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Calcium is essential for the growth of rhizobia and the formation of nitrogen-fixing root-nodules on legumes, but its precise role in these processes remains unknown. We have found that Rhizobium fredii USDA208 accumulates a major 38 kDa protein when grown in media supplemented with 0.3-2 microM Cacl2. We have purified this protein and raised polyclonal antibodies against it. The protein initially is synthesized as a 40 kDa precursor which subsequently undergoes calcium-dependent processing to give rise to the mature polypeptide. Subcellular and immunocytochemical localization studies indicate that the 38 kDa protein accumulates preferentially in the periplasmic space. Its N-terminal sequence, AETIKIGVAGPMTG, shows significant homology to the N-termini of amino acid binding proteins from the periplasm, including leucine-, isoleucine-, and valine-specific binding proteins of Pseudomonas aeruginosa and Escherichia coli and a leucine-specific binding protein of E. coli. The R. fredii protein does not, however, bind [3H]-leucine. The 38 kDa protein is encoded by the bacterial chromosome. It is absent in several rhizobia other than R. fredii, but antigenically related polypeptides are present in Escherichia coli and Erwinia carotovora subsp. carotovora.

We have detected strong homology between a 9.2-kb EcoRI restriction fragment from Rhizobium fredii USDA257 that contains nodABC and eight additional EcoRI fragments in DNA digests from this organism. A series of repetitive sequences responsible for this hybridization lies within a 0.95-kb HindIII/SalI subfragment about 1-kb upstream of nodA. This subfragment also hybridizes to multiple restriction fragments from nine other strains of R. fredii, but only one is common to all strains. The 0.95-kb subfragment does not hybridize to genomic DNA from 17 other strains of fast-growing rhizobia, but there is weak homology to two fragments from Rhizobium sp. strain NGR234. We sequenced 2,432 base pairs (bp) of the region encompassing the repetitive sequences. It contains 65 separate 8- to 11-bp inverted and direct repeats, as well as two large open reading frames (ORFs) that overlap on opposite strands. ORF1 reads in the same direction as nodABC, contains 1,071 bp, and encodes a 40.6-kD protein. It has 74% sequence homology to an ORF within the T-DNA of Agrobacterium rhizogenes and similar homology to a series of repetitive sequences from Bradyrhizobium japonicum. ORF2 (981 bp) reads in the opposite direction, encodes a 34.7-kD protein, and has partial identity with a second ORF from A. rhizogenes. We could detect no poly(A)+ nodule transcripts with homology to ORF1 and ORF2. The eight sets of repetitive sequences found in other EcoRI fragments of the genome were cloned from USDA257 on separate cosmids. Some of these cosmids appear to overlap, and two have fragments with homology to nifKDH.

We characterized five isolates of Agrobacterium tumefaciens from naturally occurring galls on Chrysanthemum morifolium. The isolates are similar, possibly identical, members of a single strain of A. tumefaciens that we designate Chry5. The strain is a biotype I, as indicated by its response to both newly described and traditional biotype tests. Chry5 produces tumors on at least 10 plant species. It is unusual in its ability to form efficiently large tumors on soybean (Glycine max), a species normally refractory to transformation. Chry5 is unable to utilize octopine or mannopine as a carbon source. Although Chry5 can catabolize a single isomer each of nopaline and succinamopine, it differs from other known nopaline and succinamopine strains in its insensitivity to agrocin 84. This pattern of opine catabolism is unique among Agrobacterium strains examined to date. All five isolates of Chry5 contain at least two plasmids, one of which shares homology with pTiB6.

