PTP1B phosphatase dampens iPSC-derived neutrophil motility and antimicrobial function.

Neutrophils are rapidly recruited to sites of infection and are critical for pathogen clearance. Therapeutic use of primary neutrophils has been limited as they have a short lifespan and are not amenable to genetic manipulation. Human induced pluripotent stem cells (iPSCs) can provide a robust source of neutrophils for infusion and are genetically tractable. However, current work has indicated that dampened intracellular signaling limits iPSC-derived neutrophil (iNeutrophil) cellular activation and antimicrobial response. Here, we show that protein tyrosine phosphatase 1B (PTP1B) inhibits intracellular signaling and dampens iNeutrophil effector function. Deletion of the PTP1B phosphatase increased PI3K and ERK signaling and was associated with increased F-actin polymerization, cell migration and phagocytosis. In contrast, other effector functions like NETosis and ROS production were reduced. PTP1B-deficient neutrophils were more responsive to A. fumigatus and displayed rapid recruitment and control of hyphal growth. Accordingly, depletion of PTP1B increased production of inflammatory factors including the neutrophil chemokine IL-8. Taken together, these findings suggest that PTP1B limits iNeutrophil motility and antimicrobial function.

Terpenoid balance in Aspergillus nidulans unveiled by heterologous squalene synthase expression.

Filamentous fungi produce numerous uncharacterized natural products (NPs) that are often challenging to characterize because of cryptic expression in laboratory conditions. Previously, we have successfully isolated novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into Aspergillus nidulans. Here, we demonstrate a twist to FAC utility wherein heterologous expression of a Pseudogymnoascus destructans FAC in A. nidulans altered endogenous terpene biosynthetic pathways. In contrast to wild type, the FAC transformant produced increased levels of squalene and aspernidine type compounds, including three new nidulenes (1- 2, and 5), and lost nearly all ability to synthesize the major A. nidulans characteristic terpene, austinol. Deletion of a squalene synthase gene in the FAC restored wild-type chemical profiles. The altered squalene to farnesyl pyrophosphate ratio leading to synthesis of nidulenes and aspernidines at the expense of farnesyl pyrophosphate-derived austinols provides unexpected insight into routes of terpene synthesis in fungi.

Fungal secondary metabolism is governed by an RNA-binding protein CsdA/RsdA complex.

Production of secondary metabolites is controlled by a complicated regulatory network in eukaryotic cells. Several layers of regulators are involved in this process, ranging from pathway-specific regulation, to epigenetic control, to global regulation. Here, we discover that interaction of an RNA-binding protein CsdA with a regulator RsdA coordinates fungal secondary metabolism. Employing a genetic deletion approach and transcriptome analysis as well as metabolomics analysis, we reveal that CsdA and RsdA synergistically regulate fungal secondary metabolism comprehensively. Mechanistically, comprehensive genetic and biochemical studies prove that RsdA and CsdA co-localize in the nucleus and physically interact to achieve their functions. In particular, we demonstrate that CsdA mediates rsdA expression by binding specific motif "GUCGGUAU" of its pre-mRNA at a post-transcriptional level. We thus uncover a mechanism in which RNA-binding protein physically interacts with, and controls the expression level of, the RsdA to coordinate fungal secondary metabolism.

Mining the Penicillium expansum Genome for Virulence Genes: A Functional-Based Approach to Discover Novel Loci Mediating Blue Mold Decay of Apple Fruit.

Blue mold, a postharvest disease of pome fruits, is caused by the filamentous fungus Penicillium expansum. In addition to the economic losses caused by P. expansum, food safety can be compromised, as this pathogen is mycotoxigenic. In this study, forward and reverse genetic approaches were used to identify genes involved in blue mold infection in apple fruits. For this, we generated a random T-DNA insertional mutant library. A total of 448 transformants were generated and screened for the reduced decay phenotype on apples. Of these mutants, six (T-193, T-275, T-434, T-588, T-625, and T-711) were selected for continued studies and five unique genes were identified of interest. In addition, two deletion mutants (Δt-625 and Δt-588) and a knockdown strain (t-434KD) were generated for three loci. Data show that the ∆t-588 mutant phenocopied the T-DNA insertion mutant and had virulence penalties during apple fruit decay. We hypothesize that this locus encodes a glyoxalase due to bioinformatic predictions, thus contributing to reduced colony diameter when grown in methylglyoxal (MG). This work presents novel members of signaling networks and additional genetic factors that regulate fungal virulence in the blue mold fungus during apple fruit decay.

Terpenoid balance in Aspergillus nidulans unveiled by heterologous squalene synthase expression.

