Iron homeostasis and secondary metabolism are controlled by the basic leucine zipper (bZIP) transcription factor HapX in many fungi. In this study, AflHapX was identified through homology research of the Aspergillus flavus genome with the bZIP protein HapX in Aspergillus fumigatus and subsequent gene deletion. We established that AflHapX functions as a regulator for adaptation to iron-excess and iron-starvation conditions. The AflhapX deletion strain (ΔAflhapX) produces less aflatoxin B1 (AFB1) compared to the wild type (WT) and complementary strain (ΔAflhapXC) on YES medium. While iron typically represses AFB1 biosynthesis in A. flavus, the deletion of AflhapX restores iron’s inhibition of AFB1 production. Furthermore, compared to WT and ΔAflhapXC, ΔAflhapX produced more sclerotia and displayed greater sensitivity to the oxidative reagents H2O2, menadione sodium bisulfite (MSB), and diamide. On peanuts, the ΔAflhapX strain produced fewer conidia and less AFB1 than WT and ΔAflhapXC. Transcriptome analysis data provided evidence that AflHapX globally regulates genes involved in aflatoxin biosynthesis, oxidative stress response, and iron homeostasis. Our results suggest that AflHapX may regulate the effect of iron on aflatoxin production.
Fungi are important chassis organisms for sustainable biomanufacturing owing to their strong secretion capacity, metabolic diversity, and adaptability to industrial processes. Driven by the rapid development of Artificial Intelligence (AI), computational approaches that enable machines to learn patterns from data and make predictions, together with omics technologies and synthetic biology, data- and model-driven strategies are increasingly reshaping fungal cell factory design and optimization. This review summarizes recent advances in AI-enabled fungal biomanufacturing, covering AI-assisted genome mining, enzyme and pathway analysis, genome editing, metabolic network modeling, and intelligent fermentation control. The review further highlights the emerging concept of the artificial intelligence virtual cell (AIVC), which integrates multi-omics data, mechanistic modeling, and machine learning-based approaches into a unified multiscale framework for simulating dynamic cellular behaviors. The technical foundations and potential applications of AIVC in fungal metabolic engineering, drug discovery, agriculture, and environmental biotechnology are reviewed. Finally, key challenges-data quality and standardization, cross-scale model integration and interpretability, and the gap between in silico prediction and experimental validation-are highlighted. This review provides a systematic perspective on the role of AI and virtual cell technologies in advancing next generation fungal biotechnology.
Verticillium dahliae is a widely distributed vascular wilt pathogen with a broad host range, infecting over 200 plant species. Nitrogen metabolism is closely associated with fungal growth and pathogenicity, but the role of glutamate synthase-mediated nitrogen assimilation in V. dahliae has not been fully clarified. Here, we examined the functions of two glutamate synthase-encoding genes, VdGogat1 and VdGogat2, in nitrogen metabolism, virulence, and stress adaptation. Deletion of VdGogat1 (ΔVdGogat1) or VdGogat2 (ΔVdGogat2) reduced host colonization and compromised penetration peg formation. The ΔVdGogat1, ΔVdGogat2, and double mutant strain (ΔVdGogat1/2) showed defects in utilizing specific nitrogen sources. VdGogat1 played a major role in ammonium utilization, whereas VdGogat2 contributed more specifically to glutamine metabolism; accordingly, the ΔVdGogat1/2 strain showed a significantly reduced capacity to utilize ammonium. In addition, loss of VdGogat1 enhanced sensitivity to nitrosative stress. Collectively, these findings indicate that VdGogat1 and VdGogat2 contribute to nitrogen utilization, penetration, and full virulence in V. dahliae.
