植物共生固氮体系对农业的可持续性发展至关重要,也是研究原核与真核生物互利共生的模式体系之一。长期以来,根瘤菌共生固氮相关研究主要集中在结瘤因子与固氮酶合成及调控等少数关键基因,但仅获得这些关键基因却不能保证细菌获得结瘤固氮能力。随着比较和功能基因组学的快速发展和应用,越来越多的研究发现根瘤菌使用了很多系统发育分支特异的遗传机制与豆科植物建立有效的共生关系,进一步揭示了双方互利共生的复杂性。本综述总结了近年来比较基因组学、遗传学以及实验进化等方面的相关研究进展,在此基础上讨论根瘤菌共生固氮能力的进化模式。     

Recruitment of novel calcium-binding proteins for root nodule symbiosis in Medicago truncatula

Legume rhizobia symbiotic nitrogen (N2) fixation plays a critical role in sustainable nitrogen management in agriculture and in the Earth's nitrogen cycle. Signaling between rhizobia and legumes initiates development of a unique plant organ, the root nodule, where bacteria undergo endocytosis and become surrounded by a plant membrane to form a symbiosome. Between this membrane and the encased bacteria exists a matrix-filled space (the symbiosome space) that is thought to contain a mixture of plant- and bacteria-derived proteins. Maintenance of the symbiosis state requires continuous communication between the plant and bacterial partners. Here, we show in the model legume Medicago truncatula that a novel family of six calmodulin-like proteins (CaMLs), expressed specifically in root nodules, are localized within the symbiosome space. All six nodule-specific CaML genes are clustered in the M. truncatula genome, along with two other nodule-specific genes, nodulin-22 and nodulin-25. Sequence comparisons and phylogenetic analysis suggest that an unequal recombination event occurred between nodulin-25 and a nearby calmodulin, which gave rise to the first CaML, and the gene family evolved by tandem duplication and divergence. The data provide striking evidence for the recruitment of a ubiquitous Ca(2+)-binding gene for symbiotic purposes.     

Legume nodulation: The host controls the party

Global demand to increase food production and simultaneously reduce synthetic nitrogen fertilizer inputs in agriculture are underpinning the need to intensify the use of legume crops. The symbiotic relationship that legume plants establish with nitrogen‐fixing rhizobia bacteria is central to their advantage. This plant–microbe interaction results in newly developed root organs, called nodules, where the rhizobia convert atmospheric nitrogen gas into forms of nitrogen the plant can use. However, the process of developing and maintaining nodules is resource intensive; hence, the plant tightly controls the number of nodules forming. A variety of molecular mechanisms are used to regulate nodule numbers under both favourable and stressful growing conditions, enabling the plant to conserve resources and optimize development in response to a range of circumstances. Using genetic and genomic approaches, many components acting in the regulation of nodulation have now been identified. Discovering and functionally characterizing these components can provide genetic targets and polymorphic markers that aid in the selection of superior legume cultivars and rhizobia strains that benefit agricultural sustainability and food security. This review addresses recent findings in nodulation control, presents detailed models of the molecular mechanisms driving these processes, and identifies gaps in these processes that are not yet fully explained.     

Medicago-Sinorhizobium symbiotic specificity evolution and the geographic expansion of Medicago

The legume genus Medicago interacts with soil bacteria commonly referred to as rhizobia, in a nitrogen fixing symbiosis. We analysed the diversity of symbiotic association specificity among the two organisms, and its evolution in the plant genus. Nitrogen fixation tests and molecular phylogenetic reconstructions revealed that the genus Medicago includes more symbiotic specificity groups than previously suggested and that plant specificity is highly unstable and has repeatedly switched along the diversification of this genus. A phylogenetic analysis including geographical data shows that bacterial geographical diversity distribution has a strong influence on the geographic distribution of plant species and their ability to colonize new areas. Multiple other modifications of specificity occurred along the diversification of the genus, presumably due to selection for specialization to a single bacterial biovar. Codivergence between plants and bacteria may also have taken place.     

