Trifolin: A Rhizobium recognition protein from white clover

A protein agglutinin, trifoliin, was purified from white clover seeds and seedling roots. Trifoliin specifically agglutinates the symbiont of clover, Rhizobium trifolii, at concentrations as low as 0.2 μg protein/ml, and binds to the surface of encapsulated R. trifolii 0403. This clover protein has a subunit with M_r ≈ 50 000, an isoelectric point of 7.3, and contains carbohydrate. Antibody to purified trifoliin binds to the root hair region of 24-h-old clover seedlings, but does not bind to alfalfa, birdsfoot trefoil or joint vetch. The highest concentration of trifoliin on a clover root is present at sites where material in the capsule of R. trifolii binds. 2-Deoxy-d-glucose elutes trifoliin from intact clover-seedling roots, suggesting that this protein is anchored to root cell walls through its carbohydrate binding sites. We propose that trifoliin on the root hair surface plays an important role in the recognition of R. trifolii by clover.     

Study of theRhizobium japonicum-soybean symbiosis

Summary Capsular polysaccharides were isolated fromRhizobium japonicum (61A76NS) and conjugated to a fluorescent dye to determine if the specificity in theRhizobium japonicum-soybean symbiosis is expressed by a component (lectin) located on soybean roots which binds to the sugars of the bacterial capsules.The conjugated Fraction A capsular polysaccharides ofR. japonicum bound only to the root hair tips of soybean seedlings. The polysaccharide would not bind specifically to the roots of clover or alfalfa seedlings. Rhodamine conjugated polysaccharides ofR. japonicum could be inhibited from binding to soybean root hairs by the addition of N-acetylgalactosamine or galactose, effective hapten inhibitors of this type of binding. This is the first report of hapten-reversible binding of an isolated rhizobial component to soybean root hairs, the differentiated epidermal cells which are subsequently infected by this nitrogen-fixing symbiont.Paper number6046 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina.     

Selective removal of seedling root hairs for studies of the Rhizobium-legume symbiosis

A freeze-fracture method has been developed for the selective removal of root hairs from white clover (Trifolium repens L.) and alfalfa (Medicago sativa L.) seedling. This procedure yields sufficient material for analysis of root hair proteins by polyacrylamide gel electrophoresis and can be adapted to study in vivo protein synthesis in these differentiated epiderman cells. Clover root hairs which have been injected by the nitrogen-fixing symbiont, Rhizobium trifolii 0403, are also detached from roots by this process, yielding appropriate material to study root responses to the bacterial symbiont during the infection process.     

Hydrogen Evolution from Alfalfa and Clover Nodules and Hydrogen Uptake by Free-Living Rhizobium meliloti

A series of Rhizobium meliloti and Rhizobium trifolii strains were used as inocula for alfalfa and clover, respectively, grown under bacteriologically controlled conditions. Replicate samples of nodules formed by each strain were assayed for rates of H₂ evolution in air, rates of H₂ evolution under Ar and O₂, and rates of C₂H₂ reduction. Nodules formed by all strains of R. meliloti and R. trifolii on their respective hosts lost at least 17% of the electron flow through nitrogenase as evolved H₂. The mean loss from alfalfa nodules formed by 19 R. meliloti strains was 25%, and the mean loss from clover nodules formed by seven R. trifolii strains was 35%. R. meliloti and R. trifolii strains also were cultured under conditions that were previously established for derepression of hydrogenase synthesis. Only strains 102F65 and 102F51 of R. meliloti showed measurable activity under free-living conditions. Bacteroids from nodules formed by the two strains showing hydrogenase activity under free-living conditions also oxidized H₂ at low rates. The specific activity of hydrogenase in bacteroids formed by either strain 102F65 or strain 102F51 of R. meliloti was less than 0.1% of the specific activity of the hydrogenase system in bacteroids formed by H₂ uptake-positive Rhizobium japonicum USDA 110, which has been investigated previously. R. meliloti and R. trifolii strains tested possessed insufficient hydrogenase to recycle a substantial proportion of the H₂ evolved from the nitrogenase reaction in nodules of their hosts. Additional research is needed, therefore, to develop strains of R. meliloti and R. trifolii that possess an adequate H₂-recycling system.     

