BACKGROUND Fungal cell wall integrity is vital for fungal pathogenesis and stress tolerance. Calcofluor white (CFW), a cell wall perturbing agent, inhibits fungal growth by binding chitin in the cell wall. The roles of CFW sensitive proteins remain insufficiently understood in pathogenic fungi. RESULTS We investigated two calcofluor white hypersensitive proteins, MaCwh1 and MaCwh43, in the entomopathogenic fungus Metarhizium acridum. Both Green fluorescent protein (GFP)‐tagged MaCwh1 and MaCwh43 localized at the endoplasmic reticulum. Our results showed that the ΔMacwh1 and ΔMacwh43 mutants were more sensitive to CFW and ultraviolet irradiation stress compared to wild‐type and complement strains. ΔMacwh1 had a stronger sensitivity to these stresses than ΔMacwh43. Both ΔMacwh1 and ΔMacwh43 mutants showed smoother cell wall surface, and drastically reduced chitin and mannose glycoprotein level in the cell wall and glycerol level in conidia compared to wild type. Insect bioassay showed significantly attenuated virulence for both ΔMacwh1 and ΔMacwh43 mutants with impaired ability in penetrating the host cuticle. RNA‐Seq analysis revealed that a large number of genes presumably involved in cell wall construction and modification, pathogenicity and stress response were down‐regulated in both ΔMacwh1 and ΔMacwh43 mutants. CONCLUSIONS These findings demonstrate that both Macwh1 and Macwh43 affect the fungal cell wall ultrastructure and contribute to the stress tolerance and pest control potential in M. acrdium. © 2020 Society of Chemical Industry

Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation (MC). Fungal conidiation can shift between the two patterns, which involved a large number of genes in the regulation of this process. In this study, we investigated the role of a dipeptidase gene pepdA in conidiation pattern shift in Metarhizium acridum , which is upregulated in MC pattern compared to typical conidiation. Results showed that disruption of the pepdA resulted in a shift of conidiation pattern from MC to typical conidiation. Metabolomic analyses of amino acids showed that the levels of 19 amino acids significantly changed in Δ pepdA mutant. The defect of MC in Δ pepdA can be rescued when nonpolar amino acids, α-alanine, β-alanine or proline, were added into s ucrose y east extract a gar (SYA) medium. Digital gene expression profiling analysis revealed that PEPDA mediated transcription of sets of genes which were involved in hyphal growth and development, sporulation, cell division, and amino acid metabolism. Our results demonstrated that PEPDA played important roles in the regulation of MC by manipulating the levels of amino acids in M. acridum . IMPORTANCE Conidia, as the asexual propagules in many fungi, are start and end of fungal lifecycle. In entomopathogenic fungi, conidia are the infective form essential for their pathogenicity. Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation. The mechanisms of the shift between the two conidiation patterns remain to be elucidated. In this study, we demonstrated that the dipeptidase PEPDA, a key enzyme from the insect-pathogenic fungus Metarhizium acridum for the hydrolysis of dipeptides, is associated with a shift of conidiation pattern. The conidiation pattern of the Δ pepdA mutant was restored when supplemented with the nonpolar amino acids rather than polar amino acids. Therefore, this report highlights that the dipeptidase PEPDA regulates MC by manipulating the levels of amino acids in M. acridum.

Conidium is the main infection unit and reproductive unit of pathogenic fungi. Exploring the mechanism of conidiation and its regulation contributes to understanding the pathogenicity of pathogenic fungi. Vib-1, a transcription factor, was reported to participate in the conidiation process. However, the regulation mechanism of Vib-1 in conidiation is still unclear. In this study, we analyzed the function of Vib-1 and its regulation mechanism in conidiation through knocking out and overexpression of Vib-1 in entomopathogenic fungus Metarhizium acridum. Results showed that the colonial growth of Mavib-1 disruption mutant (ΔMavib-1) was significantly decreased, and conidiation was earlier compared to wild type (WT), while overexpression of Mavib-1 led to a delayed conidiation especially when carbon or nitrogen sources were insufficient. Overexpression of Mavib-1 resulted in a conidiation pattern shift from microcycle conidiation to normal conidiation on nutrient-limited medium. These results indicated that Mavib-1 acted as a positive regulator in vegetative growth and a negative regulator in conidiation by affecting utilization of carbon and nitrogen sources in M. acridum. Transcription profile analysis demonstrated that many genes related to carbon and nitrogen source metabolisms were differentially expressed in ΔMavib-1 and OE strains compared to WT. Moreover, Mavib-1 affects the conidial germination, tolerance to UV-B and heat stresses, cell wall integrity, conidial surface morphology and conidial hydrophobicity in M. acridum. These findings unravel the regulatory mechanism of Mavib-1 in fungal growth and conidiation, and enrich the knowledge to conidiation pattern shift of filamentous fungi.