一氧化氮对豆科植物结瘤及固氮的影响机制

豆科植物-根瘤菌共生过程受双方基因复杂且精细的调控, 能够产生特异的根瘤结构并可将大气中的惰性氮气(N₂)转化为可被植物直接利用的氨态氮。结瘤与固氮受多种因素影响, 其中, 一氧化氮(NO)作为一种自由基反应性气体信号分子, 可参与调节植物的许多生长发育过程, 如植物的呼吸、光形态建成、种子萌发、组织和器官发育、衰老以及响应各种生物及非生物胁迫。在豆科植物中, NO不仅影响寄主与菌共生关系的建立, 还参与调控根瘤菌对氮气的固定并提高植株氮素营养利用效率。该文主要从豆科植物及共生菌内NO的产生、降解及其对结瘤、共生固氮的影响和对环境胁迫的响应, 阐述了NO调控豆科植物共生体系中根瘤形成和共生固氮过程的作用机制, 展望了NO信号分子在豆科植物共生固氮体系中的研究前景。     

No evidence for adaptation to local rhizobial mutualists in the legume Medicago lupulina

Local adaptation is a common but not ubiquitous feature of species interactions, and understanding the circumstances under which it evolves illuminates the factors that influence adaptive population divergence. Antagonistic species interactions dominate the local adaptation literature relative to mutualistic ones, preventing an overall assessment of adaptation within interspecific interactions. Here, we tested whether the legume Medicago lupulina is adapted to the locally abundant species of mutualistic nitrogen‐fixing rhizobial bacteria that vary in frequency across its eastern North American range. We reciprocally inoculated northern and southern M. lupulina genotypes with the northern (Ensifer medicae) or southern bacterium (E. meliloti) in a greenhouse experiment. Despite producing different numbers of root nodules (the structures in which the plants house the bacteria), neither northern nor southern plants produced more seeds, flowered earlier, or were more likely to flower when inoculated with their local rhizobia. We then used a pre‐existing dataset to perform a genome scan for loci that showed elevated differentiation between field‐collected plants that hosted different bacteria. None of the loci we identified belonged to the well‐characterized suite of legume–rhizobia symbiosis genes, suggesting that the rhizobia do not drive genetic divergence between M. lupulina populations. Our results demonstrate that symbiont local adaptation has not evolved in this mutualism despite large‐scale geographic variation in the identity of the interacting species.     

Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus

Nitrogen-fixing root nodules develop on legumes as a result of an interaction between host plants and soil bacteria collectively referred to as rhizobia. The organogenic process resulting in nodule development is triggered by the bacterial microsymbiont, but genetically controlled by the host plant genome. Using T-DNA insertion as a tool to identify novel plant genes that regulate nodule ontogeny, we have identified two putatively tagged symbiotic loci, Ljsym8 and Ljsym13, in the diploid legume Lotus japonicus. The sym8 mutants are arrested during infection by the bacteria early in the developmental process. The sym13 mutants are arrested in the final stages of infection, and ineffective nodules are formed. These two plant mutant lines were identified in progeny from 1112 primary transformants obtained after Agrobacterium tumefaciens T-DNA-mediated transformation of L. japonicus and subsequent screening for defects in the symbiosis with Mesorhizobium loti. Additional nontagged mutants arrested at different developmental stages were also identified and genetic complementation tests assigned all the mutations to 16 monogenic symbiotic loci segregating recessive mutant alleles. In the screen reported here independent symbiotic loci thus appeared with a frequency of ∼1.5%, suggesting that a relatively large set of genes is required for the symbiotic interaction. Received: 12 May 1998 / Accepted: 24 June 1998     

MALDI mass spectrometry‐assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula–Sinorhizobium meliloti symbiosis

Symbiotic associations between leguminous plants and nitrogen‐fixing rhizobia culminate in the formation of specialized organs called root nodules, in which the rhizobia fix atmospheric nitrogen and transfer it to the plant. Efficient biological nitrogen fixation depends on metabolites produced by and exchanged between both partners. The Medicago truncatula–Sinorhizobium meliloti association is an excellent model for dissecting this nitrogen‐fixing symbiosis because of the availability of genetic information for both symbiotic partners. Here, we employed a powerful imaging technique – matrix‐assisted laser desorption/ionization (MALDI)/mass spectrometric imaging (MSI) – to study metabolite distribution in roots and root nodules of M. truncatula during nitrogen fixation. The combination of an efficient, novel MALDI matrix [1,8–bis(dimethyl‐amino) naphthalene, DMAN] with a conventional matrix 2,5–dihydroxybenzoic acid (DHB) allowed detection of a large array of organic acids, amino acids, sugars, lipids, flavonoids and their conjugates with improved coverage. Ion density maps of representative metabolites are presented and correlated with the nitrogen fixation process. We demonstrate differences in metabolite distribution between roots and nodules, and also between fixing and non‐fixing nodules produced by plant and bacterial mutants. Our study highlights the benefits of using MSI for detecting differences in metabolite distributions in plant biology.     

