Marie-Louise Milat

A lipid transfer protein binds to a receptor involved in the control of plant defence responses

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.

The fungal elicitor cryptogein is a sterol carrier protein

Cryptogein is a protein secreted by the phytopathogenic pseudo‐fungus, Phytophthora cryptogea. It is a basic 10 kDa hydrophilic protein having a hydrophobic pocket and three disulfide bridges. These common features with sterol carrier proteins led us to investigate its possible sterol transfer activity using the fluorescent sterol, dehydroergosterol. The results show that cryptogein has one binding site with strong affinity for dehydroergosterol. Moreover, this protein catalyzes the transfer of sterols between phospholipidic artificial membranes. This is the first evidence for the existence of an extracellular sterol carrier protein and for a molecular activity of cryptogein. This property should contribute to an understanding of the role of cryptogein in plant‐microorganism interactions.

Fatty acids bind to the fungal elicitor cryptogein and compete with sterols

Cryptogein is a proteinaceous elicitor of plant defense reactions which also exhibits sterol carrier properties. In this study, we report that this protein binds fatty acids. The stoichiometry of the fatty acid–cryptogein complex is 1:1. Linoleic acid and dehydroergosterol compete for the same site, but elicitin affinity is 27 times lower for fatty acid than for sterol. We show that C7 to C12 saturated and C16 to C22 unsaturated fatty acids are the best ligands. The presence of double bonds markedly increases the affinity of cryptogein for fatty acids. A comparison between elicitins and known lipid transfer proteins is discussed.

The 1.45 Å resolution structure of the cryptogein–cholesterol complex: a close-up view of a sterol carrier protein (SCP) active site

Cryptogein is a small 10 kDa elicitor produced by the phytoparasitic oomycete Phytophthora cryptogea. The protein also displays a sterol carrier activity. The native protein crystallizes in space group P4(1)22, with unit-cell parameters a = b = 46.51, c = 134.9 A (diffraction limit: 2.1 A). Its complex with cholesterol crystallizes in space group C222(1), with unit-cell parameters a = 30.96, b = 94.8, c = 65.3 A and a resolution enhanced to 1.45 A. The large inner non-specific hydrophobic cavity is able to accommodate a large variety of 3-beta-hydroxy sterols. Cryptogein probably acts as a sterol shuttle helping the pathogen to grow and complete its life cycle.

Elicitation of tobacco cells with ergosterol activates a signal pathway including mobilization of internal calcium

Abstract Ergosterol interacts with tobacco suspension ( Nicotiana tabacum ) cells and triggers pH changes of extracellular medium, oxidative burst and synthesis of phytoalexins. Compared with the responses induced by cryptogein, a proteinaceous elicitor from Phytophthora sp., oxidative burst and ΔpH changes were weaker whereas phytoalexin accumulation was higher with ergosterol. Cryptogein stimulated an apparent continuous uptake of external calcium within 40 min, whereas no net uptake of external calcium occurred upon the addition of ergosterol. However, the elicitation with both cryptogein and ergosterol resulted in an increase of the fluorescence of calcium green 1 in cytosol. The use of several inhibitors of calcium channels (La 3+ , TMB-8, verapamil, ruthenium red, nifedipine) and a protein-kinase inhibitor (staurosporin) suggests that the elicitation with ergosterol includes the mobilization of internal calcium stores mediated by inositol 1,4,5-trisphosphate and serine/threonine protein kinases.

Beticolins, Nonpeptidic, Polycyclic Molecules Produced by the Phytopathogenic Fungus Cercospora beticola, as a New Family of Ion Channel-Forming Toxins

Beticolins are toxins produced by Cercospora beticola, a phytopathogenic fungus responsible for the leaf spot disease of sugar beet. They form a family of 20 nonpeptidic compounds (named B0 to B19) that share the same polycyclic skeleton but differ by isomeric configuration (ortho- or para-) and by a variable residue R (bridging two carbons in one of the six cycles). It has been previously shown that B0 assembles itself into a multimeric structure and forms ion channels into planar lipid bilayers (C. Goudet, A.-A. Very, M.-L. Milat, M. Ildefonse, J.-B. Thibaud, H. Sentenac, and J.-P. Blein, Plant J. 14:359-364, 1998). In the present work, we investigate pore formation by three ortho-beticolins, B0, B2, and B4, and their related (i.e., same R) para-isomers, B13, B1, and B3, respectively, using planar lipid bilayers. All beticolins were able to form ion channels with multiple conductance states, although the type of cyclization (ortho- or para-) and residue (R) result in variations of channel conductance and ionic permeability, respectively. Channel formation by beticolins is likely to be involved in the biological activity of these toxins.

