Gene editing with an oxathiapiprolin resistance selection marker reveals that PuLLP, a loricrin-like protein, is required for oospore development in Pythium ultimum

文献类型： 外文期刊

第一作者： Feng, Hui

作者： Feng, Hui;Liu, Tianli;Li, Jiaxu;Wan, Chuanxu;Ding, Feifei;Wang, Yuanchao;Zheng, Xiaobo;Ye, Wenwu;Feng, Hui;Liu, Tianli;Li, Jiaxu;Wan, Chuanxu;Ding, Feifei;Wang, Yuanchao;Zheng, Xiaobo;Ye, Wenwu;Feng, Hui;Liu, Tianli;Li, Jiaxu;Wan, Chuanxu;Ding, Feifei;Wang, Yuanchao;Zheng, Xiaobo;Ye, Wenwu;Feng, Hui

作者机构：

关键词： Oomycete; Pythium; Genetic transformation; Selection marker; Oxathiapiprolin

期刊名称：PHYTOPATHOLOGY RESEARCH （ 影响因子：3.4； 五年影响因子：4.2 ）

ISSN： 2096-5362

年卷期： 2023 年 5 卷 1 期

页码：

收录情况： SCI

摘要： Oomycetes, such as Pythium species, contain numerous devastating plant pathogens that inflict substantial economic losses worldwide. Although CRISPR/Cas9-based genome editing is available, the selection markers available for genetic transformation in these species are limited. In this study, a mutated version of the Phytophthora capsici oxysterol-binding protein-related protein 1 (PcMuORP1), known to confer oxathiapiprolin resistance, was introduced into the CRISPR/Cas9 system for in situ complementation in Pythium ultimum. We targeted PuLLP, which encodes a loricrin-like protein, and showed significant downregulation when the Puf RNA-binding protein-encoding gene PuM90 was knocked out. The PuLLP knockout mutants could not produce oospores, indicating a similar biological function as PuM90. The reintroduction of PuLLP into the knockout mutant using PcMuORP1 as a selection marker restored oospore production. Further comparisons with the conventional selection marker NPTII indicated that PcMuORP1 could be applied at a lower concentration and cost, resulting in a higher screening efficiency. Successive subculturing in the absence of selective pressure showed that PcMuORP1 had little long-term effect on the fitness of transformants. Hence, it could be reused as an alternative selection marker. This study demonstrates the successful implementation of the PcMuORP1 gene as a selection marker in the genetic transformation of Py. ultimum and reveals the loricrin-like protein PuLLP as a sexual reproduction-related factor downstream of the Puf RNA-binding protein PuM90. Overall, these results will help accelerate the functional genomic investigation of oomycetes.

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