The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR/Cas9 and Cre/loxP systems
The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR/Cas9 and Cre/loxP systems
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DOI：
10.1101/2021.10.24.465644
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发表时间：
2021-10
期刊：
影响因子：
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通讯作者：
Kohji Yamada;Toya Yamamoto;Kanon Uwasa;K. Osakabe;Y. Takano
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文献类型：
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作者：
Kohji Yamada;Toya Yamamoto;Kanon Uwasa;K. Osakabe;Y. Takano
属于炭疽菌属（Colletotrichum）的植物病原真菌对许多植物物种造成毁灭性损害。其中，炭疽菌作为模式真菌，分析植物-真菌相互作用的分子方面。虽然通过同源重组（HR）的基因中断建立了C。但是，这种方法由于其低效率而费力。在这里，我们开发的方法，有效地产生多个敲除突变体的C。轮匝肌。我们首先发现CRISPR/Cas9系统大大提高了基因靶向效率。通过瞬时引入CRISPR/Cas9载体，超过90%的获得的转化体是敲除突变体。此外，我们还优化了一个自切除Cre/loxP标记回收系统。因为所需选择性标记的有限可用性阻碍了连续的基因破坏。在该系统中，整合的选择性标记可通过由木糖诱导型启动子驱动的Cre重组酶从基因组中去除，使得相同的选择性标记能够重复用于下一次转化。使用我们的CRISPR/Cas9和Cre/loxP系统，我们试图鉴定C.圆形的假定的奎尼酸转运蛋白基因的多重破坏限制了真菌在含有奎尼酸作为唯一碳源的培养基上的生长，证实了它们作为奎尼酸转运蛋白的功能。我们的分析表明，奎尼酸收购是在真菌感染，因为这个突变体显示正常的毒力宿主植物。此外，我们成功地在菌株中构建了17个纤维二糖转运蛋白基因的突变。从本研究中建立的敲除突变体的数据，我们推断使用CRISPR/Cas9和Cre/loxP系统的重复轮基因破坏不会对真菌毒力和生长造成负面影响。因此，这些系统将是进行系统性基因打靶的有力工具。圆形的
Phytopathogenic fungi belonging to the Colletotrichum genus cause devastating damage for many plant species. Among them, Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to efficiently generate multiple knockout mutants of C. orbiculare. We first found that CRISPR/Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR/Cas9 vector, more than 90 % of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre/loxP marker recycling system for C. orbiculare because limited availability of desired selective markers hampers sequential gene disruption. In this system, integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling reuse of the same selective marker for the next transformation. Using our CRISPR/Cas9 and Cre/loxP systems, we attempted to identify functional sugar transporters in C. orbiculare. Multiple disruptions of putative quinate transporter genes restrict fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. Our analyses revealed that quinate acquisition is dispensable during fungal infection because this mutant displayed normal virulence to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants established in this study, we inferred that repetitive rounds of gene disruption using CRISPR/Cas9 and Cre/loxP systems do not cause negative effects for fungal virulence and growth. Therefore, these systems will be powerful tools to perform systematic gene targeting approach for C. orbiculare.