Effects of fusaric acid on cells from tomato cultivars resistant or susceptible toFusarium oxysporum f. sp.Lycopersici

Cell suspension cultures were set up from two tomato cultivars, one resistant, (‘Rio grande’) and one susceptible (‘63.5’) toFusarium oxysporum f. sp.lycopersici. Growth rates of the two cell cultures were comparable. Toxicity of fusaric acid, expressed as the fresh weight loss, was analyzed: It was significant in both cases after 10 h, but toxicity was twice as high for ‘63.5’ suspension cells. In the same way, electrolyte leakage caused by fusaric acid was three times more important for ‘63.5’ suspension cells. Moreover, fusaric acid treatment resulted in an acidification of the extracellular medium for ‘63.5’ suspension cells (0.4 pH unit), whereas an alkalization was observed for ‘Rio grande’ suspension cells (0.2 pH unit). Preliminary experiments suggest that fusaric acid was partially metabolized by ‘Rio grande’ suspension cells, however, no detoxified forms of fusaric acid were detected either in cells or in culture filtrates. For these two tomato cultivars, the differences in sensitivity to fusaric acid of cultivated cells correspond to the differences in plant susceptibility toFusarium oxysporum f. sp.lycopersici.

Cluster Organization and Pore Structure of Ion Channels Formed by Beticolin 3, a Nonpeptidic Fungal Toxin

Beticolin 3 (B3) belongs to a family of nonpeptidic phytotoxins produced by the fungus Cercospora beticola, which present a broad spectrum of cytotoxic effects. We report here that, at cytotoxic concentration (10 microM), B3 formed voltage-independent, weakly selective ion channels with multiple conductance levels in planar lipid bilayers. In symmetrical standard solutions, conductance values of the first levels were, respectively, 16 +/- 1 pS, 32 +/- 2 pS, and 57 +/- 2 pS (n = 4) and so on, any conductance level being roughly twice the lower one. Whether a cluster organization of elementary channels or different channel structures underlies this particular property was addressed by investigating the ionic selectivity and the pore size corresponding to the first three conductance levels. Both selectivity and pore size were found to be almost independent of the conductance level. This indicated that multiple conductance behavior resulted from a cluster organization of B3 elementary channels. According to the estimated pore size and analyses of x-ray diffraction of B3 microcrystals, a structural model for B3 elementary channels is proposed. The ability to form channels is likely to be involved in the biological activity of beticolins.

Cercospora beticola toxins III. Purification, thin-layer and high performance liquid chromatographic analyses

Abstract Cercospora beticola toxins were extracted from the mycelium and purified by flash chromatography and crystallization. TLC was used to monitor the purification. The pure compounds were used to determine the optimum conditions for HPLC analysis. The HPLC method was used to quantify these metabolites in crude extracts.

Cercospora beticola toxins. VII. Fluorometric study of their interactions with biological membranes

The interactions of two beticolins, Cercospora beticola toxins, and of their magnesium complexes with liposomes or plasma membrane were studied. The fluorometric pH titration curves of beticolins in liposomes and in plasma membranes reveal the presence of the dissociated form of beticolins. The concentration of the magnesium complex in these membranes increases at high pH. The partition coefficient of beticolin-1 on liposomes is 3-fold higher than that of beticolin-2 and the fluorescence of both compounds on liposomes is similar. The addition of magnesium to liposomes causes a 40-fold and 20-fold increase in the partition coefficient of beticolin-1 and -2, respectively, as a result of the interactions between membrane, magnesium and beticolins. Beticolins react to a delta pH across the liposome membrane but the formation of the magnesium complex completely abolishes this effect.