Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Assignment of a dubious gene cluster to melanin biosynthesis in the tomato fungal pathogen Cladosporium fulvum
Griffiths, Scott A. ; Cox, Russell J. ; Overdijk, Elysa J.R. ; Mesarich, Carl H. ; Saccomanno, Benedetta ; Lazarus, Colin M. ; Wit, Pierre J.G.M. de; Collemare, Jérôme - \ 2018
PLoS ONE 13 (2018)12. - ISSN 1932-6203

Pigments and phytotoxins are crucial for the survival and spread of plant pathogenic fungi. The genome of the tomato biotrophic fungal pathogen Cladosporium fulvum contains a predicted gene cluster (CfPKS1, CfPRF1, CfRDT1 and CfTSF1) that is syntenic with the characterized elsinochrome toxin gene cluster in the citrus pathogen Elsinoë fawcettii. However, a previous phylogenetic analysis suggested that CfPks1 might instead be involved in pigment production. Here, we report the characterization of the CfPKS1 gene cluster to resolve this ambiguity. Activation of the regulator CfTSF1 specifically induced the expression of CfPKS1 and CfRDT1, but not of CfPRF1. These co-regulated genes that define the CfPKS1 gene cluster are orthologous to genes involved in 1,3-dihydroxynaphthalene (DHN) melanin biosynthesis in other fungi. Heterologous expression of CfPKS1 in Aspergillus oryzae yielded 1,3,6,8-tetrahydroxynaphthalene, a typical precursor of DHN melanin. Δcfpks1 deletion mutants showed similar altered pigmentation to wild type treated with DHN melanin inhibitors. These mutants remained virulent on tomato, showing this gene cluster is not involved in pathogenicity. Altogether, our results showed that the CfPKS1 gene cluster is involved in the production of DHN melanin and suggests that elsinochrome production in E. fawcettii likely involves another gene cluster.

Down-regulation of cladofulvin biosynthesis is required for biotrophic growth of Cladosporium fulvum on tomato : A secondary metabolite prevents fungal biotrophy
Griffiths, Scott ; Mesarich, Carl H. ; Overdijk, Elysa J.R. ; Saccomanno, Benedetta ; Wit, Pierre J.G.M. De; Collemare, Jérôme - \ 2018
Molecular Plant Pathology 19 (2018)2. - ISSN 1464-6722 - p. 369 - 380.
Fungal biotrophy is associated with a reduced capacity to produce potentially toxic secondary metabolites (SMs). Yet, the genome of the biotrophic plant pathogen Cladosporium fulvum contains many SM biosynthetic gene clusters, with several related to toxin production. These gene clusters are, however, poorly expressed during colonisation of tomato. The sole detectable SM produced by C. fulvum during in vitro growth is the anthraquinone cladofulvin. Although this pigment is not detected in infected leaves, cladofulvin biosynthetic genes are expressed throughout the pre-penetration phase and during conidiation at the end of the infection cycle, but they are repressed during the biotrophic phase of tomato colonization. It was suggested that tight regulation of SM gene clusters is required for C. fulvum to behave as a biotrophic pathogen, while retaining potential fitness determinants for growth and survival outside its host. To address this hypothesis, we analysed the disease symptoms caused by mutant C. fulvum strains that do not produce or over-produce cladofulvin during the biotrophic growth phase. Non-producers infected tomato similar to wild type, suggesting that cladofulvin is not a virulence factor. In contrast, the cladofulvin over-producers caused strong necrosis and desiccation of tomato leaves, which in turn, arrested conidiation. Consistent with the role of pigments in survival against abiotic stresses, cladofulvin protects conidia against UV light and low temperature stress. Overall this study demonstrates that repression of cladofulvin production is required for C. fulvum to sustain its biotrophic lifestyle in tomato, while its production is important for survival outside its host.
Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization
Griffiths, Scott ; Mesarich, Carl H. ; Saccomanno, Benedetta ; Vaisberg, Abraham ; Wit, Pierre J.G.M. de; Cox, Russell ; Collemare, Jérôme - \ 2016
Proceedings of the National Academy of Sciences of the United States of America 113 (2016)25. - ISSN 0027-8424 - p. 6851 - 6856.
Cytoxicity - Emodin - Gene cluster - Nataloe-emodin - Secondary metabolism

Anthraquinones are a large family of secondary metabolites (SMs) that are extensively studied for their diverse biological activities. These activities are determined by functional group decorations and the formation of dimers from anthraquinone monomers. Despite their numerous medicinal qualities, very few anthraquinone biosynthetic pathways have been elucidated so far, including the enzymatic dimerization steps. In this study, we report the elucidation of the biosynthesis of cladofulvin, an asymmetrical homodimer of nataloe-emodin produced by the fungus Cladosporium fulvum. A gene cluster of 10 genes controls cladofulvin biosynthesis, which begins with the production of atrochrysone carboxylic acid by the polyketide synthase ClaG and the β-lactamase ClaF. This compound is decarboxylated by ClaH to yield emodin, which is then converted to chrysophanol hydroquinone by the reductase ClaC and the dehydratase ClaB. We show that the predicted cytochrome P450 ClaM catalyzes the dimerization of nataloe-emodin to cladofulvin. Remarkably, such dimerization dramatically increases nataloe-emodin cytotoxicity against mammalian cell lines. These findings shed light on the enzymatic mechanisms involved in anthraquinone dimerization. Future characterization of the ClaM enzyme should facilitate engineering the biosynthesis of novel, potent, dimeric anthraquinones and structurally related compound families.

Regulation of secondary metabolite production in the fungal tomato pathogen Cladosporium fulvum
Griffiths, S.A. ; Saccomanno, B. ; Wit, P.J.G.M. de; Collemare, J. - \ 2015
Fungal Genetics and Biology 84 (2015). - ISSN 1087-1845 - p. 52 - 61.
Cladosporium fulvum is a non-obligate biotrophic fungal tomato pathogen for which fifteen secondary metabolite (SM) gene clusters were previously identified in its genome. However, most of these SM biosynthetic pathways remain cryptic during growth in planta and in different in vitro conditions. The sole SM produced in vitro is the pigment cladofulvin. In this study, we attempted to activate cryptic pathways in order to identify new compounds produced by C. fulvum. For this purpose, we manipulated orthologues of the global regulators VeA, LaeA and HdaA known to regulate SM biosynthesis in other fungal species. In C. fulvum, deleting or over-expressing these regulators yielded no new detectable SMs. Yet, quantification of cladofulvin revealed that CfHdaA is an activator whilst CfVeA and CfLaeA seemed to act as repressors of cladofulvin production. In the wild type strain, cladofulvin biosynthesis was affected by the carbon source, with highest production under carbon limitation and traces only in presence of saccharose. Repression of cladofulvin production by saccharose was dependent on both CfVeA and CfLaeA. Deletion of CfVeA or CfLaeA caused production of sterile mycelia, whilst ¿cfhdaa deletion mutants sporulated, suggesting that cladofulvin production is not linked to asexual reproduction. Profiling the transcription of these regulators showed that CfHdaA-mediated regulation of cladofulvin production is independent of both CfVeA and CfLaeA. Our data suggest CfLaeA directly affects cladofulvin production whilst the effect of CfVeA is indirect, suggesting a role for CfLaeA outside of the Velvet complex. In conclusion, our results showed that regulation of SM production in C. fulvum is different from other fungi and indicate that manipulation of global regulators is not a universal tool to discover new fungal natural products.
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