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 106 protopasts) and low frequency of random insertions were achieved with a DNA cassette quantity of 15 μg per 106 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.