Regulation by fixed nitrogen of host-symbiont recognition in the Rhizobium-clover symbiosis

Either NO₃⁻ (16 millimolar) or NH₄⁺ (1 millimolar) completely inhibited infection and nodulation of white clover seedlings (Trifoliin repens) inoculated with Rhizobium trifolii. The binding of R. trifolii to root hairs and the immunologically detectable levels of the plant lectin, trifoliin, on the root hair surface had parallel declining slopes as the concentration of either NO₃⁻ or NH₄⁺ was increased in the rooting medium. This supports the role of trifoliin in binding R. trifolii to clover root hairs. Agglutination of R. trifolii by trifoliin from seeds was not inhibited by these levels of NO₃⁻ or NH₄⁺. The results suggest that these fixed N ions may play important roles in regulating an early recognition process in the Rhizobium-clover symbiosis, namely the accumulation of high numbers of infective R. trifolii cells on clover root hairs.     

Interaction of Azospirillum and Rhizobium Strains Leading to Inhibition of Nodulation

Rhizobium-Azospirillum interactions during establishment of Rhizobium-clover symbiosis were studied. When mixed cultures of Azospirillum and Rhizobium trifolii strains were simultaneously inoculated onto clover plants, no nodulation by R. trifolii was observed. R. trifolii ANU1030, which nodulated clover plants without attacking root hairs, i.e., does not cause root hair curling (Hac⁻), did not show inhibition of nodulation when inoculated together with Azospirillum strains. Isolation of bacteria from surface-sterilized roots showed that azospirilla could be isolated both from within root segments and from nodules. Inhibition of nodulation could be mimicked by the addition of auxins to the plant growth medium.     

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO₂ evolution or as O₂ uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 10⁷ bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO₂ production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO₂, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.     

Root Hair Deformations Associated with Fractionated Extracts from Rhizobium trifolii

Components from culture fluid and whole cells of Rhizobium trifolii were examined for effects on root hair morphology of white clover seedlings (Trifolium repens var. Ladino). Cell-free culture fluid, exopolysaccharides, supernatant fluid from the precipitation of the exopolysaccharides, capsular polysaccharides, lipopolysaccharides, and a protein fraction from culture fluids were assayed for morphogenetic effects on the root hairs of axenically grown clover seedlings. Crude fractions were chromatographed on Bio Gel A-5m (Bio-Rad Laboratories), and fractions collected were similarly assayed. Hexose, uronic acid, and protein concentrations were determined for all fractions assayed. Gel chromatography indicated the materials with deforming ability to be of high molecular weight (>10,000). For all fractions except exopolysaccharide, deforming ability was associated with a protein component. This suggested that two components were associated with deformation; both contained polysaccharides and one contained protein. Crude fractions differed in their ability to cause deformations and indicated the following relative ability (in decreasing order) to deform root hairs: cell-free culture fluid, capsular polysaccharides, protein from culture fluids, exopolysaccharide, and cell envelope. Lipopolysaccharides had no effect.     

Quantitation of Adsorption of Rhizobia in Low Numbers to Small Legume Roots

Bacteria adsorbed in low numbers to alfalfa or clover root surfaces were counted after incubation of seedlings in mineral solution with very dilute inocula (less than 10⁵ bacteria per ml) of an antibiotic-resistant strain under defined conditions. After specified washing, bacteria which remained adsorbed to roots were selectively quantitated by culturing the roots embedded in yeast extract-mannitol-antibiotic agar and counting the microcolonies along the root surface; the range was from about 1 bacterium per root (estimated as the most probable number) to 50 bacteria per cm of root length (by direct counting). This simple procedure can be used with any pair of small-rooted plant and antibiotic-resistant bacterium, requires bacterial concentrations comparable to those frequently found in soils, and yields macroscopic localization and distribution data for adsorbed bacteria over the root surface. The number of adsorbed bacteria was proportional to the size of the inoculum. One of every four Rhizobium meliloti cells adsorbed in very low numbers to alfalfa roots resulted in the formation of a nodule. Overall adsorption of various symbiotic and nonsymbiotic bacterial strains to alfalfa and clover roots did not reflect the specificities of these legumes for their respective microsymbionts, R. meliloti and R. trifolii.     

Hair Curling Induced in Heterologous Legumes and Monocots by Flavonoid-Treated Rhizobia

The curling of root hairs and the deformation response wereobserved when white clover was infected with homologous strainsof Rhizobium leguminosarum biovar trifolii 4S and 0403. In thecase of Rhizobium meliloti NZ and Rhizobium leguminosarum biovarviciae 128C53, however, curling was only induced when thesebacteria were pretreated with flavonoids: luteolin in the caseof R. meliloti and naringenin for R.I. viciae. The same resultswere obtained with oat, a monocotyledonous non-leguminous plant.The two flavonoids mentioned are secreted from the host plantsand induce the expression of genes for root hair curling (Hac)on Sym plasmid in homologous rhizobia, therefore, the curlingresponse in both white clover and oat appears to be correlatedwith the activation of the Hac genes. These results suggestthat a factor(s) that activates the Hac genes, such as 7,4'-dihydroxyflavonewhich is known as the factor required by R. I. trifolii, issecreted from the oat roots. (Received June 12, 1989; Accepted November 9, 1989)     

Bacterial polysaccharide which binds Rhizobium trifolii to clover root hairs.

