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.     

Variation within and between white clover varieties in their preference for strains of Rhizobium trifolii

Variation within as well as between varieties of white clover (Trifolium repens L.) in their selection or preference for rhizobial strains in nodulation was studied using antibiotic-resistant mutant strains of Rhizobium trifolii. Significant differences between varieties were found. Cv. S100 was nodulated almost exclusively by 75 str but there was more variation within S184 and, in particular, Pajbjerg smalbladet. In this experiment 93% of the total variance was genotypic, of which 68% was due to the difference between varieties and 25% due to variance between genotypes within varieties. Significant correlations were found between the preference for rhizobial strains of plants grown from seeds and of stolon lines vegetatively propagated from the former plants, indicating that the preference for rhizobial strains is genetically controlled by the host. A considerable drift in preference towards selection of more compatible rhizobial strains by the clonally propagated stolons compared with the original white clover plants was observed. The results are discussed from the point of view of improving symbiotic nitrogen fixation by combining highly effective and competitive Rhizobium strains with a host variety with uniform preference for those strains.     

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     

Effect of a bacteriophage on colonisation and nodulation of clover roots by paired strains of Rhizobium trifolii

The effect of a virulent bacteriophage on the competitive behaviour of paired strains of Rhizobium trifolii in the root zone of clover plants was examined. The presence of bacteriophage reduced the population density of susceptible strains on the root surface and, in nodulation, favoured resistant or even partially resistant strains which were otherwise less able to form nodules. These effects occurred with different growth temperatures, host species, and ratios of strains in the inoculum.     

Effect of a bacteriophage on the colonisation and nodulation of clover roots by a strain of Rhizobium trifolii

The presence of a virulent bacteriophage in the root zone of clover growing in seedling agar under controlled environments (14--17 and 19--23 degrees C) produced changes in the persistence and symbiotic effectiveness of a susceptible strain of Rhizobium trifolii. The phage reduced the rhizoplane population of rhizobia and led to the appearance of variant substrains which were less susceptible to the bacteriophage and mostly ineffective in symbiotic nitrogen fixation. Some were also changed in colonial morphology and nutritional requirements. At the higher temperature, the frequency of bacterial variants increased and the number of nodules due to the parent strain decreased. A large initial population of bacteriophage was able to reduce, but generally did not completely suppress, nodulation.     

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.     

Rhizobium induces marked root hair curling by redirection of tip growth: a computer simulation

Computer simulation shows that Rhizobium can induce marked curling in legume root hairs by growth induction. Essential elements are: a) the attachment of one inducing principle (e.g. one bacterium or a group of bacteria), preferably within the growth area of the root hair; b) translocation of the inductor along the growing root hari tip; and c) redirection of the original plant-driven tip growth. Also other root hair deformations, for example root hair branching and infection thread growth, can be explained with the proposed model.     

Correlation between extracellular fibrils and attachment of Rhizobium leguminosarum to pea root hair tips.

As part of a project meant to characterize molecules involved in nodulation, a semiquantitative microscopic assay was developed for measuring attachment of Rhizobium leguminosarum cells to pea root hair tips, i.e., the site at which R. leguminosarum initiates nodulation. This form of attachment, designated as cap formation, was dependent on the incubation pH and growth phase, with optimal attachment at pH 7.5 and with bacteria in the early stationary phase of growth. Addition of glucose to the growth medium delayed the initiation of the stationary phase and cap formation, suggesting a correlation between cap formation and carbon limitation. Attachment of R. leguminosarum was not inhibited by pea lectin haptens which makes it unlikely that lectins are involved under the tested conditions. Moreover, heterologous fast-growing rhizobia adhered equally well to pea root hair tips. Since the attachment characteristics of a Sym plasmid-cured derivative were indistinguishable from those of the wild-type strain, the Sym plasmidborne nodulation genes are not necessary for attachment. Sodium chloride and various other salts abolished attachment when present during the attachment assay in final concentrations of 100 mM. R. leguminosarum produced extracellular fibrils. A positive correlation between the percentage of fibrillated cells and the ability of the bacteria to form caps and to adhere to glass and erythrocytes was observed under various conditions, suggesting that these fibrils play a role in attachment of the bacteria to pea root hair tips, to glass, and to erythrocytes.     

Effects of some fungicides used against cereal pathogens on the growth of Rhizobium trifolii and its capacity to fix nitrogen in white clover

Fungicide residues in soils may affect nitrogen fixation by legumes. Effects of nine systemic fungicides, used against cereal pathogens, on Rhizobium trifolii and white clover were measured. Fenarimol and oxycarboxin inhibited growth of R. trifolii in vitro. The weight of clover plants after 10 weeks' growth in soil containing carboxin, oxycarboxin, benodanil, tridemorph and pyracarbolid was reduced. Symbiotic nitrogen fixation was decreased by carboxin, oxycarboxin and tridemorph present in soil at concentrations somewhat greater than that likely to result from several applications of the fungicide. Only tridemorph reduced nodulation. None of the compounds seems likely to affect nitrogen fixation in the field if applied at the recommended rate.     

