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The Hog1 mitogen-activated protein (MAP) kinase regulates environmental stress responses and virulence in the entomopathogenic fungus Beauveria bassiana. To further characterize this pathway, we constructed a subtraction library enriched for genes regulated by Hog1. One targeted gene, encoding a novel membrane protein, Ohmm (oxidative homeostasis membrane-protein-mitochondria), was uniquely identified as being downregulated in the ΔHog1 background during growth under non-stress and osmotic stress conditions, but upregulated under oxidative stress. Ohmm was an experimentally validated flavin-binding protein and targeted to the mitochondria. Deletion of Ohmm resulted in increased oxidative stress resistance, whereas overexpression caused an opposite phenotype. The ΔOhmm showed accumulation of reactive oxygen species with alterations in cell wall composition and compatible solute accumulation evident as compared with the wild type parent. Conidiation was reduced > 80%; however, conidia produced by the ΔOhmm strain germinated significantly faster than wild type cells. Insect bioassays using the greater wax moth revealed increased virulence for the ΔOhmm strain in both topical and intrahemocoel injection assays, indicating a negative effect of the presence of Ohmm with respect to pathogenesis. As predicted from our characterization, deletion of Ohmm in a ΔHog1 background rescued its oxidative sensitivity phenotype, confirming that Ohmm acts downstream of the Hog1 MAP-kinase.

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.

No abstract

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.