MoVast2 combined with MoVast1 regulates lipid homeostasis and autophagy in Magnaporthe oryzae

ABSTRACTMacroautophagy/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.KEYWORDS: Autophagylipid homeostasismagnaporthe oryzaeregulationTORC2 Disclosure statementDr. Daniel J. Klionsky is a Founder and Editor-in-Chief of Autophagy. All authors declare no conflict of interest.AbbreviationsATMT: Agrobacterium tumefaciens-mediated transformation; BiFC: bimolecular fluorescence complementation; COP: coat protein; Co-IP: co-immunoprecipitation; CM: complete medium; ER: endoplasmic reticulum; HMM: hidden Markov models; hpi: post-inoculation; IH: invasive hyphae; MCC: membrane compartment occupied by Can1; MCT: membrane compartment containing TORC2; PAS: phagophore assembly site; 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.Supplementary MaterialSupplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2181739Additional informationFundingThis study was supported by grants from the National Natural Science Foundation of China [32100159, 31970140 & 31972216], Key R&D projects of Zhejiang Province of China [2021C02010], Natural Science Foundation of Zhejiang province of China [LQ22C140005] and National Institutes of Health of USA [GM131919];Research and Development Project of Zhejiang Province [2021C2010];Natural Science Foundation of Zhejiang Province [LQ22C140005].