LYSMD3: A mammalian pattern recognition receptor for chitin.

Chitin, a major component of fungal cell walls, has been associated with allergic disorders such as asthma. However, it is unclear how mammals recognize chitin and the principal receptor(s) on epithelial cells that sense chitin remain to be determined. In this study, we show that LYSMD3 is expressed on the surface of human airway epithelial cells and demonstrate that LYSMD3 is able to bind chitin, as well as ß-glucan, on the cell walls of fungi. Knockdown or knockout of LYSMD3 also sharply blunts the production of inflammatory cytokines by epithelial cells in response to chitin and fungal spores. Competitive inhibition of the LYSMD3 ectodomain by soluble LYSMD3 protein, multiple ligands, or antibody against LYSMD3 also blocks chitin signaling. Our study reveals LYSMD3 as a mammalian pattern recognition receptor (PRR) for chitin and establishes its role in epithelial cell inflammatory responses to chitin and fungi.

Heparin-based hydrogel scaffolding alters the transcriptomic profile and increases the chemoresistance of MDA-MB-231 triple-negative breast cancer cells.

The tumor microenvironment plays a critical role in the proliferation and chemoresistance of cancer cells. Growth factors (GFs) are known to interact with the extracellular matrix (ECM) via heparin binding sites, and these associations influence cell behavior. In the present study, we demonstrate the ability to define signals presented by the scaffold by pre-mixing growth factors, such as epidermal growth factor, into the heparin-based (HP-B) hydrogel prior to gelation. In the 3D biomimetic microenvironment, breast cancer cells formed spheroids within 24 hours of initial seeding. Despite higher number of proliferating cells in 2D cultures, 3D spheroids exhibited a higher degree of chemoresistance after 72 hours. Further, our RNA sequencing results highlighted the phenotypic changes influenced by solid-phase GF presentation. Wnt/ß-catenin and TGF-ß signaling were upregulated in the cells grown in the hydrogel, while apoptosis, IL2-STAT5 and PI3K-AKT-mTOR signaling were downregulated. With emerging technologies for precision medicine in cancer, this nature of fine-tuning the microenvironment is paramount for cultivation and downstream characterization of primary cancer cells and rare circulating tumor cells (CTCs), and effective screening of chemotherapeutic agents.

Innate Immunity Induced by the Major Allergen Alt a 1 From the Fungus Alternaria Is Dependent Upon Toll-Like Receptors 2/4 in Human Lung Epithelial Cells.

Allergens are molecules that elicit a hypersensitive inflammatory response in sensitized individuals and are derived from a variety of sources. Alt a 1 is the most clinically important secreted allergen of the ubiquitous fungus, Alternaria. It has been shown to be a major allergen causing IgE-mediated allergic response in the vast majority of Alternaria-sensitized individuals. However, no studies have been conducted in regards to the innate immune eliciting activities of this clinically relevant protein. In this study, recombinant Alt a 1 was produced, purified, labeled, and incubated with BEAS-2B, NHBE, and DHBE human lung epithelial cells. Alt a 1 elicited strong induction of IL-8, MCP-1, and Gro-a/b/g. Using gene-specific siRNAs, blocking antibodies, and chemical inhibitors such as LPS-RS, it was determined that Alt a 1-induced immune responses were dependent upon toll-like receptors (TLRs) 2 and 4, and the adaptor proteins MYD88 and TIRAP. Studies utilizing human embryonic kidney cells engineered to express single receptors on the cell surface such as TLRs, further confirmed that Alt a 1-induced innate immunity is dependent upon TLR4 and to a lesser extent TLR2.

Modulation of Immune Signaling and Metabolism Highlights Host and Fungal Transcriptional Responses in Mouse Models of Invasive Pulmonary Aspergillosis.