Evidence that L-glutamate can act as an exogenous signal to modulate root growth and branching in Arabidopsis thaliana

The roots of many plant species are known to use inorganic nitrogen, in the form of , as a cue to initiate localized root proliferation within nutrient-rich patches of soil. We report here that, at micromolar concentrations and in a genotype-dependent manner, exogenous l-glutamate is also able to elicit complex changes in Arabidopsis root development. l-Glutamate is perceived specifically at the primary root tip and inhibits mitotic activity in the root apical meristem, but does not interfere with lateral root initiation or outgrowth. Only some time after emergence do lateral roots acquire l-glutamate sensitivity, indicating that their ability to respond to l-glutamate is developmentally regulated. Comparisons between different Arabidopsis ecotypes revealed a remarkable degree of natural variation in l-glutamate sensitivity, with C24 being the most sensitive. The aux1-7 auxin transport mutant had reduced l-glutamate sensitivity, suggesting a possible interaction between l-glutamate and auxin signaling. Surprisingly, two loss-of-function mutants at the AXR1 locus (axr1-3 and axr1-12) were hypersensitive to l-glutamate. A pharmacological approach, using agonists and antagonists of mammalian ionotropic glutamate receptors, was unable to provide evidence of a role for their plant homologs in sensing exogenous glutamate. We discuss the mechanism of l-glutamate sensing and the possible ecological significance of the observed l-glutamate-elicited changes in root architecture.     

Identification and characterisation of two distinct acid phosphatases in cell walls of roots of white clover

White clover (Trifolium repens L.) plants were grown in liquid media and subjected to phosphate starvation by removal of the sole phosphate source. After 21 d, roots were harvested and ionically-bound (1 M salt-extractable) cell wall proteins isolated. Two distinct acid phosphatases (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2), designated APase I and II, were identified using hydrophobic column chromatography with each protein consisting of two isoforms resolved by ion-exchange column chromatography. For APase I, both isoforms (IA and IB) are glycosylated (as determined by binding to a monoclonal antibody, mAb 2.23, which is specific to xylose/fucose-containing complex-type N-linked glycans, and a Galanthus nivalis (GNA) lectin which recognises terminal mannose sugars), and exist as active monomers of 52 kDa as determined by SDS-PAGE and by gel filtration. APase IA and IB have pH optima for p-nitrophenyl phosphate of 5.8 and 6.2, and pIs of 7.3 and 6.5, respectively. For APase II, both isoforms exist as active monomers of 113 kDa by SDS-PAGE and 92 kDa by gel filtration, with pH optima of 5.8 and 6.8, and pIs of 4.4 and 5.2 to 5.3 for IIA and IIB, respectively. Isoform IIA was not recognised by the two glycan probes, while IIB was recognised by mAb 2.23. The activity of all four isoforms was severely inhibited by Cu²⁺, Zn²⁺ and Mo²⁺, while each showed activity against a range of phosphate monoester substrates with highest substrate specificity (V_max/K_m) for ATP and PPi. Activity associated with APase I and II is detectable in roots isolated from plants maintained in P-containing media, but the activity of both is increased with the onset of P-deficiency. However, a temporal difference in response between APase I and II is observed over a 28-d time course of P-deprivation.     

Flavonoids induce Rhizobium leguminosarum to produce nodDABC gene-related factors that cause thick, short roots and root hair responses on common vetch.

Rhizobium leguminosarum produced a factor(s) that caused thick, short roots (Tsr phenotype) as well as root hair induction (Hai phenotype) and deformation (Had phenotype) in Vicia sativa plants upon incubation with root exudate or with one of the nod gene inducers naringenin or apigenin; this was a nodDABC gene-dependent process. Detection of the Hai and Had phenotypes was much more sensitive than that of the Tsr phenotype.     

Studies of symbiotic plasmids in Rhizobium trifolii and fast-growing bacteria that nodulate soybeans

The Rhizobium trifolii symbiotic plasmid pRt5a was transferred to the fast-growing soybean strain USDA 194. Transconjugants carrying pRt5a were not able to nodulate clovers and one of the transconjugants had lost its smallest resident plasmid and did not fix nitrogen in soybean. Transconjugants of USDA 194 carrying pRt5a were able to transfer pRt5a back to a non-nodulating R. trifolii which inherited the symbiotic properties of the R. trifolii strain from which the plasmid was derived.     

Rhizobium nodM and nodN genes are common nod genes: nodM encodes functions for efficiency of nod signal production and bacteroid maturation.

