Endophytic colonisation of Bacillus subtilis in the roots of Robinia pseudoacacia L

The endophytic colonisation of Bacillus subtilis strain GXJM08, isolated from roots of Podocarpus imbricatus B1. Enum. P1. Jav., in roots of the leguminous plant Robinia pseudoacacia L. was investigated. Ultrastructure observations showed that B. subtilis caused morphological changes in the root hair and colonised the plant through infected root hairs. The structure of the infection thread was similar to that of rhizobia, but the structure of infected cells was different. B. subtilis is also different from rhizobia and plant pathogens in terms of the formation of a peribacteroid membrane and the mode of penetration through the host cell wall. Our results provide a basis for studying development of the mutualistic symbiotic relationship between B. subtilis and plants, and a basis for studying the mechanism of the B. subtilis–plant interaction.     

Phosphate Limitation Alters <Emphasis Type="Italic">Medicago–Sinorhizobium</Emphasis> Signaling: Flavonoid Synthesis and AHL Production

The legume plant Medicago truncatula Gaertn. can establish a symbiotic interaction with Sinorhizobium meliloti. One of the most limiting factors for symbiosis is phosphate (P) deficiency. Therefore, legumes and their symbiotic partners, rhizobia, have developed mechanisms to adapt to P restriction. In the non-symbiotic state, plants would up-regulate flavonoid biosynthesis via increasing the expression of chalcone synthase (chs), catalyzing the first step of flavonoid synthesis. Simultaneously, bacterial quorum sensing (QS) pathway can regulate the expression of certain genes involved in symbiotic functions of bacteria in response to P availability as well as bacterial population. Since both flavonoids and QS signaling molecules (N-acyl homoserine lactones, AHL) play important roles in the rhizobia-legume symbiosis, we evaluated these processes in the symbiotic state under different P concentrations and bacterial populations. In this study, by using real-time PCR and HPLC, we showed the expression of pt1 (phosphate transporter 1) and chs as well as luteolin production increased, in a time dependent manner, in plants following P limitation. Nod gene inducing flavonoids can up-regulate the bacterial QS pathway which results in an increase in AHL production, possibly to enhance symbiotic behaviors of rhizobia. It has been estimated that there is a feedback loop from bacterial AHL to flavonoid production pathway in legume plants.     

Complete sequence of a Rhizobium plasmid carrying genes necessary for symbiotic association with the plant host

The soil bacteria rhizobia have the capacity to establish nitrogen-fixing symbiosis with their leguminous host plants. In most Rhizobium species the genes for nodule development and nitrogen fixation have been localized on large indigenous plasmids that are transmissible, allowing lateral transfer of symbiotic functions. A recent paper reports on the complete sequencing of the symbiotic plasmid pNGR234a from Rhizobium species NGR234⁽¹⁾, revealing not only putative new symbiotic genes but also possible mechanisms for evolution and lateral dispersal of symbiotic nitrogen-fixing abilities among rhizobia.     

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.     

Early interactions between legumes and rhizobia: disclosing complexity in a molecular dialogue

The exchange of chemical signals between soil bacteria (rhizobia) and legumes has been termed a molecular dialogue. As initially conceived in the early 1990s, it involved two main groups of molecules: nod gene-inducing flavonoids from plants and the mitogenic lipochito-oligosaccharide Nod factors of rhizobia. This review considers how subsequent research revealed the existence of a more complex set of interactions, featuring expanded roles for the original participants and contributions from additional plant and bacterial metabolites. Rhizobia respond to chemoattractants and growth-enhancing compounds in root exudates, and several plant nonflavonoids possess nod gene-inducing properties. Expression of non-nod genes is induced by flavonoids; these include encoders of a type I secreted protein and the entire type III, and possibly also type IV, secretion systems. Many other genes and proteins in rhizobia are flavonoid-inducible but their functions are largely unknown. Rhizobia produce far more Nod factor variants than was previously envisaged and their structures can be influenced by the pH of the environment. Other symbiotically active compounds or systems of rhizobia, some of them universally present, are: the surface polysaccharides, quorum-sensing N-acyl homoserine lactones, plant growth-promoting lumichrome and two-component regulatory systems.     

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.     