We cloned and analyzed nodABC from Rhizobium fredii USDA257. These genes are thought to have common functions in initiation of nitrogen-fixing nodules by all rhizobia. In USDA257, they were located in a 9.2-kb EcoRI fragment that was not closely linked to either of two copies of the regulatory gene, nodD. nodABC was present in a 3,094-base pair (bp) sequenced region, which also included a consensus nod-box promoter. The three open reading frames contained 654, 642, and 1,239 bp, respectively, and encoded deduced proteins of 21.9, 23.4, and 44.7 kD. The sequence of the nodABC region of USDA257 was generally homologous with corresponding regions from other rhizobia, but it diverged significantly in the 5' non-translated region and in the 3'terminus of nodC. nodC was not translationally coupled to nodSU, as in another soybean symbiont, Bradyrhizobium japonicum, and the deduced NodC protein was the shortest of any such proteins yet described. Site-directed mutagenesis of the 9.2-kb EcoRI fragment confirmed that nodA, nodB, and nodC are essential for nodulation of soybean, but failed to identify other linked nod genes. Daidzein, a major isoflavone from soybean roots, was the most potent of nine tested flavonoids in activating a plasmid-borne nodC::lacZ fusion. The 9.2-kb fragment complemented nodA-, nodB-, and nodC- mutants of R. meliloti to the Nod+ phenotype on Medicago sativa, M. truncatula, and Trigonella foenum-graecum. Nodule numbers, percentage of nodulated plants, and shoot dry weights, however, were considerably less than in plants inoculated with mutants complemented with nodABC from R. meliloti.

We screened a total of 1365 pea (Pisum sativum) lines for response to inoculation with Agrobacterium tumefaciens, strain B6, and characterized resistance in one cultivar, Sweet Snap. Sweet Snap seedlings were highly resistant to tumorigenesis under most conditions. Resistance was overcome at inoculum concentrations of greater than 10(9) bacteria per milliliter. At such high concentrations, very small tumors developed on Sweet Snap in response to four wide-host-range Agrobacterium strains, but tumors on other cultivars were two-to sevenfold larger than those that formed on Sweet Snap. The hypervirulent strain A281 induced larger tumors on Sweet Snap than did other Agrobacterium strains, but tumors on other genotypes were more than 100% larger than those on Sweet Snap. Physiological experiments suggested that tumorigenesis in Sweet Snap is not blocked in early stages of infection, and genetic analysis indicated that inheritance of resistance to crown gall is a quantitative trait. In addition to the observed resistance in Sweet Snap, three ;supersusceptible' genotypes, which developed very large tumors, also were identified.

Rhizobium fredii USDA257 forms nitrogen-fixing nodules on soybean cultivar Peking, but not on cultivar McCall. This pattern of nodulation persists when McCall and Peking seedlings are cultivated together in plastic growth pouches. Reciprocal grafting experiments confirm that the root genotype, and not that of the shoot, regulates such cultivar specificity. When Peking roots are grafted onto McCall seedlings, the nodulation responses of roots similarly remain unaffected. Transposon-mutant 257DH4, which is derived from USDA257, can form nitrogen-fixing nodules on McCall. Such nodulation is blocked by the presence of USDA257 in the inoculum. Grafting experiments indicate that blocking is not due to a translocatable inhibitor produced by McCall roots or triggered by their interaction with USDA257. Thus, neither freely diffusible nor graft-transmissible substances are involved in cultivar-specific interactions of soybean with R. fredii and its derivatives.

Rhizobium fredii USDA257 fails to nodulate the improved soybean [Glycine max (L.)Merr.] cultivar McCall in plastic growth pouches. Mutant 257DH4, which was derived from USDA257 by transposon mutagenesis, forms nitrogen fixing nodules under these conditions. If USDA257 is present in inocula containing the mutant, most infections are arrested prior to organization of the nodule meristem, and nodule number is reduced by 95%. The improved cultivars Essex, Harosoy, Hodgson 78, and Viçoja, as well as a supernodulating mutant of Williams, respond like McCall to inoculation with such mixtures of bacteria. Nodulation blocking on McCall can be elicited by rhizobia other than USDA257, provided that they meet two criteria: Blocking strains must themselves be able to induce cortical cells of McCall to divide, and such divisions must proceed to the stage of nodule meristem formation. Nodulation by the mutant remains sensitive to a challenge inoculation with USDA257 for only the first 6 to 12 hours after inoculation. Nodulation blocking involving mutant 257DH4 thus appears to be a rapid, generalized process.

