Development and trifoliin A-binding ability of the capsule of Rhizobium trifolii.

The age-dependent lectin-binding ability of Rhizobium trifolii 0403 capsular polysaccharide (CPS) was examined by following the development of the capsule and its ability to interact with the white clover lectin trifoliin A. Bacteria grown on agar plates for 3, 5, 7, 14, and 21 days were examined by electron microscopy and immunofluorescence microscopy with antibodies prepared against either R. trifolii 0403 CPS or trifoliin A after pretreatment with the lectin. The capsule began to develop at one pole by day 3 and completely surrounded the cells in cultures incubated for 5 days or longer. The capsular polysaccharide on cells cultured for 3 and 5 days was completely reactive with trifoliin A, became noticeably less reactive by day 7, and was only reactive with the lectin at one pole of a few cells after that time. The quantity and location of lectin receptors on bacteria of different ages directly correlated with their attachment in short-term clover root hair-binding studies. Cells from 3- or 21-day-old cultures attached almost exclusively in a polar fashion, whereas cells grown for 5 days attached to root hairs randomly and in the highest numbers. CPS isolated from a 5-day-old culture had higher lectin-binding ability than CPS from 3- and 7-day-old cultures, whereas the CPS from a 14-day-old culture had the lowest. Chemical analyses of the isolated CPS showed changes in the levels of uronic acids (as glucuronic acid), pyruvate, and O-acetyl substitutions with culture age, but the neutral sugar composition remained relatively constant. These results provide evidence that the age-dependent distribution of lectin receptors dictates the level and orientation of attachments of R. trifolii 0403 to clover root hairs.     

Alteration of surface properties in a Tn5 mutant strain of Rhizobium trifolii 0403.

A symbiotically defective mutant strain of Rhizobium trifolii, UR251, was obtained by transposon Tn5 mutagenesis of R. trifolii 0403 rif and recognized by its partially ineffective (Fix +/-) phenotype on white clover plants. UR251 had a single Tn5 insertion in plasmid DNA, a wild-type plasmid pattern, and no detectable Mu DNA sequences originally present in the vector used for Tn5 mutagenesis. Agglutination by the clover lectin trifoliin A and attachment to clover root hairs was higher with UR251 than with the wild-type strain. The capsular polysaccharide (CPS) of UR251 was altered, as shown by a slower rate of CPS depolymerization with a CPS beta-lyase, PD-I; more pyruvate and less acetate and 3-hydroxybutanoate noncarbohydrate substitutions as quantitated by 1H nuclear magnetic resonance; and a higher pyruvyl transferase activity (enzymatic pyruvylation of lipid-bound saccharides). The site of increased pyruvylation in the CPS of UR251 was on the terminal galactose of the branch of the repeating oligosaccharide unit. These results show that the level of noncarbohydrate substitutions of the CPS as well as pyruvyl transferase activity are altered in R. trifolii UR251 and that trifoliin A-binding ability and clover root hair attachment are improved in this mutant strain of R. trifolii 0403 rif.     

Alteration of the Trifoliin A-Binding Capsule of Rhizobium trifolii 0403 by Enzymes Released from Clover Roots

The effect of white clover root exudate on capsules of Rhizobium trifolii 0403 was examined. The clover lectin trifoliin A was detected in root exudate of two clover varieties by indirect immunofluorescence with antibody against this lectin purified from clover seed. Trifoliin A bound uniformly to encapsulated, heat-fixed cells during 1 h of incubation with root exudate. After 4 to 8 h of incubation, trifoliin A was only bound to one pole of the cells. Transmission electron microscopy showed that the capsule itself was altered. The disorganization of the acidic polymers of the capsule began in the equatorial center of the rod-shaped cell and then progressed toward the poles at unequal rates. Trifoliin A could no longer be detected on heat-fixed cells after 12 h of incubation with root exudate. However, trifoliin A was detected in situ on one pole of cells grown for 4 days in the clover root environment of Fahraeus slide cultures. Inhibition studies with the hapten 2-deoxy-d-glucose showed that trifoliin A in root exudate had a higher affinity for one of the cell poles. Immunoelectrophoresis was used to monitor the alteration of the extracellular polysaccharides from R. trifolii 0403 by concentrated root exudate. These polysaccharides were converted into products which eventually lost their ability to immunoprecipitate with homologous antibody. This progressive loss of antigenic reactivity proceeded more rapidly with root exudate from seedlings grown under nitrogen-free conditions than with root exudate from plants grown with 15 mM KNO(3). The root exudate, depleted of trifoliin A by immunoaffinity chromatography, was still able to alter the capsule of R. trifolii 0403. Reconstitution experiments showed that the substance(s) in root exudate which induced this alteration of the capsule was of a high molecular weight, heat labile, trypsin sensitive, and antigenically unrelated to trifoliin A. A variety of glycosidase activities were also detected in the fraction depleted of trifoliin A. These results suggest that enzymes in clover root exudate alter the trifoliin A-binding capsule in a way which would favor polar attachment of R. trifolii to clover root hairs.     

