MrDnaJ, a heat shock protein, regulates stress tolerance and virulence in the entomopathogenic fungus Metarhizium robertsii.

Heat shock proteins are essential molecular chaperones synthesized in response to environmental stresses, yet their roles in entomopathogenic genus Metarhizium remain poorly understood. Here, we identified and characterized MrDnaJ, a heat shock protein DnaJ family member that physically interacts with MrWetA, a key transcription factor in Metarhizium robertsii. MrdnaJ is highly expressed in appressoria and strongly induced by heat stress, with nuclear localization. MrdnaJ knockout mutants (ΔMrdnaJ) exhibited significantly impaired growth and reduced conidial yield under heat stress. Additionally, ΔMrdnaJ conidia displayed enhanced sensitivity to heat, UV, oxidative, osmotic, and cell wall perturbing stresses. Transcriptomic analysis revealed that deletion of MrdnaJ downregulated 605 genes which including genes encoding ROS-scavenging enzymes, heat shock proteins, cell wall components, and osmolyte synthesis. Notably, ΔMrdnaJ mutants showed substantially reduced virulence against Galleria mellonella larvae during topical infection, but maintained normal infectivity following direct injection, indicating defective cuticle penetration. Further analysis demonstrated that MrDnaJ positively regulates the expression of cuticle-degrading proteases and chitinases. Collectively, these findings demonstrate that MrDnaJ functions as a critical regulator of both stress tolerance and virulence in M. robertsii by controlling the expression of genes essential for conidial survival and host cuticle penetration. This work provides important insights into heat shock protein function in entomopathogenic fungi and identifies MrDnaJ as a potential target for enhancing the biocontrol efficacy of fungal insecticides.