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

Background: Botryosphaeria dothidea causes apple white rot and infects many tree plants. Genome data for B. dothidea are available and many pathogenesis-related genes have been predicted. However, a gene manipulation method is needed to study the pathogenic mechanism of B. dothidea.

Results: We established a gene disruption (GD) method based on gene homologous recombination (GHR) for B. dothidea using polyethylene glycol-mediated protoplast transformation. The results showed that a GHR cassette gave much higher GD efficiency than a GHR plasmid. A high GD efficiency (1.3 ± 0.14 per 10 protopasts) and low frequency of random insertions were achieved with a DNA cassette quantity of 15 μg per 10 protoplasts. Moreover, we successfully disrupted genes in two strains. Bdo_05381-disrupted transformants produced less melanin, whereas the Bdo_02540-disrupted transformant showed a slower growth rate and a stronger resistance to Congo red.

Conclusion: The established GD method is efficient and convenient and has potential for studying gene functions and the pathogenic mechanisms of B. dothidea and other coenocytic fungi.