Perception and Signal Transduction of Rhizobial NOD Factors

Referee: Dr. Gary Stacey, Director, Center for Legume Research, Department of Microbiology, M409 Walters Life Science Bldg., University of Tennessee, Knoxville, TN 37966-0845 Soil bacteria belonging to genera Rhizobium, Bradyrhizobium, Allorhizobium, Azorhizobium, Mesorhizobium, and Sinorhizobium are able to induce nodule formation on the roots of leguminous plants. In the differentiated root nodules bacteria fix as bacteroids atmospheric nitrogen and deliver it to the host plant. The interaction between bacteria and host plant starts with a complex signal exchange. After induction by plant flavonoids, rhizobia synthesize and secrete lipo-chitooligosaccharides (LCOs), known as Nod factors, which induce morphological changes and expression of early nodulin genes in the roots of host plants. Specific recognition of Nod factors by host plants and early stages of signal transduction are discussed.     

Effect of Inoculation with Various Rhizobia Strains on the Fatty Acid Composition of Peribacteroid and Bacteroid Membranes of the Nodule Symbiosomes

The fatty acid (FA) composition of bacteroid and peribacteroid membranes was studied in the symbiotic pairs differing in their nitrogen-fixing efficiency; the results are compared with the FA composition of plasmalemma and free-living rhizobia. The experiments involved lupine plants inoculated with strains of Bradyrhizobium lupini359a (Nod⁺Fix⁺) and 400 (Nod⁺Fix L) manifesting high and low nitrogen-fixing efficiency, respectively, and broad bean plants inoculated with strains of Rhizobium leguminosarum97 (Nod⁺Fix⁺) and 87 (Nod⁺Fix L) of high and low nitrogen-fixing efficiency, respectively. We showed that the rhizobia of the strains 359a and 97 were able to form nodules with peribacteroid membranes containing FA mainly or exclusively of plant origin. These strains were able to develop effective symbiotic pairs with legume plants. The use of strains 400 and 87 resulted in the formation of nodules with peribacteroid membranes containing typical bacterial (branched-chain) FAs; these strains were characterized by an ineffective symbiosis.     

Roots and Their Symbiotic Microbes: Strategies to Obtain Nitrogen and Phosphorus in a Nutrient-Limiting Environment

The association between Rhizobium and legumes and that between arbuscular mycorrhizal (AM) fungi and most land plants display a remarkable degree of similarity. Both events involve the recognition of, entrance into, and coexistence within the plant root, with the development of a specialized interface that always separates the two partners and at which nutrient exchange occurs. Molecules produced by rhizobia during the early stages of the symbiosis are related to fungal chitin, and the plant responds to both microbes with an increase in the production of flavonoids, which may assist in recognition and development of the symbioses. Many of the same plant genes are up-regulated in the two symbiotic pathways, and notably plants that are Nod^– are often defective in the AM association as well. However, there are a number of differences between the associations, and these are important for understanding the relationship between the two symbioses. The Rhizobium and AM symbioses will be compared and the question of whether the nitrogen-fixing association evolved from the much more ancient AM symbiosis will be discussed.     

Genetic diversity and symbiotic compatibility among rhizobial strains and Desmodium incanum and Lotus spp. plants

This work aimed to evaluate the symbiotic compatibility and nodulation efficiency of rhizobia isolated from Desmodium incanum, Lotus corniculatus, L. subbiflorus, L. uliginosus and L. glaber plants by cross-inoculation. Twelve reference strains and 21 native isolates of rhizobia were genetically analyzed by the BOX-PCR technique, which showed a high genetic diversity among the rhizobia studied. The isolates were also characterized based on their production of indolic compounds and siderophores, as well as on their tolerance to salinity. Fifteen of the 33 rhizobia analyzed were able to produce indolic compounds, whereas 13 produced siderophores. All the tested rhizobia were sensitive to high salinity, although some were able to grow in solutions of up to 2% NaCl. Most of the native rhizobia isolated from L. uliginosus were able to induce nodulation in all plant species studied. In a greenhouse experiment using both D. incanum and L. corniculatus plants, the rhizobia isolate UFRGS Lu2 promoted the greatest plant growth. The results demonstrate that there are native rhizobia in the soils of southern Brazil that have low host specificity and are able to induce nodulation and form active nodules in several plant species.     

