Clover root exudate contains a particulate form of the lectin, trifoliin A, which binds Rhizobium trifolii

Abstract

A particulate form of the lectin, trifoliin A, has been isolated from the root exudate of axenically grown seedlings of white clover (Trifolium repens L. cv. Louisiana Nolin). The majority of trifoliin A active in binding to R. trifolii 0403 was sedimented from root exudate by centrifugation at 15 000 g for 30 min, indicating that it was particulate. Immunofluorescence, electron and immunoelectron microscopy using antibody prepared against trifoliin A from seeds, suggested that trifoliin A was associated with the particles in root exudate that bound specifically to the acidic capsular polysaccharides of Rhizobium trifolii 0403. Electron microscopic examination also showed that trifoliin A‐colloidal gold conjugates bound to these same particles, indicating that they also have affinity for the lectin. The particles could be dislodged from intact seedlings by vigorous shaking in isotonic plant growth medium. Isolated particles fractionated by ultracentrifugation through a metrizamide gradient had a mean density of 1.12 g cm‐3. These isolated particles retained the ability to bind to R. trifolii 0403 as shown by immunofluorescence using anti‐trifoliin A antibody. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the isolated particles are a mixture of proteins including one with an approximate molecular weight of trifoliin A. The protein which electrophoretically comigrated with trifoliin A also reacted with anti‐trifoliin A antibody by western blot analysis, confirming that it was trifoliin A. The protein composition of the isolated particles did not reflect the total protein composition of the root exudate, which was far more complex. These studies indicate that the majority of trifoliin A which can bind to R. trifolii in root exudate of white clover seedlings is associated with electron‐dense particles, and that a major early interaction between R. trifolii and clover roots involves the binding of these particles containing trifoliin A to acidic polymers on the bacterial cell surface.