Glycoside hydrolase Ma3360 mediates immune evasion by Metarhizium anisopliae in insects

Entomopathogenic fungi can precisely inhibit the cellular and humoral immune responses of host insects by secreting effector proteins, allowing them to overcome the innate immune barriers of their hosts. Nodule formation is an immune response primarily mediated by insect hemocytes, which can rapidly and efficiently capture invading pathogenic fungi in the hemocoel. However, the molecular mechanisms by which fungi inhibit insect nodule formation through the secretion of effector proteins remain unclear. First, we systematically observed the process of nodule induction and breakthrough by Metarhizium anisopliae. Following injection, the spores of M. anisopliae are quickly captured by hemocytes, and within 2 h, a significant number of hemocyte aggregates encapsulating the spores (early nodules) can be observed in the hemolymph. After 6 h, larger, melanized nodules can be collected from the hemocoel. After 18 to 24 h, the encapsulated spores begin to germinate and break through the nodules. By 36 to 48 h, the hyphae produce spores through budding, rapidly spreading and occupying the hemocoel. Subsequently, using Hemocytin, a key protein in insect nodule formation, as a mediator, we conducted pull-down assays, yeast two-hybrid experiments, surface plasmon resonance, and yeast secretion assays, discovering that a secreted glycoside hydrolase family 16 protein (Ma3360) in M. anisopliae acts as an effector that facilitates the breakthrough of nodules. Further functional studies indicated that Ma3360 also promotes the growth of M. anisopliae under stress conditions and disrupts the morphological structure of hemocytes. The results of this study reveal the molecular mechanisms by which pathogenic fungi utilize effector proteins to disrupt or evade the cellular immune responses of host insects.