Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae

Area of Science:

• Plant Pathology
• Molecular Biology
• Fungal Genetics

Background:

• Verticillium dahliae is a destructive fungal pathogen causing significant agricultural losses.
• Controlling V. dahliae is challenging due to its virulence and pathogenicity.
• Existing gene screening methods for V. dahliae require faster and simpler transformation techniques.

Purpose of the Study:

• To develop a quick and easy transformation method for Verticillium dahliae.
• To establish a reliable method for gene silencing in V. dahliae using short interfering RNAs (siRNAs).
• To facilitate the investigation of genes related to V. dahliae growth, virulence, and pathogenicity.

Main Methods:

• Generation of high-quality V. dahliae protoplasts with efficient regeneration.
• Transformation of protoplasts using polyethylene glycol (PEG)-mediated delivery or electroporation.
• Gene silencing in V. dahliae using specific short interfering RNAs (siRNAs) targeting GFP and Vta2 genes.

Main Results:

• PEG-mediated transformation achieved high transformation efficiencies (up to 600 transformants/µg DNA for linear GFP cassette).
• Successful delivery and function of siRNAs in V. dahliae protoplasts, achieving up to 100% GFP gene silencing.
• Demonstrated silencing of the Verticillium transcription activator of adhesion (Vta2) gene using specific siRNAs.

Conclusions:

• A rapid and efficient transformation protocol for V. dahliae has been established.
• This method enables effective gene silencing via siRNA delivery in V. dahliae.
• The developed technique is valuable for functional genomics research in V. dahliae, aiding in understanding and controlling plant diseases.