A dual endogenous selectable marker system for Aspergillus flavus and its application in characterizing nsdD function in development and aflatoxin biosynthesis

Aspergillus flavus poses a significant threat to global food security and public health, as it not only triggers severe mold spoilage in staple crops like peanuts and corn but also produces aflatoxin, a potent carcinogen. However, genetic manipulation of this fungus is notoriously challenging due to its inherent insensitivity to conventional antibiotics. To overcome this barrier, we established a robust, fully endogenous genetic transformation system for a highly toxigenic wild-type strain, A. flavus -SD, using polyethylene glycol (PEG)-mediated protoplast transfection with two selectable markers: pyrG (uracil auxotrophy/5-FOA counterselection) and a β-tubulin E198A point mutation (carbendazim resistance). We successfully constructed the tool strains with a ΔpyrG mutation, which were used for gene knockout and overexpression, and the β-tubulin E198A mutant was used for gene complementation. To validate the operational efficacy of this platform, we performed knockout, complementation, and overexpression of the nsdD gene as a test case. This study provides a reliable technical foundation for elucidating the molecular mechanisms governing aflatoxin biosynthesis and pathogenicity, thereby facilitating the identification of novel targets for A. flavus control. IMPORTANCE This study established a novel genetic transformation system for a highly toxigenic wild-type strain, Aspergillus flavus -SD, utilizing fully endogenous selectable markers: pyrG (uracil auxotrophy/5-FOA counterselection) and a β-tubulin E198A point mutation (carbendazim resistance). This platform eliminates the need for heterologous antibiotic resistance cassettes, thereby reducing biosafety concerns while enabling iterative genome editing through marker recycling. Using this system, we demonstrated its efficacy by validating the expected phenotypic changes upon nsdD manipulation, revealing a previously unrecognized trade-off between sclerotia formation and aflatoxin biosynthesis. This work provides a valuable, compliance-friendly resource for the fungal research community and lays a foundation for identifying novel targets to disrupt fungal persistence and mycotoxin production in agricultural settings.