Developmental regulation of a Rhizobium cell surface antigen during growth of pea root nodules.

A monoclonal antibody, AFRC MAC 203, was used to examine the expression of a nodule-induced cell surface antigen associated with lipopolysaccharide in Rhizobium leguminosarum bv. viciae 3841. Silver-enhanced immunogold-labeled tissue sections revealed that, in very young tissues of pea root nodules, the nodule-induced form of lipopolysaccharide antigen was not expressed either by rhizobia in the infection thread or by bacteria recently released into the plant cell cytoplasm. In the more mature regions of the nodule, the antigen was expressed by membrane-enclosed bacteroids, including immature forms that had not yet expressed the enzyme nitrogenase and were not yet Y shaped. Immunogold labeling of thin sections revealed that the MAC 203 antigen, but not the nitrogenase, was also expressed by bacteria in infection threads situated in and between bacteroid-containing plant cells in mature nodule tissue.     

Antigenic changes in lipopolysaccharide I of Rhizobium leguminosarum bv. viciae in root nodules of Vicia sativa subsp. nigra occur during release from infection threads.

Three different monoclonal antibodies raised against the O antigen-containing lipopolysaccharide (LPS I) of free-living cells were used in an immunocytochemical study to follow the fate of LPS I on the outer membrane of Rhizobium leguminosarum bv. viciae 248 during the nodulation of Vicia sativa subsp. nigra. After immunogold labeling, the LPS I epitopes were detected on the outer membrane of bacteria present in infection threads throughout the nodule. Epitopes were not detectable on bacteria released from the infection thread. The data show that the LPS I epitopes present on rhizobia in infection droplets disappear shortly before or during endocytosis of the bacteria into the host plant cell cytoplasm. The abruptness of the change suggests an active degradation or modification of LPS I epitopes rather than only a repression of their synthesis.     

Molecular dissection of structure and function in the lipopolysaccharide of Rhizobium leguminosarum strain 3841 using monoclonal antibodies and genetic analysis

Following treatment with nitrosoguanidine, mutant derivatives of Rhizobium leguminosarum strain 3841 were isolated which failed to react with AFRC MAC 203. This monoclonal antibody normally recognizes a strain-specific lipopolysaccharide epitope which is developmentally regulated during legume nodule differentiation. Structural modification of lipopolysaccharide (LPS) was analysed by examining reactivity with a range of monoclonal antibodies with different epitope specificities, and also by analysis of LPS mobility changes after electrophoresis on polyacrylamide gels. One class of these LPS-defective mutants induced normal nitrogen-fixing (Fix+) nodules on peas (Pisum sativum), while another two classes of Fix- mutants were also identified, suggesting that a component of the LPS antigen that is part of the MAC 203 epitope is essential for normal nodule development leading to symbiotic nitrogen fixation. When grown under low-oxygen or low-pH culture conditions, one class of Fix- mutants completely lacked LPS-1 (the species that carries O antigen) and a second class showed a modified and truncated form of LPS-1. Mutants with defective LPS structure were also obtained after Tn5 mutagenesis of R. leguminosarum 3841 and all nine Fix- mutants were also found to lack the MAC 203 epitope. Three of these transposon-induced mutants synthesized a truncated form of LPS-1 that was structurally similar to that of the class of the NTG-induced mutants described above. These transposon-induced mutations, and the nitrosoguanidine-induced Fix- mutations, were closely linked and could be suppressed by the same cloned fragment of chromosomal DNA. The data presented here suggest that a precondition for normal nodule development of R. leguminosarum 3841 within pea nodules is the ability to synthesize relatively long-chain LPS-1 macromolecules under the physiological conditions encountered within the nodule. All mutants that lacked the ability to elongate LPS-1 macromolecules also failed to express the MAC 203 epitope.     

Visualization of symbiotic tissue in intact root nodules of Vicia tetrasperma using GFP-marked Rhizobium leguminosarum bv. viciae

