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. 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. 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.

中文翻译：

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木本植物病原体Botryosphaeria dothidea的有效基因破坏方法

Botryosphaeria dothidea引起苹果腐烂并感染许多树木。可得到双歧双歧杆菌的基因组数据，并且已经预测了许多与发病相关的基因。然而，需要一种基因操纵方法来研究多虫双歧杆菌的致病机理。我们建立了基于聚乙二醇介导的原生质体转化的双歧杆菌的基因同源重组（GHR）的基因破坏（GD）方法。结果表明，GHR盒比GHR质粒具有更高的GD效率。每盒106个原生质体的DNA盒量为15μg，可获得较高的GD效率（每106个protopasts为1.3±0.14）和随机插入的频率较低。而且，我们成功地破坏了两个菌株中的基因。受Bdo_05381干扰的转化子产生的黑色素减少，而破坏Bdo_02540的转化子显示出较慢的生长速度和对刚果红的较强抵抗力。所建立的GD方法高效，便捷，具有研究双歧双歧杆菌及其他表皮真菌的基因功能和致病机理的潜力。