The construction of CRISPR-Cas9 system for endophytic Phomopsis liquidambaris and its PmkkA-deficient mutant revealing the effect on rice.

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. Copyright © 2019 Elsevier Inc. All rights reserved.

Citation

Peng-Wei Huang, Qian Yang, Ya-Li Zhu, Jun Zhou, Kai Sun, Yan-Zhen Mei, Chuan-Chao Dai. The construction of CRISPR-Cas9 system for endophytic Phomopsis liquidambaris and its PmkkA-deficient mutant revealing the effect on rice. Fungal genetics and biology : FG & B. 2020 Mar;136:103301

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PMID: 31765708

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