In rhizobial symbiosis with legume plant hosts, the symbiotic tissue in the root nodules of indeterminate type is localized to the basal part of the nodule where the symbiotic zones contain infected cells (IC) interspersed with uninfected cells (UC) that are devoid of rhizobia. Although IC are easily distinguished in nodule sections using standard histochemical techniques, their observation in intact nodules is hampered by nodule tissue characteristics. Tagging of Rhizobium leguminosarum bv. viciae strain 128C30 with a constitutively expressed gene for green fluorescent protein (nonshifted mutant form cycle3) in combination with the advantages of the tiny nodules formed by Vicia tetrasperma (L.) SCHREB: . allowed for vital observation of symbiotic tissue using fluorescence microscopy. Separation of a red-shifted background channel and digital image stacking along z-axis enabled us to construct a nodule image in a classical fluorescence microscopy of nodules exceeding 1 mm in diameter. In parallel, visualization of nodule bacteria inside the symbiotic tissue by confocal microscopy at the excitation wavelength 488 nm clearly distinguished IC/UC pattern in the nodule virtual sections and revealed red-shifted fluorescence of nonrhizobial origin. This signal was located on the periphery of IC and increased with their degradation, thus suggesting accumulation of secondary metabolites, presumably flavonoids. The simultaneous detection of bacteria and secondary metabolites can be used for monitoring changes to intact nodule physiology in the model legumes. The advantage of V. tetrasperma as a suggested laboratory model for pea cross-inoculation group has been demonstrated.     

Lipid A and O-chain modifications cause Rhizobium lipopolysaccharides to become hydrophobic during bacteroid development

Modifications to the lipopolysaccharide (LPS) structure caused by three different growth conditions were investigated in the pea-nodulating strain Rhizobium leguminosarum 3841. The LPSs extracted by hot phenol-water from cultured cells fractionated into hydrophilic water and/or hydrophobic phenol phases. Most of the LPSs from cells grown under standard conditions extracted into the water phase, but a greater proportion of LPSs were extracted into the phenol phase from cells grown under acidic or reduced-oxygen conditions, or when isolated from root nodules as bacteroids. Compared with the water-extracted LPSs, the phenol-extracted LPSs contained greater degrees of glycosyl methylation and O-acetylation, increased levels of xylose, glucose and mannose and increased amounts of long-chain fatty acids attached to the lipid A moiety. The water- and phenol-phase LPSs also differed in their reactivity with monoclonal antibodies and in their polyacrylamide gel electrophoretic banding patterns. Phenol-extracted LPSs from rhizobia grown under reduced-oxygen conditions closely resembled the bulk of LPSs isolated from pea nodule bacteria (i.e. mainly bacteroids) in their chemical properties, reactivities with monoclonal antibodies and extraction behaviour. This finding suggests that, during symbiotic bacteroid development, reduced oxygen tension induces structural modifications in LPSs that cause a switch from predominantly hydrophilic to predominantly hydrophobic molecular forms. Increased hydrophobicity of LPSs was also positively correlated with an increase in the surface hydrophobicity of whole cells, as shown by the high degree of adhesion to hydrocarbons of bacterial cells isolated from nodules or from cultures grown under low-oxygen conditions. The implications of these LPS modifications are discussed for rhizobial survival and function in different soil and in planta habitats.     

Effects of Boron on Rhizobium-Legume Cell-Surface Interactions and Nodule Development

Boron (B) is an essential micronutrient for the development of nitrogen-fixing root nodules in pea (Pisum sativum). By using monoclonal antibodies that recognize specific glycoconjugate components implicated in legume root-nodule development, we investigated the effects of low B on the formation of infection threads and the colonization of pea nodules by Rhizobium leguminosarum bv viciae. In B-deficient nodules the proportion of infected host cells was much lower than in nodules from plants supplied with normal quantities of B. Moreover, the host cells often developed enlarged and abnormally shaped infection threads that frequently burst, releasing bacteria into damaged host cells. There was also an over-production of plant matrix material in which the rhizobial cells were embedded during their progression through the infection thread. Furthermore, in a series of in vitro binding studies, we demonstrated that the presence of B can change the affinity with which the bacterial cell surface interacts with the peribacteroid membrane glycocalyx relative to its interaction with intercellular plant matrix glycoprotein. From these observations we suggest that B plays an important role in mediating cell-surface interactions that lead to endocytosis of rhizobia by host cells and hence to the correct establishment of the symbiosis between pea and Rhizobium.     

Identification of the rhizobium strains in pea root nodules using genetic markers.

Pea plants were inoculated jointly with pairs of genetically marked strains of Rhizobium leguminosarum. Out of 297 modules examined 56 contained both inoculant strains. The ratios of the strains in the inoculum did not affect the frequencies of mixed nodules. Generally one of the strains consistently occupied the majority of the nodules and ithe mixed nodules comprised the majority of bacteria. Transfer of the P-group R factor, RP4, between certain strains of Rhizobium within mixed nodules was detected. In some cases the non-parental progeny comprised 10% of the rhizobia isolated from such nodules.     

