Aspergillus
flavus is an agriculturally
important and aflatoxigenic fungus, underscoring the need for alternative
antifungal strategies. Cysteine-rich antifungal proteins (AFPs) are promising
bioactive molecules, yet their recombinant production and genetic determinants
of fungal tolerance remain insufficiently characterized. Here, we investigated
AnAFP, an antifungal protein from Aspergillus niger, and evaluated its
activity against A. flavus. Bioinformatic analyses predicted an
N-terminal signal peptide, a putative intrinsically disordered region, and a
mature cysteine-rich domain structurally related to known fungal AFPs. Guided
by these features, the predicted mature region of AnAFP was expressed in Escherichia
coli and purified through Ni-NTA affinity chromatography, tag cleavage,
cation-exchange chromatography, and size-exclusion chromatography. Purified
AnAFP inhibited A. flavus growth, and comparison with PgAFP and AfAFP
confirmed antifungal activity at micromolar concentrations. To identify genes
associated with AFP tolerance, Δado1, Δdef1, and Δadk1 mutants were generated by homologous recombination. All three mutants showed
increased sensitivity to AnAFP, PgAFP, and AfAFP relative to the wild-type
strain, suggesting that ado1, def1, and adk1 may
contribute to AFP tolerance. Deletion of these genes also affected colony
growth, conidiation, sclerotial formation, stress responses, and aflatoxin
production. These findings establish a recombinant production strategy for
AnAFP and provide preliminary evidence linking ado1, def1, and adk1 to AFP sensitivity and fungal physiology more broadly in this pathogenic and
aflatoxigenic species.utf-8
Open Access