Inoculation of 2-day-old cowpea (Vigna unguiculata [L.] Walp.) seedlings with Rhizobium fredii USDA257 results in proficient nodulation of the tap root. The most abundant nodulation occurs in a region roughly corresponding to the position of the root tip at the time of inoculation. We have examined plant gene expression in this region, after inoculation with either USDA257 or a nonnodulating mutant, 257B3. After isolation of mRNA and in vitro translation, the protein products were separated by two-dimensional gel electrophoresis. Seven proteins are induced within 2.5 days after inoculation with USDA257. One additional induced protein is detectable by 3.5 days after inoculation, and three more appear by day 6. Three of the proteins that are differentially expressed at 2.5 and 3.5 days after inoculation are produced at equivalent levels after 6 days, indicating transient induction of these genes during early stages of nodule development. Several proteins were more abundant in translations of mRNA from roots that had been inoculated with the nonnodulating mutant. This was particularly true after 6 days, when nine proteins were in this class. Thus, altered plant gene expression in carefully selected, highly responsive tissue can be detected 2 days before emerging nodules are visible on the roots, and 6 to 7 days before acetylene reduction is detectable. Additionally, comparisons of ionically bound cell wall proteins isolated 6 days after inoculation revealed four that were unique to nodulating roots, suggesting that some of the nodulation-induced genes may code for structural proteins.

Rhizobium fredii USDA257 does not nodulate McCall soybean (Glycine max (L.) Merr.), but two transposon-mutants derived from it, 257DH4 and 257DH5, do. All three organisms cause curling of McCall root hairs and induce the formation of underlying cortical cell divisions. The mutants produce infection threads, and many of the meristematic foci develop into nodules. In contrast, root hairs that deform in response to USDA257 lack infection threads, and meristematic activity ceases prior to the appearance of nodule meristems. Root systems nodulated by mutant 257DH4 reduce acetylene at rates similar to those of roots nodulated by reference R. fredii strain USDA191. The presence of living cells of USDA257 in inocula leads to strong inhibition of nodulation by 257DH4 but not by 257DH5. This blocking effect depends on the ratio of USDA257 cells to 257DH4 cells in the inoculum; nodules that form contain cells of 257DH4, but not those of parental strain USDA257.

Double inoculation (15 h apart) of the soybean cultivar Williams with Bradyrhizobium japonicum I-110ARS reveals a rapid regulatory plant response that inhibits nodulation of distal portions of the primary root (M Pierce, WD Bauer 1984 Plant Physiol 73: 286-290). Only living, homologous rhizobia elicit the response. We conducted similar double inoculation experiments to test the hypothesis that this is a universal phenomenon in soybean symbioses. We investigated interactions of the cultivar McCall with the slow-growing strain Bradyrhizobium sp. 3185 (=3G4b16) and strains of the fast-growing soybean symbiont, Rhizobium fredii (USDA191 [Nod(+) on McCall] and USDA257 [Nod(-) on McCall]). Nodulation was not detectably inhibited when USDA257 was included in various combinations with an inoculum of USDA191. Strain USDA257 cohabited nodules with strain USDA191 when plants were inoculated sequentially with both strains, but USDA257 did not nodulate McCall when a sterile culture filtrate of USDA191 was added to USDA257 inoculum. There was only a slight inhibition of nodulation of distal portions of the primary root in double inoculation experiments with McCall and strain 3185. Because these results were unexpected, we repeated the experiments with Williams and strain I-110ARS. The response was similar to that observed in the McCall x 3185 interaction. Regulation of nodulation on the primary root thus appears to be variable and depend on strain X cultivar interactions.

