The endophytic fungus Phomopsis liquidambaris efficiently promotes the nitrogen metabolism and growth of host plants such as rice and peanut. However, a lack of genetic tools limits further research regarding the mechanisms of interaction between P. liquidambaris and its host plants. Herein, a CRISPR/Cas9 system for targeted gene disruption in this strain was first constructed and optimized. The knock-out efficiency increased to over 60% when the ku70 or ku80 gene (involved in nonhomologous end-joining, NHEJ) was disrupted. Furthermore, the CRISPR/Cas9 system was applied to disrupt the PmkkA gene, encoding a mitogen-activated protein kinase kinase (MAPKK) in the cell-wall integrity (CWI) MAPK pathway of the strain. The ΔPmkkA mutant strain induced higher reactive oxygen species (ROS) production, chitinase activity and glucanase activity in rice seedlings than wild-type P. liquidambaris (WT), resulting in growth inhibition and strong resistance on rice. These results suggested that the PmkkA gene is crucial during the interaction with rice and may play a role in inhibiting the immune system of host plants. The CRISPR-Cas9 system will be of great use for the study of the interaction between P. liquidambaris and its host plants.

中文翻译：

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用于内生Phomopsis liquidambaris的CRISPR-Cas9系统的构建及其PmkkA缺陷型突变体揭示了对水稻的影响。

内生真菌液体拟南芥（Phomopsis liquidambaris）有效地促进了宿主植物如水稻和花生的氮代谢和生长。但是，缺乏遗传工具限制了对液态拟青霉及其宿主植物之间相互作用机制的进一步研究。在此，首先构建和优化了用于在该菌株中靶向基因破坏的CRISPR / Cas9系统。当ku70或ku80基因（涉及非同源末端连接，NHEJ）被破坏时，敲除效率提高到60％以上。此外，将CRISPR / Cas9系统应用于破坏PmkkA基因，该基因在菌株的细胞壁完整性（CWI）MAPK途径中编码有丝分裂原激活的蛋白激酶激酶（MAPKK）。ΔPmkkA突变株诱导更高的活性氧（ROS）产生，水稻幼苗中的几丁质酶活性和葡聚糖酶活性要高于野生型液体假单胞菌（WT），从而对水稻的生长具有抑制作用和较强的抗性。这些结果表明，PmkkA基因在与水稻的相互作用中至关重要，并可能在抑制宿主植物的免疫系统中发挥作用。CRISPR-Cas9系统将在研究液态拟青霉及其宿主植物之间的相互作用方面具有重要用途。