Incubation of Bradyrhizobium japonicum with the cultured soybean cell line SB-1 resulted in the adhesion of the bacteria to the plant cells. An antiserum was raised against B. japonicum, and the 1251-laheled immunoglobulin fraction was used to quantitate the number of bacteria bound to the soybean cells. The measurement of '251-labeled antibody binding correlated well with parallel assays by microscopic observation.Using this quantitation, we have optimized the parameters of the assay in terms of time course, ratio of B. japonicum to SB-1 cells, and pH. We then explored the effects of saccharides, NaCl, EDTA, and culture age of the bacteria and SB-1 cells on B. japonicum binding under these optimal assay conditions. The results showed good correlation between conditions that govern B. japonicum binding to SB-1 cells in culture and those that regulate B. japonicum-induced nodulation in legume roots. Together, they suggest that this binding event may be important in controlling host specfficity.In previous studies we have shown that incubation of Bradyrhizobium japonicum with the cultured soybean cell line SB-1, originally derived from the roots of Glycine max, resulted in specific adhesion of the bacteria to the plant cells (10). There was correlation between Rhizobium binding and establishment of in vivo symbiosis; only homologous Rhizobium strains that normally nodulate soybean roots bound to the SB-1 cells. In all of these studies, the binding of the bacteria to the plant cell was assayed by microscopic observation. 4 days by transferring 20 ml of cultures to 50 ml of fresh lB5C medium (basic medium plus 1 mg of 2,4-dichlorophenoxyacetic acid and 2 g of casein hydrolysate [pH 6.2] per liter) (10).B. japonicum RllOd was obtained from Barry Chelm, and Rhizobium meliloti 102F28, R. leguminosarum 128C56, and R. trifolii 0403 were obtained from Frank Dazzo. Various Rhizobium strains were cultured on yeast extract-mannitolsodium gluconate medium at 30°C in a gyratory shaker at 100 rpm (4). Inocula were grown to mid-exponential phase. The concentrations of the bacteria were adjusted to 2 x 108 bacteria per ml or otherwise as stated below with 1B5C medium by direct counting under the microscope.The growth curve of B. japonicum was constructed by inoculating 0.5 ml of the bacterial suspension culture to 50 ml of fresh bacterial medium. The culture was maintained at 300C. At different time intervals, 1.0 ml of the bacterial suspension was removed and the A620 was determined.Generation of antibody against B. japonicum. B. japonicum cells at mid-exponential phase were washed three times by centrifugation (10,000 x g for 20 min) and suspension in phosphate-buffered saline (PBS; 10 mM sodium phosphate, 0.137 M NaCl, 3 mM KCl, 1 mM MgCl2, 1 mM CaCl2 [pH 7.4]). The bacteria were suspended in water and heated for 10 min in a sand bath at 97°C. This bacterial sample was kept in the freezer at -20°C in 0.2-ml aliquots (1 mg of protein per ml). One such aliquot (0.2 ml) was diluted to 1 ml with H20, 1 ml of complete Freund adjuva...
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