Hirsutella satumaensis, an endoparasitic fungus that targets Lepidoptera insects, holds significant potential for biocontrol applications. However, its molecular study has been limited due to the absence of an efficient genetic transformation system. In this study, an optimized Agrobacterium tumefaciens-mediated transformation protocol was developed for H. satumaensis using binary vectors pBARGPE1-GFP and pK2-bar, which carry the green fluorescent protein (eGFP) and phosphinothricin resistance (bar) genes, respectively. The optimal transformation conditions included a conidial concentration of 10⁵ conidia/mL, an A. tumefaciens (strain AGL-1) concentration of OD = 0.6, and a 3-day co-cultivation period with 200 μM acetosyringone, resulting in an average of 121 ± 5.07 transformants. Successful integration was confirmed by green fluorescence in the transformants. Furthermore, the ribotoxin gene hirsutellin A (HtA), specific to the genus Hirsutella, was successfully overexpressed using this system. Insect bioassays demonstrated that the gpdA promoter effectively drives HtA expression in H. satumaensis. The transformation system exhibited stable gene integration, strong fluorescence, and bioactivity. This study establishes the first genetic transformation protocol for H. satumaensis, providing a valuable tool for exploring insect-pathogen interactions and the functional roles of key genes in this entomopathogenic fungus.
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