Dermatophytosis, or tinea, is a common superficial fungal infection affecting people of all ages worldwide. It is caused by keratinophilic dermatophytes that invade the skin barrier, colonize keratinized tissues, and activate host immune responses, leading to inflammation and tissue damage. Understanding the molecular pathogenesis is critical for standardizing treatment and developing targeted therapies. Key aspects include fungal invasion mechanisms, adhesion and proliferation on skin, secreted molecules, virulence factors, colonization patterns, and host-pathogen interactions. As new dermatophyte species emerge, advanced techniques are needed to study gene expression regulation during infection, disease progression, and how fungal factors modulate the host immune response, particularly in keratinocytes. Functional annotation of dermatophyte genes and their human counterparts is essential to map these interactions. This review summarizes the most common clinical forms of dermatophytosis, discusses selected host-dermatophyte interactions and evolutionary adaptations, and highlights current strategies for diagnosis and therapy. A deeper comprehension of these mechanisms will help in designing precise antifungal drugs and improving disease management.
Morphological development in filamentous fungi critically impacts nutrient and oxygen transfer, broth rheology, and metabolite production during submerged fermentation. However, life-cycle-resolved quantitative analysis remains technically challenging because fungal morphology dynamically spans multiple spatial scales from germinating spores and branching hyphae to mycelial pellets. Here, we developed PAMP, Python-based Automated Morphology analysis Pipeline, integrating skeletonization with quantitative image analysis for high-resolution morphological profiling throughout the fungal life cycle. Using two Aspergillus niger strains, MA70.15 and D353.8, PAMP accurately captures distinct morphogenetic trajectories associated with protein secretion and citric acid production. Application to pkaC conditional-expression mutant further reveals that graded PkaC activity modulates hyphal extension, branching frequency and pellet porosity, thereby improving citric acid production. Correlation analysis identifies peripheral hyphae and pellet porosity as major morphology determinants of productivity, which were not detectable by conventional pellet-analysis tools due to their limited resolution of peripheral hyphae. PAMP aslo demonstrates robust performance across multiple filamentous fungi, including Fusarium venenatum and Acremonium chrysogenum. Overall, PAMP provides a robust platform for morphology-guided strain engineering and fermentation optimization in fungal biotechnology.
Mycelium-based composites (MBCs) are produced through a combination of fungi and lignocellulosic materials. Identifying novel combinations of fungi and lignocellulosic waste is crucial for exploring new material properties. In this study, MBCs were prepared with strains of Ganoderma orbiforme and Lentinus squarrosulus from Sri Lanka, using three different types of locally sourced lignocellulosic substrates, including Cocos nucifera sawdust, Mangifera indica sawdust, and coir pith derived from coconut husk. Mycelium inoculum grown on rice seeds was introduced to organic substrates and incubated at 28 °C for 30 d. The resulting composites were separated from the container, dried at 80 °C, and characterised for physicochemical, and microscopic properties. Results indicated that the produced MBCs exhibit properties equivalent to or superior to those of expanded polystyrene (EPS). The Ashby chart generated revealed that MBCs possess properties comparable to cork and other low-density foams, making them suitable for insulating, cushioning, and packaging applications. Among the combinations tested, MBCs made with coir pith and coconut sawdust proved to be the most effective, eco-friendly alternatives to protective packaging.
Cryptococcal meningitis (CM), caused by Cryptococcus neoformans (C. neoformans), is marked by a rapidly rising incidence and mortality. Furthermore, the WHO has designated C. neoformans as a highest-priority pathogen within its critical fungal pathogens list. Novel antifungal agents and new therapeutic strategies for CM are urgently needed. Herein, a series of novel spiroquinoxaline antifungal compounds were discovered by phenotypic screening and structural optimization. Notably, compound B1 exhibited a potent in vitro antifungal activity against C. neoformans (MIC80 = 1-4 mu g/mL). In a murine model of CM, compound B1 significantly reduced the brain fungal burden. Mechanism studies showed that compound B1 substantially suppressed key virulence factors of C. neoformans, including capsule formation, biofilm development, and melanin production, and promoted intracellular accumulation of reactive oxygen species and lipid peroxides. It is indicated that compound B1 provoked oxidative-stress-mediated cellular damage, thereby exerting antifungal activity. This study offered a novel promising lead for therapeutic development against cryptococcosis.
