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…

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

From elicitins to lipid-transfer proteins: a new insight in cell signalling involved in plant defence mechanisms.

Elicitins and lipid-transfer proteins are small cysteine-rich lipid-binding proteins secreted by oomycetes and plant cells, respectively, that share some structural and functional properties. In spite of intensive work on their structure and diversity at the protein and genetic levels, the precise biological roles of lipid-transfer proteins remains unclear, although the most recent data suggest a role in somatic embryogenesis, in the formation of protective surface layers and in defence against pathogens. By contrast, elicitins are known elicitors of plant defence, and recent work demonstrating that elicitins and lipid-transfer proteins share the same biological receptors gives a new perspe…

Modulation of the Biological Activity of a Tobacco LTP1 by Lipid Complexation

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…

Elicitins trap and transfer sterols from micelles, liposomes and plant plasma menbranes

International audience

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…

Les stimulateurs de défense des plantes. Panorama et solutions d'avenir

Prod 2018-223 SPE IPM INRA CT1; National audience; Stimuler les défenses des plantes pour qu’elles puissent se défendre : ce concept est élégant et désormais techniquement à portée de main. Les stimulateurs des défenses des plantes (SDP), trouvent en effet des applications concrètes en production même s’il reste du chemin pour élucider tous les mystères de ces éliciteurs. Ils doivent trouver leur place dans l’évolution que connait actuellement la santé du végétal (évolution des systèmes de culture, agroécologie, …) et permettent de répondre aux attentes de la société en matière de réduction de l’usage des produits phytopharmaceutiques. Fruit d’un travail collectif, cet ouvrage dresse un éta…

Acquired resistance triggered by elicitins in tobacco and other plants

Elicitins are a family of proteins excreted byPhytophthora spp. They exhibit high sequence homology but large net charge differences. They induce necrosis in tobacco plants which then become resistant to the tobacco pathogenPhytophthora parasitica var.nicotianae. In stem-treated plants, resistance was not restricted to the site of elicitin application, but could be demonstrated by petiole inoculation at all levels on the stem. Resistance was already maximum after two days and lasted for at least two weeks. It was effective not only towardsP. p. var.nicotianae infection, but also against the unrelated pathogenSclerotinia sclerotiorum. In contrast to dichloroisonicotinic acid, an artificial i…

Utilisation d'élicitines pour le transport de lipides

*INRA URD BP 86510 21065 Dijon cedex (FRA)

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

International audience

The 1.45 A 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.

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…

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 functionali…

Purifiation, crystallization and preliminary X-ray studies of sylvaticin, an elicitin-like protein from Pythium sylvaticum

International audience

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.

Specific adduction of plant lipid transfer protein by an allene oxide generated by 9-lipoxygenase and allene oxide synthase

International audience; Lipid transfer proteins (LTPs) are ubiquitous plant lipid-binding proteins that have been associated with multiple developmental and stress responses. Although LTPs typically bind fatty acids and fatty acid derivatives in a non-covalent way, studies on the LTPs of barley seeds have identified an abundantly occurring covalently modified form, LTP1b, the lipid ligand of which has resisted clarification. In the present study, this adduct was identified as the {alpha}-ketol 9-hydroxy-10-oxo-12(Z)-octadecenoic acid. Further studies on the formation of LTP1b demonstrated that the ligand was introduced by nucleophilic attack of the free carboxylate group of the Asp-7 residu…

L'histoire des élicitines : Des acquis dans la compréhension de la résistance des plantes aux maladies

National audience

Are elicitins cryptograms in plant-oomycete communications?

Stimulation of plant natural defenses is an important challenge in phytoprotection prospects. In that context, elicitins, which are small proteins secreted by Phytophthora and Pythium species, have been shown to induce a hypersensitive-like reaction in tobacco plants. Moreover, these plants become resistant to their pathogens, and thus this interaction constitutes an excellent model to investigate the signaling pathways leading to plant resistance. However, most plants are not reactive to elicitins, although they possess the functional signaling pathways involved in tobacco responses to elicitin. The understanding of factors involved in this reactivity is needed to develop agronomic applica…

The combined action of 9 lipoxygenase and galactolipase is sufficient to bring about programmed cell death during tobacco hypersensitive response

International audience; Oxylipins, derived from fatty acid hydroperoxides (FAHs), are thought to play different roles during plant pathogen interactions. During hypersensitive response (HR) some of them serve as signals necessary for defence gene activation whereas others could contribute to pathogen killing or could participate in the execution of plant programmed cell death (PCD) associated with this resistance. In order to address the role of these compounds in the latter process, we have closely observed lipid peroxidation, the first step of this metabolic pathway, under different situations which led either to accelerated or inhibited HR cell death. The oxidative process has been studi…

Oligandrin. A proteinaceous molecule produced by the mycoparasite Pythium Oligandrum induces resistance to Phytophthora parasitica infection in tomato plants

International audience

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.

Comparison of binding properties and early biological effects of elicitins in tobacco cells

Abstract Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum). They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis. In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca2+ influx, extracellular medium alkalinization, and active oxygen species production). The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins. Furthermore, elicitins compete with one another fo…

Signal perception and transduction, secondary messengers and gene activation in elicitin-triggered HR and SAR in tobacco

National audience