Heat shock arrests the synthesis of many cellular proteins and simultaneously initiates expression of a unique set of proteins, termed heat shock proteins. We have found that heat shock rapidly triggers phosphorylation of a set of proteins in soybean seedlings. Although the kinetics of phosphorylation and the heat shock response are similar, the major identified phosphorylation products do not comigrate with heat shock proteins on polyacrylamide gels. Cadmium, which is known to induce the heat shock response, stimulates phosphorylation of the same set of proteins. The rapidity of phosphorylation suggests that it may play a pivotal role in sensing and transducing elevated temperature stress in plants.

Seeds of six soybean lines (Glycine max (L.) Merr. cv. Columbia, D68-127, Norredo, Sooty, T-102, Wilson 5) have been reported to lack the 120 000 dalton soybean lectin. Immunodiffusion and radioimmunoassay using anti-soybean lectin immunoglobulin failed to detect the lectin in seeds of five lines, but D68-127 seeds contained as much soybean lectin as the control line, Harosoy 63. The D68-127 seed lectin could be purified by affinity chromatography on Sepharose-N-caproylgalactosamine, and was indistinguishable from the conventional soybean lectin by the following criteria: electrophoretic migration in acidic and alkaline buffers, subunit molecular weight and composition, analytical isoelectric focusing, gel filtration chromatography. Phosphate-buffered saline extracts of roots, hypocotyls, stems, and leaves of 3--66-day-old Norredo and Harosoy 63 plants lacked soybean lectin, as determined by hemagglutination and radioimmunoassay (detection limit: 1.4 micrograms soybean lectin/g dry weight tissue). Cotyledons of Harosoy 63 (but not Norredo) contained large quantities of the lectin, which diminished as the plants aged. 5-day-old roots and hypocotyls of 20 soybean lines did not contain soybean lectin. Roots of Columbia, Norredo, Sooty, T-102, Wilson 5, and Harosoy 63 (control) were nodulated by a variety of strains of Rhizobium japonicum and Rhizobium sp.

Peanut lectin was purified from seed meal of the Spanish and Jumbo Virginia varieties of peanut (Arachis hypogaea L.) by affinity chromatography on lactose coupled to Sepharose 4B. Polyacrylamide gel isoelectric focusing resolved the lectin preparation from Jumbo Virginia seeds into seven isolectins (pI 5.7, 5.9, 6.0, 6.2, 6.3, 6.5, and 6.7). Seed meal from the Spanish variety contained six isolectins which were indistinguishable from the pI 5.7, 5.9, 6.2, 6.3, 6.5, and 6.7 isolectins from Jumbo Virginia. Quantitative, lactose-specific hemagglutination was used to examine the lectins in tissues of both peanut varieties. In young (3- to 9-day-old) seedlings of each variety, more than 90% of the total amount of lectins detected in the plants was in the cotyledons. Most of the remainder was in hypocotyls, stems, and leaves; young roots contained no more than 4 micrograms of lectin per plant. Lectins were present in all nonroot tissues of 21- to 30-day-old seedlings, except 27-day-old Spanish hypocotyls. As cotyledons of each variety senesced, several of the more basic isolectins decreased to undetectable levels, but the acidic isolectins remained until at least 15 days after planting. Some of the seed isolectins and several apparently new lactose-binding lectins were also identified in affinity-purified extracts of 5-day-old roots and hypocotyls. Rabbit antibodies raised against the Jumbo Virginia seed isolectin preparation reacted with seed, cotyledon, and hypocotyl lectin preparations from both varieties. Analysis of seed lectin preparations from seven varieties of A. hypogaea and of a related species (A. villosulicarpa) indicated that isolectin composition in Arachis may be a characteristic of both the species and the subspecies (botanical type) to which the variety belongs.

Inoculation with Rhizobium japonicum or R. meliloti reduced the electrical transmembrane potential (E(m)) of soybean (Glycine max [L.] Merr.) root cells within 1 day. The response could be attributed to altered diffusion potential (E(D)). E(m) values return to control levels by the second day after inoculation, but again were reduced in R. meliloti-inoculated tissue on the seventh day. Increased concentrations of sodium phosphate in the perfusion solution magnified the effects of inoculation on E(m). Neither heat-killed rhizobia nor living cells of Pseudomonas fluorescens elicited the response. The E(m) and E(D) of nodule cells were nearly 20% lower than corresponding values from adjacent cortical cells of the root.

