MoVast2 combined with MoVast1 regulates lipid homeostasis and autophagy in <i>Magnaporthe oryzae</i>

Macroautophagy/autophagy is an evolutionarily conserved biological process among eukaryotes that degrades unwanted materials such as protein aggregates, damaged mitochondria and even viruses to maintain cell survival. Our previous studies have demonstrated that MoVast1 acts as an autophagy regulator regulating autophagy, membrane tension, and sterol homeostasis in rice blast fungus. However, the detailed regulatory relationships between autophagy and VASt domain proteins remain unsolved. Here, we identified another VASt domain-containing protein, MoVast2, and further uncovered the regulatory mechanism of MoVast2 in M. oryzae. MoVast2 interacted with MoVast1 and MoAtg8, and colocalized at the PAS and deletion of MoVAST2 results in inappropriate autophagy progress. Through TOR activity analysis, sterols and sphingolipid content detection, we found high sterol accumulation in the ΔMovast2 mutant, whereas this mutant showed low sphingolipids and low activity of both TORC1 and TORC2. In addition, MoVast2 colocalized with MoVast1. The localization of MoVast2 in the MoVAST1 deletion mutant was normal; however, deletion of MoVAST2 leads to mislocalization of MoVast1. Notably, the wide-target lipidomic analyses revealed significant changes in sterols and sphingolipids, the major PM components, in the ΔMovast2 mutant, which was involved in lipid metabolism and autophagic pathways. These findings confirmed that the functions of MoVast1 were regulated by MoVast2, revealing that MoVast2 combined with MoVast1 maintained lipid homeostasis and autophagy balance by regulating TOR activity in M. oryzae.巨自噬（Macroautophagy，又称自噬autophagy）是真核生物中进化保守的生物学过程，可通过降解蛋白质聚集物、受损线粒体乃至病毒等冗余物质，维持细胞存活。本课题组既往研究表明，MoVast1作为自噬调控因子，可在稻瘟病菌中调控自噬、膜张力及固醇稳态。然而，自噬与VASt结构域（VASt domain）蛋白之间的具体调控关系仍未阐明。本研究鉴定出另一种含VASt结构域的蛋白MoVast2，并进一步阐明了MoVast2在稻瘟病菌（M. oryzae）中的调控机制。MoVast2可与MoVast1及MoAtg8发生相互作用，并共定位于自噬组装位点（PAS）；敲除MoVAST2会导致自噬进程异常。通过雷帕霉素靶蛋白（Target of Rapamycin，TOR）活性分析、固醇与鞘脂含量检测，我们发现ΔMovast2突变体中固醇积累过量，而该突变体的鞘脂含量及TOR复合物1（TORC1）、TOR复合物2（TORC2）的活性均显著降低。此外，MoVast2与MoVast1存在共定位现象。在MoVAST1敲除突变体中，MoVast2的定位正常；但敲除MoVAST2会导致MoVast1的定位异常。值得注意的是，宽靶向脂质组学分析显示，ΔMovast2突变体中作为质膜（PM，Plasma Membrane）主要组分的固醇与鞘脂发生了显著变化，且这些变化参与脂质代谢与自噬通路。上述研究结果证实，MoVast1的功能受MoVast2调控；这表明MoVast2与MoVast1协同作用，通过调控TOR活性，维持稻瘟病菌的脂质稳态与自噬平衡。