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.     

Bacteriophage-induced acidic heteropolysaccharide lyases that convert the acidic heteropolysaccharides of Rhizobium trifolii into oligosaccharide units

Acidic heteropolysaccharide lyases from lysates of phages 4S and BY15 grown on Rhizobium trifolii 4S and R. trifolii 0403, respectively, were used to analyze the capsular and excreted extracellular acidic polysaccharides of R. trifolii 0403. The activities of the enzymes as measured by viscometry were enhanced by the addition of calcium. The oligosaccharide products obtained by depolymerase digestion of the polysaccharides isolated from cells grown on agar plates for 5 days were isolated by gel filtration and had a glycosyl composition of glucose, galactose, glucuronic acid, and alpha-linked 4-deoxy-L-threo-hex-4-enopyranosyluronic acid in an approximate molar ratio of 5:1:1:1. This latter component was identified by 1H-nuclear magnetic resonance spectroscopy and confirmed by UV spectroscopy, ozonolysis, and its reactivity with thiobarbituric acid. The oligosaccharide had glucose as the reducing terminus, 4-deoxy-L-threo-hex-4-enopyranosyluronic acid as the enzymatically generated nonreducing terminus, and galactose as the terminus of the branched chain. The noncarbohydrate components of the oligosaccharides were acetate, ketal-linked pyruvate, and ether-linked 3-hydroxybutyrate. The mode of action of the enzymes was by beta-elimination from a uronic acid residue with concomitant loss of the glycosyl component substituted at C-4. The 235-nm absorbing properties of the resulting terminal unsaturated sugar were used to study the kinetics of depolymerization of the capsular and excreted extracellular acidic polysaccharides, using the enzyme from phage BY15. The two substrates exhibited different kinetics of depolymerization, and the oligosaccharide products differed in the amount of noncarbohydrate substituents, indicating that the acidic capsular and excreted extracellular polysaccharides from 5-day-old cultures of R. trifolii 0403 were different.     

Specific phases of root hair attachment in the Rhizobium trifolii-clover symbiosis

The time course and orientation of attachment of Rhizobium trifolii 0403 to white clover root hairs was examined in slide cultures by light and electron microscopy. Inocula were grown for 5 days on defined BIII agar medium and represented the large subpopulation of fully encapsulated single cells which uniformly bind the clover lectin trifoliin A. When 10(7) cells or more were added per seedling, bacteria attached within minutes, forming randomly oriented clumps at the root hair tips. Several hours later, single cells attached polarly to the sides of the root hair. This sequence of attachment to clover root hairs was selective for R. trifolii at inoculum sizes of 10(7) to 4 X 10(8) per seedling, specifically inhibited if 2-deoxy-D-glucose, a hapten for trifoliin A, was present in the inoculum, and not observed when 4 X 10(8) cells were added to alfalfa seedling roots or to large clover root cell wall fragments which lacked trifoliin A but still had trifoliin A receptors. Once attached, R. trifolii 0403 became progressively less detachable with 2-deoxy-D-glucose. At smaller inoculum sizes (10(5) to 10(6) cells per seedling), there was no immediate clumping of R. trifolii at clover root hair tips, although polar binding of bacteria along the root hair surface was observed after 4 h. The interface between polarly attached bacteria and the root hair cell wall was shown to contain trifoliin A by immunofluorescence microscopy. Also, this interface was shown by transmission electron microscopy to contain electron-dense granules of host origin. Scanning electron microscopy revealed an accumulation of extracellular microfibrils associated with the lateral and polar surfaces of the attached bacteria, detectable after 12 h of incubation with seedling roots. At this same time, there was a significant reduction in the effectiveness of 2-deoxy-D-glucose in dislodging bacteria already attached to root hairs and an increase in firm attachment of bacteria to the root hair surface, which withstood the hydrodynamic shear forces of high-speed vortexing. These results are interpreted as a sequence of phases in attachment, beginning with specific reversible interactions between bacterial and plant surfaces (phase I attachment), followed by production of extracellular microfibrils which firmly anchor the bacterium to the root hair (phase 2 adhesion). Thus, attachment of R. trifolii to clover root hairs is a specific process requiring more than just the inherent adhesiveness of the bacteria to the plant cell wall.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specific phases of root hair attachment in the Rhizobium trifolii-clover symbiosis.

