Optimized Protoplasts Preparation of Fungus Magnaporthe oryzae by Novel Myxolase F Lyase

Rice blast disease is one of most devastating fungal diseases caused by Magnaporthe oryzae. The preparation and transformation of protoplasm are of vital importance for understanding the pathogenic mechanism of M. oryzae and achieving the control of rice blast disease. In this study, we aim to optimize conditions for preparing and regenerating the protoplasts of M. oryzae using a novel commercial fungal cell wall-decomposing enzyme, Myxolase F lyase. By optimizing the enzyme concentration, lysis time and pH, osmatic stabilizer and agar percentage in regeneration medium, an applicable protoplast preparation system for M. oryzae was established. Following lysis of M. oryzae mycelium with 5 mg/mL Myxolase F lyase for 3 h, a 49.7% regeneration rate was achieved using a regenerative medium consisting of 0.8 M sucrose and 1% agar at pH 7.0 (50 mM Tris-HCl buffer) under optimum conditions. Moreover, the prepared M. oryzae protoplasts were available for subsequent genetic transformation, with a regeneration rate of 54.28% using Myxolase F compared with 26.86% using the commercial enzyme. The simplified operations, robust reproducibility and improved efficiency give Myxolase F lyase an advantage over other available products, thus providing powerful tools for the efficient genetic transformation of M. oryzae.