Jean-Pierre Blein

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 perspective to understand the role played by lipid binding proteins, mainly the early recognition of intruders in plants.

Involvement of lipoxygenase-dependent production of fatty acid hydroperoxides in the development of the hypersensitive cell death induced by cryptogein on tobacco leaves

Lipid peroxidation was investigated in relation with the hypersensitive reaction in cryptogein-elicited tobacco leaves. A massive production of free polyunsaturated fatty acid (PUFA) hydroperoxides dependent on a 9-lipoxygenase (LOX) activity was characterized during the development of leaf necrosis. The process occurred after a lag phase of 12 h, was accompanied by the concomitant increase of 9-LOX activity, and preceded by a transient accumulation of LOX transcripts. Free radical-mediated lipid peroxidation represented 10% of the process. Inhibition and activation of the LOX pathway was shown to inhibit or to activate cell death, and evidence was provided that fatty acid hydroperoxides are able to mimic leaf necrotic symptoms. Within 24 h, about 50% of leaf PUFAs were consumed, chloroplast lipids being the major source of PUFAs. The results minimize the direct participation of active oxygen species from the oxidative burst in membrane lipid peroxidation. They suggest, furthermore, the involvement of lipase activity to provide the free PUFA substrates for LOX. The LOX-dependent peroxidative pathway, responsible for tissue necrosis, appears as being one of the features of hypersensitive programmed cell death.

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.

Evidence for specific, high-affinity binding sites for a proteinaceous elicitor in tobacco plasma membrane

Binding of cryptogein, a proteinaceous elicitor, was studied on tobacco plasma membrane. The binding of the [125I]cryptogein was saturable, reversible and specific with an apparent K d of 2 nM. A single class of cryptogein binding sites was found with a sharp optimum pH for binding at about pH 7.0. The high‐affinity correlates with cryptogein concentrations required for biological activity in vivo.

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.

Tobacco cells contain a protein, immunologically related to the neutrophil small G protein Rac2 and involved in elicitor-induced oxidative burst

© 1997 Federation of European Biochemical Societies.

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.

Induction of tcI 7, a gene encoding a β-subunit of proteasome, in tobacco plants treated with elicitins, salicylic acid or hydrogen peroxide1

We previously isolated, by differential display and 5′ RACE (rapid amplification of cDNA ends), cDNAs corresponding to genes activated following cryptogein treatment of tobacco cell suspensions, among them tcI 7 (tcI for obacco ryptogein nduced), a gene encoding a β‐subunit of proteasome. Here, we report that tcI 7 was up‐regulated in tobacco plants treated with elicitins (cryptogein and parasiticein) that have been shown to induce a systemic acquired resistance (SAR). Moreover, subsequent inoculation of tobacco with the pathogen Phytophthora parasitica var. nicotianae (Ppn) was shown to induce an additional activation of tcI 7 in tobacco plants pretreated with cryptogein. We also showed an up‐regulation of tcI 7 by salicylic acid (SA). Moreover, accumulation of tcI 7 transcripts after treatment with cryptogein or with SA only occurred in NahG 9− tobacco plants that do not express the salicylate hydroxylase and thus are able to accumulate SA and develop a SAR. Suppressed accumulation of tcI 7 transcripts in NahG 8+ tobacco plants after cryptogein or SA treatment correlated with the loss of SAR. H2O2 was also shown to up‐regulate tcI 7 in tobacco plants. Using gene walking by PCR we cloned and sequenced the 5′ flanking region of tcI 7 containing hypothetical regulatory sequences, especially myb and NF‐κB boxes, that could be responsible for the regulation of tcI 7 by salicylic acid and H2O2 respectively.

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.