Identification of Rhizobium leguminosarum and Rhizobium sp. (Cicer) strains using a custom Fatty Acid Methyl Ester (FAME) profile library

The potential of using fatty acid methyl ester (FAME) profiles of Rhizobium leguminosarum bv. viceae , phaseoli and trifolii , and Rhizobium sp. ( Cicer ) strains, for the identification of unknown isolates was assessed. This was achieved by developing a Rhizobium FAME library using 16 different Rhizobium strains of Rh. leguminosarum bv. viceae ( n  ₌ 5), Rh. leguminosarum bv. phaseoli ( n  ₌ 5), Rh. leguminosarum bv. trifolii ( n  ₌ 1) and Rhizobium sp. ( Cicer ) ( n  ₌ 5). Although there were considerable differences between Rh. leguminosarum biovars and strains and Rhizobium sp. ( Cicer ) strains, the variation within a particular biovar of Rh. leguminosarum was not high. Nevertheless, the feature FAME profiles of the various groups in the library allowed 75 putative rhizobia obtained from surface-sterilized nodules of field-grown lentil and pea plants to be identified.     

Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P1 from Lupin Root Nodules

Six hybridoma clones were obtained that secreted monoclonal antibodies against the aspartate aminotransferase-P1 (AAT-P1) isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. This enzyme is found constitutively in the plant cytosol fraction. The monoclonal antibodies produced were all of the immunoglobulin G1 class, recognized two distinct epitopes on the protein, and represented the major paratopes found in the immunoglobulin fraction of sera taken from mice and rabbits immunized with the pure AAT-P1 protein. One of these epitopes was unique to lupin nodule AAT-P1. The other epitope was shown to be present on enzyme from lupin bean, white clover and tobacco leaves, lupin roots and nodules, and potato tubers. Both epitopes were recognized by the appropriate monoclonal antibodies in both their native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium lupini NZP2257, Escherichia coli extracts, or with the inducible aspartate aminotransferase-P2 (AAT-P2) isoform also found in root nodules. A sandwich enzyme-linked immunosorbent assay utilizing two monoclonal antibodies recognizing the two distinct epitopes was developed and was capable of quantitating AAT-P1 in plant extracts. The limit of detection of AAT-P1 was less than 15 pg/mL and AAT-P1 protein could be quantified in the range 80 to 1000 pg/mL. Using this assay, AAT-P1 protein was shown to remain relatively constant during nodule development. Use of an AAT-P2-specific monoclonal antibody that inhibits the enzyme activity of this isoform enabled the direct determination of AAT-P1 enzyme activity in nodule extracts. Using these assays, specific activities of the individual isoforms were calculated; that of the AAT-P1 isoform was shown to be 7.5-fold higher than that of the AAT-P2 isoform.     

The effect of inoculation with Rhizobium leguminosarum on the contents of cytoplasmic protein and free amino acids in the roots of pea seedlings

The changes in the contents of protein and free amino acids in pea plants inoculated with Rhizobium leguminosarum were studied taking into account the susceptibility of roots to root nodule bacteria. The content of cytoplasmic protein during infection increased in the actively growing root region (0-5 mm) and decreased in the root regions susceptible to rhizobia (5-20 mm from the root tip). The quantitative composition of free amino acids changed essentially upon inoculation of pea seedlings with R. leguminosarum.     

Rhizobium leguminosarum bv. viciae genotypes interact with pea plants in developmental responses of nodules, roots and shoots

The variability of the developmental responses of two contrasting cultivars of pea (Pisum sativum) was studied in relation to the genetic diversity of their nitrogen-fixing symbiont Rhizobium leguminosarum bv. viciae. A sample of 42 strains of pea rhizobia was chosen to represent 17 genotypes predominating in indigenous rhizobial populations, the genotypes being defined by the combination of haplotypes characterized with rDNA intergenic spacer and nodD gene regions as markers. We found contrasting effects of the bacterial genotype, especially the nod gene type, on the development of nodules, roots and shoots. A bacterial nod gene type was identified that induced very large, branched nodules, smaller nodule numbers, high nodule biomass, but reduced root and aerial part development. The plants associated with this genotype accumulated less N in shoots, but N concentration in leaves was not affected. The results suggest that the plant could not control nodule development sustaining the energy demand for nodule functioning and its optimal growth. The molecular and physiological mechanisms that may be involved are discussed.     

