Rhizobium lipopolysaccharide modulates infection thread development in white clover root hairs.

The interaction between Rhizobium lipopolysaccharide (LPS) and white clover roots was examined. The Limulus lysate assay indicated that Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) released LPS into the external root environment of slide cultures. Immunofluorescence and immunoelectron microscopy showed that purified LPS from R. trifolii 0403 bound rapidly to root hair tips and infiltrated across the root hair wall. Infection thread formation in root hairs was promoted by preinoculation treatment of roots with R. trifolii LPS at a low dose (up to 5 micrograms per plant) but inhibited at a higher dose. This biological activity of LPS was restricted to the region of the root present at the time of exposure to LPS, higher with LPS from cells in the early stationary phase than in the mid-exponential phase, incubation time dependent, incapable of reversing inhibition of infection by NO3- or NH4+, and conserved among serologically distinct LPSs from several wild-type R. trifolii strains (0403, 2S-2, and ANU843). In contrast, infections were not increased by preinoculation treatment of roots with LPSs from R. leguminosarum bv. viciae strain 300, R. meliloti 102F28, or members of the family Enterobacteriaceae. Most infection threads developed successfully in root hairs pretreated with R. trifolii LPS, whereas many infections aborted near their origins and accumulated brown deposits if pretreated with LPS from R. meliloti 102F28. LPS from R. leguminosarum 300 also caused most infection threads to abort. Other specific responses of root hairs to infection-stimulating LPS from R. trifolii included acceleration of cytoplasmic streaming and production of novel proteins. Combined gas chromatography-mass spectroscopy and proton nuclear magnetic resonance analyses indicated that biologically active LPS from R. trifolii 0403 in the early stationary phase had less fucose but more 2-O-methylfucose, quinovosamine, 3,6-dideoxy-3-(methylamino)galactose, and noncarbohydrate substituents (O-methyl, N-methyl, and acetyl groups) on glycosyl components than did inactive LPS in the mid-exponential phase. We conclude that LPS-root hair interactions trigger metabolic events that have a significant impact on successful development of infection threads in this Rhizobium-legume symbiosis.     

Growth-phase-dependent immunodeterminants of Rhizobium trifolii lipopolysaccharide which bind trifoliin A, a white clover lectin.

The lipopolysaccharide (LPS) from Rhizobium trifolii 0403 was isolated at different stages of growth and was examined for its (i) ability to bind a white clover lectin (trifoliin A), (ii) immunochemical properties, and (iii) composition. There was significantly more binding of trifoliin A to purified LPS and cells in the early stationary phase than to cells in the exponential phase. Immunofluorescence and enzyme-linked immunosorbent assays indicated that new antigenic determinants of the LPS appeared for brief periods on cells at the end of the lag phase and again at the beginning of the stationary phase. These new antigens were not detected on cells in midexponential or late stationary phase. Monovalent fragments of immunoglobulin G antibodies raised against the unique antigenic determinants in the LPS competitively blocked the binding of trifoliin A to cells in the early stationary phase. Gas chromatographic analysis showed that the relative quantity of several glycosyl components in the LPS increased as the culture advanced from the midexponential to the early stationary phase. In addition, LPS from cells in the early stationary phase had a higher aggregate molecular weight. Quinovosamine (2-amino-2,6-dideoxyglucose) was identified by combined gas chromatography-mass spectrometry as a sugar component of the LPS which had not been previously reported. D-Quinovosamine, N-acetyl-D-quinovosamine, and its n-propyl-beta-glycoside were effective hapten sugars which inhibited the binding of trifoliin A, anti-clover root antibody, and homologous antibody to these new determinants in the LPS. White clover plants had more infected root hairs after incubation with an inoculum of cells in the early stationary phase than after incubation with cells in the midexponential phase. The profound influence of the growth phase on the composition of lectin-binding polysaccharides of Rhizobium may be a major underlying cause of conflicting data among laboratories testing the lectin-recognition hypothesis. In addition, these chemical modifications may reflect mechanisms which regulate Rhizobium-root hair recognition in this nitrogen-fixing symbiosis.     

Effect of low temperature pre-treatment on infection of clover root hairs by Rhizobium

Summary Holding 24-h seedlings ofTrifolium spp. at 3°C increased the number of root hairs infected byRhizobium when the plants were subsequently inoculated and grown at 19°C. This effect was transitory, disappearing on plants more than 7 days old.     

