Exploring the antifungal arsenal of the soil-dwelling fungus Aspergillus fischeri: Identification and functional characterization of the antifungal protein AfAfpC

Filamentous fungi encode a wide repertoire of antifungal proteins (AFPs) with potential applications for the control of fungal diseases. In this study, we report the simultaneous production of three distinct AFPs by a natural soil isolate of Aspergillus fischeri, including the previously uncharacterized class C protein AfAfpC. AfAfpC was the most abundantly secreted AFP, followed by NFAP2 and, to a lesser extent, NFAP, when grown in YPD medium, whereas AfAfpC was also detected in minimal medium and starch-based formulations. Structural prediction revealed that AfAfpC would adopt a typical β-barrel fold stabilized by disulfide bridges, consistent with other AFPs. Functional analyses showed that AfAfpC displays antifungal activity against economically relevant species of the Aspergillus and Penicillium genera, despite exhibiting a near-neutral isoelectric point and low net charge, suggesting a distinct mode of action compared to cationic AFPs. To investigate afafpC biological role, we generated A. fischeri ΔafafpC strains via CRISPR-Cas9, representing the first successful application of genome editing in this species. Phenotypic analyses revealed that afafpC disruption did not affect vegetative growth, asexual development or tolerance to AFPs. Our findings provide the first functional insight into class C afp genes, advancing our understanding of the biological significance of AFPs. Moreover, the implementation of CRISPR-Cas9 in A. fischeri enables precise studies on gene function and the engineering of strains with optimized AFP production. These results not only deepen our understanding of AFP function but also support the potential use of AFP-producing fungi in sustainable agriculture and other biotechnological applications.