The products of non-canonical isocyanide synthase (ICS) biosynthetic gene clusters (BGCs) mediate pathogenesis, microbial competition, and metal-homeostasis through metal-associated chemistry. We sought to enable research into this class of compounds by characterizing the biosynthetic potential and evolutionary history of these BGCs across the Fungal Kingdom. We amalgamated a pipeline of tools to predict BGCs based on shared promoter motifs and located 3800 ICS BGCs in 3300 genomes, making ICS BGCs the fifth largest class of specialized metabolites compared to canonical classes found by antiSMASH. ICS BGCs are not evenly distributed across fungi, with evidence of gene-family expansions in several Ascomycete families. We show that the ICS dit1/2 gene cluster family (GCF), which was prior only studied in yeast, is present in ∼30% of all Ascomycetes. The dit variety ICS exhibits greater similarity to bacterial ICS than other fungal ICS, suggesting a potential convergence of the ICS backbone domain. The evolutionary origins of the dit GCF in Ascomycota are ancient and these genes are diversifying in some lineages. Our results create a roadmap for future research into ICS BGCs. We developed a website (https://isocyanides.fungi.wisc.edu/) that facilitates the exploration and downloading of all identified fungal ICS BGCs and GCFs.

The genomes of many filamentous fungi, such as Aspergillus spp., include diverse biosynthetic gene clusters of unknown function. We previously showed that low copper levels upregulate a gene cluster that includes crmA, encoding a putative isocyanide synthase. Here we show, using untargeted comparative metabolomics, that CrmA generates a valine-derived isocyanide that contributes to two distinct biosynthetic pathways under copper-limiting conditions. Reaction of the isocyanide with an ergot alkaloid precursor results in carbon-carbon bond formation analogous to Strecker amino-acid synthesis, producing a group of alkaloids we term fumivalines. In addition, valine isocyanide contributes to biosynthesis of a family of acylated sugar alcohols, the fumicicolins, which are related to brassicicolin A, a known isocyanide from Alternaria brassicicola. CrmA homologs are found in a wide range of pathogenic and non-pathogenic fungi, some of which produce fumicicolin and fumivaline. Extracts from A. fumigatus wild type (but not crmA-deleted strains), grown under copper starvation, inhibit growth of diverse bacteria and fungi, and synthetic valine isocyanide shows antibacterial activity. CrmA thus contributes to two biosynthetic pathways downstream of trace-metal sensing.

In 1999, the first biosynthetic gene cluster (BGC), synthesizing the virulence factor DHN melanin, was characterized in Aspergillus fumigatus. Since then, 19 additional BGCs have been linked to specific secondary metabolites (SMs) in this species. Here, we provide a comprehensive timeline of A. fumigatus BGC discovery and find that initial advances centered around the commonly expressed SMs where chemical structure informed rationale identification of the producing BGC (e.g., gliotoxin, fumigaclavine, fumitremorgin, pseurotin A, helvolic acid, fumiquinazoline). Further advances followed the transcriptional profiling of a ΔlaeA mutant, which aided in the identification of endocrocin, fumagillin, hexadehydroastechrome, trypacidin, and fumisoquin BGCs. These SMs and their precursors are the commonly produced metabolites in most A. fumigatus studies. Characterization of other BGC/SM pairs required additional efforts, such as induction treatments, including co-culture with bacteria (fumicycline/neosartoricin, fumigermin) or growth under copper starvation (fumivaline, fumicicolin). Finally, four BGC/SM pairs were discovered via overexpression technologies, including the use of heterologous hosts (fumicycline/neosartoricin, fumihopaside, sphingofungin, and sartorypyrone). Initial analysis of the two most studied A. fumigatus isolates, Af293 and A1160, suggested that both harbored ca. 34–36 BGCs. An examination of 264 available genomes of A. fumigatus located only four additional new BGCs, suggesting the secondary metabolome across A. fumigatus isolates is remarkably conserved. Based on our analysis, around 20 of the genetically characterized BGCs within the A. fumigatus species complex still lack a known chemical product. Such BGCs remain the final hurdle in fully understanding the secondary metabolism in this important species.

In order to gain a comprehensive understanding of plant disease in natural and agricultural ecosystems, it is essential to examine plant disease in multi-pathogen–host systems. Ralstonia solanacearum and Fusarium oxysporum f. sp. lycopersici are vascular wilt pathogens that can result in heavy yield losses in susceptible hosts such as tomato. Although both pathogens occupy the xylem, the costs of mixed infections on wilt disease are unknown. Here, we characterize the consequences of co-infection with R. solanacearum and F. oxysporum using tomato as the model host. Our results demonstrate that bacterial wilt severity is reduced in co-infections, that bikaverin synthesis by Fusarium contributes to bacterial wilt reduction, and that the arrival time of each microbe at the infection court is important in driving the severity of wilt disease. Further, analysis of the co-infection root secretome identified previously uncharacterized secreted metabolites that reduce R. solanacearum growth in vitro and provide protection to tomato seedlings against bacterial wilt disease. Taken together, these results highlight the need to understand the consequences of mixed infections in plant disease.

Summary: Plants and soils have been moved around the world for centuries, but invasive mushrooms receive scant attention. The Amanita muscaria species complex was introduced to South Africa in the context of forestry, but its origins, ecology and recent evolution are unstudied. We sequenced the genomes of 24 Northern and Southern Hemisphere A. muscaria , built phylogenies and reconstructed its South African history. We identified the biosynthetic gene clusters (BGCs) encoding specialized metabolites (SMs). We subsequently extracted mushrooms' metabolites and used mass spectrometry data to group SMs into unique molecular families (MFs). We tested metabolites for bioactivity against diverse microbes and animals. We identify Europe as the origin of South African A. muscaria . A highly conserved group of BGCs is found in nearly all European and African genomes, and only 13 of 273 MFs are unique to South Africa. Metabolites extracted from all mushrooms kill nematodes, while microbes and flies appear unaffected. The nearly global distribution of the fly agaric results from multiple introductions of a single European clade to the Southern Hemisphere. Despite its long history in South Africa, the fungus has not lost any of its BGCs, suggesting a conservation of function(s) across multiple continents.

The products of non-canonical isocyanide synthase (ICS) biosynthetic gene clusters (BGCs) have notable bioactivities that mediate pathogenesis, microbial competition, and metal-homeostasis through metal-associated chemistry. We sought to enable research into this class of compounds by characterizing the biosynthetic potential and evolutionary history of these BGCs across the Fungal Kingdom. We developed the first genome-mining pipeline to identify ICS BGCs, locating 3,800 ICS BGCs in 3,300 genomes. Genes in these clusters share promoter motifs and are maintained in contiguous groupings by natural selection. ICS BGCs are not evenly distributed across fungi, with evidence of gene-family expansions in several Ascomycete families. We show that the ICS dit1 / 2 gene cluster family (GCF), which was thought to only exist in yeast, is present in ∼30% of all Ascomycetes, including many filamentous fungi. The evolutionary history of the dit GCF is marked by deep divergences and phylogenetic incompatibilities that raise questions about convergent evolution and suggest selection or horizontal gene transfers have shaped the evolution of this cluster in some yeast and dimorphic fungi. Our results create a roadmap for future research into ICS BGCs. We developed a website ( www.isocyanides.fungi.wisc.edu ) that facilitates the exploration, filtering, and downloading of all identified fungal ICS BGCs and GCFs.