A PEG-mediated protoplast transformation system for EGFP labeling of the shot-hole pathogen Wilsonomyces carpophilus

Wilsonomyces carpophilus is a fungal pathogen causing shot-hole disease in stone fruit trees, including wild apricot populations in the Tianshan wild fruit forests. The absence of an effective genetic transformation system has limited molecular studies of this pathogen. In this study, we established a PEG-mediated protoplast transformation system for W. carpophilus strain G052 5 m2. Key factors affecting protoplast preparation, including mycelial culture duration, enzyme combination, digestion time, and osmotic stabilizer, were evaluated. The optimal conditions consisted of 24-h-old mycelia, enzymatic digestion with 20 g/L Driselase and 25 g/L Snailase for 5 h, and 1.2 mol/L NaCl as the osmotic stabilizer, yielding up to 4.13 × 10⁶ protoplasts/mL. The PEG-mediated transformation efficiency was calculated as 1.75 transformants per microgram of plasmid DNA under the optimized conditions. Both PCR detection and EGFP fluorescence observation successfully validated the exogenous gene expression in fungal transformants. No obvious differences in colony morphology, vegetative growth, and pathogenicity were observed between transformants and the wild-type strain. This study provides the first PEG-mediated protoplast transformation system for W. carpophilus and establishes a practical molecular tool for future studies on gene function, pathogenicity mechanisms, and host-pathogen interactions in this important stone fruit pathogen.