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Tomato (Solanum lycopersicum L.) is an economically important vegetable susceptible to various fungal diseases, including leaf spot caused by Alternaria spp. from the section Alternaria. In our study, a total of 72 tomato-associated Alternaria spp. strains from Latvia, Lithuania, Estonia, and Algeria were analysed by integrating morphological data, pathogenicity assay, multi-locus phylogeny, and haplotype assignment. Recovered Alternaria spp. strains were characterized by considerable variation in phenotypic diversity, non-pathogenicity to their host of origin and absence of the AAL-toxin biosynthesis gene (ALT1). Multi-locus phylogeny of the RNA polymerase II second largest subunit (rpb2), putative F-box-domain-containing protein (ASA-10), and putative histone-like transcription factor (ASA-19) confirmed the occurrence of both A. alternata and A. arborescens species complexes along with A. longipes and A. postmessia on symptomatic tomatoes. The discordant tree topology among single-gene phylogenies suggested the occurrence of potential recombination between phylogenetic lineages in the section Alternaria, resulting in putative alternata-arborescens and alternata-longipes hybrids. DNA polymorphism analysis of the rpb2, ASA-10, and ASA-19 loci revealed a high level of genetic diversity in the section Alternaria, and the number of single nucleotide polymorphisms (SNPs) and haplotypes varied among loci and lineages studied. A total of 16 and 6 multi-locus haplotypes were assigned in alternata and arborescens lineages, respectively. Global genetic diversity analysis of A. alternata and A. arborescens strains at the rpb2 locus confirmed that major haplotypes described from tomatoes were shared among other hosts of origin., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The unfolded protein response (UPR) is a key component of fungal virulence. The prenylated xanthone γ-mangostin isolated from Garcinia mangostana (Clusiaceae) fruit pericarp, has recently been described to inhibit this fungal adaptative pathway. Considering that Calophyllum caledonicum (Calophyllaceae) is known for its high prenylated xanthone content, its stem bark extract was fractionated using a bioassay-guided procedure based on the cell-based anti-UPR assay. Four previously undescribed xanthone derivatives were isolated, caledonixanthones N-Q ( 3 , 4 , 8 , and 12 ), among which compounds 3 and 8 showed promising anti-UPR activities with IC 50 values of 11.7 ± 0.9 and 7.9 ± 0.3 μM, respectively.

Discovering new solutions for crop protection is a major challenge for the next decades as a result of the ecotoxicological impact of classical fungicides, the emergence of fungicide resistances, and the consequence of climate change on pathogen distribution. Previous work on fungal mutants deficient in the unfolded protein response (UPR) supported that targeting this pathway is a promising plant disease control strategy. In particular, we showed that the UPR is involved in fungal virulence by altering cell protection against host defense compounds, such as phytoalexins and phytoanticipins. In this study, we evaluated natural products targeting fungal IRE1 protein (UPR effector) and consequently increasing fungal susceptibility to plant defenses. Developing an in vitro cell-based screening assay allowed for the identification of seven potential IRE1 inhibitors with a focus on polyhydroxylated prenylated xanthones. Inhibition of hac 1 mRNA splicing, which is mediated by IRE1, was then validated for the most active compound, namely, γ-mangostin 3 . To study the mode of interaction between the binding site of IRE1 and active xanthones, molecular docking was also undertaken, revealing similar and novel interactions between the known inhibitor and the binding site. Eventually, active xanthones applied at subtoxic doses induced a significant reduction in necrosis size for leaves of Brassica oleracea inoculated with Alternaria brassicicola and Botrytis cinerea .

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., (© 2022. The Author(s).)

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.

Alternaria brassicicola causes black spot disease in Brassicaceae . During host infection, this necrotrophic fungus is exposed to various antimicrobial compounds, such as the phytoalexin brassinin which is produced by many cultivated Brassica species. To investigate the cellular mechanisms by which this compound causes toxicity and the corresponding fungal adaptive strategies, we first analyzed fungal transcriptional responses to short-term exposure to brassinin and then used additional functional approaches. This study supports the hypothesis that indolic phytoalexin primarily targets mitochondrial functions in fungal cells. Indeed, we notably observed that phytoalexin treatment of A. brassicicola disrupted the mitochondrial membrane potential and resulted in a significant and rapid decrease in the oxygen consumption rates. Secondary effects, such as Reactive oxygen species production, changes in lipid and endoplasmic reticulum homeostasis were then found to be induced. Consequently, the fungus has to adapt its metabolism to protect itself against the toxic effects of these molecules, especially via the activation of high osmolarity glycerol and cell wall integrity signaling pathways and by induction of the unfolded protein response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 N’Guyen, Raulo, Porquier, Iacomi, Pelletier, Renou, Bataillé-Simoneau, Campion, Hamon, Kwasiborski, Colou, Benamar, Hudhomme, Macherel, Simoneau and Guillemette.)

