Defense Responses in Grapevine Leaves Against Botrytis cinerea Induced by Application of a Pythium oligandrum Strain or Its Elicitin, Oligandrin, to Roots.

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.

Cytological Characterization of Elicitin-Induced Protection in Tobacco Plants Infected by Phytophthora parasitica or Phytoplasma.

ABSTRACT 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.

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.

Effects of 12 beticolins, Cercospora beticola toxins, on proliferation of ras-transformed adrenocortical cell.

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

Mediation of elicitin activity on tobacco is assumed by elicitin-sterol complexes.

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.

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.

Crystallization and preliminary X-ray studies of oligandrin, a sterol-carrier elicitor from Pythium oligandrum.

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.

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.

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.

Elicitins trap and transfer sterols from micelles, liposomes and plant plasma membranes.

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.

Magnesium ions promote assembly of channel-like structures from beticolin 0, a non-peptide fungal toxin purified from Cercospora beticola.

Beticolins are toxins produced by the fungus Cercospora beticola. Using beticolin 0 (B0), we have produced a strong and Mg(2+)-dependent increase in the membrane conductance of Arabidopsis protoplasts and Xenopus oocytes. In protein-free artificial bilayers, discrete deflexions of current were observed (12 pS unitary conductance in symmetrical 100 mM KCl) in the presence of B0 (approximately 10 microM) and in the presence of nominal Mg2+. Addition of 50 microM Mg2+ induced a macroscopic current which could be reversed to single channel current by chelating Mg2+ with EDTA. Both unitary and macroscopic currents were ohmic. The increase in conductance of biological membranes triggered by B0 is therefore likely to originate from the ability of this toxin to organize itself into transmembrane pores in the presence of Mg2+. The pore is poorly selective, displaying permeability ratios PCl/PK, PNa/PK and PCa/PK close to 0.3, 0.65 and 0.4, respectively. Such channel-like activity could be involved in the deleterious biological activity of the toxin, by causing the collapse of ionic and electrical gradients through biological membranes together with Ca2+ influx and scrambling of cellular signals.

Elicitins, proteinaceous elicitors of plant defense, are a new class of sterol carrier proteins.

Some phytopathogenic fungi within Phytophthora species are unable to synthesize sterols and therefore must pick them up from the membranes of their host-plant, using an unknown mechanism. These pseudo-fungi secrete elicitins which are small hydrophilic cystein-rich proteins. The results show that elicitins studied interact with dehydroergosterol in the same way, but with some time-dependent differences. Elicitins have one binding site with a similar strong affinity for dehydroergosterol. Using a non-steroid hydrophobic fluorescent probe, we showed that phytosterols are able to similarly bind to elicitins. Moreover, elicitins catalyze sterol transfer between phospholipidic artificial membranes. Our results afford the first evidence for a molecular activity of elicitins which appears to be extracellular sterol carrier proteins. This property should contribute to an understanding of the molecular mechanism involved in sterol uptake by Phytophthora. It opens new perspectives concerning the role of such proteins in plant-microorganism interactions, since elicitins trigger defence reactions in plants.

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.

Activation of the plant plasma membrane H+-ATPase. Is there a direct interaction between lysophosphatidylcholine and the C-terminal part of the enzyme?

The antagonistic effects of the fungal toxin beticolin-1 and of L-alpha-lysophosphatidylcholine (lysoPC) were investigated on the plasma membrane H+-ATPase of the plant Arabidopsis thaliana (isoform 2) expressed in yeast, using both wild-type enzyme (AHA2) and C-terminal truncated enzyme (aha2delta92). Phosphohydrolytic activities of both enzymes were inhibited by beticolin-1, with very similar 50% inhibitory concentrations, indicating that the toxin action does not involve the C-terminal located autoinhibitory domain of the proton pump. Egg lysoPC, a compound that activates the H+-ATPase by a mechanism involving the C-terminal part of the protein, was found to be able to reverse the inhibition of AHA2 by beticolin-1. The lack of effect of other detergents and the comparison of different carbon chain length lysoPCs show that the capacity to reverse the enzyme inhibition is clearly related to their ability to activate the pump. Long chain length lysoPC was also shown to reverse the inhibition of aha2delta92 by beticolin-1, which strongly suggests that lysoPC binds to the H+-ATPase on site(s) not located on its autoinhibitory domain.

Cercospora beticola toxins. Part XVII. The role of the beticolin/Mg2+ complexes in their biological activity. Study of plasma membrane H(+)-ATPase, vacuolar H(+)-PPase, alkaline and acid phosphatases.

Beticolin-1 and beticolin-2, yellow toxins produced by the phytopathogenic fungus Cercospora beticola, inhibit the plasma membrane H(+)-ATPase. Firstly, since beticolins are able to form complexes with Mg2+, the role of the beticolin/Mg2+ complexes in the inhibition of the plasma membrane proton pump has been investigated. Calculations indicate that beticolins could exist under several forms, in the H(+)-ATPase assay mixture, both free or complexed with Mg2+. However, the percentage inhibition of the H(+)-ATPase activity is correlated to the concentration of one single form of beticolin, the dimeric neutral complex Mg2H2B2, which appears to be the active form involved in the H(+)-ATPase inhibition. Secondly, since previous data suggested that beticolins could also be active against other Mg2(+)-dependent enzymes, we tested beticolin-1 on the vacuolar H(+)-PPase, which requires Mg2+ as co-substrate, and on the alkaline and acid phosphatases, which do not use Mg2+ as co-substrate. Only vacuolar H(+)-PPase is sensitive to beticolin-1, which suggests that beticolins are specific to enzymes that use a complex of Mg2+ as the substrate. The same Mg2H2B2 complex which is responsible of the plasma membrane H(+)-ATPase inhibition appears to be also involved in the inhibition of the vacuolar H(+)-PPase.

