Vol. 12, Issue 9, 2825-2834, September 2001

Mediation of Elicitin Activity on Tobacco Is Assumed by Elicitin-Sterol Complexes

Hanan Osman,^* Sébastien Vauthrin,^* Vladimir Mikes, Marie-Louise Milat,^* Franck Panabières,^§ Antoine Marais,^§ Simone Brunie, Bernard Maume,^* Michel Ponchet,^§¶ and Jean-Pierre Blein^*

 ^*Unité Mixte de Recherche 692, Laboratoire de Phytopharmacie et de Biochimie des Interactions Cellulaires, Institut National de la Recherche Agronomique, 21065 Dijon-cedex, France;  Department of Biochemistry, Faculty of Science, Masaryk University, 61137 Brno, Czech Republic;  ^§Unité Interactions Plantes-Microorganismes et Santé Végétale Institut National de la Recherche Agronomique, 06606 Antibes-cedex, France; and  Unité de Recherche Biochimie et Structure des Protéines, Institut National de la Recherche Agronomique, 78352 Jouy en Josas, France

Elicitins secreted by phytopathogenic Phytophthora spp. are proteinaceous elicitors of plant defense mechanisms and were demonstrated to load, carry, and transfer sterols between membranes. The link between elicitor and sterol-loading properties was assessed with the use of site-directed mutagenesis of the 47 and 87 cryptogein tyrosine residues, postulated to be involved in sterol binding. Mutated cryptogeins were tested for their ability to load sterols, bind to plasma membrane putative receptors, and trigger biological responses. For each mutated elicitin, the chemical characterization of the corresponding complexes with stigmasterol (1:1 stoichiometry) demonstrated their full functionality. However, these proteins were strongly altered in their sterol-loading efficiency, specific binding to high-affinity sites, and activities on tobacco cells. Ligand replacement experiments strongly suggest 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 labeled 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 cell responses is proposed to be the protein loading with a sterol molecule.