Abstract
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.
Highlights
Secondary metabolites (SMs) are compounds produced by microbes, plants and insects that are often repurposed as medicines and pesticides
The CfPKS1 gene cluster (Fig 1A) was initially predicted solely through its homology and synteny with the characterized elsinochrome gene cluster in E. fawcettii, as only minimal gene expression within this gene cluster had been observed during the growth of wild-type C. fulvum under diverse conditions [38]
To up-regulate and clearly define the CfPKS1 gene cluster, wild-type C. fulvum was transformed with a plasmid containing CfTSF1 fused to the promoter region of the nitrogen-regulated C. fulvum Avr9 gene [44]
Summary
Secondary metabolites (SMs) are compounds produced by microbes, plants and insects that are often repurposed as medicines and pesticides. C. fulvum shows limited filamentous growth on in vitro media in the dark, forming small sporulating colonies They exhibit a green-brown colour, which was linked to the production of the pigment cladofulvin [36]. The hyper-black appearance of Δcfwor colonies and the absence of detectable SMs suggested that CfPks might be involved in the production of polymerized DHN melanin in C. fulvum. Such ambiguity between elsinochrome and DHN production remains unresolved. We provide chemical evidence of the pigment produced by this pathway and assessed the role of this compound in pathogenicity and biotrophic growth of C. fulvum
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