Incidences of invasive pulmonary aspergillosis, an infection caused predominantly by Aspergillus fumigatus, have increased due to the growing number of immunocompromised individuals. While A. fumigatus is reliant upon deficiencies in the host to facilitate invasive disease, the distinct mechanisms that govern the host-pathogen interaction remain enigmatic, particularly in the context of distinct immune modulating therapies. To gain insights into these mechanisms, RNA-Seq technology was utilized to sequence RNA derived from lungs of 2 clinically relevant, but immunologically distinct murine models of IPA on days 2 and 3 post inoculation when infection is established and active disease present. Our findings identify notable differences in host gene expression between the chemotherapeutic and steroid models at the interface of immunity and metabolism. RT-qPCR verified model specific and nonspecific expression of 23 immune-associated genes. Deep sequencing facilitated identification of highly expressed fungal genes. We utilized sequence similarity and gene expression to categorize the A. fumigatus putative in vivo secretome. RT-qPCR suggests model specific gene expression for nine putative fungal secreted proteins. Our analysis identifies contrasting responses by the host and fungus from day 2 to 3 between the two models. These differences may help tailor the identification, development, and deployment of host- and/or fungal-targeted therapeutics.

The Alternaria alternata Mycotoxin Alternariol Suppresses Lipopolysaccharide-Induced Inflammation.

The Alternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) have been shown to possess genotoxic and cytotoxic properties. In this study, the ability of AOH and AME to modulate innate immunity in the human bronchial epithelial cell line (BEAS-2B) and mouse macrophage cell line (RAW264.7) were investigated. During these studies, it was discovered that AOH and to a lesser extent AME potently suppressed lipopolysaccharide (LPS)-induced innate immune responses in a dose-dependent manner. Treatment of BEAS-2B cells with AOH resulted in morphological changes including a detached pattern of growth as well as elongated arms. AOH/AME-related immune suppression and morphological changes were linked to the ability of these mycotoxins to cause cell cycle arrest at the G2/M phase. This model was also used to investigate the AOH/AME mechanism of immune suppression in relation to aryl hydrocarbon receptor (AhR). AhR was not found to be important for the immunosuppressive properties of AOH/AME, but appeared important for the low levels of cell death observed in BEAS-2B cells.

The Alternaria genomes database: a comprehensive resource for a fungal genus comprised of saprophytes, plant pathogens, and allergenic species.

BACKGROUND: Alternaria is considered one of the most common saprophytic fungal genera on the planet. It is comprised of many species that exhibit a necrotrophic phytopathogenic lifestyle. Several species are clinically associated with allergic respiratory disorders although rarely found to cause invasive infections in humans. Finally, Alternaria spp. are among the most well known producers of diverse fungal secondary metabolites, especially toxins. DESCRIPTION: We have recently sequenced and annotated the genomes of 25 Alternaria spp. including but not limited to many necrotrophic plant pathogens such as A. brassicicola (a pathogen of Brassicaceous crops like cabbage and canola) and A. solani (a major pathogen of Solanaceous plants like potato and tomato), and several saprophytes that cause allergy in human such as A. alternata isolates. These genomes were annotated and compared. Multiple genetic differences were found in the context of plant and human pathogenicity, notably the pro-inflammatory potential of A. alternata. The Alternaria genomes database was built to provide a public platform to access the whole genome sequences, genome annotations, and comparative genomics data of these species. Genome annotation and comparison were performed using a pipeline that integrated multiple computational and comparative genomics tools. Alternaria genome sequences together with their annotation and comparison data were ported to Ensembl database schemas using a self-developed tool (EnsImport). Collectively, data are currently hosted using a customized installation of the Ensembl genome browser platform. CONCLUSION: Recent efforts in fungal genome sequencing have facilitated the studies of the molecular basis of fungal pathogenicity as a whole system. The Alternaria genomes database provides a comprehensive resource of genomics and comparative data of an important saprophytic and plant/human pathogenic fungal genus. The database will be updated regularly with new genomes when they become available. The Alternaria genomes database is freely available for non-profit use at http://alternaria.vbi.vt.edu .

