Acetylation enables a dual-targeting fungal effector to inhibit insect immunity and development.

Pathogenic microbes deploy secreted effectors to subvert host immunity and physiology across kingdom boundaries, yet the molecular mechanisms that maintain effector competence during this transition remain poorly defined. Here, we identify MrNIS1, an effector from the insect-pathogenic fungus Metarhizium robertsii whose stability and secretion are governed by K24 acetylation through physical association with the acetyltransferase MrKAT1 and the deacetylase MrSIR2. Once inside the insect, MrNIS1 acts as a dual-targeting effector to disrupt distinct physiological pathways. It binds the serine protease SPS to block prophenoloxidase activation, thereby suppressing melanization-based immunity. MrNIS1 interacts with the evolutionarily conserved cholesterol transporter ERP, disrupting ecdysteroid biosynthesis and leading to impaired larval growth and cuticle synthesis. This MrNIS1-ERP interaction is functionally conserved across Lepidoptera, Coleoptera, and Diptera. Furthermore, heterologous expression in Beauveria bassiana demonstrates that the KAT1/SIR2 regulatory system is conserved across fungal species, where MrNIS1 acts as a portable virulence module to significantly enhance pathogen efficacy. Our findings reveal that lysine acetylation serves as a molecular switch that preserves effector competence during cross-kingdom transit, enabling the simultaneous targeting of multiple host physiological processes. These results elucidate a conserved strategy of fungal pathogenesis and identify potential targets for the improvement of microbial biocontrol agents.