0000000000065098
AUTHOR
Marie-louise Milat
A lipid transfer protein binds to a receptor involved in the control of plant defence responses
AbstractLipid 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.
Purification, crystallization and preliminary X-ray studies of sylvaticin, an elicitin-like protein from Pythium sylvaticum.
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.
The fungal elicitor cryptogein is a sterol carrier protein
AbstractCryptogein 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 p…
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 inh…
Active oxygen species production in tobacco cells elicited by cryptogein*
We analyse the relationship between active oxygen species (AOS) production and pH changes induced in tobacco cells by cryptogein, a fungal proteinaceous elicitor of defence mechanisms in plants. When tobacco cells were treated with cryptogein, an intracellular acidification, an alkalinization of the extracellular medium and a transient burst of AOS (H 2 O 2 ) were observed. Treatment of elicited cells with either diphenyleneiodonium (DPI), an inhibitor of the neutrophil NADPH oxidase, or Tiron, which scavenges O2 - , abolished AOS production. These data suggest the involvement of a NADPH oxidase-like enzyme leading to H 2 O 2 production through O 2 - dismutation. Although H 2 O 2 production…
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 membran…
Cercospora beticola toxins. Use of fluorescent cyanine dye to study their effects on tobacco cell suspensions
Abstract The fluorescent dye 3,3′-diethylthiadicarbocyanine iodide [diS-C 2 -(5)] was used to observe plasmalemma transmembrane potential variations of tobacco cells treated with uncoupler (FCCP), respiratory inhibitors (azide and cyanide), and H + -ATPase inhibitors (DCCD and a carbanilate derivative). These chemicals induced an increase in fluorescence, indicating a dissipation of the transmembrane potential. The [diS-C 2 -(5)] was also used to study the effects of two Cercospora beticola toxins on tobacco cells. Changes in fluorescence of [diS-C 2 -(5)] suggested that these two toxins caused a dissipation of the transmembrane potential with a different magnitude whereas kinetics of their…
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 investi…
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 sterolcontaining 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, u…
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 g…
Fatty acids bind to the fungal elicitor cryptogein and compete with sterols
Abstract 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 yellow toxins produced by Cercospora Beticola. Part VIII : Chemical equilibrium between beticolins ; structures of minor compounds : beticolin 6 and beticolin 8
Abstract a general chemical transformation of the “linear” beticolin skeleton (beticolin 2 and 4) into a “bent” beticolin skeleton (cebetin A and beticolin 3 respectively) is described. Beticolins 6 and 8, minor components of mycelial extract are also characterized as minor compounds resulting from these transformations.
INHIBITION OF CELLULAR GROWTH AND STEROID 11β-HYDROXYLATION INRAS-TRANSFORMED ADRENOCORTICAL CELLS BY THE FUNGAL TOXINS BETICOLINS
Abstract The proliferation of GM16 and 4CDTras-transformed newborn rat adrenocortical (RTAC) cells and Y1 mouse adrenal tumor cells was inhibited by beticolins, the fungal toxins extracted fromCercospora beticola, at submicromolar concentrations in a dose-dependent manner. Inhibitory concentrations for half the maximum inhibition were 150, 75 and 25 n M for beticolin-1 and 230, 150 and 50 n M for beticolin-2 in GM16, 4CDT and Y1 cells respectively. Beticolins strongly inhibited the production of 11β-hydroxysteroids on the second and third days of treatment in a dose-dependent manner between 0.1 and 1 μ M . Beticolins were shown by confocal microscopy to be localized in cytoplasmic organelle…
Crystallization and preliminary X-ray studies of oligandrin, a sterol-carrier elicitor fromPythium 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.
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…
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…