Filamentous fungi produce numerous uncharacterized natural products (NPs) that are often challenging to characterize due to cryptic expression in laboratory conditions. Previously, we have successfully isolated novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into Aspergillus nidulans. Here, we demonstrate a new twist to FAC utility wherein heterologous expression of a Pseudogymnoascus destructans FAC in A. nidulans altered endogenous terpene biosynthetic pathways. In contrast to wildtype, the FAC transformant produced increased levels of squalene and aspernidine type compounds, including three new nidulenes (1-2, 5), and lost nearly all ability to synthesize the major A. nidulans characteristic terpene, austinol. Deletion of a squalene synthase gene in the FAC restored wildtype chemical profiles. The altered squalene to farnesyl pyrophosphate ratio leading to synthesis of nidulenes and aspernidines at the expense of farnesyl pyrophosphate derived austinols provides unexpected insight into routes of terpene synthesis in fungi.

A glimpse into the fungal metabolomic abyss: Novel network analysis reveals relationships between exogenous compounds and their outputs.

Fungal specialized metabolites are a major source of beneficial compounds that are routinely isolated, characterized, and manufactured as pharmaceuticals, agrochemical agents, and industrial chemicals. The production of these metabolites is encoded by biosynthetic gene clusters that are often silent under standard growth conditions. There are limited resources for characterizing the direct link between abiotic stimuli and metabolite production. Herein, we introduce a network analysis-based, data-driven algorithm comprising two routes to characterize the production of specialized fungal metabolites triggered by different exogenous compounds: the direct route and the auxiliary route. Both routes elucidate the influence of treatments on the production of specialized metabolites from experimental data. The direct route determines known and putative metabolites induced by treatments and provides additional insight over traditional comparison methods. The auxiliary route is specific for discovering unknown analytes, and further identification can be curated through online bioinformatic resources. We validated our algorithm by applying chitooligosaccharides and lipids at two different temperatures to the fungal pathogen Aspergillus fumigatus. After liquid chromatography-mass spectrometry quantification of significantly produced analytes, we used network centrality measures to rank the treatments' ability to elucidate these analytes and confirmed their identity through fragmentation patterns or in silico spiking with commercially available standards. Later, we examined the transcriptional regulation of these metabolites through real-time quantitative polymerase chain reaction. Our data-driven techniques can complement existing metabolomic network analysis by providing an approach to track the influence of any exogenous stimuli on metabolite production. Our experimental-based algorithm can overcome the bottlenecks in elucidating novel fungal compounds used in drug discovery.

The bZIP-type transcription factors NapA and RsmA modulate the volumetric ratio and the relative superoxide ratio of mitochondria in Aspergillus nidulans.

Basic leucine zipper (bZIP) transcription factors are crucial components of differentiation, cellular homeostasis and the environmental stress defense of eukaryotes. In this work, we further studied the consequence of gene deletion and overexpression of two bZIP transcription factors, NapA and RsmA, on superoxide production, mitochondrial morphology and hyphal diameter of Aspergillus nidulans. We have found that reactive oxygen species production was influenced by both gene deletion and overexpression of napA under tert-butylhydroperoxide (tBOOH) elicited oxidative stress. Furthermore, gene expression of napA negatively correlated with mitochondrial volumetric ratio as well as sterigmatocystin production of A. nidulans. High rsmA expression was accompanied with elevated relative superoxide ratio in the second hyphal compartment. A negative correlation between the expression of rsmA and catalase enzyme activity or mitochondrial volumetric ratio was also confirmed by statistical analysis. Hyphal diameter was independent on either rsmA and napA expression as well as 0.2 mM tBOOH treatment.

The KdmB-EcoA-RpdA-SntB (KERS) chromatin regulatory complex controls development, secondary metabolism and pathogenicity in Aspergillus flavus.

The filamentous fungus Aspergillus flavus is a plant and human pathogen predominantly found in the soil as spores or sclerotia and is capable of producing various secondary metabolites (SM) such as the carcinogenic mycotoxin aflatoxin. Recently, we have discovered a novel nuclear chromatin binding complex (KERS) that contains the JARID1-type histone demethylase KdmB, a putative cohesion acetyl transferase EcoA, a class I type histone deacetylase RpdA and the PHD ring finger reader protein SntB in the model filamentous fungus Aspergillus nidulans. Here, we show the presence of the KERS complex in A. flavus by immunoprecipitation-coupled mass spectrometry and constructed kdmBΔ and rpdAΔ strains to study their roles in fungal development, SM production and histone post-translational modifications (HPTMs). We found that KdmB and RpdA couple the regulation of SM gene clusters with fungal light-responses and HPTMs. KdmB and RpdA have opposing roles in light-induced asexual conidiation, while both factors are positive regulators of sclerotia development through the nsdC and nsdD pathway. KdmB and RpdA are essential for the productions of aflatoxin (similar to findings for SntB) as well as cyclopiazonic acid, ditryptophenaline and leporin B through controlling the respective SM biosynthetic gene clusters. We further show that both KdmB and RpdA regulate H3K4me3 and H3K9me3 levels, while RpdA also acts on H3K14ac levels in nuclear extracts. Therefore, the chromatin modifiers KdmB and RpdA of the KERS complex are key regulators for fungal development and SM metabolism in A. flavus.

