Responses to hydric stress in the seed-borne necrotrophic fungus Alternaria brassicicola

Alternaria brassicicola is a necrotrophic fungus causing black spot disease and is an economically important seed-borne pathogen of cultivated brassicas. Seed transmission is a crucial component of its parasitic cycle as it promotes long-term survival and dispersal. Recent studies, conducted with the Arabidopsis thaliana/A. brassicicola pathosystem, showed that the level of susceptibility of the fungus to water stress strongly influenced its seed transmission ability. In this study, we gained further insights into the mechanisms involved in the seed infection process by analyzing the transcriptomic and metabolomic responses of germinated spores of A. brassicicola exposed to water stress. Then, the repertoire of putative hydrophilins, a group of proteins that are assumed to be involved in cellular dehydration tolerance, was established in A. brassicicola based on the expression data and additional structural and biochemical criteria. Phenotyping of single deletion mutants deficient for fungal hydrophilin-like proteins showed that they were affected in their transmission to A. thaliana seeds, although their aggressiveness on host vegetative tissues remained intact

Identification of Glycolytic Enzyme Polypeptides on the Two-Dimensional Protein Map of Saccharomyces cerevisiae and Application to the Study of Some Wine Yeasts

Using a modification of the basic two-dimensional polyacrylamide gel electrophoresis technique, the polypeptides of the protein map of Saccharomyces cerevisiae involved in glycolysis were investigated. This study resulted in a reassignment of two of the seven glycolytic enzyme polypeptides previously identified (Ludwig et al., Mol. Cell. Biol. 2:117-126, 1982), those corresponding to phosphoglycerate kinase and to alcohol dehydrogenase. It also resulted in the identification of two additional glycolytic polypeptides, the enolase B monomer and the glyceraldehyde phosphate dehydrogenase B monomer. The glycolytic enzymes polypeptides so identified were investigated in 5 laboratory strains (all S. cerevisiae) and in 11 commerical strains used for wine making (S. cerevisiae and Saccharomyces bayanus). It appeared highly significant that a particular electrophoretic variant of the glyceraldehyde phosphate dehydrogenase B monomer was found only in the wine yeasts. Furthermore, it was strongly suggested that S. cerevisiae and S. bayanus strains are distinguishible on the basis of a different electrophoretic migration of the enolase B monomer

Characterization of New Small-Spored <i>Alternaria</i> Species Isolated from <i>Solanaceae</i> in Algeria

Although large-spored Alternaria species of the section Porri are considered to be the major agents responsible for leaf spot and blight of Solanaceae, small-spored Alternaria species are also frequently isolated from symptomatic tissues. A survey of the north-western regions of Algeria during the 2017–2018 growing seasons revealed that amongst the 623 Alternaria isolates from tomato, potato, pepper, eggplant and black nightshade, 8% could not be morphologically assigned to either section Porri or section Alternaria. In order to more precisely determine the taxonomic position of these isolates, detailed morphological characterizations and multi-locus phylogenetic analyses were performed. Based on these analyses, the isolates were grouped into four main clades: section Ulocladioides, section Infectoriae, including two new species, section Embellisioides, and section Eureka, including one new species. These isolates were also characterized for their virulence under green-house conditions. They were able to produce leaf spot symptoms on tomato plants but with variable levels

Data from: Characterization of glutathione transferases involved in the pathogenicity of Alternaria brassicicola

Background: Glutathione transferases (GSTs) represent an extended family of multifunctional proteins involved in detoxification processes and tolerance to oxidative stress. We thus anticipated that some GSTs could play an essential role in the protection of fungal necrotrophs against plant-derived toxic metabolites and reactive oxygen species that accumulate at the host-pathogen interface during infection. Results: Mining the genome of the necrotrophic Brassica pathogen Alternaria brassicicola for glutathione transferase revealed 23 sequences, 17 of which could be clustered into the main classes previously defined for fungal GSTs and six were ‘orphans’. Five isothiocyanate-inducible GSTs from five different classes were more thoroughly investigated. Analysis of their catalytic properties revealed that two GSTs, belonging to the GSTFuA and GTT1 classes, exhibited GSH transferase activity with isothiocyanates (ITC) and peroxidase activity with cumene hydroperoxide, respectively. Mutant deficient for these two GSTs were however neither more susceptible to ITC nor less aggressive than the wild-type parental strain. By contrast mutants deficient for two other GSTs, belonging to the Ure2pB and GSTO classes, were distinguished by their hyper-susceptibility to ITC and low aggressiveness against Brassica oleracea. In particular AbGSTO1 could participate in cell tolerance to ITC due to its glutathione-dependent thioltransferase activity. The fifth ITC-inducible GST belonged to the MAPEG class and although it was not possible to produce the soluble active form of this protein in a bacterial expression system, the corresponding deficient mutant failed to develop normal symptoms on host plant tissues. Conclusions: Among the five ITC-inducible GSTs analyzed in this study, three were found essential for full aggressiveness of A. brassicicola on host plant. This, to our knowledge is the first evidence that GSTs might be essential virulence factors for fungal necrotrophs