Discord between morphological and phylogenetic species boundaries: incomplete lineage sorting and recombination results in fuzzy species boundaries in an asexual fungal pathogen.

BACKGROUND: Traditional morphological and biological species concepts are difficult to apply to closely related, asexual taxa because of the lack of an active sexual phase and paucity of morphological characters. Phylogenetic species concepts such as genealogical concordance phylogenetic species recognition (GCPSR) have been extensively used; however, methods that incorporate gene tree uncertainty into species recognition may more accurately and objectively delineate species. Using a worldwide sample of Alternaria alternata sensu lato, causal agent of citrus brown spot, the evolutionary histories of four nuclear loci including an endo-polygalacturonase gene, two anonymous loci, and one microsatellite flanking region were estimated using the coalescent. Species boundaries were estimated using several approaches including those that incorporate uncertainty in gene genealogies when lineage sorting and non-reciprocal monophyly of gene trees is common. RESULTS: Coalescent analyses revealed three phylogenetic lineages strongly influenced by incomplete lineage sorting and recombination. Divergence of the citrus 2 lineage from the citrus 1 and citrus 3 lineages was supported at most loci. A consensus of species tree estimation methods supported two species of Alternaria causing citrus brown spot worldwide. Based on substitution rates at the endo-polygalacturonase locus, divergence of the citrus 2 and the 1 and 3 lineages was estimated to have occurred at least 5, 400 years before present, predating the human-mediated movement of citrus and associated pathogens out of SE Asia. CONCLUSIONS: The number of Alternaria species identified as causing brown spot of citrus worldwide using morphological criteria has been overestimated. Little support was found for most of these morphospecies using quantitative species recognition approaches. Correct species delimitation of plant-pathogenic fungi is critical for understanding the evolution of pathogenicity, introductions of pathogens to new areas, and for regulating the movement of pathogens to enforce quarantines. This research shows that multilocus phylogenetic methods that allow for recombination and incomplete lineage sorting can be useful for the quantitative delimitation of asexual species that are morphologically indistinguishable. Two phylogenetic species of Alternaria were identified as causing citrus brown spot worldwide. Further research is needed to determine how these species were introduced worldwide, how they differ phenotypically and how these species are maintained.

Role of the Alternaria alternata blue-light receptor LreA (white-collar 1) in spore formation and secondary metabolism.

Alternaria alternata is a filamentous fungus that causes considerable loss of crops of economically important feed and food worldwide. It produces more than 60 different secondary metabolites, among which alternariol (AOH) and altertoxin (ATX) are the most important mycotoxins. We found that mycotoxin production and spore formation are regulated by light in opposite ways. Whereas spore formation was largely decreased under light conditions, the production of AOH was stimulated 2- to 3-fold. ATX production was even strictly dependent on light. All light effects observed could be triggered by blue light, whereas red light had only a minor effect. Inhibition of spore formation by light was reversible after 1 day of incubation in the dark. We identified orthologues of genes encoding the Neurospora crassa blue-light-perceiving white-collar proteins, a cryptochrome, a phytochrome, and an opsin-related protein in the genome of A. alternata. Deletion of the white-collar 1 (WC-1) gene (lreA) resulted in derepression of spore formation in dark and in light. ATX formation was strongly induced in the dark in the lreA mutant, suggesting a repressing function of LreA, which appears to be released in the wild type after blue-light exposure. In addition, light induction of AOH formation was partially dependent on LreA, suggesting also an activating function. A. alternata ΔlreA was still able to partially respond to blue light, indicating the action of another blue-light receptor system.

Allerdictor: fast allergen prediction using text classification techniques.