Immunofluorescence, quantitative immunoprecipitation, and inhibition of bacterial agglutination and passive hemagglutination indicate that cross-reactive antigenic determinants are present on the surface of Rhizobium trifolii and clover roots. These determinants are immunochemically unique to this Rhizobium-legume cross-inoculation group. The multivalent lectin trifoliin and antibody to the clover root antigenic determinants bind competitively to two acidic heteropolysaccharides isolated from capsular material of R. Trifolii 0403. The major polysaccharide is an antigen which lacks heptose, 2-keto-3-deoxyoctulosonic acid, and endotoxic lipid A. The minor polysaccharide in the capsular material of R. Trifolii 0403 contains the same antigen in addition to heptose, 2-keto-3-deoxyoctonate, and lipid A. The acidic polysaccharides of two strains of R. trifolii share the clover r-ot cross-reactive antigenic determinant despite other differences in their carbohydrate composition. Studies with monovalent antigen-binding fragments of anti-clover root antibody and Azotobacter vinelandii hybrid transformants carrying the unique antigenic determinant suggest that these polysaccharides bind R. trifolii to the clover root hair tips which contain trifoliin.     

Adsorption ofRhizobium meliloti to alfalfa roots: Dependence on divalent cations and pH

Adsorption ofRhizobium meliloti L5-30 in low numbers to alfalfa (Medicago sativa L.) roots was dependent on the presence of divalent cations, and required neutral pH. Adsorption was proportional to Ca and/or Mg concentrations up to 1.5 mM. Ca was not substituted by Sr, Ba or Mn. Adsorption was abolished and viability decreased at pH6. When lowering pH, higher Ca concentrations were required to attain similar adsorption levels, indicating a marked interactive effect between Ca and H ions. Pretreatment of the roots with Ca and low pH did not affect subsequent adsorption of the bacteria. However, Ca pretreatment ofR. meliloti sustained further adsorption at low Ca levels and low pH substantially affected their ability to adsorb. Low pH appears to affect the stability of binding causing desorption of the previously bound bacteria. The presence of saturating concentrations of heterologousR. leguminosarum bv.trifolii A118, did not prevent the expression of divalent cations and pH requirements, as well as their interaction. Our results suggest that rhizobial binding to the root surface already shows the Ca and pH dependence of alfalfa nodulation, which was generally associated to some event prior to rhizobial penetration of root hairs.     

Modified Fluorescent Technique, Using Rhodamine, for Studies of Rhizobium japonicum-Soybean Symbiosis

Rhodamine-conjugated capsular polysaccharides isolated from Rhizobium japonicum 61A76NS were used to examine binding between the labeled polysaccharides and soybean roots. Fluorescein-labeled polysaccharides were not satisfactory because soybean root hairs autofluoresce in the fluorescein region.     

Production of Cellulose Microfibrils by Rhizobium

Electron microscope examination of Rhizobium spp. revealed microfibrils produced by flocculating strains but not by nonflocculating strains. The microfibrils from R. trifolii (NA30) were isolated and identified as cellulose by enzymatic, X-ray diffraction, and infrared spectral analyses. Both infective and noninfective strains of R. trifolii flocculated and produced microfibrils. More infection threads were observed in clover root hairs growing in the presence of flocs in comparison with root hairs where single bacterial cells predominated.     

The Effect of the Agrobacterium tumefaciens attR Mutation on Attachment and Root Colonization Differs between Legumes and Other Dicots

Infections of wound sites on dicot plants by Agrobacterium tumefaciens result in the formation of crown gall tumors. An early step in tumor formation is bacterial attachment to the plant cells. AttR mutants failed to attach to wound sites of both legumes and nonlegumes and were avirulent on both groups of plants. AttR mutants also failed to attach to the root epidermis and root hairs of nonlegumes and had a markedly reduced ability to colonize the roots of these plants. However, AttR mutants were able to attach to the root epidermis and root hairs of alfalfa, garden bean, and pea. The mutant showed little reduction in its ability to colonize these roots. Thus, A. tumefaciens appears to possess two systems for binding to plant cells. One system is AttR dependent and is required for virulence on all of the plants tested and for colonization of the roots of all of the plants tested except legumes. Attachment to root hairs through this system can be blocked by the acetylated capsular polysaccharide. The second system is AttR independent, is not inhibited by the acetylated capsular polysaccharide, and allows the bacteria to bind to the roots of legumes.     