Antifungal persistence has emerged as an important survival strategy that may contribute to treatment failure, yet its prevalence and phenotypic characteristics in Aspergillus fumigatus populations remain incompletely defined. Here, we systematically assessed voriconazole persistence in a collection of 225 azole-susceptible A. fumigatus isolates derived from clinical (n = 208) and environmental (n = 17) sources in China. Persistence-associated behavior was evaluated using complementary broth microdilution-based germination assays, agar disk diffusion assays, and time-kill analyses. Approximately 10% of azole-susceptible isolates reproducibly exhibited persistence-associated phenotypes. Environmental conditions strongly influenced persistence-associated growth, as iron chelation selectively suppressed persistence. Importantly, persistent isolates showed an increased tendency to acquire azole resistance during prolonged antifungal exposure. Together, this study provides an isolate-based assessment of antifungal persistence in A. fumigatus, highlighting its phenotypic diversity and potential role in resistance evolution. These findings underscore the persistence of antifungal susceptibility as an additional dimension, with important implications for therapy and resistance development.
The genus Inocybe, a highly diverse lineage within the Inocybaceae, contains more than 1000 species worldwide. Species of this genus have been studied in many parts of China since the beginning of this century. However, the species diversity of the genus in southwestern China remains largely unknown. In the past few years, the authors made several forays in the Gaoligong Mountains, western Yunnan, making many collections of Inocybe species. Based on morphological studies and a three-locus (ITS-nrLSU-rpb2) phylogeny, ten novel species have been recognized, namely I. hirsuticeps, I. ceratina, I. longistipes, I. gracilipes, I. flocculosipes, I. flavocrocea, I. rufosquamosa, I. aculeata, I. dulongjiangensis, and I. squamulomarginata. They are described and characterized with morphological features and phylogenetic evidence.
Extracellular vesicles (EVs) play critical roles in the cross-kingdom trafficking of molecules during species interactions. At present, the isolation of pure, intact EVs from filamentous fungi remains challenging. Here, we provide an effective method for the isolation and purification of fungal EVs. This method includes a detailed protocol for isolating EVs from a typical filamentous fungus, Arthrobotrys oligospora, via differential-speed ultracentrifugation protocols. We found that F100 is the most precise method for specific EVs isolation when specific EVs biomarkers are available. Furthermore, proteomic analysis of the purified EVs revealed potential novel mechanisms underlying the regulation of fungal secondary metabolism and host interactions by these vesicles. These findings provide robust methodological support and a solid theoretical foundation for future functional studies of fungal-derived extracellular vesicles.
Polyamines accumulate in wheat spikes during infection by Fusarium graminearum, the primary causal agent of Fusarium head blight (FHB). Notably, putrescine acts as a potent inducer of deoxynivalenol (DON) biosynthesis. Despite exogenous polyamine uptake is essential for fungal fitness and DON biosynthesis, the responsible transporters remain unknown. Here, we identified 13 genes in F. graminearum homologous to Saccharomyces cerevisiae polyamine uptake transporters, including Agp2, Dur3, and Hol1 orthologs. Surprisingly, neither single deletions nor a 13-gene (13K) deletion mutant impaired polyamine utilization, in vivo DON production, or virulence. Furthermore, exogenous polyamines fully rescued growth in polyamine-auxotrophic mutants, indicating these genes are not the primary transporters. Instead, the 13K mutant displayed reduced DON biosynthesis and enhanced conidiation under specific in vitro low-inorganic-salt conditions, correlated with downregulated farnesyl diphosphate (FPP) pathway genes. Notably, this effect was absent during wheat infection. Our findings demonstrate that these homologs primarily regulate nutrient adaptation and secondary metabolism in vitro rather than mediating major exogenous polyamine uptake, revealing an unexpected functional divergence from their yeast counterparts in nutrient-related physiology. [GRAPHICS]
Previous transcriptome analysis suggested that proteases of the microbial antagonist Pythium oligandrum and protease inhibitors of its host/prey Pythium myriotylum were important in their interactions. P. oligandrum protease activity was induced when cultured with inactivated oomycete mycelial powder, and exo-proteomic analysis showed significantly higher abundance of 25 proteases in cultures with mycelial powder. Two interaction-upregulated P. myriotylum Kazal-type serine protease inhibitors (Pm_KSPI-7 and Pm_KSPI-11) were heterologously produced, and Pm_KSPI-11, but not Pm_KSPI-7, inhibited P. oligandrum protease activities. Pm_KSPI-11 was a strong inhibitor of porcine trypsin activity (K-i = similar to 6.5 nmol/L), while neither trypsin nor chymotrypsin activity was inhibited by Pm_KSPI-7. P1-P2 loop residues and protein modeling analyses suggested that the lysine P1 residue in Pm_KSPI-11 may explain the differences in inhibitory properties. Pm_KSPI-11 supplementation of skim milk agar reduced P. oligandrum growth, but supplementation of cV8 agar did not substantially reduce P. oligandrum antagonism towards P. myriotylum. The role of Pm_KSPI-11 may be in reducing the ability of P. oligandrum to derive nutrition from peptides rather than directly counter-antagonizing P. oligandrum in a confrontation. Understanding potential P. myriotylum counter-antagonism abilities, such as protease inhibitors, may improve the biological control of crop diseases caused by P. myriotylum in more complex environments.