The time course and orientation of attachment of Rhizobium trifolii 0403 to white clover root hairs was examined in slide cultures by light and electron microscopy. Inocula were grown for 5 days on defined BIII agar medium and represented the large subpopulation of fully encapsulated single cells which uniformly bind the clover lectin trifoliin A. When 10(7) cells or more were added per seedling, bacteria attached within minutes, forming randomly oriented clumps at the root hair tips. Several hours later, single cells attached polarly to the sides of the root hair. This sequence of attachment to clover root hairs was selective for R. trifolii at inoculum sizes of 10(7) to 4 X 10(8) per seedling, specifically inhibited if 2-deoxy-D-glucose, a hapten for trifoliin A, was present in the inoculum, and not observed when 4 X 10(8) cells were added to alfalfa seedling roots or to large clover root cell wall fragments which lacked trifoliin A but still had trifoliin A receptors. Once attached, R. trifolii 0403 became progressively less detachable with 2-deoxy-D-glucose. At smaller inoculum sizes (10(5) to 10(6) cells per seedling), there was no immediate clumping of R. trifolii at clover root hair tips, although polar binding of bacteria along the root hair surface was observed after 4 h. The interface between polarly attached bacteria and the root hair cell wall was shown to contain trifoliin A by immunofluorescence microscopy. Also, this interface was shown by transmission electron microscopy to contain electron-dense granules of host origin. Scanning electron microscopy revealed an accumulation of extracellular microfibrils associated with the lateral and polar surfaces of the attached bacteria, detectable after 12 h of incubation with seedling roots. At this same time, there was a significant reduction in the effectiveness of 2-deoxy-D-glucose in dislodging bacteria already attached to root hairs and an increase in firm attachment of bacteria to the root hair surface, which withstood the hydrodynamic shear forces of high-speed vortexing. These results are interpreted as a sequence of phases in attachment, beginning with specific reversible interactions between bacterial and plant surfaces (phase I attachment), followed by production of extracellular microfibrils which firmly anchor the bacterium to the root hair (phase 2 adhesion). Thus, attachment of R. trifolii to clover root hairs is a specific process requiring more than just the inherent adhesiveness of the bacteria to the plant cell wall.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Characterization of the anomalous infection and nodulation of subterranean clover roots by Rhizobium leguminosarum 1020

Anomalous nodulation of Trifolium subterraneum (subterranean clover) roots by Rhizobium leguminosarum 1020 was examined as a model of modified host-specificity in a Rhizobium-legume symbiosis. Consistent with previous reports, these nodules (i) appeared most often at sites of secondary root emergence, (ii) were ineffective in nitrogen fixation and (iii) were as numerous as nodules formed by an effective Rhizobium trifolii strain. R. leguminosarum 1020, grown on agar plates or in the clover root environment, did not bind the white clover lectin, trifoliin A. This strain did not attach in high numbers, and did not induce shepherd's crooks or infection threads, in subterranean clover root hairs. However, R. leguminosarum 1020 did cause branching, moderate curling and other deformations of root hairs. The bacteria probably entered the clover root through breaks in the epidermis at sites of lateral root emergence. The anomalous nodulation was inhibited by nitrate. Only trace amounts of leghaemoglobin were detected in the nodules by Western blot analysis. The nodules were of the meristematic type and initially contained well-developed infection, bacteroid and senescent zones. Infection threads were readily found in the infection zone of the nodule. However, the bacteroid-containing tissue senesced more rapidly than in the effective symbiosis between subterranean clover and R. trifolii 0403. This anomalous nodulation of subterranean clover by R. leguminosarum 1020 suggests a naturally-occurring alternative route of infection that allows Rhizobium to enlarge its host range.     

