Summary Laccases are widely present in bacteria, fungi, plants and invertebrates and involved in a variety of physiological functions. Here, we report that Beauveria bassiana, an economic important entomopathogenic fungus, secretes a laccase 2 (BbLac2) during infection that detoxifies insect immune response‐generated reactive oxygen species (ROS) and interferes with host immune phenoloxidase (PO) activation. BbLac2 is expressed in fungal cells during proliferation in the insect haemocoel and can be found to distribute on the surface of haemolymph‐derived in vivo fungal hyphal bodies or be secreted. Targeted gene‐knockout of BbLac2 increased fungal sensitivity to oxidative stress, decreased virulence to insect, and increased host PO activity. Strains overexpressing BbLac2 showed increased virulence, with reduced host PO activity and lowered ROS levels in infected insects. In vitro assays revealed that BbLac2 could eliminate ROS and oxidize PO substrates (phenols), verifying the enzymatic functioning of the protein in detoxification of cytotoxic ROS and interference with the PO cascade. Moreover, BbLac2 acted as a cell surface protein that masked pathogen associated molecular patterns (PAMPs), enabling the pathogen to evade immune recognition. Our data suggest a multifunctional role for fungal pathogen‐secreted laccase 2 in evasion of insect immune defenses.

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Summary MADS‐box transcription factor Mcm1 plays crucial roles in regulating mating processes and pathogenesis in some fungi. However, its roles are varied in fungal species, and its function remains unclear in entomopathogenic fungi. Here, Mcm1 orthologue, Bbmcm1, was characterized in a filamentous entomopathogenic fungus, Beauveria bassiana. Disruption of Bbmcm1 resulted in a distinct reduction in growth with abnormal conidiogenesis, and a significant decrease in conidial viability with abnormal germination. ΔBbmcm1 displayed impaired cell integrity, with distorted cell wall structure and altered cell wall component. Abnormal cell cycle was detected in ΔBbmcm1 with longer G2/M phase but shorter G1/G0 and S phases in unicellular blastospores, and sparser septa in multicellular hyphae, which might be responsible for defects in development and differentiation due to the regulation of cell cycle‐involved genes, as well as the corresponding cellular events‐associated genes. Dramatically decreased virulence was examined in ΔBbmcm1, with impaired ability to escape haemocyte encapsulation, which was consistent with markedly reduced cuticle‐degrading enzyme production by repressing their coding genes, and downregulated fungal effector protein‐coding genes, suggesting a novel role of Mcm1 in interaction with host insect. These data indicate that Mcm1 is a crucial regulator of development, cell integrity, cell cycle and virulence in insect fungal pathogens.

Summary Adaptation to low‐oxygen (LO) environment in host tissues is crucial for microbial pathogens, particularly fungi, to successfully infect target hosts. However, the underlying mechanisms responsible for hypoxia tolerance in most pathogens are poorly understood. A mitochondrial protein, BbOhmm, is demonstrated to limit oxidative stress resistance and virulence in the insect fungal pathogen, Beauveria bassiana. Here, we found that BbOhmm negatively affected hypoxic adaptation in the insect haemocoel while regulating respiration‐related events, heme synthesis and mitochondrial iron homeostasis. A homologue of the mammalian sterol regulatory element‐binding proteins (SREBPs), BbSre1, was shown to be involved in BbOhmm‐mediated LO adaptation. Inactivation of BbSre1 resulted in a significant increase in sensitivity to hypoxic and oxidative stress. Similar to ΔBbOhmm, ΔBbSre1 or the ΔBbOhmmΔBbSre1 double mutant accumulated high levels of heme and mitochondrial iron, regulating the similar pathways during hypoxic stress. BbSre1 transcriptional activity and nuclear import were repressed in ΔBbOhmm cells and affected by intracellular reactive oxygen species (ROS) and oxygen levels. These findings have led to a new model in which BbOhmm affects ROS homeostasis in combination with available oxygen to control the transcriptional activity of BbSre1, which in turn mediates LO adaptation by regulating mitochondrial iron homeostasis, heme synthesis and respiration‐implicated genes.

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