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Lipid transfer proteins (LTPs) and elicitins are both able to load and transfer lipidic molecules and share some structural and functional properties. While elicitins are known as elicitors of plant defence mechanisms, the biological function of LTP is still an enigma. We show that a wheat LTP1 binds with high affinity sites. Binding and in vivo competition experiments point out that these binding sites are common to LTP1 and elicitins and confirm that they are the biological receptors of elicitins. A mathematical analysis suggests that these receptors could be represented by an allosteric model corresponding to an oligomeric structure with four identical subunits. ß

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.

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Using elicitins, proteins secreted by some phytopathogenic Oomycetes (Phytophthora) known to be able to transfer sterols between phospholipid vesicles, the transfer of sterols between micelles, liposomes and biological membranes was studied. Firstly, a simple fluorometric method to screen the sterol-carrier capacity of proteins, avoiding the preparation of sterol-containing phospholipidic vesicles, is proposed. The transfer of sterols between DHE micelles (donor) and stigmasterol or cholesterol micelles (acceptor) was directly measured, as the increase in DHE fluorescence signal. The results obtained with this rapid and easy method lead to the same conclusions as those previously reported, using fluorescence polarization of a mixture of donor and acceptor phospholipid vesicles, prepared in the presence of different sterols. Therefore, the micelles method can be useful to screen proteins for their sterol carrier activity. Secondly, elicitins are shown to trap sterols from purified plant plasma membranes and to transfer sterols from micelles to these biological membranes. This property should contribute to understand the molecular mechanism involved in sterol uptake by Phytophthora. It opens new perspectives concerning the role of such proteins in plant-microorganism interactions.