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.     

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.     

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.     

Isolation and characterization ofSporomusa acidovorans sp. nov., a methylotrophic homoacetogenic bacterium

Exopolysaccharides (EPS) of nodulating strains of Rhizobium trifolii and Rhizobium leguminosarum added to red clover seedlings before inoculation reduced the number of nodules. The inhibition of the nodulation was correlated with the amount of EPS. The preparations of EPS from mutants defective in early stages of nodulation (Roa^- or Hac^-) did not affect the nodulation, whereas EPS from mutants deficient in late stages (post Hac^-) exerted an inhibitory effect.Inactive preparation of EPS contained less O-acetyl groups and pyruvic acid residues. Deacetylation and depyruvylation of EPS from R. trifolii Nod⁺ abolished it inhibitory effect. It was concluded that noncarbohydrate substitutions (acetate, pyruvate) are involved in EPS effect.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - LPS lipopolysaccharide - Nod nodulation - Fix nitrogen fixation - Hac root hairs curling - Roa root adhesion     

Reexamination of the presence and linkage of 3-hydroxybutyryl substituents in the acidic capsular polysaccharide of Rhizobium trifolii 0403.

We resolved previous conflicting results concerning the presence of 3-hydroxybutyryl substituents on the extracellular acidic polysaccharide from Rhizobium trifolii 0403. These substituents were indeed present in the polysaccharide and in the oligosaccharide fragments obtained by hydrogen fluoride solvolysis of the extracellular and capsular polysaccharides of the bacteria grown on plates. The 3-hydroxybutyrate substituent could be removed from the polysaccharide by 10 mM sodium deuteroxide without evidence of elimination, indicating that this substituent was ester linked.     

Host-range related structural features of the acidic extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum

Proton nuclear magnetic resonance (1H NMR) and fast atom bombardment mass spectrometric analyses were performed on enzymatically derived oligosaccharides from the acidic excreted polysaccharides (EPS) from representative bacterial strains of the pea-nodulating symbiont, Rhizobium leguminosarum (128C53, 128C63, and 300) and the clover-nodulating symbiont, Rhizobium trifolii (NA-30, ANU843, 0403, TA-1, LPR5035, USDA20.102, and 4S). The results revealed structural similarities and differences between EPS of these two species. Octasaccharide units containing galactose, glucuronic acid, alpha-L-threo-hex-4-enopyranosyluronic acid, and glucose in a molar ratio of 1:1:1:5 were obtained from the EPS of the three R. leguminosarum strains and had the same primary glycosyl sequence and location of pyruvate, acetate, and 3-hydroxybutyrate substituents. About 80% of the galactose residues were acylated with 3-hydroxybutyrate, and there were two acetyl groups per repeating unit distributed between the 2 glucose residues of the main chain-derived sequence of the octasaccharides. In contrast, the R. trifolii strains had varied EPS structures, each of which differed from the common R. leguminosarum EPS structure. The EPS from one group of R. trifolii strains (0403 and LPR5035) most closely resembled the R. leguminosarum EPS but differed in that a lower number of galactose and glucose residues were substituted by 3-hydroxybutyryl and acetyl groups, respectively. The EPS from a second group of R. trifolii strains (ANU843, TA-1, and NA-30) was even more different than the R. leguminosarum EPS. These R. trifolii octasaccharides bore a single acetyl group on O-3 of the glucuronic acid residue. In addition, the level of acylation by 3-hydroxybutyryl groups was 50% of that present in the R. leguminosarum EPS. The remaining two strains of R. trifolii (USDA20.102 and 4S) had very different patterns of acylation to each other and to all of the other strains. The EPS from strain USDA20.102 practically lacked 3-hydroxybutyryl groups and had a unique degree and pattern of acetylation. The oligomers from the EPS of R. trifolii strain 4S completely lacked 3-hydroxybutyryl groups and galactose. The latter EPS contained only one O-1-carboxyethylidene group and had a different degree and pattern of acetylation. Interestingly, these two latter strains differ from the other R. trifolii strains in nodulation rates on rare clover species in the Trifolium cross-inoculation group. Thus, we define several groups of R. trifolii based upon their EPS structures and establish their similarities and distinct differences with the EPS of R. leguminosarum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Presence of trifoliin A,a rhizobium-binding lectin,in clover root exudate