While pectate lyases are crucial for pathogen colonization via pectin degradation, their roles in endophytic fungi remain largely unexplored. This study characterizes FlPL1, a pectate lyase from the endophyte Fusarium lateritium. FlPL1 was highly expressed during Nicotiana benthamiana colonization and exhibited confirmed pectin-degrading activity. Deleting FlPL1 did not affect hyphal growth but impaired pectin utilization, decreased colonization efficiency, and diminished the fungus's plant growth-promoting effects. Furthermore, FlPL1 loss intensified host immune responses by upregulating the jasmonic acid (JA) pathway (increasing JA-Ile and Me-JA) while reducing auxin (IAA and IBA) levels, triggering an antagonistic JA-auxin interplay. Additionally, FlPL1 alone could independently induce a JA-dependent host immune response. Ultimately, FlPL1 facilitates F. lateritium colonization and promotes plant growth by degrading pectin and modulating host hormone balances to suppress immunity. This study unveils the novel dual regulatory function of an endophyte-derived pectate lyase, providing new insights into endophytic colonization mechanisms. [GRAPHICS] .
Talaromyces marneffei is a lethal human pathogenic fungus endemic to Southeast Asia. While the previously reported mycovirus Talaromyces marneffei partitivirus 1 (TmPV1) enhances fungal virulence, we report the discovery of a second mycovirus, Talaromyces marneffei narnavirus 1 (TmNV1), which induces hypovirulence. Through viral curing experiments, we demonstrated that TmNV1 acted as a key virulence attenuator. Specifically, the TmNV1-infected strain exhibited increased sensitivity to Congo red and increased susceptibility to macrophage killing compared to the isogenic virus-free strain. In a murine model, infection with the TmNV1-infected strain resulted in significantly improved survival rates, reduced fungal burden, and attenuated inflammation. Interestingly, TmPV1 was found to synergistically amplify most of the hypovirulent effects associated with TmNV1. Cell wall remodelling, abnormal vacuole dynamics, and altered intracellular reactive oxygen species (ROS) production in macrophages were observed in TmNV1-/TmNV1-TmPV1-infected strains, which may be associated with their hypovirulent effects. Collectively, our findings provide new insights into the viral determinants of T. marneffei pathogenesis and reveal a critical virus-virus interaction within this important human fungal pathogen. [GRAPHICS]
Pathogen effectors mediate infection and pathogenesis, and extensive research has been conducted on this topic. In contrast, studies on endophyte effectors remain limited, and their functions and mechanisms of action are still not well understood. Here, we report a novel effector identified from the endophytic Fusarium lateritium. Knockout of this effector reduced the colonization rate of the endophyte in the host plant by approximately 70%; therefore, we named it Colonization-Related Effector Protein 1 (CREP1). We found that CREP1 strongly activates host plant immunity and induces cell necrosis in Nicotiana benthamiana leaves. Disruption of CREP1 resulted in significant downregulation of immune defense-related genes in the host plant, accompanied by an approximately 50% decrease in the plant growth-promoting ability of the endophytic fungus. Further investigation revealed that CREP1 interacts with the plant cell-surface recognition receptor protein NbEIX2 to regulate plant immunity. These findings demonstrate that CREP1 is a necrosis-inducing effector in F. lateritium that modulates plant immunity through its interaction with NbEIX2, affecting the colonization and growth-promoting capacity of the endophytic fungus in plants.