Inhibition of cellular growth and steroid 11 beta-hydroxylation in ras-transformed adrenocortical cells by the fungal toxins beticolins.

The proliferation of GM16 and 4CDT ras-transformed newborn rat adrenocortical (RTAC) cells and Y1 mouse adrenal tumor cells was inhibited by beticolins, the fungal toxins extracted from Cercospora beticola, at submicromolar concentrations in a dose-dependent manner. Inhibitory concentrations for half the maximum inhibition were 150, 75 and 25 nM for beticolin-1 and 230, 150 and 50 nM for beticolin-2 in GM16, 4CDT and Y1 cells respectively. Beticolins strongly inhibited the production of 11 beta-hydroxysteroids on the second and third days of treatment in a dose-dependent manner between 0.1 and 1 microM. Beticolins were shown by confocal microscopy to be localized in cytoplasmic organelles about 30-40 min after treatment. This finding favors a direct action of beticolins on mitochondrial steroid 11 beta-hydroxylase albeit another less direct mechanism involving a cytoplasmic signaling pathway cannot be excluded.

Cercospora beticola Toxins (X. Inhibition of Plasma Membrane H+-ATPase by Beticolin-1).

Beticolin-1 is a toxin produced by the fungus Cercospora beticola. The chemical structure of this toxin was previously elucidated. The effects of beticolin-1 on purified corn root plasma membrane H+-ATPase were studied in a solubilized form or were reconstituted into liposome membranes. The ATP hydrolysis activity of the purified solubilized enzyme was inhibited by micromolar concentrations of beticolin-1, and this inhibition was noncompetitive with respect to ATP. When this purified enzyme was inserted into liposome membranes, a competitive inhibition of the H+-ATPase hydrolysis activity by beticolin-1 was observed. The effect of beticolin-1 on the formation of H+-ATPase-phosphorylated intermediate was also studied. With the purified enzyme in its solubilized form, the level of phosphorylated intermediate was not affected by the presence of beticolin-1, whereas micromolar concentrations of the toxin led to a marked inhibition of its formation when the enzyme was reconstituted into liposomes. These data suggest that (a) the plasma membrane H+-ATPase is a direct target for beticolin-1, and (b) the kinetics of inhibition and the effect on the phosphorylated intermediate are linked and both depend on the lipid environment of the enzyme.

Relationship between Active Oxygen Species, Lipid Peroxidation, Necrosis, and Phytoalexin Production Induced by Elicitins in Nicotiana.

Excised leaves of Nicotiana tabacum var Xanthi and Nicotiana rustica were treated with cryptogein and capsicein, basic and acidic elicitins, respectively. Both compounds induced leaf necrosis, the intensity of which depended on concentration and duration of treatment. N. tabacum var Xanthi was the most sensitive species and cryptogein was the most active elicitin. Lipid peroxidation in elicitin-treated Nicotiana leaves was closely correlated with the appearance of necrosis. Elicitin treatments induced a rapid and transient burst of active oxygen species (AOS) in cell cultures of both Nicotiana species, with the production by Xanthi cells being 6-fold greater than that by N. rustica. Similar maximum AOS production levels were observed with both elicitins, but capsicein required 10-fold higher concentrations than those of cryptogein. Phytoalexin production was lower in response to both elicitins in N. tabacum var Xanthi cells than in N. rustica cells, and capsicein was the most efficient elicitor of this response. In cryptogein-treated cell suspensions, phytoalexin synthesis was unaffected by diphenyleneiodonium, which inhibited AOS generation, nor was it affected by tiron or catalase, which suppressed AOS accumulation in the extracellular medium. These results suggest that AOS production, lipid peroxidation, and necrosis are directly related, whereas phytoalexin production depends on neither the presence nor the intensity of these responses.

Cercospora beticola toxins. Part VI: preliminary studies of protonation and complexation equilibria.

The biological activity of Cercospora beticola toxins might be enhanced by the complex formation with magnesium. Therefore, protonation and complexation equilibria of beticolins were studied. Beticolins carry three dissociable functions (H3B) two of which dissociate at a physiological pH. In the presence of magnesium, the neutralisation and protonation curves provide evidence for the formation of complexes. At physiological pH, the uncharged complex, Mg2H2B2, is the predominant form. The nonionised forms of free beticolin-1 and -2 fluoresce in a 50% dioxan-water solution and their emission maxima shift to higher wavelengths in water. The dianion HB(2-) is non-fluorescent both in water and in less polar media. The formation of the Mg2H2B2 complex which strongly fluoresces in nonpolar media is confirmed by a marked increase in fluorescence at 520 nm and by a shift of the excitation maximum.

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.