Nod factors stimulate seed germination and promote growth and nodulation of pea and vetch under competitive conditions

Nod factors are lipochitooligosaccharide (LCO) produced by soil bacteria commonly known as rhizobia acting as signals for the legume plants to initiate symbiosis. Nod factors trigger early symbiotic responses in plant roots and initiate the development of specialized plant organs called nodules, where biological nitrogen fixation takes place. Here, the effect of specific LCO originating from flavonoid induced Rhizobium leguminosarum bv. viciae GR09 culture was studied on germination, plant growth and nodulation of pea and vetch. A crude preparation of GR09 LCO significantly enhanced symbiotic performance of pea and vetch grown under laboratory conditions and in the soil. Moreover, the effect of GR09 LCOs seed treatments on the genetic diversity of rhizobia recovered from vetch and pea nodules was presented.     

Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P(2) from Lupin Root Nodules

Twenty-one monoclonal antibodies were raised against the aspartate aminotransferase-P₂ isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. Induction of this isoenzyme is positively correlated with the onset of N₂ fixation in effective root nodules and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. The monoclonal antibodies produced were all of the IgG class, recognized five different epitopes on the protein, and represented greater than 90% of the available epitopes. These epitopes were not unique to lupin nodule aspartate aminotransferase-P₂ but were shown to be present on the enzyme from tobacco leaves and potato. Four of the epitopes were conformational with a fifth epitope recognized by the appropriate monoclonals in both its native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium Iupini NZP2257 extracts. Antibodies against two epitopes showed some cross-reaction with the constitutive aspartate aminotransferase-P₁ isoenzyme also found in lupin root nodules. However, affinity of these monoclonals for AAT-P₁ was three orders of magnitude lower than for AAT-P₂. Monoclonals against the other epitopes appeared to be specific for aspartate aminotransferase-P₂.     

Strain Identification in Rhizobium Using Intrinsic Antibiotic Resistance

The variation in intrinsic resistance to low levels of eight antibiotics was used as an identifying characteristic for 26 Rhizobium leguminosarum strains. The pattern of antibiotic resistance of each strain was a stable property by which rhizobia isolated from root nodules of inoculated Pisum sativum could be recognized. The antibiotic tests for strain identification with R. leguminosarum were applied to R. phaseoli . It was necessary to include reference cultures in tests with this species, as the tests most suitable for the R. leguminosarum strains showed some variability with R. phaseoli .     

Mixed Inoculations with Effective and Ineffective Strains of Rhizobium leguminosarum

An ineffective Rhizobium leguminosarum strain capable of forming green nodules of similar size and number as normally effective strains was tested for its ability to compete with an effective strain in nodule formation on the pea. The ineffective strain was found to be more competitive and influenced the pattern of nodulation by the effective strain on the same root system. Nodules containing both strains were pink and able to reduce acetylene.     

The influence of lipopolysaccharides and glucans from two Rhizobium leguminosarum bv. viciae strains on the formation and efficiency of their symbioses with pea plants

The influence of lipopolysaccharides (LPS), glucans, and their unseparated complexes on nodulation activity of rhizobia and efficiency of their symbioses with pea plants was studied in vegetation experiments. Two Rhizobium leguminosarum bv. viciae strains which differed in their symbiotic properties were used: strain 31 (fix+, efficient, moderately virulent, moderately competitive), and strain 248b (fix-, inefficient, highly virulent, highly competitive). Preparations of LPS-glucan complex and the respective LPS from the highly virulent strain 248b increased the nodulation activity of both strains by 10-26%. Analogous preparations from a less virulent strain 31 did not have this ability. Unseparated LPS-glucan complexes from these strains increased the productivity of plants infected with the efficient strain by 18-23% but did not change it in plants inoculated with the other, inefficient strain. No significant influence of LPS preparations on the symbiosis productivity was observed. Glucans from both strains enhanced the nodulation ability of the highly virulent strain by 36-56%. In addition, treatment of pea plants with glucan from strain 248b increased nitrogen fixation by root nodules by 27% in plants inoculated with strain 31. In general, the formation and efficiency of the symbiosis of R. leguminosarum bv. viciae with pea plants was more influenced by preparations from strain 248b, highly virulent but deficient in nitrogen fixation, than by preparations from the nitrogen fixation-proficient but less virulent strain 31.     

