Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China.
Department of Plant Pathology and Key Laboratory of Integrated Management of Crop Disease and Pests (Ministry of Education), Nanjing Agricultural University, Nanjing, China.
PLoS Pathog. 2023 Apr 21;19(4):e1011346. doi: 10.1371/journal.ppat.1011346. eCollection 2023 Apr.
Oomycetes are a group of filamentous microorganisms that include some of the biggest threats to food security and natural ecosystems. However, much of the molecular basis of the pathogenesis and the development in these organisms remains to be learned, largely due to shortage of efficient genetic manipulation methods. In this study, we developed modified transformation methods for two important oomycete species, Phytophthora infestans and Plasmopara viticola, that bring destructive damage in agricultural production. As part of the study, we established an improved Agrobacterium-mediated transformation (AMT) method by prokaryotic expression in Agrobacterium tumefaciens of AtVIP1 (VirE2-interacting protein 1), an Arabidopsis bZIP gene required for AMT but absent in oomycetes genomes. Using the new method, we achieved an increment in transformation efficiency in two P. infestans strains. We further obtained a positive GFP transformant of P. viticola using the modified AMT method. By combining this method with the CRISPR/Cas12a genome editing system, we successfully performed targeted mutagenesis and generated loss-of-function mutations in two P. infestans genes. We edited a MADS-box transcription factor-encoding gene and found that a homozygous mutation in MADS-box results in poor sporulation and significantly reduced virulence. Meanwhile, a single-copy avirulence effector-encoding gene Avr8 in P. infestans was targeted and the edited transformants were virulent on potato carrying the cognate resistance gene R8, suggesting that loss of Avr8 led to successful evasion of the host immune response by the pathogen. In summary, this study reports on a modified genetic transformation and genome editing system, providing a potential tool for accelerating molecular genetic studies not only in oomycetes, but also other microorganisms.
卵菌是一类丝状微生物,其中包括一些对粮食安全和自然生态系统构成最大威胁的物种。然而,这些生物体的发病机制和发育的许多分子基础仍有待了解,这主要是由于缺乏有效的遗传操作方法。在这项研究中,我们为两种重要的卵菌物种——致病疫霉和葡萄生单轴霉——开发了改良的转化方法,这两种菌在农业生产中造成了严重破坏。作为研究的一部分,我们通过原核表达在根癌农杆菌中建立了一个改良的农杆菌介导转化(AMT)方法,表达了拟南芥 bZIP 基因 AtVIP1(VirE2 互作蛋白 1),该基因对于 AMT 是必需的,但在卵菌基因组中缺失。使用新方法,我们提高了两种致病疫霉菌株的转化效率。我们进一步使用改良的 AMT 方法获得了阳性 GFP 转化的葡萄生单轴霉菌株。通过将该方法与 CRISPR/Cas12a 基因组编辑系统相结合,我们成功地对两个致病疫霉菌基因进行了靶向诱变,并产生了功能丧失突变。我们编辑了一个 MADS 盒转录因子编码基因,发现 MADS 盒的纯合突变导致产孢能力差,毒力显著降低。同时,我们靶向了致病疫霉中的一个单拷贝无毒力效应子编码基因 Avr8,并发现编辑转化体在携带同源抗性基因 R8 的马铃薯上具有毒性,表明 Avr8 的缺失导致病原菌成功逃避了宿主的免疫反应。总之,本研究报告了一种改良的遗传转化和基因组编辑系统,为加速卵菌以及其他微生物的分子遗传学研究提供了一种潜在的工具。