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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.

Fungal secondary metabolites (SMs) have captured the interest of natural products researchers in academia and industry for decades. In recent years, the high rediscovery rate of previously characterized metabolites is making it increasingly difficult to uncover novel compounds. Additionally, the vast majority of fungal SMs reside in genetically intractable fungi or are silent under normal laboratory conditions in genetically tractable fungi. The fungal natural products community has broadly overcome these barriers by altering the physical growth conditions of the fungus and heterologous/homologous expression of biosynthetic gene cluster regulators or proteins. The protocols described here summarize vital methodologies needed when researching SM production in fungi. We also summarize the growth conditions, genetic backgrounds, and extraction protocols for every published SM in Aspergillus fumigatus, enabling readers to easily replicate the production of previously characterized SMs. Readers will also be equipped with the tools for developing their own strategy for expressing and extracting SMs from their given fungus or a suitable heterologous model system. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Making glycerol stocks from spore suspensions Alternate Protocol 1: Creating glycerol stocks from non-sporulating filamentous fungi Basic Protocol 2: Activating spore-suspension glycerol stocks Basic Protocol 3: Extracting secondary metabolites from Aspergillus spp grown on solid medium Alternate Protocol 2: Extracting secondary metabolites from Aspergillus spp using ethyl acetate Alternate Protocol 3: High-volume metabolite extraction using ethyl acetate Alternate Protocol 4: Extracting secondary metabolites from Aspergillus spp in liquid medium Support Protocol: Creating an overlay culture Basic Protocol 4: Extracting DNA from filamentous fungi Basic Protocol 5: Creating a DNA construct with double-joint PCR Alternate Protocol 5: Creating a DNA construct with yeast recombineering Basic Protocol 6: Transformation of Aspergillus spp Basic Protocol 7: Co-culturing fungi and bacteria for extraction of secondary metabolites.

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&nbsp, 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.