The development and structure of root nodules on bambara groundnut [Voandzeia (Vigna)subterranea]

The infection of Vigna subterranea (formerly Voandzeia subterranea) by Bradyrhizobium strain MAO 113 (isolated from V. subterranea) was examined by light and transmission electron microscopy. Bacteria accumulated on the epidermis close to root hairs, and subsequently entered the latter via infection threads. Most of the steps involved in nodule formation were generally characteristic of determinate nodules, such as those which form on the closely related V. radiata. For example, nodule meristems were induced beneath the root epidermis adjacent to infected root hairs, but prior to infection of the meristem by rhizobia. Moreover, after the infection of some of the meristematic cells by the infection threads, and the release of the rhizobia into membrane-bound vesicles, the infection process ceased and dissemination of the rhizobia was by division of already-infected host cells. However, there were some aspects of this process in V. subterranea which have been more commonly described in indeterminate nodules. These include long infection threads entering a number of cells within the meristems simultaneously and a matrix within infection threads which was strongly labelled with immunogold monoclonal antibodies, MAC236 and MAC265, which recognize epitopes on an intercellular glycoprotein. The MAC236 and MAC265 antibodies also recognized material in the unwalled infection droplets surrounding bacteria which were newly-released from the infection threads. The amount of labelling shown was more characteristic of the long infection threads seen in indeterminate nodules such as pea (Pisum sativum) and Neptunia plena. The structure of mature V. subterranea nodules was similar to that described for other determinate nodules such as Glycine max, Vigna unguiculata and V.radiata, i.e. they were spherical and the infected zone consisted of both infected and uninfected cells. Surrounding the infected tissue was an inner cortex of uninfected cell layers containing the putative components of an oxygen diffusion barrier (including glycoprotein-occluded intercellular spaces), and an outer cortex with cells containing calcium oxalate crystals.     

The development and structure of root nodules on bambara groundnut [Voandzeia (Vigna)subterranea]

The infection of Vigna subterranea (formerly Voandzeia subterranea) by Bradyrhizobium strain MAO 113 (isolated from V. subterranea) was examined by light and transmission electron microscopy. Bacteria accumulated on the epidermis close to root hairs, and subsequently entered the latter via infection threads. Most of the steps involved in nodule formation were generally characteristic of determinate nodules, such as those which form on the closely related V. radiata. For example, nodule meristems were induced beneath the root epidermis adjacent to infected root hairs, but prior to infection of the meristem by rhizobia. Moreover, after the infection of some of the meristematic cells by the infection threads, and the release of the rhizobia into membrane-bound vesicles, the infection process ceased and dissemination of the rhizobia was by division of already-infected host cells. However, there were some aspects of this process in V. subterranea which have been more commonly described in indeterminate nodules. These include long infection threads entering a number of cells within the meristems simultaneously and a matrix within infection threads which was strongly labelled with immunogold monoclonal antibodies, MAC236 and MAC265, which recognize epitopes on an intercellular glycoprotein. The MAC236 and MAC265 antibodies also recognized material in the unwalled infection droplets surrounding bacteria which were newly-released from the infection threads. The amount of labelling shown was more characteristic of the long infection threads seen in indeterminate nodules such as pea (Pisum sativum) and Neptunia plena. The structure of mature V. subterranea nodules was similar to that described for other determinate nodules such as Glycine max, Vigna unguiculata and V.radiata, i.e. they were spherical and the infected zone consisted of both infected and uninfected cells. Surrounding the infected tissue was an inner cortex of uninfected cell layers containing the putative components of an oxygen diffusion barrier (including glycoprotein-occluded intercellular spaces), and an outer cortex with cells containing calcium oxalate crystals.     

The relationship between nodulin gene expression and the Rhizobium nod genes in Vicia sativa root nodule development

The role of the Rhizobium nod genes in the induction of nodulin gene expression was examined by analyzing nodules formed on vetch roots by bacterial strains containing only the nod region. Introduction of an 11-kb cloned nod region of the R. leguminosarum sym plasmid pRL1JI into sym plasmid-cured rhizobia conferred on the recipient strains the ability to induce nodules in which all nodulin genes were expressed. This proves that from the sym plasmid only the nod region is involved in the induction of nodulin gene expression. A transconjugant of Agrobacterium carrying the same nod region induces nodules in which only early nodulin gene expression is detected. Thus, the nod region is essential for the induction of early nodulin gene expression. In this case, nodule cytology may indicate that a defense response of the plant interferes with the induction of late nodulin gene expression. Indirect evidence is presented that indeed the Rhizobium nod genes are also in some way involved in the induction of the expression of late noduling genes. The combination between histological data and pattern of nodulin gene expression furthermore reveals a correlation between nodule structure and nodulin gene expression. This correlation may aid in speculations about the functions of nodulins.