Molecular Characterization of the Cercosporin Biosynthetic Pathway in the Fungal Plant Pathogen Cercospora nicotianae

Abstract

Perylenequinones are a class of photoactivated polyketide mycotoxins produced by fungal plant pathogens that notably produce reactive oxygen species with visible light. The best-studied perylenequinone is cercosporin-a product of the Cercospora species. While the cercosporin biosynthetic gene cluster has been described in the tobacco pathogen Cercospora nicotianae, little is known of the metabolite's biosynthesis. Furthermore, in vitro investigations of the polyketide synthase central to cercosporin biosynthesis identified the naphthopyrone nor-toralactone as its direct product-an observation in conflict with published biosynthetic proposals. Here, we present an alternative biosynthetic pathway to cercosporin based on metabolites characterized from a series of biosynthetic gene knockouts. We show that nor-toralactone is the key polyketide intermediate and the substrate for the unusual didomain protein CTB3. We demonstrate the unique oxidative cleavage activity of the CTB3 monooxygenase domain in vitro. These data advance our understanding of perylenequinone biosynthesis and expand the biochemical repertoire of flavindependent monooxygenases.

Genome Sequencing and Analysis of the Hypocrellin-Producing Fungus Shiraia bambusicola S4201

“…the sources of HA substrates, and the genes marked with red on the right were upregulated in the transcriptome analysis we performed (Zhao et al, 2016). The proposed functions of the proteins encoded by the HA biosynthesis gene cluster are based on the biosynthesis studies of cercosporin (Newman and Townsend, 2016) and elsinochrome C (Chooi et al, 2017). The orange dotted lines represent the substrates for HA biosynthesis.…”

“…Comparing the CTB and HA cluster genes, NADPH-dependent oxidoreductase (CTB6) and FAD-dependent monooxygenase (CTB7) appeared to be unique to the CTB cluster. Considering the function of these enzymes, it makes sense that they are absent in the HA cluster, given that these structural features are non-existent (Newman and Townsend, 2016). Future studies are needed to determine whether these genes participate in HA biosynthesis.…”

The AVR4 effector is involved in cercosporin biosynthesis and likely affects the virulence of Cercospora cf. flagellaris on soybean

“…A biosynthetic pathway for cercosporin was also proposed (Chen et al, 2007). However, recent studies presented alternative biosynthetic pathways obtained by the characterization of metabolites from a series of biosynthetic pathway gene knockouts, as well as the discovery of several additional genes required for cercosporin biosynthesis through a comparative genomic and conserved gene cluster analysis (de Jonge et al, 2017(de Jonge et al, , 2018Newman and Townsend, 2016;Shim and Dunkle, 2003).…”

Gene cluster conservation identifies melanin and perylenequinone biosynthesis pathways in multiple plant pathogenic fungi

“…The majority of assessed species from the genus Colletotrichum , a large genus of crop and/or ornamental plant pathogens (Perfect et al, ), were shown to harbour full‐ to partial‐length CTB gene clusters, of which the post‐harvest apple fruit pathogen Co. fioriniae was shown to produce cercosporin (de Jonge et al, ). The core gene of the Cercospora CTB gene cluster is the NR‐PKS gene CTB1 (Newman and Townsend, ), which is flanked by nine genes that encode decorating enzymes (CTB2, CTB3, CTB5, CTB6, CTB7, CTB9, CTB10, CTB11 and CTB12) (de Jonge et al, ). Besides those 10 genes essential for toxin formation, the cluster also encodes a zinc finger transcription factor (CTB8) for regulation of cluster gene expression, and two major facilitator superfamily (MFS) transporters; CTB4 that is necessary for toxin secretion and the cercosporin facilitator protein (CFP) involved in toxin auto‐resistance (Chen et al, ; Choquer et al, ; de Jonge et al, ).…”