Membrane fractions from seedlings of four soybean [Glycine max (L.) Merr.] lines were examined by radioimmunoassay and hemagglutination assay for the 120,000 dalton soybean lectin. Two of the lines (Sooty and T-102) are genotypically lele and lack buffer-soluble soybean lectin; the remaining two lines (Beeson and Harosoy 63) are Le and produce seeds that contain the lectin (Su et al. 1980 Biochim. Biophys. Acta 629: 292-304). Both Triton X-100 (0.5% v/v) and nonidet P-40 (0.05% v/v) solubilized soybean lectin from membrane fractions of Le cotyledons. Triton X-100 interfered substantially with the assay of protein and hemagglutinating activity and was unacceptable for use in quantitative measurements. The nonidet P-40-solubilized soybean lectin from Le cotyledons was consistently present both in washed 13,000g and 82,500g membrane fractions, but it accounted for less than 1.5% of the total (buffer-soluble plus membrane-bound) soybean lectin. The membrane lectin was purified by the affinity chromatography procedure devised for soluble soybean lectin, and it was immunologically indistinguishable from authentic soybean lectin. Membrane fractions from Le cotyledons contained insignificant amounts of radioisotope-labeled soybean lectin that had been added during homogenization, and purified membrane fractions did not bind the lectin in the presence of the hapten, d-galactose. These controls make it unlikely that the membrane soybean lectin was of cytoplasmic origin. Soybean lectin and other hemagglutinins were not present in buffer-soluble or membrane fractions from lele cotyledons or from roots and hypocotyls of any of the lines.

We developed a quantitative assay to measure tumorigenesis on roots and root crowns, the natural sites of Agrobacterium tumefaciens infection. Efficiency of tumor formation and tumor weight on seedlings of Pisum sativum ;Little Marvel' were directly proportional to the logarithm of inoculum concentration. Depth of wounding prior to inoculation also significantly influenced tumor weight but not efficiency. Mean weight of tumors that developed in response to inoculation with strain B6 varied significantly among 34 different commercial cultivars. Tumors on the most susceptible cultivar, Target, were more than tenfold heavier than those formed on the least susceptible cultivar, Sweet Snap. Efficiency of tumorigenesis on ;Sweet Snap' was also relatively low: only 64% of inoculated seedlings developed tumors compared with 89 to 100% efficiencies for all other cultivars.

Adsorption of Agrobacterium tumefaciens cells to potato tuber disks reached equilibrium after coincubation for about 30 min. More than 10% of the number of bacteria bound at equilibrium were adsorbed within 30 s. Adsorption isotherms obtained at three temperatures showed that the heat of adsorption was nearly zero.

Interactions of soybean with Bradyrhizobium japonicum 123 (serogroup 123) and 138 (serogroup c1) were used to examine the relationship between early infection rates, competition for nodulation, and patterns of nodule occupancy. Both strains formed more infections in autoclaved soil (sterile soil) than in untreated soil (unsterile soil). Inoculation did not increase numbers of infection threads in unsterile soil-grown plants, where infection of proximal portions of primary roots was complete by 5 days after planting. Both strains infected and nodulated at similar rates in sterile soil. Nodules were always clustered on the upper root system, regardless of inoculation and soil treatment. Sixty-seven percent of the nodules of uninoculated plants grown in unsterile soil were occupied by rhizobia belonging to serogroups other than 123 or c1. Inoculation with strain 123 or 138 increased occupancy by that strain at the expense of residency by other rhizobia. Eighty-three percent of all nodules on plants dually inoculated with both strains in sterile soil contained strain 138. The corresponding value for plants inoculated in unsterile soil was 31%. Neither inoculum strain dominated occupancy of first-formed nodules in unsterile soil. It appears that north central Missouri soil may not have populations of highly competitive serogroup 123 and that early infection and nodulation rates do not contribute to the competitive success of strain 138.

