Suppression of Drosophila antifungal immunity by a parasite effector via blocking GNBP3 and GNBP-like 3, the dual receptors for ß-glucans.

The tactics used by animal pathogens to combat host immunity are largely unclear. Here, we report the depiction of the virulence-required effector Tge1 deployed by the entomopathogen Metarhizium robertsii to suppress Drosophila antifungal immunity. Tge1 can target both GNBP3 and GNBP-like 3 (GL3), and the latter can bind to ß-glucans like GNBP3, whereas the glucan binding by both receptors can be attenuated by Tge1. As opposed to the surveillance GNBP3, GL3 is inducible in Drosophila depending on the Toll pathway via a positive feedback loop mechanism. Losses of GNBP3 and GL3 genes result in the deregulations of protease cascade, Spätzle maturation, and antimicrobial gene expressions in Drosophila upon fungal challenges. Fly survival assays confirm that GL3 plays a more essential role than GNBP3 in combating fungal infections. In addition to evidencing the gene-for-gene interactions between fungi and insects, our data advance insights into Drosophila antifungal immunity.

Activation of microlipophagy during early infection of insect hosts by Metarhizium robertsii.

The requirement of macroautophagic/autophagic machinery for filamentous fungal development and pathogenicity has been recognized, but the underlying effects and mechanisms remain elusive. The insect pathogenic fungus Metarhizium robertsii infects hosts by cuticular penetration through the formation of the infection structure appressoria. Here, we show that autophagic fluxes were highly activated during the appressorial formation of M. robertsii. Genome-wide deletion of the autophagy-related genes and insect bioassays identified 10 of 23 encoded MrATG genes with requirements for topical fungal infection of insect hosts. Besides the defect in forming appressoria on insects (two null mutants), these virulence-reduced mutants were largely impaired in penetrating cellophane membrane and insect cuticles, suggesting their failures in generating proper appressorium turgor. We found that the conidial storage of lipid droplets (LDs) had no obvious difference between strains, but autophagic LD degradation was impaired in different mutants. After induction of cell autophagy by nitrogen starvation, we found that LD entry into vacuoles was unaffected in the selected mutant cells with potential failures in forming autophagosomes. The finding therefore reveals a microlipophagy machinery employed in this fungus and that the direct engulfment of LDs occurs without inhibition by the downstream defective lipolysis. Our data first unveil the activation and contribution of microlipophagy to fungal infection biology. The obtained technique may benefit future detection of microlipophagy in different organisms by examining vacuolar or lysosomal engulfment of LDs in core autophagic gene deletion mutants.Abbreviations: AIM: Atg8-family interacting motif; ATG: autophagy-related; CM: complete medium; CMAC: 7-amino-4-chloromethylcoumarin; DTT: dithiothreitol; ER: endoplasmic reticulum; GFP: green fluorescent protein; LD, lipid droplet; MM: minimum medium; MM-N: minimum medium without nitrogen source; PDA: potato dextrose agar; PMSF: phenylmethylsulfonyl fluoride; RFP: red fluorescent protein; SDB: Sabouraud dextrose broth; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TAG: triacylglycerol; TEM: transmission electron microscopy; WT, wild type.

Production of Diverse Beauveriolide Analogs in Closely Related Fungi: a Rare Case of Fungal Chemodiversity.

Fungal chemodiversity is well known in part due to the production of diverse analogous compounds by a single biosynthetic gene cluster (BGC). Usually, similar or the same metabolites are produced by closely related fungal species under a given condition, the foundation of fungal chemotaxonomy. Here, we report a rare case of the production of the cyclodepsipeptide beauveriolides (BVDs) in three insect-pathogenic fungi. We found that the more closely related fungi Beauveria bassiana and Beauveria brongniartii produced structurally distinct analogs of BVDs, whereas the less-close relatives B. brongniartii and Cordyceps militaris biosynthesized structurally similar congeners under the same growth condition. It was verified that a conserved BGC containing four genes is responsible for BVD biosynthesis in three fungi, including a polyketide synthase (PKS) for the production of 3-hydroxy fatty acids (FAs) with chain length variations. In contrast to BVD production patterns, phylogenetic analysis of the BGC enzymes or enzyme domains largely resulted in the congruence relationship with fungal speciation. Feeding assays demonstrated that an FA with a chain length of eight carbon atoms was preferentially utilized, whereas an FA with a chain longer than 10 carbon atoms could not be used as a substrate for BVD biosynthesis. Insect survival assays suggested that the contribution of BVDs to fungal virulence might be associated with the susceptibility of insect species. The results of this study enrich the knowledge of fungal secondary metabolic diversity that can question the reliability of fungal chemotaxonomy.IMPORTANCE Fungal chemotaxonomy is an approach to classify fungi based on the fungal production profile of metabolites, especially the secondary metabolites. We found an atypical example that could question the reliability of fungal chemical classifications in this study, i.e., the more closely related entomopathogenic species Beauveria bassiana and Beauveria brongniartii produced structurally different congeners of the cyclodepsipeptide beauveriolides, whereas the rather divergent species B. brongniartii and Cordyceps militaris biosynthesized similar analogs under the same growth condition. The conserved biosynthetic gene cluster (BGC) containing four genes present in each species is responsible for beauveriolide production. In contrast to the compound formation profiles, the phylogenies of biosynthetic enzymes or enzymatic domains show associations with fungal speciation. Dependent on the insect species, production of beauveriolides may contribute to fungal virulence against the susceptible insect hosts. The findings in this study augment the diversity of fungal secondary metabolisms.