An Efficient Genetic Manipulation Protocol for Dark Septate Endophyte Falciphora Oryzae

Abstract Falciphora oryzae is a dark septate endophyte (DSE) isolated from wild rice roots (Oryza sativa L.). It was classified as a non-clavicitaceous endophyte. The fungus colonizes rice roots, showing a significant increase in agronomic parameters with plant biomass, rice blast resistance, yield, and quality. The construction of the genetic manipulation system is critical to study the relationship between F. oryzae and O. sativa. In the present study, the protoplast preparation and transformation system of F. oryzae was investigated. The key parameters affecting the efficiency of protoplast production, such as osmotic pressure stabilizer, enzyme digestion conditions, and fungal age, were studied. The results showed that F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9mol L-1 KCl solution and 10 mg mL−1 glucanase at 30℃ with shaking 80 rpm for 2-3 h. When the protoplasts were plated on a regenerations-agar (RgA) medium containing 1M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired GFP-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.The genetic manipulation system of endophytic fungus facilitates the further exploration the interaction between the endophytic fungus and their hosts.