Philippe Simoneau

Dual Roles of Reactive Oxygen Species and NADPH Oxidase RBOHD in an Arabidopsis-Alternaria Pathosystem

Arabidopsis (Arabidopsis thaliana) NADPH oxidases have been reported to suppress the spread of pathogen- and salicylic acid-induced cell death. Here, we present dual roles of RBOHD (for respiratory burst oxidase homolog D) in an Arabidopsis-Alternaria pathosystem, suggesting either initiation or prevention of cell death dependent on the distance from pathogen attack. Our data demonstrate that a rbohD knockout mutant exhibits increased spread of cell death at the macroscopic level upon inoculation with the fungus Alternaria brassicicola. However, the cellular patterns of reactive oxygen species accumulation and cell death are fundamentally different in the AtrbohD mutant compared with the wild type. Functional RBOHD causes marked extracellular hydrogen peroxide accumulation as well as cell death in distinct, single cells of A. brassicicola-infected wild-type plants. This single cell response is missing in the AtrbohD mutant, where infection triggers spreading-type necrosis preceded by less distinct chloroplastic hydrogen peroxide accumulation in large clusters of cells. While the salicylic acid analog benzothiadiazole induces the action of RBOHD and the development of cell death in infected tissues, the ethylene inhibitor aminoethoxyvinylglycine inhibits cell death, indicating that both salicylic acid and ethylene positively regulate RBOHD and cell death. Moreover, A. brassicicola-infected AtrbohD plants hyperaccumulate ethylene and free salicylic acid compared with the wild type, suggesting negative feedback regulation of salicylic acid and ethylene by RBOHD. We propose that functional RBOHD triggers death in cells that are damaged by fungal infection but simultaneously inhibits death in neighboring cells through the suppression of free salicylic acid and ethylene levels.

Emergence shapes the structure of the seed microbiota.

ABSTRACT Seeds carry complex microbial communities, which may exert beneficial or deleterious effects on plant growth and plant health. To date, the composition of microbial communities associated with seeds has been explored mainly through culture-based diversity studies and therefore remains largely unknown. In this work, we analyzed the structures of the seed microbiotas of different plants from the family Brassicaceae and their dynamics during germination and emergence through sequencing of three molecular markers: the ITS1 region of the fungal internal transcribed spacer, the V4 region of 16S rRNA gene, and a species-specific bacterial marker based on a fragment of gyrB. Sequence analyses revealed important variations in microbial community composition between seed samples. Moreover, we found that emergence strongly influences the structure of the microbiota, with a marked reduction of bacterial and fungal diversity. This shift in the microbial community composition is mostly due to an increase in the relative abundance of some bacterial and fungal taxa possessing fast-growing abilities. Altogether, our results provide an estimation of the role of the seed as a source of inoculum for the seedling, which is crucial for practical applications in developing new strategies of inoculation for disease prevention.

Conventional and real-time PCR-based assay for detecting pathogenic Alternaria brassicae in cruciferous seed.

Alternaria brassicae is an important seedborne pathogenic fungus responsible for the black spot disease of crucifers. Sanitary control of commercial seed is necessary to limit the spread of this pathogen. Current detection methods, based on culture and morphological identification of the fungus, are time consuming, laborious, and not always reliable. Therefore, a polymerase chain reaction (PCR)-based assay was developed with A. brassicae-specific primers designed on the basis of the sequence of two clustered genes potentially involved in pathogenicity. Two sets of primers were selected for conventional and real-time PCR, respectively. In both cases, A. brassicae was specifically detected using DNA extracted from seed. The real-time PCR-based method presented here can be automated easily and preliminary results indicate that it is efficient for quantitative estimation of seed infection.

Characterization of mutations in the two-component histidine kinase gene AbNIK1 from Alternaria brassicicola that confer high dicarboximide and phenylpyrrole resistance

Highly iprodione- and fludioxonil-resistant field and laboratory isolates of A. brassicicola were found to be either moderately sensitive or tolerant to osmotic stress. AbNIK1, a two-component histidine kinase gene, was isolated from a fungicide-sensitive strain. The predicted protein possessed the six tandem amino acid repeats at the N-terminal end, which is a landmark of osmosensor histidine kinases from filamentous fungi. A comparison of the nucleic acid sequences of the AbNIK1 gene from fungicide-sensitive and fungicide-resistant isolates revealed the presence of mutations in six of the seven resistant strains analyzed. Null mutants were all found to be moderately sensitive to osmotic stress, indicating that they are similar to Neurospora crassa Type I os-1 mutants. Only one mutation, corresponding to a single amino acid change within the H-box of the kinase domain, was found in an osmotolerant strain. These results suggest that AbNIK1p participates in osmoregulation and that expression of the fully functional enzyme is essential for dicarboximide and phenylpyrrole antifungal activities.

