Mediation of elicitin activity on tobacco is assumed by elicitin-sterol complexes

Elicitins secreted by phytopathogenic Phytophthora spp. are proteinaceous e licitors of plant defense mechanisms and were demonstrated to load, carry, and transfer sterols between membranes. The link between elicitor and stero l-loading properties was assessed with the use of site-directed mutagenesis of the 47 and 87 cryptogein tyrosine residues, postulated to be involved i n sterol binding. Mutated cryptogeins were tested for their ability to load sterols, bind to plasma membrane putative receptors, and trigger biologica l responses. For each mutated elicitin, the chemical characterization of th e corresponding complexes with stigmasterol (1:1 stoichiometry) demonstrate d their full functionality. However, these proteins were strongly altered i n their sterol-loading efficiency, specific binding to high-affinity sites, and activities on tobacco cells. Ligand replacement experiments strongly s uggest that the formation of a sterol-elicitin complex is a requisite step before elicitins fasten to specific binding sites. This was confirmed with the use of two sterol-preloaded elicitins. Both more rapidly displaced labe led cryptogein from its specific binding sites than the unloaded proteins. Moreover, the binding kinetics of elicitins are related to their biological effects, which constitutes the first evidence that binding sites could be the biological receptors. The first event involved in elicitin-mediated cel l responses is proposed to be the protein loading with a sterol molecule.