Trifolin: a Rhizobium recognition protein from white clover

A protein agglutinin, trifoliin, was purified from white clover seeds and seedling roots. Trifoliin specifically agglutinates the symbiont of clover, Rhizobium trifolii, at concentrations as low as 0.2 microgram protein/ml, and binds to the surface of encapsulated R. trifolii 0403. This clover protein has a subunit with Mr approximately 50 000, an isoelectric point of 7.3, and contains carbohydrate. Antibody to purified trifoliin binds to the root hair region of 24-h-old clover seedlings, but does not bind to alfalfa, birdsfoot trefoil or joint vetch. The highest concentration of trifoliin on a clover root is present at sites where material in the capsule of R. trifolii binds. 2-Deoxy-D-glucose elutes trifoliin from intact clover-seedling roots, suggesting that this protein is anchored to root cell walls through its carbohydrate binding sites. We propose that trifoliin on the root hair surface plays an important role in the recognition of R. trifolii by clover.     

Adsorption of bacteria to roots as related to host specificity in the Rhizobium-clover symbiosis.

Quantitative microscope techniques were utilized to examine the adsorption of rhizobial cells to clover root hairs. Adsorption of cells of noninfective strains of Rhizobium trifolii or infective R. meliloti strains to clover root hairs was four to five times less than that of the infective R. trifolii strains. Attachment of the rod-shaped bacteria to clover root cells occurred in a polar, end-on fashion. Viable or heat-killed R. trifolii cells precoated with a clover lectin having 2-deoxyglucose specificity had increased adsorption to clover roots. Adsorption of bacteria to roots was not increased if the clover lectin was inactivated by heat or 2-deoxyglucose treatment prior to incubation with R. trifolii. Adsorption of R. trifolii to clover root hairs was inhibited by 2-deoxyglucose (30 mM) but not by 2-deoxygalactose or alpha-D-glucose. Adsorption of R. meliloti cells to alfalfa root hairs was not affected by 2-deoxyglucose at that concentration. These results suggest that expression of host specificity in the Rhizobium-clover symbiosis involves a preferential adsorption of infective cells to clover root hairs through a 2-deoxyglucose-sensitive receptor site.     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

Nodules Initiated by Rhizobium meliloti Exopolysaccharide Mutants Lack a Discrete, Persistent Nodule Meristem

Infection of alfalfa with Rhizobium meliloti exo mutants deficient in exopolysaccharide results in abnormal root nodules that are devoid of bacteria and fail to fix nitrogen. Here we report further characterization of these abnormal nodules. Tightly curled root hairs or shepherd's crooks were found after inoculation with Rm 1021-derived exo mutants, but curling was delayed compared with wild-type Rm 1021. Infection threads were initiated in curled root hairs by mutants as well as by wild-type R. meliloti, but the exo mutant-induced threads aborted within the peripheral cells of the developing nodule. Also, nodules elicited by Rm 1021-derived exo mutants were more likely to develop on secondary roots than on the primary root. In contrast with wild-type R. meliloti-induced nodules, the exo mutant-induced nodules lacked a well defined apical meristem, presumably due to the abortion of the infection threads. The relationship of these findings to the physiology of nodule development is discussed.     

Subnanomolar concentrations of membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii are fully capable of eliciting symbiosis-related responses on white clover

Axenic seedling bioassays were performed on white clover, vetch, and alfalfa to assess the variety and dose responses of biological activities exhibited by membrane chitolipooligosaccharides (CLOSs) from wild type Rhizobium leguminosarum bv. trifolii ANU843. Subnanomolar concentrations of CLOSs induced deformation of root hairs (Had) and increased the number of foci of cortical cell divisions (Ccd) in white clover, some of which developed into nodule meristems. In contrast, ANU843 CLOSs were unable to induce Had in alfalfa and required a 10⁴-fold higher threshold concentration to induce this response in vetch. Also, ANU843 CLOSs were not mitogenic on either of these non-host legumes. In addition, CLOS action also increased chitinase activity in white clover root exudate. Thus, the membrane CLOSs from wild type R. leguminosarum bv. trifolii are fully capable of eliciting various symbiosis-related responses in white clover in the same concentration range as extracellular CLOSs of other rhizobia on their respective legume hosts. These results and our earlier studies indicate that membrane CLOSs represent one of many different classes of bioactive metabolites made by R. leguminosarum bv. trifolii which elicit more intense symbiosis-related responses in white clover than in other legumes. Therefore, CLOSs evidently play an important role in symbiotic development, but they may not be the sole determinant of host-range in the Rhizobium-clover symbiosis.Abbreviations Ccd cortical cell division - CLOS chitolipooligosaccharide - Had root hair deformation