Diversity and Biogeography of Rhizobia Isolated from Root Nodules of <Emphasis Type="Italic">Glycine max</Emphasis> Grown in Hebei Province,China

A total of 215 rhizobial strains were isolated and analyzed with 16S rRNA gene, 16S–23S intergenic spacer, housekeeping genes atpD, recA, and glnII, and symbiotic genes nifH and nodC to understand the genetic diversity of soybean rhizobia in Hebei province, China. All the strains except one were symbiotic bacteria classified into nine genospecies in the genera of Bradyrhizobium and Sinorhizobium. Surveys on the distribution of these rhizobia in different regions showed that Bradyrhizobium japonicum and Bradyrhizobium elkanii strains were found only in neutral to slightly alkaline soils whereas Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense-related strains and strains of five Sinorhizobium genospecies were found in alkaline–saline soils. Correspondence and canonical correspondence analyses on the relationship of rhizobial distribution and their soil characteristics reveal that high soil pH, electrical conductivity, and potassium content favor distribution of the B. yuanmingense and the five Sinorhizobium species but inhibit B. japonicum and B. elkanii. High contents of available phosphorus and organic matters benefit Sinorhizobium fredii and B. liaoningense-related strains and inhibit the others groups mentioned above. The symbiotic gene (nifH and nodC) lineages among B. elkanii, B. japonicum, B. yuanmingense, and Sinorhizobium spp. were observed in the strains, signifying that vertical gene transfer was the main mechanism to maintain these genes in the soybean rhizobia. However, lateral transfer of symbiotic genes commonly in Sinorhizobium spp. and rarely in Bradyrhizobium spp. was also detected. These results showed the genetic diversity, the biogeography, and the soil determinant factors of soybean rhizobia in Hebei province of China.     

The Rhizobium sp. strain NGR234 systemically suppresses arbuscular mycorrhizal root colonization in a split‐root system of barley (Hordeum vulgare)

Nitrogen‐fixing bacteria (rhizobia) form a nodule symbiosis with legumes, but also induce certain effects on non‐host plants. Here, we used a split‐root system of barley to examine whether inoculation with Rhizobium sp. strain NGR234 on one side of a split‐root system systemically affects arbuscular mycorrhizal (AM) root colonization on the other side. Mutant strains of NGR234 deficient in Nod factor production (strain NGRΔnodABC), perception of flavonoids (strain NGRΔnodD1) and secretion of type 3 effector proteins (strain NGRΩrhcN) were included in this study. Inoculation resulted in a systemic reduction of AM root colonization with all tested strains. However, the suppressive effect of strain NGRΩrhcN was less pronounced. Moreover, levels of salicylic acid, an endogenous molecule related to plant defense, were increased in roots challenged with rhizobia. These data indicate that barley roots perceived NGR234 and that a systemic regulatory mechanism of AM root colonization was activated. The suppressive effect appears to be Nod factor independent, but enhanced by type 3 effector proteins of NGR234.     

Cell cycle regulation in the course of nodule organogenesis in Medicago

The molecular mechanisms of de novo meristem formation, cell differentiation and the integration of the cell cycle machinery into appropriate stages of the developmental programmes are still largely unknown in plants. Legume root nodules, which house nitrogen-fixing rhizobia, are unique plant organs and their development may serve as a model for organogenetic processes in plants. Nodules form and are essential for the plant only under limitation of combined nitrogen in the soil. Moreover, their development is triggered by external mitogenic signals produced by their symbiotic partners, the rhizobia. These signals, the lipochitooligosaccharide Nod factors, act as host-specific morphogens and induce the re-entry of root cortical cells into mitotic cycles. Maintenance of cell division activity leads to the formation of a persistent nodule meristem from which cells exit continuously and enter the nodule differentiation programme, involving multiple cycles of endoreduplication and enlargement of nuclear and cell volumes. While the small diploid 2C cells remain uninfected, the large polyploid cells can be invaded and, after completing the differentiation programme, host the nitrogen-fixing bacteroids. This review summarizes the present knowledge on cell cycle reactivation and meristem formation in response to Nod factors and reports on a novel plant cell cycle regulator that can switch mitotic cycles to differentiation programmes.     