Characterization of glutathione transferases involved in the pathogenicity of Alternaria brassicicola

Background Glutathione transferases (GSTs) represent an extended family of multifunctional proteins involved in detoxification processes and tolerance to oxidative stress. We thus anticipated that some GSTs could play an essential role in the protection of fungal necrotrophs against plant-derived toxic metabolites and reactive oxygen species that accumulate at the host-pathogen interface during infection. Results Mining the genome of the necrotrophic Brassica pathogen Alternaria brassicicola for glutathione transferase revealed 23 sequences, 17 of which could be clustered into the main classes previously defined for fungal GSTs and six were ‘orphans’. Five isothiocyanate-inducible GSTs from five different classes were more thoroughly investigated. Analysis of their catalytic properties revealed that two GSTs, belonging to the GSTFuA and GTT1 classes, exhibited GSH transferase activity with isothiocyanates (ITC) and peroxidase activity with cumene hydroperoxide, respectively. Mutant deficient for these two GSTs were however neither more susceptible to ITC nor less aggressive than the wild-type parental strain. By contrast mutants deficient for two other GSTs, belonging to the Ure2pB and GSTO classes, were distinguished by their hyper-susceptibility to ITC and low aggressiveness against Brassica oleracea. In particular AbGSTO1 could participate in cell tolerance to ITC due to its glutathione-dependent thioltransferase activity. The fifth ITC-inducible GST belonged to the MAPEG class and although it was not possible to produce the soluble active form of this protein in a bacterial expression system, the corresponding deficient mutant failed to develop normal symptoms on host plant tissues. Conclusions Among the five ITC-inducible GSTs analyzed in this study, three were found essential for full aggressiveness of A. brassicicola on host plant. This, to our knowledge is the first evidence that GSTs might be essential virulence factors for fungal necrotrophs

Isolation of 12 polymorphic microsatellite loci in the phytopathogenic fungus Alternaria brassicicola

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Dehydrin-like proteins in the necrotrophic fungus Alternaria brassicicola have a role in plant pathogenesis and stress response.

In this study, the roles of fungal dehydrin-like proteins in pathogenicity and protection against environmental stresses were investigated in the necrotrophic seed-borne fungus Alternaria brassicicola. Three proteins (called AbDhn1, AbDhn2 and AbDhn3), harbouring the asparagine-proline-arginine (DPR) signature pattern and sharing the characteristic features of fungal dehydrin-like proteins, were identified in the A. brassicicola genome. The expression of these genes was induced in response to various stresses and found to be regulated by the AbHog1 mitogen-activated protein kinase (MAPK) pathway. A knock-out approach showed that dehydrin-like proteins have an impact mainly on oxidative stress tolerance and on conidial survival upon exposure to high and freezing temperatures. The subcellular localization revealed that AbDhn1 and AbDhn2 were associated with peroxisomes, which is consistent with a possible perturbation of protective mechanisms to counteract oxidative stress and maintain the redox balance in AbDhn mutants. Finally, we show that the double deletion mutant ΔΔabdhn1-abdhn2 was highly compromised in its pathogenicity. By comparison to the wild-type, this mutant exhibited lower aggressiveness on B. oleracea leaves and a reduced capacity to be transmitted to Arabidopsis seeds via siliques. The double mutant was also affected with respect to conidiation, another crucial step in the epidemiology of the disease

Characterization of NRPS and PKS genes involved in the biosynthesis of SMs in Alternaria dauci including the phytotoxic polyketide aldaulactone

International audienceAbstract Alternaria dauci is a Dothideomycete fungus, causal agent of carrot leaf blight. As a member of the Alternaria genus, known to produce a lot of secondary metabolite toxins, A. dauci is also supposed to synthetize host specific and non-host specific toxins playing a crucial role in pathogenicity. This study provides the first reviewing of secondary metabolism genetic basis in the Alternaria genus by prediction of 55 different putative core genes. Interestingly, aldaulactone, a phytotoxic benzenediol lactone from A. dauci , was demonstrated as important in pathogenicity and in carrot partial resistance to this fungus . As nothing is known about aldaulactone biosynthesis, bioinformatic analyses on a publicly available A. dauci genome data set that were reassembled, thanks to a transcriptome data set described here, allowed to identify 19 putative secondary metabolism clusters. We exploited phylogeny to pinpoint cluster 8 as a candidate in aldaulactone biosynthesis. This cluster contains AdPKS7 and AdPKS8 , homologs with genes encoding a reducing and a non-reducing polyketide synthase. Clusters containing such a pair of PKS genes have been identified in the biosynthesis of resorcylic acid lactones or dihydroxyphenylacetic acid lactones. AdPKS7 and AdPKS8 gene expression patterns correlated with aldaulactone production in different experimental conditions. The present results highly suggest that both genes are responsible for aldaulactone biosynthesis