Fungal diseases seriously threaten global crop production, highlighting the need for fungicides with novel modes of action. Here, we show that shikonin exerts antifungal activity by targeting dihydroorotase (DHOase). Shikonin significantly inhibited mycelial growth, spore germination, and appressorium formation in representative phytopathogenic fungi in vitro. Biophysical analyses showed that shikonin directly binds to the DHOase of Magnaporthe oryzae (MoPyr4), with dissociation constants of 3.084, 1.22, and 1.26 μM determined by MST, SPR, and ITC, respectively. Molecular docking and mutagenesis identified R90, N178, and H320 as key binding residues. Consistently, deletion of MoPYR4 in M. oryzae reduced sensitivity to shikonin. In planta, shikonin restricted invasive hyphal growth in rice cells and showed both protective and curative activities against rice blast disease. Shikonin also bound to and inhibited the DHOases of Fusarium graminearum and Fusarium oxysporum, indicating broad-spectrum antifungal potential. These findings identify fungal DHOase as a promising target for fungicide development.

Nematode-trapping fungi such as Arthrobotrys flagrans employ sophisticated strategies to immobilize and digest their prey. A critical, yet poorly understood, step in this interaction is the induction of prey paralysis following capture. Here, we characterize putative effector D (PefD), a small secreted protein from A. flagrans. We demonstrate that pefD expression is specifically upregulated during the early and middle stages of infection of Caenorhabditis elegans (12-24 h post inoculation) and PefD localizes to the infection bulb within the nematode. Deletion of pefD significantly delayed the paralysis of C. elegans, whereas its overexpression accelerated the process. Through heterologous pan-neuronal expression in C. elegans, we show that PefD directly attenuates host locomotion and significantly enhances the activity of the sleep-promoting RIS interneuron. Unlike other effectors such as NipA or CyrA, PefD specifically modulates neuronal activity patterns without inducing general neurodegeneration. Our results identify PefD as a key virulence factor that hijacks the endogenous sleep induction circuitry of the host to facilitate rapid prey immobilization.

Microbes frequently exist as biofilm-embedded multi-species communities where their interactions may establish or exacerbate chronic infection. Recently, fungi and bacteria have been associated with various human tumor microenvironments, suggesting that dynamic cross-kingdom interactions may directly or indirectly contribute to tumor-associated processes. Here, we aimed to investigate whether cell-free supernatants from mono- and dual-species biofilms of the commonly associated fungus Candida albicans and bacterium Staphylococcus aureus could alter human monocyte responses that promote a tumor-related genetic signature in dysplastic oral epithelial (DOK) cells. Treatment of THP-1 monocytes with S. aureus cell-free supernatant increased the production of proinflammatory cytokines (IL-8, IL-1β, and TNF) and CD86 expression. However, exposure to cell-free supernatants from dual-species biofilm suppressed these responses. To determine the fungal virulence factors responsible, C. albicans mutants deleted for genes involved in adhesion (als3Δ/Δ), hyphal growth (efg1Δ/Δ cph1Δ/Δ), or candidalysin production (ece1Δ/Δ) were assessed during co-culture. While candidalysin was dispensable, loss of hyphal growth or the adhesin Als3p phenocopied effects of S. aureus mono-culture treatment. Conditioned medium from THP-1 cells initially challenged with mono- or dual-biofilm cell-free supernatants was applied to DOK cells to assess TP53 and BCL2 gene expression. Conditioned medium from S. aureus treated THP-1 cells led to decreased epithelial TP53, but increased BCL2 expression, which was reversed by the presence of wild-type C. albicans. These phenotypes were similarly dependent on C. albicans hyphal growth during dual-biofilm co-culture. Collectively, our results reveal that fungal-bacterial interactions may shape the monocyte-epithelial axis by orchestrating immune responses that enhance tumor-associated gene expression in dysplastic oral epithelial cells.

Chitin deacetylase 3 of Cryptococcus neoformans produces chitosans with low degree and random pattern of acetylation.