Trifoliin A, a Rhizobium-binding glycoprotein from white clover, was detected in sterile clover root exudate by a sensitive immunofluorescence assay employing encapsulated cells of Rhizobium trifolii 0403 heat-fixed to microscope slides. Its presence in root exudate was further examined by immunoaffinity chromatography. The binding of trifoliin A to cells was specifically inhibited by the hapten, 2-deoxyglucose. Significantly higher quantities of trifoliin A were detected in root exudate of seedlings grown hydroponically in nitrogen-free medium than in rooting medium containing 15 mM NO, a concentration which completely suppressed root hair infection by the nitrogen-fixing symbiont. The presence of trifoliin A in root exudate may make it possible for recognition processes to occur before the microsymbiont attaches to its plant host.     

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.     

Lectin involvement in root-hair tip adhesion as related to the Rhizobium-clover symbiosis

Contact of adjacent root hairs of seedlings of white clover ( Trifolium repens L. cv. Ladino and Louisiana Nolin) led to cell-cell adhesion of root hair tips. The involvement of the root lectin, trifoliin A, in this phenomen was examined in slide cultures of axenically grown seedlings. Trifoliin A was detected by indirect immunofluorescence on root hair tips, which had adhered to one another. Seedlings grown under conditions which specifically reduce the levels of this lectin on the root surface (e.g., in the presence of 15 m M NO₃– or 5 m M 2-deoxy- d -glucose) had significantly fewer adhesions of root hair tips. In addition, flushing the slide cultures with 20 m M 2-deoxy- d -glucose resulted in an immediate 4-fold reduction in frequency of tip adhesions. These results are consistent with the lectin cross-bridging model, which predicts that cell-cell adhesions would occur when trifoliin A on root hair tips contacts complementary glycosylated receptors on neighboring root hairs.     

Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizobium-clover association.

Cross-reactive antigens of clover roots and Rhizobium trifolii were detected on their cell surfaces by tube agglutination, immunofluorescent, and radioimmunoassay techniques. Anti-clover root antiserum had a higher agglutinating titer with infective strains of R. trifolii than with noninfective strains. The root antiserum previously adsorbed with noninfective R. trifolii cells remained reactive only with infective cells, including infective revertants. When adsorbed with infective cells, the root antiserum was reactive with neither infective nor noninfective cells. Other Rhizobium species incapable of infecting clover did not demonstrate surface antigens cross-reactive with clover. Radioimmunoassay indicated twice as much antigenic cross-reactivity of clover roots and R. trifolii 403 (infective) than R. trifolii Bart A (noninfective). Immunofluorescence with anti-R. trifolii (infective) antiserum was detected on the exposed surface of the root epidermal cells and diminished at the root meristem. The immunofluorescent crossreaction on clover roots was totally removed by adsorption of anti-R. trifolii (infective) antiserum with encapsulated infective cells but not with noninfective cells. The cross-reactive capsular antigens from R. trifolii strains were extracted and purified. The ability of these antigens to induce clover root hair deformation was much greater when they were obtained from the infective than noninfective strains. The cross-reactive capsular antigen of R. trifolii 403 was characterized as a high-molecular-weight (greater than 4.6 times 10(6) daltons), beta-linked, acidic heteropolysaccharide containing 2-deoxyglucose, galactose, glucose, and glucuronic acid. A soluble, nondialyzable, substance (clover lectin) capable of binding to the cross-reactive antigen and agglutinating only infective cells of R. trifolii was extracted from white clover seeds. This lectin was sensitive to heat, Pronase, and trypsin. inhibition studies indicated that 2-deoxyglucose was the most probable haptenic determinant of the cross-reactive capsular antigen capable of binding to the root antiserum and the clover lectin. A model is proposed suggesting the preferential adsorption of infective versus noninfective cells of R. trifolii on the surface of clover roots by a cross-bridging of their common surface antigens with a multivalent clover lectin.