Sequence similarity data used in Fig 1A.

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（Oomycetes）是一类丝状微生物，其中不乏对粮食安全与自然生态系统构成重大威胁的类群。然而，这类生物的致病与发育过程的分子机制仍有诸多待阐明之处，这在很大程度上受制于高效遗传操作方法的匮乏。本研究针对农业生产中造成毁灭性危害的两种重要卵菌物种——致病疫霉（Phytophthora infestans）与葡萄霜霉菌（Plasmopara viticola），开发了优化后的转化方法。作为研究的一部分，我们通过在根癌农杆菌（Agrobacterium tumefaciens）中原核表达AtVIP1（VirE2互作蛋白1）——一种农杆菌介导转化（Agrobacterium-mediated transformation, AMT）所需、但卵菌基因组中缺失的拟南芥bZIP基因——建立了改进型AMT方法。借助该新方法，我们在两株致病疫霉中实现了转化效率的提升。我们还通过优化后的AMT方法，获得了葡萄霜霉菌的绿色荧光蛋白（GFP）阳性转化子。将该方法与CRISPR/Cas12a基因组编辑系统相结合后，我们成功实现了靶向诱变，并在致病疫霉的两个基因中获得了功能丧失型突变体。我们编辑了一个MADS-box转录因子编码基因，研究发现该基因的纯合突变会导致孢子形成能力下降，并显著降低病原菌的毒力。与此同时，我们靶向编辑了致病疫霉中的单拷贝无毒效应因子编码基因Avr8，编辑后的转化子在携带其同源抗性基因R8的马铃薯上表现出毒力，这表明Avr8的缺失使病原菌成功逃逸了宿主的免疫应答。综上，本研究报道了一套优化后的遗传转化与基因组编辑系统，为加速卵菌乃至其他微生物的分子遗传学研究提供了潜在工具。