Franck Panabières

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.

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.

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.

Intraspecific polymorphism of Phytophthora parasitica revealed by analysis of mitochondrial DNA restriction fragment length polymorphism

Intraspecific variability of 87 Phytophthora parasitica isolates including P. parasitica var nicotianae sensu Tucker was analysed using RFLP markers of mitochondrial (mt) DNA. Eight mt DNA haplotypes were distinguished among the 87 fungal isolates, and these were compared for geographic origin and pathogenicity on tobacco. Isolates from the West Indies and Australia, displayed variability in their mt DNA haplotypes, but overall, one mitochondrial DNA haplotype predominated and had the broadest geographical distribution, suggesting that the present fungal population derives mainly from the diffusion of that one mt DNA lineage. Mitochondrial RFLPs did not show any correlation with pathogenicity. No obvious trends in genetical divergence could be demonstrated between isolates of P. parasitica and P. parasitica var nicotianae .

Intraspecific Variation in Phytophthora citrophthora from Citrus Trees in Eastern Corsica

Isolates of Phytophthora pathogenic to citrus crops on Eastern Corsica and associated with gummosis were identified by PCR-RFLP of internal transcribed spacers (ITS) sequences and characterized by the random amplified microsatellites (RAMS) technique. A sample of 114 isolates collected from diseased trunks and fruits, and from soil, were overwhelmingly Phytophthora citrophthora. Further analysis indicated that the P. citrophthora population was not homogeneous in citrus groves. There were two groups, with a few (4%) atypical isolates in two marginal groups. The major groups have been re-examined in the light of mating behaviour, RFLPs of mitochondrial DNA and sequence comparisons of ITS regions of rDNA. They were found distinct with all these criteria and perhaps constitute distinct taxa. The results indicate that important modifications occurred in the population structure of P. citrophthora over time in Corsican groves. These changes may have impact on the recent outbreaks of gummosis.

Plant‐induced cell death in the oomycete pathogen Phytophthora parasitica

The activation of programmed cell death in the host during plant–pathogen interactions is an important component of the plant disease resistance mechanism. In this study we show that activation of programmed cell death in microorganisms also regulates plant–pathogen interactions. We found that a form of vacuolar cell death is induced in the oomycete Phytophthora parasitica– the agent that causes black shank disease in Nicotiana tabacum– by extracellular stimuli from resistant tobacco. The single‐celled zoospores underwent cell death characterized by dynamic membrane rearrangements, cell shrinkage, formation of numerous large vacuoles in the cytoplasm and degradation of cytoplasmic components before plasma membrane disruption. Phytophthora cell death required protein synthesis but not caspase activation, and was associated with the production of intracellular reactive oxygen species. This characterization of plant‐mediated cell death signalling in pathogens will enhance our understanding of the biological processes regulating plant–pathogen interactions, and improve our ability to control crop diseases.

Responses of Tobacco to Elicitins, Proteins from Phytophthora SPP. Eliciting Acquired Resistance

With the exception of Phytophthora parasitica var. nicotianae (Ppn), the tobacco black-shank causing agent, Phytophthoras give rise to non-host interactions with tobacco. The resulting local hypersensitive response (HR) is accompanied by necrotic spots on the leaves at distance from the infection site [1]. Low molecular weight proteins are excreted by these Phytophthoras, both in planta and in vitro. They form a family of highly homologous holoproteins, called elicitins [2]. Tobacco plants treated with purified elicitins develop necrotic symptoms similar to those induced by the live fungus, and become resistant to further inoculation with Ppn [3]. Elicitin-treated tobacco represent an attractive model for the analysis of HR and its relation to acquired resistance.

Phylogenetic relationship of Phytophthora cryptogea Pethybr. & Laff and P. drechsleri Tucker.

The phylogeny and taxonomy of Phytophthora cryptogea and Phytophthora drechsleri has long been a matter of controversy. To re-evaluate this, a worldwide collection of 117 isolates assigned to either P. cryptogea, P. drechsleri or their sister taxon, Phytophthora erythroseptica were assessed for morphological, physiological (pathological, cultural, temperature relations, mating) and molecular traits. Multiple gene phylogenetic analysis was performed on DNA sequences of nuclear (internal transcribed spacers (ITS), ß-tubulin, translation elongation factor 1α, elicitin) and mitochondrial (cytochrome c oxidase subunit I) genes. Congruence was observed between the different phylogenetic data sets and established that P. drechsleri and P. cryptogea are distinct species. Isolates of P. drechsleri form a monophyletic grouping with low levels of intraspecific diversity whereas P. cryptogea is more variable. Three distinct phylogenetic groups were noted within P. cryptogea with an intermediate group providing strong evidence for introgression of previously isolated lineages. This evidence suggests that P. cryptogea is an operational taxonomic unit and should remain a single species. Of all the morphological and physiological traits only growth rate at higher temperatures reliably discriminated isolates of P. drechsleri and P. cryptogea. As a homothallic taxon, P. erythroseptica, considered the cause of potato pink rot, is clearly different in mating behaviour from the other two species. Pathogenicity, however, was not a reliable characteristic as all isolates of the three species formed pink rot in potato tubers. The phylogenetic evidence suggests P. erythroseptica has evolved from P. cryptogea more recently than the split from the most recent common ancestor of all three species. However, more data and more isolates of authentic P. erythroseptica are needed to fully evaluate the taxonomic position of this species.

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.

A FAMILY OF REPEATED DNA IN THE GENOME OF THE OOMYCETE PLANT PATHOGEN PHYTOPHTHORA CRYPTOGEA

Abstract We have identified a family of repetitive sequences, called PCISs (Phytophthora Cryptogea Insertion Sequences), in the genome of Phytophthora cryptogea. They vary greatly in size (in a 100–1200u2009bp range) and appear to represent 3′ terminal fragments of a larger element. Two subfamilies were characterised on the basis of nucleotide sequences. PCISs exhibit insertion polymorphism, as well as a very low sequence divergence. Most copies are flanked by terminal direct repeats, suggesting that their spreading involves insertion events rather than rearrangements. PCISs are found in very few copies in Phytophthora cinnamomi. The spreading of these sequences within the genus Phytophthora is discussed.