Development of PEG-Mediated Genetic Transformation for Pine-Inhabiting Fungus Pestalotiopsis disseminata

Abstract

Pestalotiopsis disseminata widely inhabits pines, but the lack of an efficient genetic transformation system has limited its functional genomic studies. Here, we optimized protoplast preparation and established a PEG-mediated transformation and GFP-labeling platform for strain ZXD579. The selected conditions used mycelia cultured for 24 h and digested for 3 h at 28 °C with 1% lysing enzyme, 0.1% driselase, and 1% lysozyme in 0.6 M KCl supplemented with 50 mM CaCl2. The protoplast yield reached (1.51 ± 0.15) × 107 protoplasts/mL, and the regeneration rate exceeded 90%. Hygromycin B at 130 μg/mL completely inhibited wild-type growth and was suitable for transformant selection. The pCT74-eGFP plasmid carrying the reporter gene and hygromycin B phosphotransferase gene (hph) was introduced into protoplasts using PEG. Putative transformants were examined by PCR and fluorescence microscopy; GFP fluorescence was detected in hyphae and conidia. Transformation efficiency was 4.2 ± 0.6 transformants per μg plasmid DNA, and 60% of 15 randomly selected hygromycin-resistant transformants were PCR-positive. GFP-labeled transformants showed no significant difference in vegetative growth and exhibited comparable preliminary pathogenicity phenotypes relative to the wild-type strain. This protocol provides a foundation for gene manipulation in P. disseminata.