McCarthy Hannah M, Tarallo Mariana, Mesarich Carl H, McDougal Rebecca L, Bradshaw Rosie E
BioProtection Aotearoa, School of Natural Sciences, Massey University, Palmerston North 4472, New Zealand.
BioProtection Aotearoa, School of Agriculture and Environment, Massey University, Palmerston North 4472, New Zealand.
Plants (Basel). 2022 Apr 8;11(8):1016. doi: 10.3390/plants11081016.
Dothistroma needle blight, caused by , has increased in incidence and severity over the last few decades and is now one of the most important global diseases of pines. Disease resistance breeding could be accelerated by knowledge of pathogen virulence factors and their host targets. However, this is hindered due to inefficient targeted gene disruption in , which is required for virulence gene characterisation. Here we report the first successful application of CRISPR/Cas9 gene editing to a Dothideomycete forest pathogen, Disruption of the dothistromin pathway regulator gene , with a known phenotype, was performed using nonhomologous end-joining repair with an efficiency of > 90%. Transformants with a range of disruption mutations in were produced. Disruption of , a gene encoding a secreted cell death elicitor, was also achieved using CRISPR/Cas9, by using a specific donor DNA repair template to aid selection where the phenotype was unknown. In this case, 100% of screened transformants were identified as disruptants. In establishing CRISPR/Cas9 as a tool for gene editing in , our research could fast track the functional characterisation of candidate virulence factors in and helps set the foundation for development of this technology in other forest pathogens.
由[病原菌名称未给出]引起的散斑壳针孢叶枯病，在过去几十年里发病率和严重程度都有所增加，现已成为全球松树最重要的病害之一。了解病原菌的毒力因子及其宿主靶点有助于加速抗病育种。然而，由于在[病原菌名称未给出]中进行靶向基因破坏的效率低下，这一过程受到了阻碍，而靶向基因破坏是毒力基因表征所必需的。在此，我们报告了CRISPR/Cas9基因编辑首次成功应用于座囊菌纲森林病原菌[病原菌名称未给出]。利用非同源末端连接修复，以大于90%的效率对已知表型的多聚乙酰毒素途径调节基因[基因名称未给出]进行了破坏。产生了一系列在[基因名称未给出]中有不同破坏突变的转化体。通过使用特定的供体DNA修复模板来辅助筛选未知表型的转化体，利用CRISPR/Cas9也实现了对编码分泌型细胞死亡诱导因子的基因[基因名称未给出]的破坏。在这种情况下，100%的筛选转化体被鉴定为破坏体。在确立CRISPR/Cas9作为[病原菌名称未给出]基因编辑工具的过程中，我们的研究可以快速追踪[病原菌名称未给出]中候选毒力因子的功能表征，并为该技术在其他森林病原菌中的开发奠定基础。