Apple bitter rot, caused by Colletotrichum gloeosporioides, leads to widespread yield loss and quality decline. Fungal virulence effectors are essential determinants of pathogenicity. However, the molecular mechanisms underlying C. gloeosporioides effector-mediated pathogenesis in apple remain largely elusive. Here, we found that the transcription factor MdWRKY100 binds to the promoter of the chitinase gene MdChi5, directly promoting its transcription and thereby improving apple bitter rot resistance. Meanwhile, a VQ-motif containing protein 4-like in apple, MdMVQ4, interacts with MdWRKY100, enhancing apple resistance to C. gloeosporioides. We identified the effector CgCAP1 in C. gloeosporioides via yeast two-hybrid (Y2H) and confirmed its interaction with MdMVQ4 using luciferase complementation imaging, bimolecular fluorescence complementation, and co-immunoprecipitation assays. In addition, knockout and complementation assays in C. gloeosporioides demonstrated that CgCAP1 functions as a critical virulence effector. Furthermore, the interaction of CgCAP1 with MdMVQ4 weakened the interaction between MdMVQ4 and MdWRKY100, leading to a reduction in chitinase activity during C. gloeosporioides infection and reduced apple bitter rot resistance. Together, these findings show how C. gloeosporioides utilizes CgCAP1 to suppress chitinase activity in apple, revealing the molecular interaction between the pathogen and its host.

SUMMARYThe skin harbors a diverse fungal community that contributes to both epidermal homeostasis and inflammatory disease. Historically, studies of cutaneous fungi focused primarily on opportunistic infections in immunocompromised hosts. Advances in sequencing technologies and metagenomic analyses have revealed that commensal yeasts of the skin microbiome likely influence host physiology and cutaneous disease severity. In this review, we summarize the current knowledge of host-fungal interactions at the skin epithelium, with particular emphasis on the yeast genera Malassezia and Candida. We discuss how fungal colonization shapes epidermal biology through direct interactions with keratinocytes and immune cells, highlighting fungal virulence factors such as secreted proteases and candidalysin, as well as host-sensing pathways. We further examine how these interactions contribute to inflammatory skin diseases, particularly atopic dermatitis and psoriasis, and how fungi participate in polymicrobial networks with bacteria and viruses to alter susceptibility to infection. Finally, we discuss how emerging therapeutic strategies change the fungal composition on skin. These advances suggest the importance of fungi as active regulators of skin immunity and emphasize key knowledge gaps that need to be addressed in future studies to better understand how they contribute to cutaneous diseases.

Aspergillus fumigatus forms adherent biofilms that contribute to its ability to persist and cause disease. However, significant strain diversity exists with regard to the morphology of A. fumigatus biofilms. A distinct colony morphotype associated with increased disease progression and low oxygen fitness, termed H-MORPH, was recently described. An additional defining feature of the H-MORPH biofilm morphotype is reduced in vitro adherence to surfaces. While reduced fungal strain adherence is most commonly associated with reductions in virulence, H-MORPH strains exhibit increased virulence relative to the well-studied N-MORPH reference strain AF293. Here we discover that the UDP-glucose 4-epimerase, Uge3, plays an important role in H-MORPH central carbon metabolism complementary to its role in production of the extracellular matrix polysaccharide galactosaminogalactan (GAG). In H-MORPH strains, this metabolic role for Uge3 becomes central to virulence. These data highlight A. fumigatus strain specific mechanisms of fungal carbon metabolism related to biofilm matrix production and fungal virulence.

Cryptococcus neoformans is the leading cause of fungal meningoencephalitis. Cellular heterogeneity during cryptococcal infection contributes to host adaptation and fungal pathogenesis. C. neoformans titan cells and seed cells represent enlarged and small-sized morphotypes, respectively, which exhibit distinct transcriptional profiles and can be induced by environmental factors. In this work, we describe a distinct small morphotype of C. neoformans, referred to as ovoid cells. The formation of ovoid cells is promoted by host-related conditions such as nutrient limitation and elevated CO2 levels, which was observed during the late stage of cryptococcal infection. In addition to their smaller size compared to typical yeast cells, ovoid cells highly express OSP1, a marker distinguishes ovoid cells from other small morphotypes, including seed cells and titanides. These cells exhibit an increased budding and proliferation rate, which is consistent with transcriptome data that ovoid cells upregulate cell cycle related genes. We further demonstrate that the glucose repression signaling pathway and the cAMP/PKA pathway are involved in ovoid cell formation in C. neoformans. Ovoid cells show reduced fungal virulence but enhanced tolerance under long-term fluconazole treatment, indicating their important role in the balancing virulence and antifungal tolerance within C. neoformans populations.

