An efficient genetic manipulation protocol for dark septate endophyte Falciphora oryzae

Area of Science:

• Mycology
• Plant Pathology
• Molecular Biology

Background:

• Endophytic fungi like Falciphora oryzae play crucial roles in plant health and development.
• Genetic manipulation of endophytic fungi is essential for understanding their functions and harnessing their potential.
• Establishing efficient transformation systems is a prerequisite for genetic studies.

Purpose of the Study:

• To develop and optimize a protoplast preparation and transformation system for the endophytic fungus Falciphora oryzae.
• To enable the genetic modification of F. oryzae for future research applications.

Main Methods:

• Optimized enzyme digestion conditions using KCl and glucanase at specific temperatures and durations.
• Identified optimal regeneration media containing sucrose for protoplast re-growth.
• Transformed F. oryzae protoplasts using the pKD6-GFP vector for visual selection.

Main Results:

• Achieved high protoplast yield and effective transformation rates using optimized enzyme treatment (0.9 M KCl, 10 mg/mL glucanase, 30°C, 80 rpm, 2-3 h).
• Determined that 1 M sucrose in regeneration agar yielded the highest protoplast re-growth rate.
• Successfully generated stable green fluorescent protein (GFP)-expressing transformants of F. oryzae.
• Confirmed stable and intense GFP expression in fungal hyphae during symbiosis within rice roots.

Conclusions:

• Successfully established a robust protoplast transformation system for Falciphora oryzae.
• This system provides a valuable tool for future genetic research and manipulation of F. oryzae.
• The developed system opens avenues for genetic studies in other endophytic fungi.