The histone demethylase KdmB is part of a trimeric protein complex and mediates virulence and mycotoxin production in Penicillium expansum.

Epigenetic modification of chromosome structure has increasingly been associated with alterations in secondary metabolism and sporulation defects in filamentous fungal pathogens. Recently, the epigenetic reader protein SntB was shown to govern virulence, spore production and mycotoxin synthesis in the fruit pathogen Penicillium expansum. Through immunoprecipitation-coupled mass spectrometry, we found that SntB is a member of a protein complex with KdmB, a histone demethylase and the essential protein RpdA, a histone deacetylase. Deletion of kdmB phenocopied some but not all characteristics of the ΔsntB mutant. KdmB deletion strains exhibited reduced lesion development on Golden Delicious apples and this was accompanied by decreased production of patulin and citrinin in host tissue. In addition, ΔkdmB mutants were sensitive to several cell wall stressors which possibly contributed to the decreased virulence observed on apples. Slight differences in spore production and germination rates of ΔkdmB mutants in vitro did not impact overall diameter growth in culture.

Conserved copper regulation of the antimicrobial isocyanide brassicicolin A in Alternaria brassicicola.

Phytopathogenic Alternaria species are renown for production of toxins that contribute to virulence on host plants. Typically, these toxins belong to well-known secondary metabolite chemical classes including polyketides, non-ribosomal peptides and terpenes. However, the purported host toxin brassicicolin A produced by A. brassicicola is an isocyanide, a chemical class whose genetics and encoding gene structure is largely unknown. The chemical structure of brassicicolin A shows it to have similarity to the recently characterized fumicicolins derived from the Aspergillus fumigatus isocyanide synthase CrmA. Examination of the A. brassicicola genome identified AbcrmA, a putative homolog with 64% identity to A. fumigatus CrmA. Deletion of AbcrmA resulted in loss of production of brassicicolin A. Contrary to reports that brassicicolin A is a host-specific toxin, the ΔAbcrmA mutants were equally virulent as the wildtype on Brassica hosts. However, in line with results of A. fumigatus CrmA generated metabolites, we find that brassicicolin A increased 360-fold under copper limited conditions. Also, like A. fumigatus CrmA derived metabolites, we find brassicicolin A to be a broad-spectrum antimicrobial. We speculate that CrmA-like isocyanide synthase products provide the producing fungi a fitness advantage in copper depleted environments.

Vibrio gazogenes-dependent disruption of aflatoxin biosynthesis in Aspergillus flavus: the connection with endosomal uptake and hyphal morphogenesis.

Aflatoxins, a family of fungal secondary metabolites, are toxic and carcinogenic compounds that pose an enormous threat to global food safety and agricultural sustainability. Specifically agricultural products in African, Southeast Asian and hot and humid regions of American countries suffer most damage from aflatoxin producing molds due to the ideal climate conditions promoting their growth. Our recent studies suggest that Vibrio gazogenes (Vg), an estuarine bacterium non-pathogenic to plants and humans, can significantly inhibit aflatoxin biosynthesis in the producers. In this study, we investigated the mechanism underlying Vg-dependent aflatoxin inhibition using the prominent aflatoxin producer, Aspergillus flavus. We show that aflatoxin inhibition upon Vg treatment was associated with fungal uptake of Vg-prodigiosin, a red pigment, which was consistently visible inside fungal hyphae during treatment. The association of prodigiosin with aflatoxin inhibition was further evident as Serratia marcescens, another prodigiosin producer, significantly inhibited aflatoxin, while non-producers like Escherichia coli, Staphylococcus aureus, Vibrio harveyi, and Vibrio fischeri did not. Also, pure prodigiosin significantly inhibited aflatoxin biosynthesis. Endocytosis inhibitors, filipin and natamycin, reduced the Vg-prodigiosin uptake by the fungus leading to a significant increase in aflatoxin production, suggesting that uptake is endocytosis-dependent. The Vg treatment also reduced hyphal fusion (>98% inhibition) and branching, which are both endosome-dependent processes. Our results, therefore, collectively support our theory that Vg-associated aflatoxin inhibition is mediated by an endocytosis-dependent uptake of Vg-prodigiosin, which possibly leads to a disruption of normal endosomal functions.

