Abstract
As a type of multicopper oxidase, laccases play multiple biological roles in entomopathogenic fungi, enhancing their survival, development, and pathogenicity. However, the mechanisms by which laccases operate in these fungi remain under-researched. In this study, we identified two laccase-encoding genes, Mr-lac3 and Mr-lcc2, from Metarhizium robertsii, both of which are highly expressed during conidiation. Knocking out Mr-lac3 and Mr-lcc2 resulted in a significant increase in the conidial yields of M. robertsii. Furthermore, the relative expression levels of upstream regulators associated with the conidiation pathway were markedly up-regulated in ΔMr-lac3 and ΔMr-lcc2 compared to the wild-type strain during conidiation, indicating that Mr-lac3 and Mr-lcc2 negatively regulate conidia formation. qRT-PCR analyses revealed that Mr-lac3 and Mr-lcc2 are regulated by the pigment synthesis gene cluster, including Mr-Pks1, Mr-EthD, and Mlac1, and they also provide feedback regulation to jointly control pigment synthesis. Additionally, ΔMr-lac3 and ΔMr-lcc2 significantly reduced the trehalose content in conidia and increased the sensitivity to cell wall-perturbing agents, such as Congo red and guaiacol, which led to a marked decrease in tolerance to abiotic stresses. In conclusion, the laccases Mr-lac3 and Mr-lcc2 negatively regulate conidia formation while positively regulating conidial maturation, thereby enhancing tolerance to abiotic stresses and pathogenicity.