Transcriptional responses to exposure to the brassicaceous defence metabolites camalexin and allyl‐isothiocyanate in the necrotrophic fungus Alternaria brassicicola

SUMMARY Alternaria brassicicola is the causative agent of black spot disease of Brassicaceae belonging to the genera Brassica and Raphanus. During host infection, A. brassicicola is exposed to high levels of antimicrobial defence compounds such as indolic phytoalexins and glucosinolate breakdown products. To investigate the transcriptomic response of A. brassicicola when challenged with brassicaceous defence metabolites, suppression subtractive hybridization (SSH) was performed to generate two cDNA libraries from germinated conidia treated either with allyl isothiocyanate (Al-ITC) or with camalexin. Following exposure to Al-ITC, A. brassicicola displayed a response similar to that experienced during oxidative stress. Indeed, a substantial subset of differentially expressed genes was related to cell protection against oxidative damage. Treatment of A. brassicicola conidia with the phytoalexin camalexin appeared to activate a compensatory mechanism to preserve cell membrane integrity and, among the camalexin-elicited genes, several were involved in sterol and sphingolipid biosynthesis. The transcriptomic analysis suggested that protection against the two tested compounds also involved mechanisms aimed at limiting their intracellular accumulation, such as melanin biosynthesis (in the case of camalexin exposure only) and drug efflux. From the Al-ITC and the camalexin differentially expressed genes identified here, 25 were selected to perform time-course studies during interactions with brassicaceous hosts. In planta, up-regulation of all the selected genes was observed during infection of Raphanus sativus whereas only a subset were over-expressed during the incompatible interaction with Arabidopsis thaliana ecotype Columbia.

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.

The Group III Two-Component Histidine Kinase of Filamentous Fungi Is Involved in the Fungicidal Activity of the Bacterial Polyketide Ambruticin

ABSTRACT We have shown that the plant pathogen Alternaria brassicicola exhibited very high susceptibility to ambruticin VS4 and to a lesser extent to the phenylpyrrole fungicide fludioxonil. These compounds are both derived from natural bacterial metabolites with antifungal properties and are thought to exert their toxicity by interfering with osmoregulation in filamentous fungi. Disruption of the osmosensor group III histidine kinase gene AbNIK1 (for A. brassicola NIK1) resulted in high levels of resistance to ambruticin and fludioxonil, while a mutant isolate characterized by a single-amino-acid substitution in the HAMP domain of the kinase only exhibited moderate resistance. Moreover, the natural resistance of Saccharomyces cerevisiae to these antifungal molecules switched to sensitivity in strains expressing AbNIK1p. We also showed that exposure to fludioxonil and ambruticin resulted in abnormal phosphorylation of a Hog1-like mitogen-activated protein kinase (MAPK) in A. brassicicola. Parallel experiments carried out with wild-type and mutant isolates of Neurospora crassa revealed that, in this species, ambruticin susceptibility was dependent on the OS1-RRG1 branch of the phosphorelay pathway downstream of the OS2 MAPK cascade but independent of the yeast Skn7-like response regulator RRG2. These results show that the ability to synthesize a functional group III histidine kinase is a prerequisite for the expression of ambruticin and phenylpyrrole susceptibility in A. brassicicola and N. crassa and that, at least in the latter species, improper activation of the high-osmolarity glycerol-related pathway could explain their fungicidal properties.

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.

Laser nephelometry applied in an automated microplate system to study filamentous fungus growth.

By contrast with photometry (i.e., the measurement of light transmitted through a particle suspension), nephelometry is a direct method of measuring light scattered by particles in suspension. Since the scattered light intensity is directly proportional to the suspended particle concentration, nephelometry is a promising method for recording microbial growth and especially for studying filamentous fungi, which cannot be efficiently investigated through spectrophotometric assays. We describe herein for the first time a filamentous fungi-tailored procedure based on microscale liquid cultivation and automated nephelometric recording of growth, followed by extraction of relevant variables (lag time and growth rate) from the obtained growth curves. This microplate reader technique is applicable for the evaluation of antifungal activity and for large-scale phenotypic profiling.