Monascus pigments (MPs) are important azaphilone-type polyketide pigments, yet yield control, stability, and safety pose challenges. Using an epigenetic derepression strategy, we constructed an Ash2 deletion mutant in the acidophilic fungus Talaromyces purpurogenus OUCMDZ-019, with Ash2 being a COMPASS subunit involved in H3K4 methylation. Transcriptomic analysis revealed broad transcriptional changes, including marked upregulation of an MPs-related BGC, accompanied by pronounced metabolome remodeling. Integrating genome mining, transcriptomics, and GNPS molecular networking enabled targeted isolation of twelve MP derivatives, including four new azaphilones (talarpurpurones A-D), whose absolute configurations were confirmed by spectroscopy. pH-dependent transformation experiments clarified the behavior of azaphilic addition, providing insight into pigment interconversion and composition drift during fermentation. Talarpurpurone A exhibited high color value and stable red coloration from pH 3 to 11, outperforming commercial preparations. These pigments displayed selected moderate activity against Gram-positive bacteria and lipid-lowering activity. Notably, bioinformatic and fermentation analysis supported the absence of citrinin biosynthesis in OUCMDZ-019. Overall, these results suggest that Ash2 deletion is associated with azaphilone pigment diversification in Talaromyces under the tested conditions and provide a citrinin-free fungal strain for MPs production.
Ganoderic acids (GAs), the major lanostane-type triterpenoids in Ganoderma lucidum (G. lucidum), possess diverse pharmacological activities but are produced at low levels, limiting their applications. Transcriptional regulation of GA biosynthesis remains poorly understood. In this study, we functionally characterized the GlZn(2)Cys(6)-type transcription factor GlZn(2)Cys(6)_82 and elucidated its role in GA biosynthesis. Overexpression of GlZn(2)Cys(6)_82 significantly increased total triterpenoids during the mycelial stage and promoted the accumulation of multiple individual GAs at the primordia stage, whereas silencing resulted in markedly reduced GA levels. qRT-PCR analyses revealed that GlZn(2)Cys(6)_82 positively regulates key biosynthetic genes, including HMGR, SQS, and LS, with SQS showing the strongest transcriptional induction. Subcellular localization assays demonstrated that GlZn(2)Cys(6)_82 is a nuclear protein, and yeast one-hybrid analyses confirmed its direct binding to the SQS promoter, establishing SQS as a downstream target. Comparative transcriptome profiling further showed that GlZn(2)Cys(6)_82 modulates genes involved in the mevalonate pathway and secondary metabolism. Collectively, our findings identify GlZn(2)Cys(6)_82 as a positive regulator of GA biosynthesis and provide a basis for metabolic engineering to increase GA production. [GRAPHICS]
Trichoderma reesei is an important industrial cellulase producer, but its protein secretion is limited by elongated hyphae during bioreactor cultivation. Physical and process-based controls are widely applied to modulate fungal morphology, but genetic strategies remain poorly explored. Here, we show that deletion of kinesin gene kin1 alters hyphal growth and boosts cellulase production in T. reesei. Deletion of kin1 led to pronounced morphological changes: shorter, highly branched hyphae, compact colony architecture, and increased biomass. Under cellulase-inducing conditions, the Delta kin1 strain exhibited higher cellulase titers and activities, improving biomass saccharification efficiency. Importantly, this enhanced performance was maintained during bioreactor fermentation, likely due to increased shear resistance from the altered morphology. Transcriptome analysis revealed that expression levels of major cellulase genes were largely unaffected by kin1 gene deletion, indicating that enhanced enzyme production occurs independently of transcriptional upregulation. Together, our findings uncover a previously unrecognized link between kinesin-mediated hyphal morphology and protein secretion in T. reesei. The kin1-deletion phenotype provides a genetic strategy for improving cellulase production in other filamentous fungi.
