Pascal Poupard

Cell wall integrity and high osmolarity glycerol pathways are required for adaptation of Alternaria brassicicola to cell wall stress caused by brassicaceous indolic phytoalexins

Camalexin, the characteristic phytoalexin of Arabidopsis thaliana, inhibits growth of the fungal necrotroph Alternaria brassicicola. This plant metabolite probably exerts its antifungal toxicity by causing cell membrane damage. Here we observed that activation of a cellular response to this damage requires cell wall integrity (CWI) and the high osmolarity glycerol (HOG) pathways. Camalexin was found to activate both AbHog1 and AbSlt2 MAP kinases, and activation of the latter was abrogated in a AbHog1 deficient strain. Mutant strains lacking functional MAP kinases showed hypersensitivity to camalexin and brassinin, a structurally related phytoalexin produced by several cultivated Brassica species. Enhanced susceptibility to the membrane permeabilization activity of camalexin was observed for MAP kinase deficient mutants. These results suggest that the two signalling pathways have a pivotal role in regulating a cellular compensatory response to preserve cell integrity during exposure to camalexin. AbHog1 and AbSlt2 deficient mutants had reduced virulence on host plants that may, at least for the latter mutants, partially result from their inability to cope with defence metabolites such as indolic phytoalexins. This constitutes the first evidence that a phytoalexin activates fungal MAP kinases and that outputs of activated cascades contribute to protecting the fungus against antimicrobial plant metabolites.

A wound- and ethephon-inducible PR-10 gene subclass from apple is differentially expressed during infection with a compatible and an incompatible race of Venturia inaequalis

Abstract A salicylate-inducible PR-10 gene subclass from apple, designated as APa, was characterized. The APa genes were shown to be highly induced in leaves by abiotic stimuli (ethephon, wounding), as well as by inoculation with a virulent or an avirulent strain of Venturia inaequalis. The up-regulation of APa transcripts occurred earlier in the compatible situation than in the incompatible one, but at higher levels in the latter case. At the protein level, a PR-10 polypeptide band (18 kDa) was detected in both interactions, whereas an additional band (17 kDa) was markedly revealed only after infection with the avirulent strain. Immunodetection of PR-10 proteins on leaf prints indicated their vascular localization and an intensification of the signal in the incompatible condition. Overexpression of APa gene products after infection with the avirulent strain was concomitant with the occurrence of resistance symptoms. For two genes of the APa subclass (Ypr10∗Md.b and Ypr10∗Md.d), specific patterns of expression were observed: both Ypr10∗Md.b and Ypr10∗Md.d were transcriptionally activated by ethephon or following inoculation with the avirulent strain of V. inaequalis, whereas after treatment with a salicylate analogue, only Ypr10∗Md.d was induced. This suggests that at least a defense pathway involving ethylene is activated in the incompatible situation.

Expression of a Bet v 1 homologue gene encoding a PR 10 protein in birch roots: induction by auxin and localization of the transcripts by in situ hybridization

In birch roots (Betula pendula Roth), two members of the Bet v 1 gene family which encode PR 10 proteins have previously been characterized. One of these members, named Bet v 1-sc1, is significantly induced in response to biotic or abiotic factors. We have analysed the expression of Bet v 1-sc1 in birch roots treated either with 1 M indole-3-acetic acid (IAA) or 1 M kinetin using reverse transcription–polymerase chain reaction (RT–PCR), northern blotting and competitive PCR. High accumulation of the Bet v 1-sc1 transcripts was recorded only after auxin application, while kinetin had no effect. By in situ hybridization, we have investigated the localization of Bet v 1-sc1 mRNA in birch roots after induction of the gene by root treatment with 1 M IAA. Using root tip sections, we showed that Bet v 1-sc1 is significantly expressed in the apical meristem and the procambium. In sections taken in the zone producing lateral roots, the presence of Bet v 1-sc1 was found at sites of emerging secondary root primordia. This first report of localization of Bet v 1-sc1 expression suggests that this gene could be involved in the processes leading to lateral root initiation.

