A VASt-domain protein regulates autophagy, membrane tension, and sterol homeostasis in rice blast fungus

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.

固醇（sterols）是一类对基础生物学过程及膜动态平衡至关重要的脂质。这类分子在内质网（endoplasmic reticulum, ER）中合成，并在内质网与质膜（plasma membrane, PM）之间双向运输。然而，目前人们对稻瘟病菌（Magnaporthe oryzae）中固醇的转运机制及其与巨自噬/自噬的关联仍知之甚少。本研究通过免疫共沉淀（co-immunoprecipitation）在稻瘟病菌中鉴定得到一种含StAR相关脂质转移蛋白VAD1同源结构域（VASt结构域，VAD1 Analog of StAR-related lipid transfer）的蛋白MoVast1。敲除MoVAST1会导致分生孢子缺陷、附着胞发育受损以及致病力下降。由于MoVast1缺失会导致质膜中固醇大量积累，稻瘟病菌雷帕霉素靶蛋白（MoTor, target of rapamycin in Magnaporthe oryzae）的活性受到抑制。定点诱变实验表明，902位苏氨酸（T）位点对MoVast1的定位与功能至关重要。通过菲洛平（filipin）或荧光脂质张力报告探针Flipper-TR（fluorescent lipid tension reporter）染色、自噬流检测、MoAtg8脂化修饰以及药物敏感性实验，我们发现MoVast1是一种新型自噬抑制因子，可通过调节固醇含量监测质膜张力，进而调控MoTor的活性。脂质组学与转录组学分析进一步证实，MoVast1是脂质代谢与自噬通路的重要调控因子。本研究揭示并鉴定了一种对稻瘟病菌致病力至关重要的新型固醇转运蛋白。
缩写说明：AmB：两性霉素B（amphotericin B）；ATMT：根癌农杆菌介导转化（Agrobacterium tumefaciens-mediated transformation）；CM：完全培养基（complete medium）；dpi：接种后天数（days post-inoculation）；ER：内质网（endoplasmic reticulum）；Flipper-TR：荧光脂质张力报告探针（fluorescent lipid tension reporter）；GO：基因本体论（Gene ontology）；hpi：接种后小时数（hours post-inoculation）；IH：侵入菌丝（invasive hyphae）；KEGG：京都基因与基因组百科全书（kyoto encyclopedia of genes and genomes）；MoTor：稻瘟病菌雷帕霉素靶蛋白（target of rapamycin in Magnaporthe oryzae）；PalmC：棕榈酰肉碱（palmitoylcarnitine）；PM：质膜（plasma membrane）；SD-N：无氨基酸与硫酸铵的合成限定培养基（synthetic defined medium without amino acids and ammonium sulfate）；TOR：雷帕霉素靶蛋白（target of rapamycin）；VASt：StAR相关脂质转移蛋白VAD1同源结构域（VAD1 Analog of StAR-related lipid transfer）；YFP：黄色荧光蛋白（yellow fluorescent protein）。