PCR primers used in this study.

Conidial production is a critical factor determining the efficacy of entomopathogenic fungi as biocontrol agents. Autophagy, a fundamental cellular degradation process, plays an essential role in regulating fungal conidiation. However, the modulation of autophagy through acetylation, particularly concerning the autophagy-related protein ATG4, remains poorly understood in fungi. Here, we investigate the roles of the deacetylase MrSIR2–3 and the acetyltransferase MrKAT1 in Metarhizium robertsii, focusing on their impacts on autophagy and conidiation. Our findings demonstrate that deletion of MrSIR2–3 (ΔMrsir2–3) leads to elevated autophagy levels, whereas loss of MrKAT1 (ΔMrkat1) suppresses autophagy initiation; both alterations consequently impair conidiation. Interaction assays further reveal that the key autophagy factor MrATG4 is regulated by opposing acetylation and deacetylation mediated by MrKAT1 and MrSIR2–3, potentially via modification of lysine residues K69 and/or K77. This dynamic acetylation balance is essential for maintaining autophagy homeostasis and ensuring efficient conidiation. Collectively, our results provide novel insights into how the acetylation of ATG4 modulates autophagy, advancing our understanding of conidiation regulation in entomopathogenic fungi and highlighting potential targets for enhancing fungal biocontrol efficacy.