An efficient gene disruption method for the woody plant pathogen Botryosphaeria dothidea

Area of Science:

• Mycology
• Plant Pathology
• Molecular Biology

Background:

• Botryosphaeria dothidea is a significant pathogen causing apple white rot and affecting various tree species.
• While genomic data and predicted pathogenesis-related genes are available, a method for gene manipulation is lacking.
• Understanding the pathogenic mechanisms of B. dothidea requires effective gene manipulation tools.

Purpose of the Study:

• To establish an efficient gene disruption (GD) method for Botryosphaeria dothidea.
• To enable functional studies of genes involved in the pathogenicity of B. dothidea.
• To facilitate research into the molecular mechanisms of fungal diseases.

Main Methods:

• Development of a gene disruption (GD) method utilizing gene homologous recombination (GHR).
• Employing polyethylene glycol-mediated protoplast transformation for B. dothidea.
• Optimization of DNA cassette quantity for high GD efficiency and low random insertion frequency.

Main Results:

• Achieved high gene disruption efficiency (1.3 ± 0.14 per 10^6 protoplasts) using a GHR cassette.
• Demonstrated significantly higher GD efficiency with cassettes compared to plasmids.
• Successfully disrupted genes Bdo_05381 (reduced melanin) and Bdo_02540 (slower growth, increased Congo red resistance) in two strains.

Conclusions:

• The established GD method is efficient and convenient for B. dothidea.
• This method holds significant potential for studying gene function in B. dothidea.
• The developed technique can be applied to investigate pathogenic mechanisms in B. dothidea and other coenocytic fungi.