Factors responsible for the curling and branching of clover root hairs by Rhizobium

Summary The branching of clover root hairs was due to a factor (or factors) readily extracted from cells of Rhizobium trifolii and found in seedling solution supporting clover roots inoculated with this organism. Part of the active material was retained within a dialysis sac but a smaller, escaping fraction evoked the same response. The branching fraction was stable at 100°C and, partly, to nuclease and periodate, but was unstable when left in contact with the cells and when treated with trypsin. The dried residue of active filtrate contained 2% nucleic acid, 20% protein and about 34% carbohydrate which included antigenic lipopolysaccharide (approximately 1.6%). The branching response could not be attributed to either of these polysaccharides or to indole acetic acid. The capacity to cause branching was absent from all non-invasive mutants, but was increased in ineffective invasive forms, compared with the effective parent strain.Moderate curling was quantitatively much less striking and was generally found in the same preparations as showed branching. Notable exceptions were the lack of significant moderate curling in the filtered inoculated seedling solution and retention of this capacity in avirulent mutants.Marked curling occurred only when viable virulent rhizobia were present. Avirulent mutants were incapable of causing marked curling but the response to ineffective variants was rather better than to their effective parent strains. A dialysis membrane separating bacteria and plant roots prevented marked curling. re]19750214     

Infection and nodulation of clover by nonmotile Rhizobium trifolii.

Nonmotile mutants of Rhizobium trifolii were isolated to determine whether bacterial motility is required for the infection and nodulation of clover. The nonmotile mutants were screened for their ability to infect and nodulate clover seedlings in Fahraeus glass slide assemblies, plastic growth pouches, and vermiculite-sand-filled clay pots. In each system, the nonmotile mutants were able to infect and nodulate clover.     

Modulation of development, growth dynamics, wall crystallinity, and infection sites in white clover root hairs by membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii.

We used bright-field, time-lapse video, cross-polarized, phase-contrast, and fluorescence microscopies to examine the influence of isolated chitolipooligosaccharides (CLOSs) from wild-type Rhizobium leguminosarum bv. trifolii on development of white clover root hairs, and the role of these bioactive glycolipids in primary host infection. CLOS action caused a threefold increase in the differentiation of root epidermal cells into root hairs. At maturity, root hairs were significantly longer because of an extended period of active elongation without a change in the elongation rate itself. Time-series image analysis showed that the morphological basis of CLOS-induced root hair deformation is a redirection of tip growth displaced from the medial axis as previously predicted. Further studies showed several newly described infection-related root hair responses to CLOSs, including the localized disruption of the normal crystallinity in cell wall architecture and the induction of new infection sites. The application of CLOS also enabled a NodC- mutant of R. leguminosarum bv. trifolii to progress further in the infection process by inducing bright refractile spot modifications of the deformed root hair walls. However, CLOSs did not rescue the ability of the NodC- mutant to induce marked curlings or infection threads within root hairs. These results indicate that CLOS Nod factors elicit several host responses that modulate the growth dynamics and symbiont infectibility of white clover root hairs but that CLOSs alone are not sufficient to permit successful entry of the bacteria into root hairs during primary host infection in the Rhizobium-clover symbiosis.     

Ultrastructure of Rhizobium-induced infection threads in clover root hairs.

Ultrastructural studies of Rhizobium-induced infection threads in clover root hairs show that the infection thread is initiated by an invagination process. Root hair wall growth is redirected at a localized point, resulting in the formation of an open pore. There is no direct penetration through the wall, and the bacteria remain extracellular within the root hair.     

Bacterial endophytes and root hairs

Aims

This study aimed to measure the effect of plant diversity on N uptake in grasslands and to assess the mechanisms contributing to diversity effects.

Methods

Annual N uptake into above- and belowground organs and soil nitrate pools were measured in the Jena experiment on a floodplain soil with mixtures of 2–16 species and 1–4 functional groups, and monocultures. In mixtures, the deviation of measured data from data expected from monoculture performance was calculated to assess the contribution of complementarity/facilitation and selection.

Results

N uptake varied from <1 to 45 g?N m^?2 yr^?1, and was higher in grasslands with than without legumes. On average, N uptake was higher in mixtures (21?±?1 g?N m^?2 yr^?1) than monocultures (13?±?1 g?N m^?2 yr^?1), and increased with species richness in mixtures. However, compared to N uptake expected from biomass proportions of species in mixtures, N uptake of mixtures was only slightly higher and a significant surplus N uptake was confined to mixtures containing legumes and non-legumes.

