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Reactive carbonyl and oxygen species (RCS/ROS), often generated as metabolic byproducts, particularly under conditions of pathology, can cause direct damage to proteins, lipids, and nucleic acids. Glyoxal oxidases (Gloxs) oxidize aldehydes to carboxylic acids, generating hydrogen peroxide (H₂O₂). Although best characterized for their roles in lignin degradation, Glox in plant fungal pathogens are known to contribute to virulence, however, the mechanism underlying such effects are unclear. Here, we show that Glox in the insect pathogenic fungus,Metarhizium acridum, is highly expressed in mycelia and during formation of infection structures (appressoria), with the enzyme localizing to the cell membrane.MaGloxtargeted gene disruption mutants showed RCS and ROS accumulation, resulting in cell toxicity, induction of apoptosis and increased autophagy, inhibiting normal fungal growth and development. The ability of theMaGloxmutant to scavenge RCS was significantly reduced, and the mutant exhibited increased susceptibility to aldehydes, oxidative and cell wall perturbing agents but not toward osmotic stress, with altered cell wall contents. The ΔMaGloxmutant was impaired in its ability to penetrate the host cuticle and evade host immune defense resulting in attenuated pathogenicity. Overexpression ofMaGloxpromoted fungal growth and conidial germination, increased tolerance to H₂O₂, but had little to other phenotypic effects. Transcriptomic analyses revealed downregulation of genes related to cell wall synthesis, conidiation, stress tolerance, and host cuticle penetration in the ΔMaGloxmutant. These findings demonstrate thatMaGlox-mediated scavenging of RCS is required for virulence, and contributes to normal fungal growth and development, stress resistance.