MOTIVATION: Accurately identifying and eliminating allergens from biotechnology-derived products are important for human health. From a biomedical research perspective, it is also important to identify allergens in sequenced genomes. Many allergen prediction tools have been developed during the past years. Although these tools have achieved certain levels of specificity, when applied to large-scale allergen discovery (e.g. at a whole-genome scale), they still yield many false positives and thus low precision (even at low recall) due to the extreme skewness of the data (allergens are rare). Moreover, the most accurate tools are relatively slow because they use protein sequence alignment to build feature vectors for allergen classifiers. Additionally, only web server implementations of the current allergen prediction tools are publicly available and are without the capability of large batch submission. These weaknesses make large-scale allergen discovery ineffective and inefficient in the public domain. RESULTS: We developed Allerdictor, a fast and accurate sequence-based allergen prediction tool that models protein sequences as text documents and uses support vector machine in text classification for allergen prediction. Test results on multiple highly skewed datasets demonstrated that Allerdictor predicted allergens with high precision over high recall at fast speed. For example, Allerdictor only took ∼6 min on a single core PC to scan a whole Swiss-Prot database of ∼540 000 sequences and identified <1% of them as allergens. AVAILABILITY AND IMPLEMENTATION: Allerdictor is implemented in Python and available as standalone and web server versions at http://allerdictor.vbi.vt.edu CONTACT: lawrence@vbi.vt.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Cyclophilin 20-3 relays a 12-oxo-phytodienoic acid signal during stress responsive regulation of cellular redox homeostasis.

The jasmonate family of phytohormones plays central roles in plant development and stress acclimation. However, the architecture of their signaling circuits remains largely unknown. Here we describe a jasmonate family binding protein, cyclophilin 20-3 (CYP20-3), which regulates stress-responsive cellular redox homeostasis. (+)-12-Oxo-phytodienoic acid (OPDA) binding promotes CYP20-3 to form a complex with serine acetyltransferase 1, which triggers the formation of a hetero-oligomeric cysteine synthase complex with O-acetylserine(thiol)lyase B in chloroplasts. The cysteine synthase complex formation then activates sulfur assimilation that leads to increased levels of thiol metabolites and the buildup of cellular reduction potential. The enhanced redox capacity in turn coordinates the expression of a subset of OPDA-responsive genes. Thus, we conclude that CYP20-3 is a key effector protein that links OPDA signaling to amino acid biosynthesis and cellular redox homeostasis in stress responses.

Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible.

A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, at levels well above background nonspecific entry. We also summarize host cell entry experiments with a wide diversity of oomycete and fungal effectors with RxLR or RxLR-like motifs that have been independently carried out by the seven different labs that coauthored this letter. Finally we discuss possible technical reasons why specific cell entry may have been not detected by Wawra et al. (2013).

Signatures of recombination in clonal lineages of the citrus brown spot pathogen, Alternaria alternata sensu lato.

Most Alternaria spp. are considered asexual but recent molecular evolution analyses of Alternaria mating-type genes show that the mating locus is under strong purifying selection, indicating a possible role in sexual reproduction. The objective of this study was to determine the mode of reproduction of an Alternaria alternata sensu lato population causing citrus brown spot in central Florida. Mating type of each isolate was determined, and isolates were sequenced at six putatively unlinked loci. Three genetically distinct subpopulations (SH1, SH4A, and SH4B) were identified using network and Bayesian population structure analyses. Results demonstrate that most subpopulations of A. alternata associated with citrus are clonal but some have the ability to extensively recombine through a cryptic sexual cycle or parasexual cycle. Although isolates were sampled in close physical proximity (≈2,500-m² area), we were able to reject a random mating model using multilocus gametic disequilibrium tests for two subpopulations, SH1 and SH4B, suggesting that these subpopulations were predominantly asexual. However, three recombination events were identified in SH1 and SH4B and localized to individuals of opposite mating type, possibly indicating meiotic recombination. In contrast, in the third subpopulation (SH4A), where only one mating type was present, extensive reticulation was evident in network analyses, and multilocus gametic disequilibrium tests were consistent with recombination. Recombination among isolates of the same mating type suggests that a nonmeiotic mechanism of recombination such as the parasexual cycle may be operating in this subpopulation. The level of gene flow detected among subpopulations does not appear to be sufficient to prevent differentiation, and perhaps future speciation, of these A. alternata subpopulations.

