An efficient genetic manipulation protocol for dark septate endophyte Falciphora oryzae.

Objective: To investigate the protoplast preparation and transformation system of endophytic fungus Falciphora oryzae.

Results: F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9 M KCl solution and 10 mg mL glucanase at 30 °C with shaking at 80 rpm for 2-3 h. When the protoplasts were plated on a regenerations-agar medium containing 1 M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired green fluorescent protein-expressing transformants by transforming the pKD6-GFP vector into protoplasts. Further, the GFP expression in fungal hyphae possessed good stability and intensity during symbiosis in rice roots.

Conclusions: This study provided a protoplast transformation system of F. oryzae, creating opportunities for future genetic research in other endophytic fungi.