PEG-mediated transformation: a tool for random integration or targeted gene replacement in Colletotrichum camelliae

Colletotrichum camelliae is a highly virulent fungal pathogen responsible for anthracnose in tea plants, which poses a major threat to tea production. The development of a reliable genetic transformation system for C. camelliae is a fundamental prerequisite for elucidating its molecular pathogenic mechanisms and advancing functional genomic research in this pathogen.

In this study, polyethylene glycol (PEG)-mediated protoplast transformation was optimized for genome modification in C. camelliae. Protoplasts were efficiently generated through enzymatic digestion of young mycelia using a mixture containing 20 mg/mL driselase, 15 mg/mL lyticase, and 15 mg/mL snailase at 28 °C for 3 h. The optimized protocol was validated by introducing the plasmid pKD7-RED, carrying a red fluorescent protein (RFP) coding sequence, into the fungal genome, and further applied for targeted disruption of the cutinase gene via PEG-mediated transformation coupled with homologous recombination.

The optimized enzymatic digestion protocol yielded a high quantity of viable protoplasts, which was critical for successful transformation. Fluorescence microscopy confirmed stable RFP expression across five consecutive generations, during conidial germination on hydrophobic surfaces, and throughout the infection process on tea leaves, demonstrating the heritability and stability of transgene expression. Furthermore, targeted disruption of the cutinase gene showed that it had no effect on the pathogenicity of C. camelliae under wounded inoculation conditions.

These findings demonstrate that PEG-mediated transformation is an efficient and versatile approach for both random insertional mutagenesis and targeted gene deletion in C. camelliae. This established genetic manipulation system lays a solid technical foundation for future functional genomic studies of C. camelliae pathogenicity and the development of novel strategies for tea anthracnose control.