Transcription factor Amr1 induces melanin biosynthesis and suppresses virulence in Alternaria brassicicola.

Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen. Several A. brassicicola genes have been characterized as affecting pathogenesis of Brassica species. To study regulatory mechanisms of pathogenesis, we mined 421 genes in silico encoding putative transcription factors in a machine-annotated, draft genome sequence of A. brassicicola. In this study, targeted gene disruption mutants for 117 of the transcription factor genes were produced and screened. Three of these genes were associated with pathogenesis. Disruption mutants of one gene (AbPacC) were nonpathogenic and another gene (AbVf8) caused lesions less than half the diameter of wild-type lesions. Unexpectedly, mutants of the third gene, Amr1, caused lesions with a two-fold larger diameter than the wild type and complementation mutants. Amr1 is a homolog of Cmr1, a transcription factor that regulates melanin biosynthesis in several fungi. We created gene deletion mutants of Δamr1 and characterized their phenotypes. The Δamr1 mutants used pectin as a carbon source more efficiently than the wild type, were melanin-deficient, and more sensitive to UV light and glucanase digestion. The AMR1 protein was localized in the nuclei of hyphae and in highly melanized conidia during the late stage of plant pathogenesis. RNA-seq analysis revealed that three genes in the melanin biosynthesis pathway, along with the deleted Amr1 gene, were expressed at low levels in the mutants. In contrast, many hydrolytic enzyme-coding genes were expressed at higher levels in the mutants than in the wild type during pathogenesis. The results of this study suggested that a gene important for survival in nature negatively affected virulence, probably by a less efficient use of plant cell-wall materials. We speculate that the functions of the Amr1 gene are important to the success of A. brassicicola as a competitive saprophyte and plant parasite.

A zinc-finger-family transcription factor, AbVf19, is required for the induction of a gene subset important for virulence in Alternaria brassicicola.

Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen with a broad host range within the family Brassicaceae. It produces secondary metabolites that marginally affect virulence. Cell wall-degrading enzymes (CDWE) have been considered important for pathogenesis but none of them individually have been identified as significant virulence factors in A. brassicicola. In this study, knockout mutants of a gene, AbVf19, were created and produced considerably smaller lesions than the wild type on inoculated host plants. The presence of tandem zinc-finger domains in the predicted amino acid sequence and nuclear localization of AbVf19-reporter protein suggested that it was a transcription factor. Gene expression comparisons using RNA-seq identified 74 genes being downregulated in the mutant during a late stage of infection. Among the 74 downregulated genes, 28 were putative CWDE genes. These were hydrolytic enzyme genes that composed a small fraction of genes within each family of cellulases, pectinases, cutinases, and proteinases. The mutants grew slower than the wild type on an axenic medium with pectin as a major carbon source. This study demonstrated the existence and the importance of a transcription factor that regulates a suite of genes that are important for decomposing and utilizing plant material during the late stage of plant infection.

Effector diversification within compartments of the Leptosphaeria maculans genome affected by Repeat-Induced Point mutations.

Fungi are of primary ecological, biotechnological and economic importance. Many fundamental biological processes that are shared by animals and fungi are studied in fungi due to their experimental tractability. Many fungi are pathogens or mutualists and are model systems to analyse effector genes and their mechanisms of diversification. In this study, we report the genome sequence of the phytopathogenic ascomycete Leptosphaeria maculans and characterize its repertoire of protein effectors. The L. maculans genome has an unusual bipartite structure with alternating distinct guanine and cytosine-equilibrated and adenine and thymine (AT)-rich blocks of homogenous nucleotide composition. The AT-rich blocks comprise one-third of the genome and contain effector genes and families of transposable elements, both of which are affected by repeat-induced point mutation, a fungal-specific genome defence mechanism. This genomic environment for effectors promotes rapid sequence diversification and underpins the evolutionary potential of the fungus to adapt rapidly to novel host-derived constraints.

