Establishment of the genetic transformation of Cordyceps chanhua (Hypocreales: Cordycipitaceae) and the functional research of the emopamil-binding protein Ccebp2163 gene.

Cordyceps chanhua (Hypocreales: Cordycipitaceae) is a renowned entomopathogenic fungus used as herbal medicine in China. It has been developed as a biopesticide. Previous studies by our research group found that the expression level of the responsive gene Ccebp2163 in C. chanhua was significantly upregulated under induction by the insect hormone the 20-hydroxyecdysone (20E). To improve fungal virulence, an optimized Agrobacterium tumefaciens-mediated transformation system was established for C. chanhua. The 20E-responsive gene Ccebp2163 was overexpressed in C. chanhua, and its insecticidal activity of the 20E-responsive gene Ccebp2163-mediated C. chanhua against larvae of G. mellonella. The minimum inhibitory concentration of hygromycin against C. chanhua was determined to be 300 μg/mL, with 1/2 CZP identified as the suitable transformation medium. Optimized parameters were: Agrobacterium resuspension OD660 = 0.15, co-culture temperature 20 °C, co-culture duration 60 h, acetosyringone concentration 200 μM, and spore suspension concentration 1 × 104 conidia/mL. The transformants expressing Ccebp2163 exhibited an approximately 16-fold increase in gene expression levels. The overexpression (OE) of Ccebp2163 did not significantly affect the morphology, growth rate, or sporulation capacity of C. chanhua, but it enhanced the spore germination rate. Bioassays using the larval dipping method on G. mellonella revealed that the survival rate of larvae treated with OE-Ccebp2163 was significantly lower than that of the WT group. OE-Ccebp2163 suppressed the upregulation of 20E concentration in the host during the mid to late stages of infection. OE-Ccebp2163 infection significantly altered the gut microbiota structure of G. mellonella, with notable changes in the abundance of Proteus, Enterococcus, Salmonella, Commensalibacter, Enterobacter, and Vagococcus. Klebsiella was detected in large quantities exclusively during the mid-infection stage of OE-Ccebp2163. These alterations indicate that gut bacteria responded pronouncedly to Ccebp2163 at mid-infection, with gradual community restoration later.