文献类型： 外文期刊

作者： Wang, Luyao;Zhao, Fei;Liu, Haohao;Chen, Han;Zhang, Fan;Li, Suhua;Sun, Tongjun;Nekrasov, Vladimir;Huang, Sanwen;Dong, Suomeng

作者机构：

期刊名称： PLOS PATHOGENS

ISSN： 1553-7366

年卷期： 2023 年 19 卷 4 期

页码：

收录情况： SCIE（2023版）

摘要： Author summaryOomycetes are a unique group of animal and plant pathogens that include some of the biggest threats to food security and natural ecosystems. However, much of the pathogenesis and development in these organisms remains to be learned, largely due to shortage of efficient genetic manipulation methods for a long time. In this manuscript, we developed modified Agrobacterium-mediated genetic transformation strategies for Phytophthora infestans, a notorious oomycete species that caused Irish Famine in the 19(th) century, and Plasmopara viticola, the causal agent of grapevine downy mildew that is known as a highly destructive disease of grapevines in all grape-growing areas of the world. Using prokaryotic expression of a plant protein in A. tumefaciens, we achieved considerable increase in transformation rate in different P. infestans strains, and also acquired one positive transformant of P. viticola, a oomycete species that is extremely hard-to-transform and cannot be grown as an axenic culture. Using our improved transformation protocol, combined with the CRISPR/Cas12a system, we performed genome editing and created loss-of-function alleles in P. infestans. In summary, our study reports on modified genetic transformation methods, for two important oomycete species, that have the potential to accelerate the molecular genetic study of many other microorganisms. 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.

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