External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells.

Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues.

TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen.

The regulation of intracellular levels of reactive oxygen species (ROS) is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P)-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus DeltatmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola DeltatmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus DeltatmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola DeltatmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the DeltatmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola DeltatmpL background. Collectively, we have discovered a novel protein involved in the virulence of both plant and animal fungal pathogens. Our results strongly suggest that dysregulation of oxidative stress homeostasis in the absence of TmpL is the underpinning cause of the developmental and virulence defects observed in these studies.

Recognition of fungal protease activities induces cellular activation and eosinophil-derived neurotoxin release in human eosinophils.

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of asthma and in immunity to certain organisms. Associations between exposure to an environmental fungus, such as Alternaria, and asthma have been recognized clinically. Protease-activated receptors (PARs) are G protein-coupled receptors that are cleaved and activated by serine proteases, but their roles in innate immunity remain unknown. We previously found that human eosinophils respond vigorously to Alternaria organisms and to the secretory product(s) of Alternaria with eosinophils releasing their proinflammatory mediators. In this study, we investigated the roles of protease(s) produced by Alternaria and of PARs expressed on eosinophils in their immune responses against fungal organisms. We found that Alternaria alternata produces aspartate protease(s) and that human peripheral blood eosinophils degranulate in response to the cell-free extract of A. alternata. Eosinophils showed an increased intracellular calcium concentration in response to Alternaria that was desensitized by peptide and protease ligands for PAR-2 and inhibited by a PAR-2 antagonistic peptide. Alternaria-derived aspartate protease(s) cleaved PAR-2 to expose neo-ligands; these neo-ligands activated eosinophil degranulation in the absence of proteases. Finally, treatment of Alternaria extract with aspartate protease inhibitors, which are conventionally used for HIV-1 and other microbes, attenuated the eosinophils' responses to Alternaria. Thus, fungal aspartate protease and eosinophil PAR-2 appear critical for the eosinophils' innate immune response to certain fungi, suggesting a novel mechanism for pathologic inflammation in asthma and for host-pathogen interaction.

Biosynthesis and role in virulence of the histone deacetylase inhibitor depudecin from Alternaria brassicicola.

Depudecin, an eleven-carbon linear polyketide made by the pathogenic fungus Alternaria brassicicola, is an inhibitor of histone deacetylase (HDAC). A chemically unrelated HDAC inhibitor, HC toxin, was earlier shown to be a major virulence factor in the interaction between Cochliobolus carbonum and its host, maize. In order to test whether depudecin is also a virulence factor for A. brassicicola, we identified the genes for depudecin biosynthesis and created depudecin-minus mutants. The depudecin gene cluster contains six genes (DEP1 to DEP6), which are predicted to encode a polyketide synthase (AbPKS9 or DEP5), a transcription factor (DEP6), two monooxygenases (DEP2 and DEP4), a transporter of the major facilitator superfamily (DEP3), and one protein of unknown function (DEP1). The involvement in depudecin production of DEP2, DEP4, DEP5, and DEP6 was demonstrated by targeted gene disruption. DEP6 is required for expression of DEP1 through DEP5 but not the immediate flanking genes, thus defining a coregulated depudecin biosynthetic cluster. The genes flanking the depudecin gene cluster but not the cluster itself are conserved in the same order in the related fungi Stagonospora nodorum and Pyrenophora tritici-repentis. Depudecin-minus mutants have a small (10%) but statistically significant reduction in virulence on cabbage (Brassica oleracea) but not on Arabidopsis. The role of depudecin in virulence is, therefore, less dramatic than that of HC toxin.