ABSTRACT Pythium oligandrum is known to display antagonistic activities against several species of pathogenic fungi. It also produces an elicitor of plant defense named oligandrin, which belongs to the elicitin family (10-kDa proteins synthesized by Phytophthora and Pythium species). Here, the potential of P. oligandrum or its purified elicitin to limit the progression of B. cinerea on grapevine leaf and the resulting plant-microorganism interactions are described. P. oligandrum or oligandrin were applied to roots, and changes in the ultrastructure and at the molecular level were examined. When B. cinerea was applied to leaves of pretreated plants, leaf invasion was limited and the protection level reached about 75%. On leaf tissues surrounding B. cinerea inoculation, modifications of cuticle thickness, accumulation of phenolic compounds, and cell wall apposition were observed, indicating that grapevine can be considered reactive to elicitins. No macroscopic hypersensitive reaction associated with the elicitation treatment was observed. At the molecular level, the expression of three defense-related genes (LTP-1, beta-1,3-glucanase, and stilbene synthase) was studied. RNAs isolated from B. cinerea-infected leaves of grapevine challenged or not with P. oligandrum or oligandrin were analyzed by real-time reverse transcription-polymerase chain reaction. In grapevine leaves, LTP-1 gene expression was enhanced in response to oligandrin, and RNA transcript levels of beta-1,3-glucanase and stilbene synthase increased in response to all treatments with different magnitude. Taken together, these results open new discussion on the concept of plant reactivity to elicitins, which has until now, been mainly based on plant hypersensitive responses.

Sylvaticin belongs to the elicitin family. These 10 kDa oomycetous proteins induce a hypersensitive response in plants, including necrosis and cell death, but subsequently leading to a non-specific systemic acquired resistance (SAR) against other pathogens. Sylvaticin has been crystallized using PEG 2000 MME as a precipitant agent in the presence of nickel chloride. The crystals belong to space group C2, with unit-cell parameters a = 99.29, b = 25.67, c = 67.45 A, beta = 99.66 degrees. Diffraction data were recorded to 2.1 A resolution at a synchrotron-radiation source.

Plant lipid transfer proteins (LTPs) are small, cysteine-rich proteins secreted into the extracellular space. They belong to the pathogenesis-related proteins (PR-14) family and are believed to be involved in several physiological processes including plant disease resistance, although their precise biological function is still unknown. Here, we show that a recombinant tobacco LTP1 is able to load fatty acids and jasmonic acid. This LTP1 binds to specific plasma membrane sites, previously characterized as elicitin receptors, and is shown to be involved in the activation of plant defense. The biological properties of this LTP1 were compared with those of LTP1-linolenic and LTP1-jasmonic acid complexes. The binding curve of the LTP1-linolenic acid complex to purified tobacco plasma membranes is comparable to the curve obtained with LTP1. In contrast, the LTP1-jasmonic acid complex shows a strongly increased interaction with the plasma membrane receptors. Treatment of tobacco plants with LTP1-jasmonic acid resulted in an enhancement of resistance toward Phytophthora parasitica. These effects were absent upon treatment with LTP1 or jasmonic acid alone. This work presents the first evidence for a biological activity of a LTP1 and points out the crucial role of protein-specific lipophilic ligand interaction in the modulation of the protein activity.

Elicitins, small proteins secreted by Phytophthora and Pythium spp., display the ability to induce plant resistance toward pathogens. Ultrastructural investigations of cryptogein-treated tobacco plants evidenced host defense responses such as (i) formation of a calcium pectate gel in intercellular spaces of parenchymas, (ii) impregnation of pectin by phenolic compounds in intercellular spaces of phloem bundles, and (iii) accumulation of phloem proteins (P proteins) in the lumen of leaf sieve elements. These cytological modifications lead to the enhancement of physical barriers that prevent pathogen ingress and restrict host tissue colonization when cryptogein-treated tobacco plants were challenged with the pathogen Phytophthora parasitica. Wall appositions also were observed at most sites of penetration of hyphae. Moreover, growing hyphae exhibited severe morphological damages, suggesting a modified toxic environment. The same induction of P proteins in mature sieve tubes of tobacco leaves was obtained with oligandrin treatment, another elicitin. Cryptogein or oligandrin treatment prevented symptom expression in phytoplasma-infected tobacco plants in contrast with nontreated tobacco plants. Moreover, P protein plugs and occlusion of pore sites by callose were evidenced in sieve elements of treated plants. Both these phloem modifications might prevent the in planta movement of phloem-restricted microorganisms.

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 (La3+, 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.

Egosterol is an elicitor molecule activating plant defense reaction via PLA2 and PKC when compared with cryptogein activating PLC and PKC pathway.

Low molecular lipid substances represent specific elicitors of plant defence reaction. We studied the elicitation mechanism by means of several inhibitors of calcium channels (La3+, verapamil, nifedipin, TMB-8 and ruthenium red) and fluorescence confocal microscopy with calcium green and compared the effect on the signal pathways induced by cryptogein, a typical fungal proteinaceous elicitor the receptor of which is localized on the plasma membrane, with ergosterol. The results suggest that the binding of ergosterol to an unknown receptor results in a mobilization of internal calcium stores (probably IP3 dependent) rather than in the effect on the plasma membrane channels.