A leaf spot pathogen Alternaria sp. was recovered from jimson weed, tomato, parsley, and coriander collected during surveys of blight diseases on Solanaceae and Apiaceae in Algeria. This species produced large conidial body generating long apical beaks that tapered gradually from a wide base to a narrow tip and short conidiophores originating directly from the agar surface. This species exhibited morphological traits similar to that reported for Alternaria crassa . The identification of seven strains from different hosts was confirmed by sequence analyses at the glyceraldehyde-3-phosphate dehydrogenase, RNA polymerase second largest subunit, and translation elongation factor 1-alpha loci. Further the pathogen was evaluated on jimson weed, coriander, parsley, and tomato plants, and this fungus was able to cause necrotic lesions on all inoculated plants. A. crassa is reported for the first time as a new species of the Algerian mycoflora and as a new potential pathogen for cultivated hosts., (© The Korean Society of Plant Pathology.)

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.

Background: Flavin-dependent monooxygenases are involved in key biological processes as they catalyze a wide variety of chemo-, regio- and enantioselective oxygenation reactions. Flavoprotein monooxygenases are frequently encountered in micro-organisms, most of which require further functional and biocatalytic assessment. Here we investigated the function of the AbMak1 gene, which encodes a group A flavin monooxygenase in the plant pathogenic fungus Alternaria brassicicola , by generating a deficient mutant and examining its phenotype., Results: Functional analysis indicates that the AbMak1 protein is involved in cell wall biogenesis and influences the melanization process. We documented a significant decrease in melanin content in the Δ abmak1 strain compared to the wild-type and complemented strains. We investigated the cell wall morphology and physical properties in the wild-type and transformants using electron and atomic force microscopy. These approaches confirmed the aberrant morphology of the conidial wall structure in the Δ abmak1 strain which had an impact on hydrophilic adhesion and conidial surface stiffness. However, there was no significant impairment in growth, conidia formation, pathogenicity or susceptibility to various environmental stresses in the Δ abmak1 strain., Conclusion: This study sheds new light on the function of a fungal flavin-dependent monooxygenase, which plays an important role in melanization.

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.

This study describes the gel-free phosphoproteomic analysis of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea grown in vitro under nonlimiting conditions. Using a combination of strong cation exchange and IMAC prior to LC-MS, we identified over 1350 phosphopeptides per fungus representing over 800 phosphoproteins. The preferred phosphorylation sites were found on serine (>80%) and threonine (>15%), whereas phosphorylated tyrosine residues were found at less than 1% in A. brassicicola and at a slightly higher ratio in B. cinerea (1.5%). Biological processes represented principally among the phoshoproteins were those involved in response and transduction of stimuli as well as in regulation of cellular and metabolic processes. Most known elements of signal transduction were found in the datasets of both fungi. This study also revealed unexpected phosphorylation sites in histidine kinases, a category overrepresented in filamentous ascomycetes compared to yeast. The data have been deposited to the ProteomeXchange database with identifier PXD000817 (http://proteomecentral.proteomexchange.org/dataset/PXD000817)., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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.

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.

In this study, activation of the Jak/Stat signaling pathway was followed upon growth hormone (GH) stimulation, using the rat osteosarcoma cell-line UMR-106.01 that expresses high affinity GH receptors. The results show a GH-induced and sustained phosphorylation of Jak2 and Stat5 on tyrosine residues. The tyrosine phosphorylation status of Jak2 was increased in a dose-dependent manner. In contrast to Jak2, tyrosine phosphorylation of Stat5, also elicited at 42 ng/ml GH, remained unchanged when GH concentration was raised up to 4200 ng/ml. DNA binding activity of Stat5 was also observed in response to GH. However, GH was unable to cause transactivation of reporter gene constructs harboring Stat5 binding sites (the GHREII from the rat spi 2.1 gene promoter, and the LHRE from the rat beta-casein gene promoter), except in cells transiently transfected with either Stat5 cDNAs or the rat GHR cDNA. Altogether the results suggest that UMR-106.01 cells exhibit original features of the GH-dependent Jak/Stat signaling pathway.