Conclusions

In our study, high N uptake of species rich mixtures was mainly due to dominance of productive species and facilitation by legumes whereas complementarity among non-legumes was of minor relevance.     

Ultrastructure of Rhizobium japonicum in relation to its attachment to root hairs.

In Rhizobium japonicum strain Nitragin 61A76, morphologically distinct types of bacteria were found to occur in yeast extract-mannitol broth cultures, at both mid-log and stationary phases. Of these only the capsular form, characterized by a smooth cell envelope, storage granules (glycogen and poly-beta-hydroxybutyric acid), and an amorphous extracellular capsule, bound soybean lectin. The binding site was localized in the capsular material. Less than 1% of the bacterial population differentiated into these capsular forms, which were also able to attach to the soybean root hair surface.     

Root hair deformation in the white clover/Rhizobium trifolii symbiosis

Rhizobium trifolii most frequently infects its host white clover (Trifolium repens L.) by means of infection threads formed in markedly curled root hairs. Rhizobium infections are classified as either lateral or apical based on whether they originate in the branches or at the apex of the root hairs. A quantitative estimate of lateral and apical infection in the region of the host root (Trifolium repens L. cv. Regal Ladino) that possessed mature and immature root hairs at the time of inoculation with Rhizobium trifolii TAI (CSIRO, Canberra City, Australia) indicated that lateral infection occurred more frequently in the mature root hair region of the root. Apical infections were more common in the immature root hair region. Cell free filtrates collected from R. trifolii cultured in association with the host roots induced branching in white clover root hairs. A partially purified preparation of the branching factor was obtained from freeze-dried filtrates by ethanol extraction and ion exchange chromatography. Preliminary studies on the characteristics of these substances suggest that some are dialyzable and heat stable white others are non-dialyzable and heat labile. The dialyzable, heat-stable compounds contain neutral sugars and range between 1200 to 10000 daltons in size. In roots that were exposed to low concentrations (6–25 μg-ml^?1) of these partially purified deformation factors before inoculation, the developmentally mature root hairs were deformed at the time of inoculation. Nodules appeared in the mature and immature root hair region of these plants at the same time. In plants exposed to water, nodules were observed in the immature root hair region and mature root hair regions 3 and 5 days after inoculation, respectively. Based on these results, we conclude that the nodule development was hastened in the plants exposed to the root hair-deforming substances because the mature root hairs of these plants were made infectible at the time of inoculation by this exposure.     

Stimulation of clover root hair infection by lectin-binding oligosaccharides from the capsular and extracellular polysaccharides of Rhizobium trifolii

A polysaccharide depolymerase isolated from the phage lysate of Rhizobium trifolii 4S was used to fragment capsular polysaccharides (CPS) and extracellular polysaccharides (EPS) of R. trifolii 0403 into oligosaccharides. These products were analyzed for clover lectin (trifoliin A)-binding ability, effect on infection of white clover root hairs, and changes in glycosyl and noncarbohydrate composition with culture age. The oligosaccharides from CPS of cultures grown on agar plates for 3, 5, and 7 days exhibited lectin-binding ability at levels similar to those of the corresponding intact CPS. The intact EPS did not bind to clover lectin, although the oligosaccharide fragments from EPS did. In contrast, oligosaccharides from deacetylated CPS had less than half the lectin-binding ability of the native polysaccharide substrate. The CPS from 5-day-old cultures, its corresponding oligosaccharide fragments, and the oligosaccharide fragments of EPS from 5-day-old cultures, all at a concentration of 2.5 micrograms per seedling, stimulated infection thread formation in root hairs of clover seedlings inoculated with R. trifolii 0403. Thus, this bacteriophage-induced polysaccharide depolymerase converted the acidic CPS and EPS of R. trifolii 0403 into biologically active oligosaccharides capable of binding trifoliin A and stimulating root hair infection. The amount of the noncarbohydrate substitutions (pyruvate, acetate, and ether-linked 3-hydroxybutyrate) in the CPS oligosaccharides changed with culture age as shown by 1H-nuclear magnetic resonance spectroscopy. The binding of trifoliin A, therefore, appears to be sensitive to changes in the degree of substitution of noncarbohydrate substitutions in the CPS of R. trifolii 0403.     