We evaluated the symbiotic phenotypes of nodulation-restrictive and normal soybean isolines by inoculating Clark (genotypically Rj(1)Rj(1)) and mutant Clark-rj(1) (genotypically rj(1)rj(1)) seedlings in plastic growth pouches. Nodules first appeared on Clark seedlings inoculated with Bradyrhizobium japonicum USDA 94 after 6 days. The mean number of nodules per plant was 13.9 +/- 0.8 after 24 days. In contrast, Clark-rj(1) seedlings first nodulated at 12 days, and the mean number of nodules per plant was only 1.7 +/- 0.3 at 24 days. Segments from infectible zones of primary roots, i.e. near the position occupied by the root tip at the time of inoculation, were sectioned serially. Clark roots contained cortical cell divisions and a few infection threads in question mark-shaped root hairs by 2 days after inoculation. Typical nodules developed soon thereafter. Analogous serially sectioned segments from Clark-rj(1) roots lacked these responses. This prompted us to section nodules and adjacent tissues from other parts of Clark and Clark-rj(1) roots. Clark roots contained cortical cell divisions, many associated with infected root hairs. Cortical cell divisions occasionally were present in Clark-rj(1), and a few infection threads were visible in surface cells. The presence of infection threads within Clark-rj(1) nodules was confirmed by transmission electron microscopy. Thus, although B. japonicum USDA 94 fails to elicit the wild-type spectrum of responses in the infectible zones of primary roots, it can infect Clark-rj(1) via infection threads.

Several strains of Rhizobium japonicum have been reported to consist of mixtures of stable derivativess having distinct colony morphologies and physiological characteristics. We isolated derivatives from strains of R. japonicum and systematically compared them with previously isolated derivatives with respect to the utilization of biochemicals, antibiotic sensitivity, and soyben lectin binding. With the exception of a pair of derivatives from 3Ilb110, one of which utilized pyruvate and one of which did not, sibling derivatives had essentially identical biochemical utilization patterns. The sibling derivatives of parental strains 3Ilb110 and 3Ilb140 exhibited marked variation in their sensitivities to several antibiotics, including gentamicin, sul famethoxazole, and tetracycline. Compared with the derivative with small colony morphology, derivatives with large colony morphology were in general more sensitive to these antibiotics. With one exception, the binding of soybean lectin to the derivatives was quantitatively the same as that to the parental strain. The anomaly was 110-Y which, in contrast to its parental strain and sibling derivatives, failed to bind detectable amounts of the lectin. 110-Y, as well as all the other derivatives and parental strains, nodulated Disoy soybean.

Seeds of 102 lines of Glycine max (L.) Merr., the soybean, were screened quantitatively for the presence of the 120,000-dalton soybean lectin. Wide variation in the content of this lectin was noted, and five lines of soybean whose seed totally lacked the lectin were identified. Roots of all five lines were effectively nodulated by several strains of Rhizobium japonicum, thus indicating that the 120,000-dalton soybean seed lectin is probably not required for the initiation of soybean-Rhizobium symbiosis.

Three different assay procedures have been used to quantitate the levels of soybean (Glycine max [L.] Merr.) lectin in various tissues of soybean plants. The assays used were a standard hemagglutination assay, a radioimmunoassay, and an isotope dilution assay. Most of the lectin in seeds was found in the cotyledons, but lectin was also detected in the embryo axis and the seed coat. Soybean lectin was present in all of the tissues of young seedlings, but decreased as the plants matured and was not detectable in plants older than 2 to 3 weeks. Soybean lectin isolated from seeds of several soybean varieties were identical when compared by several methods.

