Lack of a direct nitrate-trifoliin A interaction in the Rhizobium-clover symbiosis

Abstract

FRANK B. DAZZO and ESTELLE M. HRABAK Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824 USA Received 22 July 1982 Key words Clover Combined nitrogen Lectin Nitrate Nitrogen-13 Rhizobium trifolii Symbiosis Trifoliin A Trifolium repens Summary Nitrate added at critical concentrations to plant growth medium inhibits the infection of legume roots by Rhizobium. The direct interaction of nitrate and trifoliin A, a Rhizobium-recognition lectin from white clover (Trifolium repens L.), was examined as a possible basis for this regulation. Selective molecular ultrafiltration studies to detect ligand-protein interactions showed that radioactive 13NO3- did not bind directly to trifoliin A when incubated at two molar ratios. Immunoprecipitation of trifoliin A by its homologous antibody was unaffected by 15 mM NO3 -. In addition, there was no apparent reduction in attachment of R. trifolii 0403 to root hairs of clover seedlings during l h of incubation in the presence of 15 mM NO 3 -. These results show that nitrate inhibition of these early steps of the infection process is not due to a direct interaction of nitrate with trifoliin A or its glycosylated receptors. Introduction Combined nitrogen is one of the many environmental factors which can suppress the development of the nitrogen-fixing Rhizobium-legume symbiosis. For instance, R. trifolii 0403 does not form root nodules on white clover grown in the presence of 16 mM NO3 - 1. The early steps of the infection process, including microsymbiont attachment to host root hairs 1,1 z and root hair deformation and infection 7,10,12, are inhibited when seedlings are grown in hydroponic culture containing critical concentrations of nitrate. If the rhizobia infect the root hairs before these levels of nitrate are added, then nodules will form and prematurely senesce 7,12 The biochemical basis for the inhibitory effect of combined nitrogen on the infection of clover root hairs by R. trifolii is just beginning to be understood. Supplying combined nitrogen (NO3 -, NH~ +) to growing roots over a period of 12 h~4 days diminishes both the immunologically detectable levels of trifoliin A, a Rhizobium-binding lectin on clover root hairs, and the selective attachment of R. trifolii 0403 to these root hairs 1. Thus, the presence of combined nitrogen during plant growth plays an important role in regulating these early recognition steps. Hypotheses to explain how nitrate supply regulates levels of trifoliin A on clover root surfaces include i) direct ligand binding of nitrate to trifoliin A resulting in its inactivation, ii) alteration of 'wall receptors' involved in accumulation of trifoliin A on the root surface and iii) inhibition of de novo 259