Dipeptidase PEPDA Is Required for the Conidiation Pattern Shift in Metarhizium acridum

Abstract

Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation (MC). Fungal conidiation can shift between the two patterns, which involves 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 ΔpepdA mutant. The defect of MC in ΔpepdA can be rescued when nonpolar amino acids, α-alanine, β-alanine, or proline, were added into sucrose yeast extract agar (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 the start and end of the fungal life cycle. 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 ΔpepdA 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.

Keywords: conidiation pattern shift; dipeptidases; entomopathogenic fungi; microcycle conidiation.

FIG 1

Conidiation of fungal strains. Conidiation of wild-type, ΔpepdA, and ΔpepdA::pepdA strains was observed under microscopy and photographed at different growth periods. Black arrows, conidia produced via MC; red arrows, new produced conidia on the tip of hyphae via typical conidiation from mycelia. All strains were inoculated on SYA medium and cultured at 28°C.

FIG 2

Conidial yield of fungal strains. (A) Conidial yield of different fungal strains on SYA medium at 28°C for 3, 5, 7, 9, 11, 13, and 15 days. Error bars represent standard deviation. Different lowercase letters on bars denote significant differences between samples (P < 0.05). (B) Colony diameter at different cultural days on SYA medium.

FIG 3

Analysis of amino acids quantification and peptidase activity. (A) LC-MS quantitative analysis of amino acids in wild type and ΔpepdA. Log₂^(ΔpepdA/WT) stands for the relative levels of amino acids in ΔpepdA compared to WT (P < 0.05). Samples were detected in equal amounts of 10 replicates. (B) Dipeptidase activity in M. acridum. The dipeptide of Ala-Gln was used as the substrate. The enzyme activity of WT was considered 100%. The relative activity was defined as the percentage of activity comparing to that of WT. Different letters on bars denote significant differences between samples (P < 0.01).

FIG 4

Growth and conidiation with amino acid supplementation. Nonpolar amino acids (A) and polar amino acids (B) were added in the SYA medium at different concentrations and observed under microscope after grown at 28°C for 24 h. Black arrows, conidia produced via MC; red arrows, new produced conidia on the tip of hyphae via typical conidiation.

FIG 5

DGE analysis. (A) Differentially expressed genes analysis between ΔpepdA and WT. Column in green indicates upregulated gene in ΔpepdA compared to WT, and downregulated gene is in yellow. (B) GO annotation of the DEGs from ΔpepdA and WT. The x axis indicates the subcategories, the left y axis represents the number of unigenes, and the right y axis indicates the percentage of a specific category of unigenes.

FIG 6

Venn diagram showing the number of shared DEGs between 436 upregulated genes in MC (9) and 27 downregulated genes in ΔPepDA. Shown in the parentheses is the total number of upregulated or downregulated genes. Up arrows indicate upregulated genes in MC pattern reported previously (green), and down arrows indicate downregulated genes in ΔPepDA (red).