Loss of the mammalian G-protein coupled receptor, G2A, modulates severity of invasive pulmonary aspergillosis.

Background: Aspergillus fumigatus is a well-known opportunistic pathogen that causes a range of diseases including the often-fatal disease, invasive pulmonary aspergillosis (IPA), in immunocompromised populations. The severity of IPA is dependent on both host- and pathogen-derived signaling molecules that mediate host immunity and fungal growth. Oxylipins are bioactive oxygenated fatty acids known to influence host immune response and Aspergillus developmental programs. Aspergillus synthesizes 8-HODE and 5,8-diHODE that have structural similarities to 9-HODE and 13-HODE, which are known ligands of the host G-protein-coupled receptor G2A (GPR132). Materials and methods: Oxylipins were extracted from infected lung tissue to assess fungal oxylipin production and the Pathhunter ß-arrestin assay was used to assess agonist and antagonist activity by fungal oxylipins on G2A. An immunocompetent model of A. fumigatus infection was used to assess changes in survival and immune responses for G2A-/- mice. Results: Here we report that Aspergillus oxylipins are produced in lung tissue of infected mice and in vitro ligand assays suggest 8-HODE is a G2A agonist and 5,8-diHODE is a partial antagonist. To address the hypothesis that G2A could be involved in the progression of IPA, we assessed the response of G2A-/- mice to A. fumigatus infection. G2A-/- mice showed a survival advantage over wild-type mice; this was accompanied by increased recruitment of G2A-/- neutrophils and increased levels of inflammatory markers in A. fumigatus-infected lungs. Conclusions: We conclude that G2A suppresses host inflammatory responses to Aspergillus fumigatus although it remains unclear if fungal oxylipins are involved in G2A activities.

The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference.

For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

Mining for a new class of fungal natural products: the evolution, diversity, and distribution of isocyanide synthase biosynthetic gene clusters.

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.

Aspergillus fumigatus transcription factor ZfpA regulates hyphal development and alters susceptibility to antifungals and neutrophil killing during infection.

Hyphal growth is essential for host colonization during Aspergillus infection. The transcription factor ZfpA regulates A. fumigatus hyphal development including branching, septation, and cell wall composition. However, how ZfpA affects fungal growth and susceptibility to host immunity during infection has not been investigated. Here, we use the larval zebrafish-Aspergillus infection model and primary human neutrophils to probe how ZfpA affects A. fumigatus pathogenesis and response to antifungal drugs in vivo. ZfpA deletion promotes fungal clearance and attenuates virulence in wild-type hosts and this virulence defect is abrogated in neutrophil-deficient zebrafish. ZfpA deletion also increases susceptibility to human neutrophils ex vivo while overexpression impairs fungal killing. Overexpression of ZfpA confers protection against the antifungal caspofungin by increasing chitin synthesis during hyphal development, while ZfpA deletion reduces cell wall chitin and increases caspofungin susceptibility in neutrophil-deficient zebrafish. These findings suggest a protective role for ZfpA activity in resistance to the innate immune response and antifungal treatment during A. fumigatus infection.

Mining for a New Class of Fungal Natural Products: The Evolution, Diversity, and Distribution of Isocyanide Synthase Biosynthetic Gene Clusters.

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.

LaeA-Regulated Fungal Traits Mediate Bacterial Community Assembly.

Potent antimicrobial metabolites are produced by filamentous fungi in pure culture, but their ecological functions in nature are often unknown. Using an antibacterial Penicillium isolate and a cheese rind microbial community, we demonstrate that a fungal specialized metabolite can regulate the diversity of bacterial communities. Inactivation of the global regulator, LaeA, resulted in the loss of antibacterial activity in the Penicillium isolate. Cheese rind bacterial communities assembled with the laeA deletion strain had significantly higher bacterial abundances than the wild-type strain. RNA-sequencing and metabolite profiling demonstrated a striking reduction in the expression and production of the natural product pseurotin in the laeA deletion strain. Inactivation of a core gene in the pseurotin biosynthetic cluster restored bacterial community composition, confirming the role of pseurotins in mediating bacterial community assembly. Our discovery demonstrates how global regulators of fungal transcription can control the assembly of bacterial communities and highlights an ecological role for a widespread class of fungal specialized metabolites. IMPORTANCE Cheese rinds are economically important microbial communities where fungi can impact food quality and aesthetics. The specific mechanisms by which fungi can regulate bacterial community assembly in cheeses, other fermented foods, and microbiomes in general are largely unknown. Our study highlights how specialized metabolites secreted by a Penicillium species can mediate cheese rind development via differential inhibition of bacterial community members. Because LaeA regulates specialized metabolites and other ecologically relevant traits in a wide range of filamentous fungi, this global regulator may have similar impacts in other fungus-dominated microbiomes.