Systematic infectome\u2013phenome profiling reveals cryptococcal infection-associated proteins driving immune system remodeling and immunization potential

The cell wall integrity is crucial for fungal development, adaptation to adverse environments, and virulence. Here, a transcription factor (TF) heterodimer, BbOsrR2-BbOsrR3, in complex with a cyclin BbClp1 was found to orchestrate cell wall homeostasis in an entomopathogenic fungus Beauveria bassiana. The ΔBbOsrR2 mutant displayed increased sensitivity to cell wall stress, with reduced β-1,3-glucan and chitin contents. These opposite phenotypes were examined in the ΔBbOsrR3 and ΔBbClp1 mutants. The two TFs, BbOsR2 and BbOsrR3, targeted distinct genes in response to cell wall stress. Phosphorylation of the two TFs was interdependent and regulated by BbClp1, which was linked to their transcription activities. Two BbOsrR2 targets, BbGlu and BbMgn, were found to negatively and positively mediate cell wall integrity, and conidial germination and virulence, respectively, which greatly contributed to the BbOsrR2 and BbOsrR3-controlled cell wall homeostasis, and fungal development and virulence. These results demonstrate a regulatory network orchestrating fungal cell wall homeostasis and virulence.

Candidozyma auris (C. auris) is an emerging multidrug-resistant fungal pathogen, and its dissemination to the bloodstream and deep-seated organs is associated with high mortality. The limited antifungal armory and pipelines against C. auris pose a major challenge in disease management. Addressing this threat requires a deeper understanding of fungal virulence mechanisms that promote persistence and of host factors that drive susceptibility. Previous in vitro studies showed that iron enhances C. auris resistance to azoles and echinocandins, whereas iron chelation mitigates this effect. Here, we demonstrate that C. auris does not utilize cell-free heme or induce hemolysis but instead extracts and uses iron from transferrin to support growth and virulence. Deletion of the SIT1 siderophore transporter in C. auris attenuated fungal growth and reduced renal injury, while increased transferrin-iron saturation worsened disease outcomes in immunocompetent mice, highlighting the importance of transferrin-bound iron uptake. Mechanistically, C. auris exploits transferrin-bound iron to enhance ergosterol biosynthesis and activate antioxidant defenses, promoting resistance to neutrophil- and caspofungin-mediated killing. These findings identify elevated transferrin saturation as a novel host susceptibility risk factor for disseminated C. auris infection and reveal how iron availability reshapes fungal physiology to drive infection persistence.

Fungal pathogens utilize specialized cellular systems to overcome host defenses, yet no dedicated pathogenic organelles have been previously defined in fungi. Here, we describe a transient, membrane-bound vesicle (100-250 nm in diameter) that forms specifically within the infection structures of a nematode-trapping fungus Dactylellina haptotyla, which we term the virulensome. Density-gradient isolation yielded virulensome-enriched fractions that killed 81.6% of Caenorhabditis elegans larvae within 24 h and perforated the nematode cuticle. Quantitative proteomics revealed 486 core components, including 31 peptidases and 23 glycoside hydrolases. Knockout of each of three representative proteins-an α-mannosidase (DHGH1), a metalloendopeptidase (DHMEPE1), and a WSC-domain protein-reduced virulensome abundance by up to 80% and lowered nematode mortality by 4-fold. Conversely, recombinant DHGH1 or DHMEPE1 killed >90% of nematodes in vitro. Live-cell super-resolution imaging showed virulensomes polarizing toward the host interface, disintegrating within infection bulbs, and releasing toxic effector cargos into the nematode body. These findings define the virulensome that packages and delivers destructive enzymes in a targeted manner, revealing a previously unrecognized mechanism of pathogenesis.

Copper is an essential micronutrient required for survival in all kingdoms of life, as it is used as a catalytic cofactor for many essential processes in the cell. In turn, this reactivity of copper ions makes elevated levels of free copper toxic to the cell. This dual nature of copper-essential for life but toxic at elevated levels-is used by our innate immune system in a process called nutritional immunity to combat and kill invading pathogens. In this work, we explore how the fungal human pathogen Cryptococcus neoformans senses high copper stress, a copper microenvironment encountered within the host lung. We identified a specific cysteine-rich motif within the copper-responsive transcription factor Cuf1 to be essential for high copper stress sensing. Mutation of this motif led to an impaired high copper stress adaptation, which did not affect the fitness of the yeast but did impact the containment and distribution of yeast cells inside the host lung.