Insight into the evolutionary history of symbiotic genes of Robinia pseudoacacia rhizobia deriving from Poland and Japan

The phylogeny of symbiotic genes of Robinia pseudoacacia (black locust) rhizobia derived from Poland and Japan was studied by comparative sequence analysis of nodA, nodC, nodH, and nifH loci. In phylogenetic trees, black locust symbionts formed a branch of their own suggesting that the spread and maintenance of symbiotic genes within Robinia pseudoacacia rhizobia occurred through vertical transmission. There was 99–100% sequence similarity for nodA genes of Robinia pseudoacacia nodulators, 97–98% for nodC, and 97–100% for nodH and nifH loci. A considerable sequence conservation of sym genes shows that the symbiotic apparatus of Robinia pseudoacacia rhizobia might have evolved under strong host plant constraints. In the nodA and nodC gene phylograms, Robinia pseudoacacia rhizobia grouped with Phaseolus sp. symbionts, although they were not closely related to our isolates based on 16S rRNA genes, and with Mesorhizobium amorphae. nifH gene phylogeny of our isolates followed the evolutionary history of 16S rDNA and Robinia pseudoacacia rhizobia grouped with Mesorhizobium genus species. Nodulation assays revealed that Robinia pseudoacacia rhizobia effectively nodulated their native host and also Amorpha fruticosa and Amorpha californica resulting in a significant enhancement of plant growth. The black locust root nodules are shown to be of indeterminate type.     

Control of the expression of bacterial genes involved in symbiotic nitrogen fixation

Several genera of N₂-fixing bacteria establish symbiotic associations with plants. Among these, the genus Rhizobium has the most significant contribution, in terms of yield, in many important crop plants. The establishment of the Rhizobium-legume symbiosis is a very complex process involving many genes which need to be co-ordinately regulated. In the first instance, plant signal molecules, known to be flavonoids, trigger the expression of host-specific genes in the bacterial partner through the action of the regulatory NodD protein. In response to these signals, Rhizobium bacteria synthesize lipo-oligosaccharide molecules which in turn cause cell differentiation and nodule development. Once the nodule has formed, Rhizobium cells differentiate into bacteroids and another set of genes is activated. These genes, designated nif and fix, are responsible for N₂ fixation. In this system, several regulatory proteins are involved in a complex manner, the most important being NifA and a two component (FixK and FixL) regulatory system. Our knowledge about the establishment of these symbioses has advanced recently, although there are many questions yet to be solved.     

The Effect of Bacillus subtilis on in vitro Growth and Pathogenicity of Agrobacterium tumefaciens

Five isolates of Bacillus subtilis isolated from the soil, were found to be antagonistic to 6 isolates of Agrobacterium tumefaciens in vitro. Inoculation of B. subtilis in wounded castor bean plants 30 min before or simultaneous inoculation with A. tumefaciens resulted in excellent control of the crown gall symptoms on the host within 50 days of inoculation. Application of B. subtilis 30 min after inoculation with A. tumefaciens did not result in appreciable disease reduction. Treatment of the tested plants by B. subtilis did not induce any phytotoxic injury or growth retarding side effects. It appears that B. subtilis could potentially be incorporated for crown gall control. However, further tests are needed to test this biological control agent with other plant species especially fruits, nuts and vine nursery stock.     

Simulation of bacteria-plant coevolution in the mutualistic symbiosis

We present the mathematical model for coevolution of root nodule bacteria (rhizobia) and leguminous plants which is based on the partners’ positive feedbacks resulted from their metabolic integration. The model parameters were introduced which determine: (1) coordinated changes in plant and bacterial population structures; (2) increase of fitness (reproductive potentials) in both partners as dependent on the symbiotic efficiency determined by proportion of N₂-fixing rhizobia strain in root nodules. Computer experiments demonstrated that microevolution of the simulated system may follow either oscillatory or quasi-monotonous regime as dependent of frequency-dependent selection (FDS) in plant population. Negative FDS occurring in the bacterial population during competition for nodulation in combination with the positive partners’ feedback may lead to anchoring the bacterial mutations which lead either to acquisition of mutualistic traits or to changes in specificity of their expression. Anchoring of the mutualistic strains occurs most successfully in the quasi-monotonous regime and results in the improvement of genetic stability in symbiotic system.