Lifestyle alternatives for rhizobia: mutualism, parasitism, and forgoing symbiosis

Strains of rhizobia within a single species can have three different genetically determined strategies. Mutualistic rhizobia provide their legume hosts with nitrogen. Parasitic rhizobia infect legumes, but fix little or no nitrogen. Nonsymbiotic strains are unable to infect legumes at all. Why have rhizobium strains with one of these three strategies not displaced the others? A symbiotic (mutualistic or parasitic) rhizobium that succeeds in founding a nodule may produce many millions of descendants. The chances of success can be so low, however, that nonsymbiotic rhizobia can have greater reproductive success. Legume sanctions against nodules that fix little or no nitrogen favor more mutualistic strains, but parasitic strains that use plant resources only for their own reproduction may do well when they share nodules with mutualistic strains.     

Regulation of legume nodulation by acidic growth conditions

Legumes represent some of the most important crop species worldwide. They are able to form novel root organs known as nodules, within which biological nitrogen fixation is facilitated through a symbiotic interaction with soil-dwelling bacteria called rhizobia. This provides legumes with a distinct advantage over other plant species, as nitrogen is a key factor for growth and development. Nodule formation is tightly regulated by the plant and can be inhibited by a number of external factors, such as soil pH. This is of significant agricultural and economic importance as much of global legume crops are grown on low pH soils. Despite this, the precise mechanism by which low pH conditions inhibits nodule development remains poorly characterized.     

Nod genes and Nod signals and the evolution of the Rhizobium legume symbiosis.

The establishment of the nitrogen-fixing symbiosis between rhizobia and legumes requires an exchange of signals between the two partners. In response to flavonoids excreted by the host plant, rhizobia synthesize Nod factors (NFs) which elicit, at very low concentrations and in a specific manner, various symbiotic responses on the roots of the legume hosts. NFs from several rhizobial species have been characterized. They all are lipo-chitooligosaccharides, consisting of a backbone of generally four or five glucosamine residues N-acylated at the non-reducing end, and carrying various O-substituents. The N-acyl chain and the other substituents are important determinants of the rhizobial host specificity. A number of nodulation genes which specify the synthesis of NFs have been identified. All rhizobia, in spite of their diversity, possess conserved nodABC genes responsible for the synthesis of the N-acylated oligosaccharide core of NFs, which suggests that these genes are of a monophyletic origin. Other genes, the host specific nod genes, specify the substitutions of NFs. The central role of NFs and nod genes in the Rhizobium-legume symbiosis suggests that these factors could be used as molecular markers to study the evolution of this symbiosis. We have studied a number of NFs which are N-acylated by alpha,beta-unsaturated fatty acids. We found that the ability to synthesize such NFs does not correlate with taxonomic position of the rhizobia. However, all rhizobia that produce NFs such nodulate plants belonging to related tribes of legumes, the Trifolieae, Vicieae, and Galegeae, all of them being members of the so-called galegoid group. This suggests that the ability to recognize the NFs with alpha-beta-unsaturated fatty acids is limited to this group of legumes, and thus might have appeared only once in the course of legume evolution, in the galegoid phylum.     

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.     

Breeding for better symbiosis

The present review gives a critical assessment of the literature dealing with symbiosis between rhizobia and legumes and between AM fungi and most plants. Associative N₂ fixation (even though strictly speaking not a symbiotic relationship) does have some characteristics of symbiosis due to mutualistic dependence and usefulness of the relationship, and is therefore covered in this review. Nodulation in the rhizobia–legume symbiosis may be limited by an insufficient amount of the nod-gene inducers released from seed and/or roots. However, there is genotypic variation in the germplasm of legume species in all components of the signalling pathway, suggesting a prospect for improving nodulation by selecting and/or transforming legume genotypes for increased exudation of flavonoids and other signalling compounds. Deciphering chromosomal location as well as cloning nod, nif and other genes important in nodulation and N₂ fixation will allow manipulation of the presence and expression of these genes to enhance the symbiotic relationship. Increased efficacy of symbiotic N₂ fixation can be achieved by selecting not only the best host genotypes but by selecting the best combination of host genotype and nodule bacteria. As flavonoids exuded by legume seedlings may not only be nod-gene inducers, but also stimulants for hyphal growth of the AM fungi, selecting and/or transforming plants to increase exudation of these flavonoids may result in a double benefit for mycorrhizal legumes. Mutants unable to sustain mycorrhizal colonisation are instrumental in understanding the colonisation process, which may ultimately pay off in breeding for the more effective symbiosis. In conclusion, targeted efforts to breed genotypes for improved N₂ fixation and mycorrhizal symbiosis will bring benefits in increased yields of crops under a wide range of environmental conditions and will contribute toward sustainability of agricultural ecosystems in which soil-plant-microbe interactions will be better exploited.