Earlier, we showed that Rhizobium meliloti nodM codes for glucosamine synthase and that nodM and nodN mutants produce strongly reduced root hair deformation activity and display delayed nodulation of Medicago sativa (Baev et al., Mol. Gen. Genet. 228:113-124, 1991). Here, we demonstrate that nodM and nodN genes from Rhizobium leguminosarum biovar viciae restore the root hair deformation activity of exudates of the corresponding R. meliloti mutant strains. Partial restoration of the nodulation phenotypes of these two strains was also observed. In nodulation assays, galactosamine and N-acetylglucosamine could substitute for glucosamine in the suppression of the R. meliloti nodM mutation, although N-acetylglucosamine was less efficient. We observed that in nodules induced by nodM mutants, the bacteroids did not show complete development or were deteriorated, resulting in decreased nitrogen fixation and, consequently, lower dry weights of the plants. This mutant phenotype could also be suppressed by exogenously supplied glucosamine, N-acetylglucosamine, and galactosamine and to a lesser extent by glucosamine-6-phosphate, indicating that the nodM mutant bacteroids are limited for glucosamine. In addition, by using derivatives of the wild type and a nodM mutant in which the nod genes are expressed at a high constitutive level, it was shown that the nodM mutant produces significantly fewer Nod factors than the wild-type strain but that their chemical structures are unchanged. However, the relative amounts of analogs of the cognate Nod signals were elevated, and this may explain the observed host range effects of the nodM mutation. Our data indicate that both the nodM and nodN genes of the two species have common functions and confirm that NodM is a glucosamine synthase with the biochemical role of providing sufficient amounts of the sugar moiety for the synthesis of the glucosamine oligosaccharide signal molecules.     

Temporal and spatial order of events during the induction of cortical cell divisions in white clover by Rhizobium leguminosarum bv. trifolii inoculation or localized cytokinin addition

We examined the timing and location of several early root responses to Rhizobium leguminosarum bv. trifolii infection, compared with a localized addition of cytokinin in white clover, to study the role of cytokinin in early signaling during nodule initiation. Induction of ENOD40 expression by either rhizobia or cytokinin was similar in timing and location and occurred in nodule progenitor cells in the inner cortex. Inoculation of rhizobia in the mature root failed to induce ENOD40 expression and cortical cell divisions (ccd). Nitrate addition at levels repressing nodule formation inhibited ENOD40 induction by rhizobia but not by cytokinin. ENOD40 expression was not induced by auxin, an auxin transport inhibitor, or an ethylene precursor. In contrast to rhizobia, cytokinin addition was not sufficient to induce a modulation of the auxin flow, the induction of specific chalcone synthase genes, and the accumulation of fluorescent compounds associated with nodule initiation. However, cytokinin addition was sufficient for the localized induction of auxin-induced GH3 gene expression and the initiation of ccd. Our results suggest that rhizobia induce cytokinin-mediated events in parallel to changes in auxin-related responses during nodule initiation and support a role of ENOD40 in regulating ccd. We propose a model for the interactions of cytokinin with auxin, ENOD40, flavonoids, and nitrate during nodulation.     

Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides

The expression of the auxin responsive reporter construct, GH3:gusA, was examined in transgenic white clover plants to assess changes in the auxin balance during the earliest stages of root nodule formation. Reporter gene expression was monitored at marked locations after the application of bacteria or signal molecules using two precise inoculation techniques: spot-inoculation and a novel method for ballistic microtargeting. Changes in GH3:gusA expression were monitored after the inoculation of Rhizobium leguminosarum biovar trifolii, non-host rhizobia, lipo-chitin oligosaccharides (LCOs), chitin oligosaccharides, a synthetic auxin transport inhibitor (naphthylphthalamic acid; NPA), auxin, the ENOD40-1 peptide or different flavonoids. The results show that clover-nodulating rhizobia induce a rapid, transient and local downregulation of GH3:gusA expression during nodule initiation followed by an upregulation of reporter gene expression at the site of nodule initiation. Microtargeting of auxin caused a local and acropetal upregulation of GH3:gusA expression, whereas NPA caused local and acropetal downregulation of expression. Both spot-inoculation and microtargeting of R. l. bv. trifolii LCOs or flavonoid aglycones induced similar changes to GH3:gusA expression as NPA. O-acetylated chitin oligosaccharides caused similar changes to GH3:gusA expression as R. l. bv. trifolii spot-inoculation, but only after delivery by microtargeting. Non-O-acetylated chitin oligosaccharides, flavonoid glucosides or the ENOD40-1 peptide failed to induce any detectable changes in GH3:gusA expression. GH3:gusA expression patterns during the later stages of nodule and lateral root development were similar. These results support the hypothesis that LCOs and chitin oligosaccharides act by perturbing the auxin flow in the root during the earliest stages of nodule formation, and that endogenous flavonoids could mediate this response.     

The response of white clover to different strains ofRhizobium trifolii in hill land reseeding: A second trial

Summary S. 184 white clover was surface seeded into natural molinia pasture on wet stagnogley soil containing no indigenousRhizobium trifolii. Seedlings were ‘spray inoculated’ after emergence with each of three strains ofR. trifolii. The best of these treatments produced an eight fold improvement in dry matter in the seeding year, followed by a 28% improvement in the following year. The results confirm the potential benefits which may be achieved by inoculating clover with suitable strains of rhizobia. The data are compared with a previously reported trial on an adjacent site where benefits were much greater in the first year. The difference is attributed to the overall advantages conferred in the present trial by much higher seedling populations and less severe competition from native species in the establishment phase.