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Abstract This study investigates the chemical inhibition of asexual development in Aspergillus nidulans to enhance the mechanical properties of mycelial materials. We hypothesized that suppressing conidiation using the ornithine decarboxylase inhibitor α-difluoromethylornithine (DFMO) would increase material strength by inhibiting asexual development, promoting denser hyphal packing. Mycelial materials were grown in DFMO concentrations (0, 0.05, 0.5, and 5 mM), and conidiation and ultimate tensile strength (UTS) were measured. Results showed a dose-dependent reduction in conidiation, with significant decreases at all DFMO levels (P ≤ 0.05). While lower DFMO concentrations (0.05 and 0.5 mM) did not significantly alter UTS, 5 mM DFMO treatment doubled the material’s tensile strength compared to controls (P ≤ 0.05). Scanning electron microscopy confirmed reduced developmental structures in DFMO-treated samples, supporting the hypothesis. The non-linear relationship between conidiation suppression and strength improvement suggests additional mechanisms, such as hyphal morphology or cell wall changes, may contribute. These findings demonstrate that chemical modulation of fungal development can rationally tune mycelial material properties, offering a systematic approach for biomaterial engineering. 

ABSTRACT This study explores how suppressing asexual development inAspergillus nidulansenhances the mechanical properties of mycelial materials. Using four aconidial mutants(ΔbrlA, ΔflbA, ΔfluG, andfadA^G42R) that lack asexual development and a control strain (A28) that undergoes typical asexual development, we found that the absence of asexual development significantly improves mechanical strength. All mutants exhibited higher ultimate tensile strength (UTS) than the control, with ΔfluGand ΔbrlA(fluffy nonsporulating, FNS phenotype) showing the highest UTS. Additionally,fadA^G42Rand ΔflbA(fluffy autolytic dominant, FAD phenotype) demonstrated significantly higher strain at failure (SF), linked to increased autolysis and lower dry cell mass compared to the control and FNS mutants. Solid-state NMR analysis revealed that autolysis in FAD mutants disrupts galactofuranose-related metabolic processes, altering cell wall composition and contributing to higher elasticity. These findings suggest that suppressing asexual development enhances mycelial material strength, while autolysis mechanisms influence elasticity. This research highlights the potential for genetic manipulation in fungi to engineer advanced mycelial-based materials with tailored mechanical properties. 

ABSTRACT This study investigates a previously unreported stress signal transduced as crosstalk between the cell wall integrity (CWI) pathway and the septation initiation network (SIN). Echinocandins, which target cell wall synthesis, are widely used to treat mycoses. Their efficacy, however, is species specific. Our findings suggest that this is due largely to CWI–SIN crosstalk and the ability of filamentous species to fortify with septa in response to echinocandin stress. To better understand this crosstalk, we used a microscopy-based assay to measure septum density, aiming to understand the septation response to cell wall stress. The echinocandin micafungin, an inhibitor of β-(1,3)-glucan synthase, was employed to induce this stress. We observed a strong positive correlation between micafungin treatment and septum density in wild-type strains. This finding suggests that CWI activates SIN under cell wall stress, increasing septum density to protect against cell wall failure. More detailed investigations, with targeted knockouts of CWI and SIN signaling proteins, enabled us to identify crosstalk occurring between the CWI kinase, MpkA, and the SIN kinase, SepH. This discovery of the previously unknown crosstalk between the CWI and SIN pathways not only reshapes our understanding of fungal stress responses, but also unveils a promising new target pathway for the development of novel antifungal strategies. IMPORTANCEEchinocandin-resistant species pose a major challenge in clinical mycology by rendering one of only four lines of treatment, notably one of the two that are well-tolerated, ineffective in treating systemic mycoses of these species. Previous studies have demonstrated that echinocandins fail against highly polarized fungi because they target only apical septal compartments. It is known that many filamentous species respond to cell wall stress with hyperseptation. In this work, we show that echinocandin resistance hinges on this dynamic response, rather than on innate septation alone. We also describe, for the first time, the signaling pathway used to deploy the hyperseptation response. By disabling this pathway, we were able to render mycelia susceptible to echinocandin stress. This work enhances our microbiological understanding of filamentous fungi and introduces a potential target to overcome echinocandin-resistant species. 

Abstract Under synchronized conidiation, over 2500 gene products show differential expression, including transcripts for bothbrlAandabaA, which increase steadily over time. In contrast, during wall-stress induced by the echinocandin micafungin, thebrlAtranscript is upregulated while theabaAtranscript is not. In addition, whenmpkA(last protein kinase in the cell wall integrity signaling pathway) is deleted,brlAexpression is not upregulated in response to wall stress. Together, these data imply BrlA may play a role in a cellular stress-response which is independent of the canonical BrlA-mediated conidiation pathway. To test this hypothesis, we performed a genome-wide search and found 332 genes with a putative BrlA response element (BRE) in their promoter region. From this set, we identified 28 genes which were differentially expressed in response to wall-stress, but not during synchronized conidiation. This set included seven gene products whose homologues are involved in transmembrane transport and 14 likely to be involved in secondary metabolite biosynthesis. We selected six of these genes for further examination and find that they all show altered expression behavior in thebrlAdeletion strain. Together, these data support the idea that BrlA plays a role in various biological processes outside asexual development. ImportanceTheAspergillus nidulanstranscription factor BrlA is widely accepted as a master regulator of conidiation. Here, we show that in addition to this function BrlA appears to play a role in responding to cell-wall stress. We note that this has not been observed outsideA. nidulans. Further, BrlA-mediated conidiation is highly conserved acrossAspergillusspecies, so this new functionality is likely relevant in otherAspergilli. We identified several transmembrane transporters that have altered transcriptional responses to cell-wall stress in abrlAdeletion mutant. Based on our observation, together with what is known about thebrlAgene locus’ regulation, we identifybrlAβas the likely intermediary in function ofbrlAin the response to cell-wall stress.