| Summary: | Mycelial ageing is associated with ROS and autophagy in <i>Lentinula edodes</i>. However, the underlying cellular and molecular mechanisms between ROS and autophagy remain obscure. This study induced autophagy in <i>L. edodes</i> mycelia through exogenous H<sub>2</sub>O<sub>2</sub> treatment. Results showed that 100 μM H<sub>2</sub>O<sub>2</sub> treatment for 24 h significantly inhibited mycelial growth. H<sub>2</sub>O<sub>2</sub> caused the depolarisation of MMP and accumulation of TUNEL-positive nuclei, which was similar to the ageing phenotype of <i>L. edodes</i> mycelia. Transcriptome analysis showed that differentially expressed genes were enriched in the mitophagic, autophagic, and MAPK pathways. <i>LeAtg8</i> and <i>LeHog1</i> were selected as hub genes. RNA and protein levels of LeATG8 increased in the H<sub>2</sub>O<sub>2</sub>-treated mycelia. Using fluorescent labelling, we observed for the first time the classic ring structure of autophagosomes in a mushroom, while 3D imaging suggested that these autophagosomes surrounded the nuclei to degrade them at specific growth stages. Phospho-LeHOG1 protein can translocate from the cytoplasm to the nucleus to regulate mycelial cells, resisting ROS-induced oxidative stress. Furthermore, LeATG8 expression was suppressed when LeHOG1 phosphorylation was inhibited. These results suggest that the LeATG8-dependent autophagy in <i>L. edodes</i> mycelial is closely associated with the activity or even phosphorylation of LeHOG1.
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