Mr-lac3 and Mr-lcc2 in Metarhizium robertsii Regulate Conidiation and Maturation, Enhancing Tolerance to Abiotic Stresses and Pathogenicity

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, <i>Mr-lac3</i> and <i>Mr-lcc2</i>, from <i>Metarhizium robertsii</i>, both of which are highly expressed during conidiation. Knocking out <i>Mr-lac3</i> and <i>Mr-lcc2</i> resulted in a significant increase in the conidial yields of <i>M. robertsii</i>. Furthermore, the relative expression levels of upstream regulators associated with the conidiation pathway were markedly up-regulated in <i>ΔMr-lac3</i> and <i>ΔMr-lcc2</i> compared to the wild-type strain during conidiation, indicating that <i>Mr-lac3</i> and <i>Mr-lcc2</i> negatively regulate conidia formation. qRT-PCR analyses revealed that <i>Mr-lac3</i> and <i>Mr-lcc2</i> are regulated by the pigment synthesis gene cluster, including <i>Mr-Pks1</i>, <i>Mr-EthD</i>, and <i>Mlac1</i>, and they also provide feedback regulation to jointly control pigment synthesis. Additionally, <i>ΔMr-lac3</i> and <i>ΔMr-lcc2</i> 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.