Transformation and gene-disruption in the apple-pathogen, Neonectria ditissima

Area of Science:

• Plant Pathology
• Mycology
• Molecular Biology

Background:

• Apple production faces significant threats from Neonectria ditissima, the causal agent of European canker.
• Current management strategies for European canker are labor-intensive and lack eradication methods.
• Limited understanding of the N. ditissima infection process hinders effective control.

Purpose of the Study:

• To modify and improve the existing genetic transformation technique for N. ditissima.
• To enhance the efficiency of gene targeting and modification in N. ditissima.
• To develop tools for better understanding the pathogenicity of N. ditissima.

Main Methods:

• Upgraded the 2003 genetic transformation protocol using commercially available enzymes for protoplast generation.
• Incorporated hydroxyurea into the protoplast generation buffer.
• Utilized homologous recombination for gene disruption and green fluorescent protein (GFP) for ectopic expression.

Main Results:

• Successfully generated viable protoplasts capable of foreign DNA uptake and regeneration.
• Developed targeted gene-disruption mutants and ectopic mutants expressing GFP.
• Hydroxyurea significantly increased the generation of knockout mutants via homologous recombination.

Conclusions:

• A novel and improved genetic transformation protocol for N. ditissima has been established.
• The enhanced protocol facilitates efficient gene editing, including knockout mutant generation.
• Pathogenicity assays confirmed that GFP-mutants retain infectivity, enabling further study of the European canker disease.