MoLcb3参与调控稻瘟病菌鞘脂平衡和胁迫反应

MoLcb3 contributes to sphingolipid balance and stress responses in Magnaporthe oryzae

摘要:

鞘氨醇1-磷酸(sphingosine-1-phosphate, S1P)是一种具有生物活性的鞘脂，因其参与各种生物过程的调节和许多疾病的发展而引人注目，鞘氨醇1-磷酸磷酸酶(S1P phosphatase, S1PP)在控制S1P胞内代谢起着重要作用，而其在植物病原真菌中的生物学功能尚无报道。【目的】 探究稻瘟病菌(Magnaporthe oryzae)鞘氨醇1-磷酸磷酸酶在形态分化、致病过程和维持鞘脂平衡的作用。【方法】 利用同源重组方法敲除稻瘟病菌鞘氨醇1-磷酸磷酸酶编码基因MoLCB3，获得ΔMolcb3突变体，并通过表型分析、基因互补、脂质代谢组学分析等对MoLcb3的生物学功能进行研究，同时在ΔMolcb3突变体中敲除稻瘟病菌鞘氨醇激酶(sphingosine kinase, SK) MoLcb4，进一步探究磷酸酶MoLcb3和激酶MoLcb4之间的关系。【结果】 敲除MoLCB3基因导致稻瘟病菌菌丝生长速率和产孢量显著下降，影响分生孢子畸形率和附着胞初期形成，ΔMolcb3突变体完全丧失对大麦的致病性。ΔMolcb3突变体在应对高渗胁迫、细胞壁完整性胁迫、高温胁迫，以及真菌脂质合成抑制剂三唑酮和多球壳菌素时，与野生型有显著差异，说明MoLcb3参与上述胁迫反应和脂质合成代谢。ΔMolcb3ΔMolcb4双敲突变体可基本互补ΔMolcb3突变体所有表型缺陷。另外，脂质代谢组学分析显示，与野生型相比，ΔMolcb3突变体部分脂质含量有显著差异，例如游离脂肪酸、神经酰胺、磷脂酰肌醇等。【结论】 鞘氨醇1-磷酸磷酸酶MoLcb3在菌丝生长、产孢、孢子萌发、致病性、胁迫应激反应和维持脂质稳态等过程中起着重要作用，此外敲除MoLCB4基因能缓解MoLcb3缺失带来的影响。本研究的结果为进一步阐明稻瘟病菌鞘脂代谢通路以及真菌脂质生物合成抑制剂的开发提供新的思路。

关键词:稻瘟病菌;MoLcb3;鞘脂;功能分析;脂质代谢

Abstract:

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid notable for its involvement in the regulation of biological processes and the development of diseases. Sphingosine-1-phosphate phosphatase (S1PP) plays a role in regulating the intracellular metabolism of S1P, while the biological roles of S1PP in plant pathogenic fungi have not been reported. [Objective] To explore the role of S1PP in the morphological differentiation, pathogenic process, and maintenance of sphingolipid balance of Magnaporthe oryzae. [Methods] We employed homologous recombination to delete the S1PP gene MoLCB3 from M. oryzae and characterized the obtained mutant ΔMolcb3 was by phenotypic analysis, gene complementation, and lipid metabolomics. Furthermore, we deleted the sphingosine kinase (SK) gene MoLcb4 from ΔMolcb3 to explore the relationship between MoLcb3 and MoLcb4. [Results] The deletion of MoLCB3 resulted in significant decreases in the mycelial growth rate and spore production and affected conidial malformation and initial appressorium formation. ΔMolcb3 completely lost the pathogenicity to barley. Moreover, the ΔMolcb3 mutant were significantly different from the wild type in responding to hyperosmic stress, cell wall integrity stress, high temperature stress, and fungal lipid synthesis inhibitors triadimefon and myriocin, suggesting that MoLcb3 was involved in these stress responses and lipid anabolism. Interestingly, the double mutant ΔMolcb3ΔMolcb4 basically compensated for all phenotypic defects of ΔMolcb3. In addition, lipid metabolomics showed that compared with the wild type, ΔMolcb3 presented significantly different levels of lipids, such as free fatty acids, ceramides, and phosphatidyl inositol. [Conclusion] MoLcb3 plays an important role in the mycelial growth, sporulation, spore germination, pathogenicity, stress responses, and lipid homeostasis. In addition, knockout of MoLCB4 can cushion the effects of MoLcb3 deletion. The results of this study provide new ideas for elucidating the sphingolipid metabolic pathway of M. oryzae and the development of inhibitors of fungal lipid biosynthesis.

Key words:Magnaporthe oryzae;MoLcb3;sphingolipid;functional analysis;lipid metabolism