Abstract
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 125I-labeled immunoglobulin fraction was used to quantitate the number of bacteria bound to the soybean cells. The measurement of 125I-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 specificity.
Highlights
Incubation of Bradyrhizobium japonicum with the cultured soybean cell line SB-1 resulted in the adhesion of the bacteria to the plant cells
We 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
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]
Summary
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). The immunoglobulin bound on the column was eluted with 0.1 M glycine (pH 3.0) and dialyzed against PBS, and the precipitate was removed by centrifugation This immunoglobulin fraction was designated anti-Brj. Anti-Brj was iodinated by the chloramine-T method of Langone [15].
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