Laboratory of Mycology, College of Plant Protection, China Agricultural University, Beijing, 100193, China.
Key Laboratory of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing, 100193, China.
BMC Biotechnol. 2020 Mar 5;20(1):14. doi: 10.1186/s12896-020-00608-z.
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 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.
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.
聚生小丛壳（Botryosphaeria dothidea）可引起苹果白腐病，并感染许多树木植物。已获得聚生小丛壳的基因组数据，并预测了许多与发病机制相关的基因。然而，需要一种基因操作方法来研究聚生小丛壳的致病机制。
我们使用聚乙二醇介导的原生质体转化，为聚生小丛壳建立了基于基因同源重组（GHR）的基因敲除（GD）方法。结果表明，GHR 盒比 GHR 质粒具有更高的 GD 效率。当 DNA 盒的量为每个原生质体 15μg 时，GD 效率（每个 10 个原生质体中有 1.3±0.14 个）和随机插入的频率都很高。此外，我们成功地在两个菌株中敲除了基因。Bdo_05381 敲除转化体产生的黑色素较少，而 Bdo_02540 敲除转化体的生长速度较慢，对刚果红的抗性更强。
建立的 GD 方法高效便捷，有望用于研究聚生小丛壳和其他多核真菌的基因功能和致病机制。