Specific activation of PR-10 pathogenesis-related genes in apple by an incompatible race of Venturia inaequalis

Apple (Malus × domestica Borkh. cv. Golden Delicious) trees were inoculated with fungal pathogens, corresponding either to an incompatible strain of Venturia inaequalis or to non-host pathogens (Venturia pyrina, Alternaria brassicicola) in order to characterize the regulation of PR-10 genes in these different situations in relationship to symptom development. Macro-and microscopic observations of the plant-fungus interactions revealed typical symptoms of resistance with V. inaequalis and symptoms of hypersensitivity (HR) on around 5 % of leaves with V. pyrina. No HR was microscopically observed with A. brassicicola. In the non-host situations, the PR-10 expression in leaves at the transcriptional level was not (A. brassicicola) or very slightly (V. pyrina) activated. This strongly suggests that PR-10 is not a component of the non-host resistance in the interactions studied here. By contrast specific up-regulation of PR-10 was evident after inoculation with V. inaequalis.

Partial Resistance of Carrot to Alternaria dauci Correlates with In Vitro Cultured Carrot Cell Resistance to Fungal Exudates

Although different mechanisms have been proposed in the recent years, plant pathogen partial resistance is still poorly understood. Components of the chemical warfare, including the production of plant defense compounds and plant resistance to pathogen-produced toxins, are likely to play a role. Toxins are indeed recognized as important determinants of pathogenicity in necrotrophic fungi. Partial resistance based on quantitative resistance loci and linked to a pathogen-produced toxin has never been fully described. We tested this hypothesis using the Alternaria dauci – carrot pathosystem. Alternaria dauci, causing carrot leaf blight, is a necrotrophic fungus known to produce zinniol, a compound described as a non-host selective toxin. Embryogenic cellular cultures from carrot genotypes varying in resistance against A. dauci were confronted with zinniol at different concentrations or to fungal exudates (raw, organic or aqueous extracts). The plant response was analyzed through the measurement of cytoplasmic esterase activity, as a marker of cell viability, and the differentiation of somatic embryos in cellular cultures. A differential response to toxicity was demonstrated between susceptible and partially resistant genotypes, with a good correlation noted between the resistance to the fungus at the whole plant level and resistance at the cellular level to fungal exudates from raw and organic extracts. No toxic reaction of embryogenic cultures was observed after treatment with the aqueous extract or zinniol used at physiological concentration. Moreover, we did not detect zinniol in toxic fungal extracts by UHPLC analysis. These results suggest that strong phytotoxic compounds are present in the organic extract and remain to be characterized. Our results clearly show that carrot tolerance to A. dauci toxins is one component of its partial resistance.

Aldaulactone – an original phytotoxic secondary metabolite involved in the aggressiveness of Alternaria dauci on carrot

Qualitative plant resistance mechanisms and pathogen virulence have been extensively studied since the formulation of the gene-for-gene hypothesis. The mechanisms involved in the quantitative traits of aggressiveness and plant partial resistance are less well-known. Nevertheless, they are prevalent in most plant-necrotrophic pathogen interactions, including the Daucus carota–Alternaria dauci interaction. Phytotoxic metabolite production by the pathogen plays a key role in aggressiveness in these interactions. The aim of the present study was to explore the link between A. dauci aggressiveness and toxin production. We challenged carrot embryogenic cell cultures from a susceptible genotype (H1) and two partially resistant genotypes (I2 and K3) with exudates from A. dauci strains with various aggressiveness levels. Interestingly, A. dauci-resistant carrot genotypes were only affected by exudates from the most aggressive strain in our study (ITA002). Our results highlight a positive link between A. dauci aggressiveness and the fungal exudate cell toxicity. We hypothesize that the fungal exudate toxicity was linked with the amount of toxic compounds produced by the fungus. Interestingly, organic exudate production by the fungus was correlated with aggressiveness. Hence, we further analyzed the fungal organic extract using HPLC, and correlations between the observed peak intensities and fungal aggressiveness were measured. One observed peak was closely correlated with fungal aggressiveness. We succeeded in purifying this peak and NMR analysis revealed that the purified compound was a novel 10-membered benzenediol lactone, a polyketid that we named ‘aldaulactone’. We used a new automated image analysis method and found that aldaulactone was toxic to in vitro cultured plant cells at those concentrations. The effects of both aldaulactone and fungal organic extracts were weaker on I2-resistant carrot cells compared to H1 carrot cells. Taken together, our results suggest that: (i) aldaulactone is a new phytotoxin, (ii) there is a relationship between the amount of aldaulactone produced and fungal aggressiveness, and (iii) carrot resistance to A. dauci involves mechanisms of resistance to aldaulactone.