In this study, multifunctional polymer-stabilized CuO-ZnO hybrid nanocomplexes were developed through a green synthesis approach and systematically evaluated for their antimicrobial and protein delivery potential. Bimetallic CuO-ZnO nanoparticles were biosynthesized using extracts of Cotoneaster horizontalis, Salvia officinalis, and Laurus nobilis, followed by integration into chitosan/nanochitosan-P(MMA-co-MAA) copolymer matrices. The resulting nanocomplexes were characterized using SEM, DLS, FT-IR, and XRD analyses, confirming successful nanoparticle formation, homogeneous dispersion, and predominantly amorphous structures, with particle sizes ranging from 62 to 138nm. Among the formulations, nanochitosan-based systems exhibited improved structural compactness and reduced polydispersity. Molecular docking simulations revealed strong and stable interactions between the Cu-Zn alloy-modified polymeric carriers and bovine serum albumin, as well as remarkably high binding affinities toward key bacterial and fungal virulence proteins, including Sortase A, TolC, and CYP51. These findings suggested enhanced multivalent interaction capabilities of the nanocomplexes. In vitro antimicrobial assays corroborated the computational predictions, demonstrating pronounced antibacterial and antifungal activities (15-22mm against E. coli, 17-24mm against S. aureus and 21-25mm against A. niger) with low minimum inhibitory concentrations (0.50mg/mL for E. coli and 0.25mg/mL for S. aureus). Furthermore, in vitro release studies using bovine serum albumin and human insulin as model biomolecules revealed sustained and controlled release profiles over seven days, with minimal burst effects. Nanochitosan-based systems exhibited slightly slower release kinetics, attributed to denser polymeric networks and stronger biomolecule-carrier interactions. The incorporation of P(MMA-co-MAA) further contributed to pH-responsive and diffusion-controlled release behavior. Collectively, this study presents an integrated experimental and computational strategy for designing biocompatible, green-synthesized CuO-ZnO nanocomplexes with dual antimicrobial and drug delivery functionalities. The developed platform may hold strong potential for biomedical applications, including drug delivery systems, wound dressings, and implant-associated infection control.

The fungal pathogen Parastagonospora nodorum utilizes necrotrophic effectors (NEs) to cause chlorosis and necrosis on wheat. NE expression is mediated by an assortment of transcription factors (TFs), the most well-characterized of which is PnPf2. Orthologs of PnPf2 regulate virulence in phytopathogenic fungi across the Ascomycete fungal lineage, yet their protein architecture remains functionally uncharacterized. These orthologs are characterized by the archetypal N-terminal Zn2Cys6 zinc-finger DNA-binding domain, a conserved yet poorly characterized middle homology region (MHR), and a C-terminal disordered region. We investigated the role of each of the three domains through PnPf2 truncation mutants in situ. Variable deletions of the C-terminal disordered region resulted in a reduction of SnToxA and SnTox3 expression in planta. The MHR domain is indispensable for host virulence and SnToxA and SnTox3 regulation and also facilitates PnPf2 homodimerization. Using the homodimer-mediating MHR as a bait in library-scale protein-protein interaction yeast two-hybrid assays, we identified a putative COP9-signalosome protein, PnCsn6, as a likely interacting partner. PnCsn6 is essential for disease symptoms during P. nodorum infection on wheat, and we observed NE dysregulation in PnCsn6-deletion mutants. This study provides the first domain-level functional characterization of the virulence-regulating TF ortholog Pf2. Our results demonstrate the essential role of the MHR domain in mediating protein-protein interactions and effector regulation. We have also unveiled evidence that suggests that PnCsn6 contributes to pathogenicity and may point to an important signaling pathway that could work in tandem with PnPf2 for fungal pathogenesis. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Heat shock proteins (HSPs) are highly conserved molecular chaperones that play a key role in maintaining protein homeostasis and cellular survival under stress conditions. Clinically relevant human pathogenic fungi include opportunistic fungi, dimorphic fungi, dermatophytes, Mucorales, and other pathogenic groups. HSPs, including Hsp90, Hsp70, Hsp60, Hsp40, and Hsp110, are essential for the correct nascent protein folding, aggregation prevention, and degradation of misfolded polypeptides. Fungal pathogens frequently encounter environmental and host-imposed stresses, including oxidative stress, temperature fluctuations, and antifungal treatments. This review synthesizes and critically analyzes current evidence on the role of HSP families in essential processes linked to fungal virulence, including morphogenetic transitions, biofilm formation, maintenance of cell wall integrity, and interactions with host immune cells. Beyond their canonical chaperone functions, HSPs act as central mediators in pathogenic processes, such as morphogenesis transitions, biofilm formation, cell wall integrity, and interactions with host immune cells. Hsp90 stabilizes key signaling proteins involved in stress responses, morphogenesis, and antifungal resistance, while Hsp60 and Hsp70 contribute to mitochondrial function, cell wall integrity, and immune modulation. Disruption of these chaperones impairs growth, reduces virulence, and increases susceptibility to antifungal agents. The rise of antifungal resistance underscores the urgent need for new therapeutic strategies. Targeting fungal HSPs has emerged as a promising approach due to their essential roles in stress tolerance and pathogenesis. Hsp90 inhibitors, including geldanamycin derivatives and other small molecules, have demonstrated the ability to impair fungal growth, reduce virulence traits, and sensitize resistant strains to conventional antifungal drugs. Combining HSP inhibitors with existing antifungal drugs represents a potential strategy to overcome resistance and improve treatment outcomes. This review summarizes the current knowledge on HSPs in pathogenic fungi, focusing on their roles in stress adaptation, virulence, host-pathogen interaction, antifungal resistance, and their potential as targets for novel antifungal therapies.