Cells of tumorigenic Agrobacterium tumefaciens strain B6 were labeled with [35S]methionine and used to estimate bacterial adsorption to potato tuber discs. After 10 min at pH 7.2, about 5 X 10(5) bacteria adsorb to each 9.0-mm disc. Lengthening the incubation period to 90 min increases adsorption to about 11 X 10(5) bacteria per disc. Bacterial adsorption is reduced under both alkaline and acid pH conditions and increases 2.3-fold if the number of bacterial cells in the inoculum is doubled. Adsorption is increased if citrus pectin, polygalacturonic acid, or demethylated pectin is included in the inoculum; methylated polygalacturonic acid is inactive. The activity of citrus pectin is abolished, however, if the compound is applied to the discs as a pretreatment and then removed prior to inoculation. Lipopolysaccharide preparations from five of six A. tumefaciens strains have no effect on bacterial adsorption. The sixth preparation, from nontumorigenic strain NT-1, reduces adsorption by about 20%.

Highly purified soybean lectin (SBL) was labeled with fluorescein isothiocyanate (FITC-SBL) or tritium ((3)H-SBL) and repurified by affinity chromatography. FITC-SBL was found to bind to living cells of 15 of the 22 Rhizobium japonicum strains tested. The lectin did not bind to cells of the other seven R. japonicum strains, or to cells of any of the nine Rhizobium strains tested which do not nodulate soybean. The binding of the lectin to the SBL-positive strains of R. japonicum was shown to be specific and reversible by hapten inhibition with d-galactose or N-acetyl-d-galactosamine.The lectin-binding properties of the SBL-positive R. japonicum strains were found to change substantially with culture age. The percentage of cells in a population exhibiting fluorescence after exposure to FITC-SBL varied between 0 and 70%. The average number of SBL molecules bound per cell varied between 0 and 2 x 10(6). While most strains had their highest percentage of SBL-positive cells and maximum number of SBL-binding sites per cell in the early and midlog phases of growth, one strain had a distinctly different pattern. The SBL-negative strains did not bind lectin at any stage of growth.Quantitative binding studies with (3)H-SBL indicated that the affinity constant for binding of SBL to its receptor sites on R. japonicum is approximately 4 x 10(7)m(-1). Many of the binding curves were biphasic. An inhibitor of SBL binding was found to be present in R. japonicum culture filtrates.

Potato tuber disks were submerged in suspensions containing 10 to 10 cells of Agrobacterium tumefaciens B6 per ml. After 60 min, the disks were rinsed and homogenized, and portions of the homogenates were plated to measure the number of adsorbed bacteria. At low initial bacterial concentrations (10/ml), 5 to 23% of the bacteria adsorbed. At higher bacterial concentrations, the corresponding value was approximately 1.2%. Adsorption was a reversible equilibrium process. Binding saturation was not achieved, and adsorbed bacteria were confined to monolayers on the surfaces of tissue prepared for scanning electron microscopy. Adsorption of strain B6 to potato tuber tissues is described accurately by the Freundlich adsorption isotherm and may be a nonspecific phenomenon.

A lectin from seeds of the winged bean, Psophocarpus tetragonolobus (L.)DC, has been purified to homogeneity by affinity chromatography on a Bio-Gel lactobionate adsorbent. The lectin is rich in acidic amino acids, and after isoelectric focusing in polyacrylamide gel, appeared as a single band of pI5.5 The molecular weight of the lectin is 46 000 +/- 2000 as determined by analytical ultracentrifugation. Dodecyl sulfate gel electrophoresis detected a single type of subunit with a molecular weight of 29 000 +/- 3000. The lectin has specificity for receptors containing D-galactose and agglutinates trypsinized human red blood cells of all three ABO types. Of the tested inhibitors of hemagglutination caused by winged bean lectin, p-nitrophenyl-beta-D-galactoside was the most potent. Lactose was the most active oligosaccharide tested, and other oligosaccharides containing alpha-linked D-galactose were much less inhibitory.