The three dimensional structure of Candida cylindracea Cholesterol Esterase (ChE) in complex with the polymeric detergent C12H26-(O-CH2-CH2)n has been determined at 1.4 Å resolution in space group P1 using low temperature synchrotron data.The structure was refined by CNS and SHELX methods to R=0.14 and Rfree=0.17.The 8074 protein, 112 carbohydrate, 46 detergent, 1078 water nonhydrogen atoms and 8054 hydrogen atoms were localized.The ChE structure is a dimer with four spatially separated interfacial contact areas and four openings to an internal interfacial cavity.Carbohydrates were located in electron density maps on the side chains of Asn314 and Asn351 in both ChE monomers.There were two well defined N-acetyl-glucosoamine moieties at each site.For each monomer a 23 atom detergent fragment was unambiguously fitted to electron density in the elongated hydrophobic gorge of the enzyme acive site.Two alternative side chain conformations were detected for fourteen ChE residues with nearly equivalent occupancy values of 0.5+-0.1.

As for natural sterols, only ergosterol is recognized very specifically and sensitively (nM) by plants cells. Ergosterol interacts with tobacco suspension cells and trigger 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, DpH and phytoalexin accumulation were weaker 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 either cryptogein or ergosterol resulted in an increase of the fluorescence of calcium green 1 in cytosol. The use of several inhibitors of calcium channels (La3+, TMB-8, verapamil, ruthenium red, nifedipine) and a protein-kinase inhibitors (staurosporin, NPC-15437, H-89) suggests that the elicitation with ergosterol includes the mobilization of internal calcium stores in vacuoles mediated by IP3 and some protein kinases.

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.

AIM:To explore different effects of 12 beticolins, Cercospora beticola toxins, on ras-transformed adrenocortical cell growth inhibition and their functional mechanism.METHODS:Beticolin-induced inhibition was measured with survival cell number determined by an automated photocolorimetric method. The penetration of beticolin was examined by confocal microscopy. Ras protein determined by Lowry method were separated by 14 % SDS-PAGE and electroblotted to Immobilon-P transfer membrane and detected with pan-Ras (Ab-3) monoclonal antibody. The Ca2+ chelation by beticolin was investigated using a calcium ionophore.RESULTS:Cell growth inhibition was found dose- and time-dependently at submicromolar level for beticolin-1, -2, and -13 (IC50 </= 250 nmol/L) and for beticolin-0, 6, and -11 (400 nmol/L < IC50 </= 500 nmol/L). The inhibition by beticolin-1 was immediate, independent of cell culture step and not reversible for 3-day treatment. Beticolin-3 and -4 were slightly active (1 micromol/L < IC50 </= 2 micromol/L) and beticolin-7, -9, -12, and -5 were inactive at micromolar level. The beticolin-induced cell growth inhibition was correlated with the hydrophobicity of these compounds. Beticolin-1 fluorescence in RTAC cells was detected by confocal microscopy whereas beticolin-3 and -12 were not even after a 24 h incubation period. Beticolin-1-induced cell growth inhibition was partially reverted by calcium ionophore suggesting a role of intracellular Ca2+ chelation by beticolin-1 on cell growth inhibition. Furthermore, beticolin-1 blocked up Ras p21 translocation to membrane and induced accumulation of Ras in the cytosol as an inactive form by different ways.CONCLUSION:Beticolins with high hydrophobicity inhibit tumorigenic cell proliferation by different ways.

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.

Ergosterol is a fungal steroid elicitor of the plant defence reaction in tobacco and spruce cells. This interaction is accompanied by the synthesis of active oxygen species and changes of membrane permeability to H+ during 10 min. The effect of calcium channel inhibitors and the distribution of Ca by confocal microscopy were studied. These results suggest that the binding of ergosterol to an unknown receptor results in a mobilization of internal calcium stores (probably IP3 dependent) rather than in the effect on the plasma membrane channels

Elicitins secreted by phytopathogenic Phytophthoraspp. 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.

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.

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. Véry, 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 planarlipid bilayers. All beticolins were able to form ionchannels 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.

Oligandrin is a 10 kDa acidic protein produced by the fungus micromycete Pythium oligandrum and is a member of the alpha-elicitin group, with sterol- and lipid-carrier properties. Oligandrin has been crystallized at 290 K using PEG 4000 as a precipitant. A cholesterol complex was obtained under the same conditions. The space group of the crystals at low temperature (100 K) is C222, with unit-cell parameters a = 94.0, b = 171.1, c = 55.3 A. Four molecules are present in the asymmetric unit. Data from the free and cholesterol-complexed forms were recorded at synchrotron sources to resolutions of 2.4 (uncomplexed) and 1.9 A (complexed), respectively.

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.