ABSTRACT

Sterols are a class of lipids critical for fundamental biological processes and membrane dynamics. These molecules are synthesized in the endoplasmic reticulum (ER) and are transported bi-directionally between the ER and plasma membrane (PM). However, the trafficking mechanism of sterols and their relationship with macroautophagy/autophagy are still poorly understood in the rice blast fungus Magnaporthe oryzae. Here, we identified the VAD1 Analog of StAR-related lipid transfer (VASt) domain-containing protein MoVast1 via co-immunoprecipitation in M. oryzae. Loss of MoVAST1 resulted in conidial defects, impaired appressorium development, and reduced pathogenicity. The MoTor (target of rapamycin in M. oryzae) activity is inhibited because MoVast1 deletion leads to high levels of sterol accumulation in the PM. Site-directed mutagenesis showed that the 902 T site is essential for localization and function of MoVast1. Through filipin or Flipper-TR staining, autophagic flux detection, MoAtg8 lipidation, and drug sensitivity assays, we uncovered that MoVast1 acts as a novel autophagy inhibition factor that monitors tension in the PM by regulating the sterol content, which in turn modulates the activity of MoTor. Lipidomics and transcriptomics analyses further confirmed that MoVast1 is an important regulator of lipid metabolism and the autophagy pathway. Our results revealed and characterized a novel sterol transfer protein important for M. oryzae pathogenicity.

Abbreviations: AmB: amphotericin B; ATMT: Agrobacterium tumefaciens-mediated transformation; CM: complete medium; dpi: days post-inoculation; ER: endoplasmic reticulum; Flipper-TR: fluorescent lipid tension reporter; GO: Gene ontology; hpi: hours post-inoculation; IH: invasive hyphae; KEGG: kyoto encyclopedia of genes and genomes; MoTor: target of rapamycin in Magnaporthe oryzae; PalmC: palmitoylcarnitine; PM: plasma membrane; SD-N: synthetic defined medium without amino acids and ammonium sulfate; TOR: target of rapamycin; VASt: VAD1 Analog of StAR-related lipid transfer; YFP, yellow fluorescent protein.

摘要

甾醇是一类对基本生物过程和膜动力学至关重要的脂质。这些分子在内质网 (ER) 中合成，并在内质网 (ER) 和质膜 (PM) 之间双向运输。然而，在稻瘟病菌稻瘟病菌中甾醇的运输机制及其与巨自噬/自噬的关系仍然知之甚少。在这里，我们通过米菌中的免疫共沉淀鉴定了含有 StAR 相关脂质转移 (VASt) 结构域的蛋白质 MoVast1 的 VAD1 类似物。MoVAST1的缺失导致分生孢子缺陷、附着胞发育受损和致病性降低。MoTor（雷帕霉素在米霉中的靶点）) 活性受到抑制，因为 MoVast1 缺失导致 PM 中高水平的甾醇积累。定点诱变表明902 T 位点对于MoVast1 的定位和功能至关重要。通过 filipin 或 Flipper-TR 染色、自噬通量检测、MoAtg8 脂化和药物敏感性测定，我们发现 MoVast1 作为一种新的自噬抑制因子，通过调节甾醇含量来监测 PM 中的张力，进而调节发动机。脂质组学和转录组学分析进一步证实 MoVast1 是脂质代谢和自噬途径的重要调节因子。我们的研究结果揭示并表征了一种对米分枝杆菌致病性很重要的新型甾醇转移蛋白。

缩写： AmB：两性霉素B；ATMT：根癌农杆菌介导的转化；CM：完全培养基；dpi：接种后天数；ER：内质网；Flipper-TR：荧光脂质张力报告器；GO：基因本体；hpi：接种后的小时数；IH：侵入性菌丝；KEGG：京都基因和基因组百科全书；MoTor：稻瘟病菌中雷帕霉素的靶点；PalmC：棕榈酰肉碱；PM：质膜；SD-N：不含氨基酸和硫酸铵的合成确定培养基；TOR：雷帕霉素靶点；VASt：StAR 相关脂质转移的 VAD1 类似物；YFP，黄色荧光蛋白。