Identifying Fungal Secondary Metabolites and Their Role in Plant Pathogenesis.

Pathogenic fungi are the main infectious agents of plants. Secondary metabolites produced by these fungi, also recognized as natural products, are key mediators of plant-fungal interactions. Knowledge on the biosynthesis of these metabolites, the accessibility to fungal genome sequences, and the development of gene disruption techniques open up opportunities to identify many more of these metabolites both in vitro and in planta. This methodology chapter gives a detailed systematic approach aiming to discover new natural products from phytopathogenic fungi and characterize their role in triggering plant cell death and plant disease. This approach takes advantage of the global regulation of fungal secondary metabolite production by regulatory proteins reported in various fungal species.

Pangenomics of the death cap mushroom Amanita phalloides, and of Agaricales, reveals dynamic evolution of toxin genes in an invasive range.

The poisonous European mushroom Amanita phalloides (the "death cap") is invading California. Whether the death caps' toxic secondary metabolites are evolving as it invades is unknown. We developed a bioinformatic pipeline to identify the MSDIN genes underpinning toxicity and probed 88 death cap genomes from an invasive Californian population and from the European range, discovering a previously unsuspected diversity of MSDINs made up of both core and accessory elements. Each death cap individual possesses a unique suite of MSDINs, and toxin genes are significantly differentiated between Californian and European samples. MSDIN genes are maintained by strong natural selection, and chemical profiling confirms MSDIN genes are expressed and result in distinct phenotypes; our chemical profiling also identified a new MSDIN peptide. Toxin genes are physically clustered within genomes. We contextualize our discoveries by probing for MSDINs in genomes from across the order Agaricales, revealing MSDIN diversity originated in independent gene family expansions among genera. We also report the discovery of an MSDIN in an Amanita outside the "lethal Amanitas" clade. Finally, the identification of an MSDIN gene and its associated processing gene (POPB) in Clavaria fumosa suggest the origin of MSDINs is older than previously suspected. The dynamic evolution of MSDINs underscores their potential to mediate ecological interactions, implicating MSDINs in the ongoing invasion. Our data change the understanding of the evolutionary history of poisonous mushrooms, emphasizing striking parallels to convergently evolved animal toxins. Our pipeline provides a roadmap for exploring secondary metabolites in other basidiomycetes and will enable drug prospecting.

Paralogous FgIDO genes with differential roles in tryptophan catabolism, fungal development and virulence in Fusarium graminearum.

Indoleamine 2,3-dioxygenase (Ido) is a tryptophan-degrading enzyme that is widely distributed across species. Ido catalyzes the first step of tryptophan (TRP) degradation and drives the de novo synthesis of nicotinamide adenine dinucleotide (NAD+) coenzymes via the kynurenine (KYN) pathway. The budding yeast Saccharomyces cerevisiae possesses a single IDO gene (BNA2) that is responsible for NAD+ synthesis, whereas a number of fungal species contain multiple IDO genes. However, the biological roles of IDO paralogs in plant pathogens remain unclear. In the current study, we identified three FgIDOs from the wheat head blight fungus Fusarium graminearum. FgIDOA/B/C expression was significantly induced upon TRP treatment. Targeted disruption of FgIDOA and/or FgIDOB caused different levels of NAD+ auxotrophy, thus resulting in pleotropic phenotypic defects. Loss of FgIDOA resulted in abnormal conidial morphology, reduced mycelial growth, decreased virulence in wheat heads and reduced deoxynivalenol accumulation. Exogenous addition of KYN or various intermediates involved in the KYN pathway rescued auxotrophy of the mutants. Metabolomics analysis revealed shifts toward alternative TRP degradation pathways to melatonin and indole derivatives in mutants lacking FgIDOB. Upregulation of partner genes in auxotrophic mutants and the capacity to rescue the auxotroph by overexpressing a partner gene indicated functional complementation among FgIDOA/B/C. Taken together, the results of this study provide insights into differential roles in paralogous FgIDOs and how fungal TRP catabolism modulates fungal development and virulence.