Bacteriocinogeny of Rhizobium trifolii.

Rhizobium trifolii strains differing in cell and colony morphology, streptomycin resistance, phage sensitivity pattern and infectivity to clover plants produced bacteriocins sensitive to proteases. Elimination of bacteriocin production ability wtih SDS and rifampicin treatment indicates that this feature is plasmid controlled. Elimination of bacteriocinogenic plasmid did not influence other features of R. trifolii.     

Polyol metabolism by Rhizobium trifolii.

In Rhizobium trifolii 7000, the polyols myo-inositol, xylitol, ribitol, D-arabitol, D-mannitol, D-sorbital, and dulcitol are metabolized by inducible nicotinamide adenine dinucleotide-dependent polyol dehydrogenases. Five different polyol dehydrogenases were recognized: inositol dehydrogenase, specific for inositil; ribitol dehydrogenase, specific for ribitol; D-arabitol dehydrogenase, which oxidized D-arabitol, D-mannitol, and D-sorbitol; xylitol dehydrogenase, which oxidized xylitol and D-sorbitol; and dulcitol dehydrogenase, which oxidized dulcitol, ribitol, xylitol, and sorbitol. Apart from inositil and xylitol, all of the polyols induced more than one polyol dehydrogenase and polyol transport system, but the heterologous polyol dehydrogenases and polyol transport systems were not coordinately induced by a particular polyol. With the exception of xylitol, all of the polyols tested served as growth substrates. A mutant of trifolii 7000, which was constitutive for dulcitol dehydrogenase, could also grow on xylitol.     

Genetic transformation in Rhizobium trifolii.

Markers controlling the synthesis of amino acids and organic bases as well as streptomycin resistance and sensitivity to acriflavine were transformed in Rhizobium trifolii. The results indicate that the str marker was transformed independently of leu, his, ade and trp markers. Co-transformation of leu and utra markers ranged from 3 to 7%, whereas that of thi and acr was 10%.     

Regulation of nitrogen fixation in Rhizobium spp. Isolation of mutants of Rhizobium trifolii which induce nitrogenase activity

This communication describes the isolation and characterization of mutants of Rhizobium trifolii which can induce nitrogenase activity in defined liquid medium. Two procedures were used for the isolation of these mutants from R. trifolii strain DT-6: (1) following chemical mutagenesis, slow growing mutants were selected which were unable to utilize NH+4 as sole source of nitrogen; (2) as spontaneous mutants resistant to the glutamate analogue L-methionine-DL-sulfoximine. Mutants (DT-71, DT-125) isolated by these procedures induced nitrogenase activity in the free-living state, whereas the parent strain lacked this property. Induction of nitrogenase activity in these mutants occurred during the late exponential phase of growth when the rate of protein synthesis was decreasing. The addition of NH+4 to a medium containing glutamate as the nitrogen-source resulted in a 50--70% reduction (repression?) of nitrogenase activity; in contrast, the rate of protein synthesis or the rate of respiration was not influenced by exogenous NH+4. Biochemical analysis showed that these mutants (strains DT-71 and DT-125) have defects in both nitrogen and carbon metabolism. The levels of glutamate synthase (both NADP+ -and NAD+ -dependent activities) and glutamate dehydrogenase (NAD+-dependent activity) were markedly lower. In addition, the mutants were found to have no detectable ribitol dehydrogenase or beta-galactosidase activity. These findings are discussed in relation to a mechanism of regulation of symbiotic nitrogen fixation.     

Competitiveness of Rhizobium leguminosarum bv. trifolii strains in mixed inoculation of clover (Trifolium pratense)

Rhizobium leguminosarum by. trifolii (Rlt) establishes beneficial root nodule symbiosis with clover. Twenty Rlt strains differentially marked with antibiotic-resistance markers were investigated in terms of their competitiveness and plant growth promotion in mixed inoculation of clover in laboratory experiments. The results showed that the studied strains essentially differed in competition ability. These differences seem not to be dependent on bacterial multiplication in the vicinity of roots, but rather on complex physiological traits that affect competitiveness. The most remarkable result of this study is that almost half of the total number of the sampled nodules was colonized by more than one strain. The data suggest that multi-strain model of nodule colonization is common in Rhizobium-legume symbiosis and reflects the diversity ofrhizobial population living in the rhizosphere.