Apicidin, an emerging mycotoxin with potent histone deacetylase inhibitory and cytotoxic activities, is increasingly detected in wheat and rice in Jiangsu Province, China. Through a population-level screening, we identified a subset of Fusarium asiaticum strains, the dominant pathogen in wheat-rice rotation regions, as the primary producers of apicidin. Using telomere-to-telomere genome assemblies, we identified a biosynthetic gene cluster of 12 genes (FaAps-BGC) essential for apicidin production. Comparative genomic and phylogenomic analyses revealed a complex evolutionary history of FaAps-BGC, characterized by a discontinuous distribution across Fusarium species and extensive structural variation. Large-scale population genome sequencing further showed that loss of apicidin production was associated with either deletion of the entire FaAps-BGC or pseudogenization of the non-ribosomal peptide synthetase (NRPS) backbone gene, and correlated with reduced virulence in both wheat and rice. Functional analyses showed that deletion of the pathway-specific transcription factor gene FaAps2 led to coordinated silencing of other Aps genes within the FaAps-BGC, abolished apicidin biosynthesis, and significantly reduced virulence on both wheat and rice. Mechanistically, apicidin promotes infection by disrupting host plasma membrane integrity, inducing reactive oxygen species (ROS) accumulation, and compromising host defense response in a host-dependent manner. Together, these findings establish apicidin as a key virulence determinant in F. asiaticum and highlight how genomic variation in secondary metabolite biosynthetic pathways shapes fungal virulence in cereal cropping systems.

Physiological mechanisms driving virulence in insect-pathogenic fungi.

Multifaceted biocontrol mechanisms of Bacillus amyloliquefaciens F028 against Botrytis cinerea in postharvest tomato fruit.

Ustilaginoidea virens secretes numerous proteins during infection, the mechanisms regulating plant immunity and fungal virulence remain largely unknown. Herein, we identified a cortical patch protein, UvCPP1, from the U. virens secretome, as a cell death-inducing protein. UvCPP1 is secreted into rice florets via extracellular vesicles during U. virens infection. It activates plant basal immunity, as evidenced by reactive oxygen species (ROS) burst, callose deposition, and upregulation of defense-related genes. Notably, the UvCPP1(1-136) is sufficient to elicit immune responses comparable to the full-length UvCPP1. UvCPP1-induced cell death in Nicotiana benthamiana depends on brassinosteroid-insensitive 1 (BRI1)-associated receptor kinase 1 (BAK1) but is independent of suppressor of BIR1-1 (SOBIR1) and nucleotide-binding leucine-rich repeat (NLR) co-receptors. The transcript level of UvCPP1 is significantly upregulated during infection. Both knockout and overexpression of UvCPP1 significantly attenuated fungal pathogenicity, demonstrating that UvCPP1 functions as a virulence factor to promote infection and is recognized by plants. Heterologous expression of UvCPP1 significantly enhances rice immunity without compromising agronomic traits, characterized by increased ROS accumulation, callose deposition, and mitogen-activated protein kinase (MAPK) phosphorylation. UvCPP1-overexpressing lines exhibit broad-spectrum resistance against multiple pathogens. RNA-seq analysis revealed that UvCPP1 expression activates defense-related genes, phytohormone signaling pathways, and diterpenoid biosynthesis. Although UvCPP1 is indispensable for the full virulence of U.virens, it simultaneously activates plant immune responses. The characteristics of UvCPP1 highlight its potential application as a molecular tool for breeding disease-resistant crops through immune priming, offering a sustainable strategy for false smut management.