Michel Ponchet

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.

Are elicitins cryptograms in plant-Oomycete communications?

Abstract. 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 applications. In this review, it is proposed that elicitins could interact with regulating cell wall proteins before they reach the plasma membrane. Consequently, the plant reactivity or nonreactivity status could result from the equilibrium reached during this interaction. The possibility of overexpressing the elicitins directly from genomic DNA in Pichia pastoris allows site-directed mutagenesis experiments and structure/function studies. The recent discovery of the sterol carrier activity of elicitins brings a new insight on their molecular activity. This constitutes a crucial property, since the formation of a sterol-elicitin complex is required to trigger the biological responses of tobacco cells and plants. Only the elicitins loaded with a sterol are able to bind to their plasmalemma receptor, which is assumed to be an allosteric calcium channel. Moreover, Phytophthora and Pythium do not synthesize the sterols required for their growth and their fructification, and elicitins may act as shuttles trapping the sterols from the host plants. Sequence analysis of elicitin genes from several Phytophthora species sheds unexpected light on the phylogenetic relationships among the genus, and suggests that the expression of elicitins is under tight regulatory control. Finally, general involvement of these lipid transfer proteins in the biology of Pythiaceae, and in plant defense responses, is discussed. A possible scheme for the coevolution between Phytophthora and tobacco plants is approached.

Pathogen-Induced Elicitin Production in Transgenic Tobacco Generates a Hypersensitive Response and Nonspecific Disease Resistance

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. We have developed a strategy for creating novel disease resistance traits whereby transgenic plants respond to infection by a virulent pathogen with the production of an elicitor. To this end, we generated transgenic tobacco plants harboring a fusion between the pathogen-inducible tobacco hsr203J gene promoter and a Phytophthora cryptogea gene encoding the highly active elicitor cryptogein. Under noninduced conditions, the transgene was silent, and no cryptogein could be detected in the transgenic plants. In contrast, infection by the virulent fungus P. parasitica var nicotianae stimulated cryptogein production that coincided with the fast induction of several defense genes at and around the infection sites. Induced elicitor production resulted in a localized necrosis that resembled a P. cryptogea–induced hypersensitive response and that restricted further growth of the pathogen. The transgenic plants displayed enhanced resistance to fungal pathogens that were unrelated to Phytophthora species, such as Thielaviopsis basicola, Erysiphe cichoracearum, and Botrytis cinerea. Thus, broad-spectrum disease resistance of a plant can be generated without the constitutive synthesis of a transgene product.

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 inducer of systemic acquired resistance, which was increasingly effective with doses ranging from 0.25 to 5Μmole per plant, the basic elicitin cryptogein exhibited a threshold effect, inducing near total resistance and extensive leaf necrosis above 0.1 nmole per plant. Between 1 and 5 nmole, acidic elicitins (capsicein and parasiticein) protected tobacco plants with hardly any necrotic symptom. Elicitins exhibited similar effects in various tobacco cultivars andNicotiana species, although with quantitative differences, but induced neither necrosis nor protection in other SolanaceÆ (tomato, petunia and pepper). Among 24 additional species tested belonging to 18 botanical families, only some BrassicaceÆ, noticeably rape, exhibited symptoms in response to elicitins, in a cultivar-specific manner. Elicitins appear to be natural specific triggers for systemic acquired resistance and provide a tool for unraveling the mechanisms leading to its establishment.

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.

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.

RNase activity prevents the growth of a fungal pathogen in tobacco leaves and increases upon induction of systemic acquired resistance with elicitin.

The hypersensitive response and systemic acquired resistance (SAR) can be induced in tobacco (Nicotiana tabacum L.) plants by cryptogein, an elicitin secreted by Phytophthora cryptogea. Stem application of cryptogein leads to the establishment of acquired resistance to subsequent leaf infection with Phytophthora parasitica var nicotianae, the agent of the tobacco black shank disease. We have studied early events that occur after the infection and show here that a tobacco gene encoding the extracellular S-like RNase NE is expressed in response to inoculation with the pathogenic fungus. Upon induction of SAR with cryptogein, the accumulation of NE transcripts coincided with a rapid induction of RNase activity and with the increase in the activity of at least two different extracellular RNases. Moreover, exogenous application of RNase activity in the extracellular space of leaves led to a reduction of the fungus development by up to 90%, independently of any cryptogein treatment and in the absence of apparent necrosis. These results indicate that the up-regulation of apoplastic RNase activity after inoculation could contribute to the control of fungal invasion in plants induced to SAR with cryptogein.

Characterization of border species among Pythiaceae: several Pythium isolates produce elicitins, typical proteins from Phytophthora spp.

Elicitins, holoproteins which act as inducers of hypersensitivity on tobacco, were considered as a characteristic of Phytophthora. They are also produced, along with glycosylated isoforms, by three species belonging to the related genus Pythium, Py. vexans, Py. oedochilum and Py. marsipium, while other Pythium species do not possess such proteins. Various elicitin-like sequences were determined, bringing novel features to the elicitin family, such as an histidine residue and C-terminal extensions on the deduced peptide sequences. As the unique elicitin content of these species supports a distinct location among Pythiaceae, we suggest the separation of vexans, Py. oedochilum and Py. marsipium from Pythium and consider them as linking species between Phytophthora and Pythium.

Hydroxycinnamoyl acid amides and aromatic amines in the inflorescences of some araceae species

Abstract Hydroxycinnamoyl acid amides (HCAs) were found to be important components in the inflorescences of different Araceae species. HCAs occurred in large amount in spathes and in the male and female flowers, and were totally absent from the sterile flowers, commonly found on Araceae spadices. Differences in the distribution of HCAs were noted between male and female flowers. Thus the amount of neutral HCAs was always greater in the male than in the female flowers and the female flowers generally contained more basic HCAS. In the inflorescences of some Araceae species in the Monsteroideae and Philodendroideae (genera Monstera, Raphidophora and Philodendron), the aromatic amines tyramine and dopamine were very abundant, with concentrations ranging from 1 to 4 mg of each amine per g fr. wt.

Proteinaceous Elicitors of Plant Defense Responses

The role of microbial proteins as signal molecules activating plant defense responses is now well recognized. The properties of several recently characterized proteinaceous elicitors are reviewed. Apart from endopolygalacturonases which release endogenous elicitors from plant cell walls, proteinaceous elicitors seem to interact primarly with the plant plasma membrane. Several of them elicit symptoms similar to those of the hypersensitive response. Most proteinaceous elicitors are secreted, and some can be translocated in plants. Progress is made in isolating genes encoding these elicitors. Some evidences on the role of proteinaceous elicitors in host-pathogen specific interactions are presented.