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

Abstract

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.

Involvement of lipid-protein complexes in plant-microorganism interactions

“…Thirdly, a kinetic analysis of the elicitin binding curves, using the allosteric model of Monod [58], confirms that these receptors could be represented by an allosteric model corresponding to an oligomeric structure with four identical subunits [59]. Taking these results into account, we proposed that the elicitin receptor could be a ligand-dependent calcium channel constituted of a quadrimeric complex as shown in Figure 3, which summarizes the initial molecular events involving activation of elicitin by sterol loading that drive the elicitor function [56,59].Finally, all the elicitins tested are able to bind to the same sites (with a similar affinity), suggesting that they are recognized by the same receptors, although they induce differential cell and plant responses [40]. These apparently contradictory observations remained to be explained.…”

“…The elicitin binding to the receptor triggers an allosteric change of its subunits, probably associated with a phosphorylation event [56]. Thirdly, a kinetic analysis of the elicitin binding curves, using the allosteric model of Monod [58], confirms that these receptors could be represented by an allosteric model corresponding to an oligomeric structure with four identical subunits [59].…”

“…These results indicate that the formation of a sterol-elicitin complex is a prerequisite step before elicitin binds to high affinity proteins, which thus constitute their biological receptors. Consequently, this complex formation is the first event involved in the elicitin-plant cell interaction [56] and it. It leads to propose a receptor-organization model (Figure 3).…”

“…These mutations result in the decrease of all the assayed activities. Moreover, strong correlations have been established between sterol affinity and biological activities, and between the rate of elicitins loading with sterols and their capability to bind specific high affinity proteins, located on plasmalemma [56]. The characteristics of the binding kinetics of the mutated elicitins to their putative receptors pointed out to a cooperative phenomenon.…”

“…This conformational modification allows the binding of other loaded/unloaded elicitin molecules to the receptor. But only loaded elicitins can trigger biological responses [56]. …”