Four of 14 strains of Rhizobium japonicum from soybean nodulated peanut (Arachis hypogaea L. cultivar Jumbo Virginia), and 3 of 8 Rhizobium sp. strains from peanut nodulate soybean (Glycine max (L.) Merr. cultivar Harosoy 63). Cells of three peanut rhizobia bound fluorescent- and radioisotope-labeled soybean lectin. Two of these strains failed to nodulate soybean, and conversely, two peanut strains that nodulated soybean did not bind to soybean lectin. Both culture medium and age had pronounced effects of the number of peanut rhizobia cells that bound fluorescent-labeled soybean lectin. Harosoy 63 soybean root exudates stimulated the growth of peanut rhizobia, but had no consistent influence on the number of cells that bound soybean lectin. Although extracellular soybean lectin receptors were present in culture fluids from each of the peanut rhizobia whose cells bound the lectin, the titer of receptors was greatest for strains 3G4b5. The affinity constants for the adherence of soybean lectin to Rhizobium sp. 3G4b5 cells from cultures of various ages ranged from 4.2 X 10(6) to 4.9 X 10(6) M-1, and the number of lectin binding sites per cell decreased as cells aged. Cells of the soybean and peanut rhizobia did not bind fluorescent- or radioisotope-labeled peanut lectin. The results indicate that there is no relationship between the ability of peanut and soybean rhizobia to nodulate the reciprocal host plant and their ability to bind to the lectin of that plant.

Roots of soybean (Glycine max [L.] Merr. cv Hardee) and cowpea (Vigna unguiculata [L.] Walp. cv Pink Eye Purple Hull) were immersed in suspensions containing 10(4)Rhizobium cells per milliliter of a nitrogen-free solution. After 30 to 120 minutes the roots were rinsed, and the distal 2-centimeter segments excised and homogenized. Portions of the homogenates then were plated on a yeast-extract mannitol medium for bacterial cell counts. The adsorption capacities of four slow-growing rhizobia and a fast-growing R. meliloti strain varied considerably. Adsorption was independent of plant species and of the abilities of the Rhizobium strains to infect and nodulate. R. lupini 96B9 had the greatest adsorption capacity, and Rhizobium sp. 3G4b16 the least. Rhizobium sp. 229, R. japonicum 138, and R. meliloti 102F51 were intermediate, except on cowpea, where the adsorption of strain 102F51 was similar to that of strain 3G4b16. The initial adsorption rates of bacteria cultured in synthetic media and in the presence of soybean roots were about the same. Addition of soybean lectin to the bacterial inoculum failed to influence initial adsorption rates. Both treatments, however, reduced the numbers of bacteria that bound after incubation with roots for 120 minutes. The relationship between the logarithm of the number of strain 138 cells bound per soybean root segment and the logarithm of the density of bacteria in the inoculum was linear over five orders of magnitude. Binding of strain 138 to soybean roots was greatest at room temperature (27 degrees C) and substantially attenuated at both 4 and 37 degrees C. Although R. lupini 96B9 strongly rejected a model hydrophobic plastic surface, there were no simple correlations between bacterial binding to model hydrophobic and hydrophilic plastic surfaces and bacterial adsorption to roots.

Chromosomal virulence (chv) mutants of Agrobacterium tumefaciens have been reported to be deficient in binding to cells of zinnia, tobacco, and bamboo. The mutants are nonpathogenic on stems of Kalanchoë, sunflower, tomato, Jerusalem artichoke, and tobacco, but they cause tumors on tubers of Solanum tuberosum. We used a root cap cell binding assay to test ability of cells from individual plants of 13 different plant species to bind parent or chv mutant bacteria. The same plants were then inoculated to test for disease response. Cells from nine of the plant species were grossly deficient in their abilities to bind mutant bacteria, and the plants inoculated with mutant bacteria failed to form tumors. In contrast, root cap cells as well as root hairs and root surfaces of S. tuberosum, S. okadae, and S. hougasii bound chv mutant bacteria as well as wild type. Nevertheless, S. tuberosum roots inoculated with mutant bacteria did not develop tumors. Although S. okadae plants inoculated with mutant bacteria formed a few tumors, and S. hougasii developed as many tumors in response to chv mutants as in response to the parent strain, the tumors induced by mutant bacteria were smaller.