Disruption of a C69-Family Cysteine Dipeptidase Gene Enhances Heat Shock and UV-B Tolerances in Metarhizium acridum

Abstract

In fungi, peptidases play a crucial role in adaptability. At present, the roles of peptidases in ultraviolet (UV) and thermal tolerance are still unclear. In this study, a C69-family cysteine dipeptidase of the entomopathogenic fungus Metarhizium acridum, MaPepDA, was expressed in Escherichia coli. The purified enzyme had a molecular mass of 56-kDa, and displayed a high activity to dipeptide substrate with an optimal Ala-Gln hydrolytic activity at about pH 6.0 and 55 • C. It was demonstrated that MaPepDA is an intracellular dipeptidase localized in the cytosol, and that it is expressed during the whole fungal growth. Disruption of the MaPepDA gene increased conidial germination, growth rate, and significantly improved the tolerance to UV-B and heat stress in M. acridum. However, virulence and conidia production was largely unaffected in the MaPepDA mutant. Digital gene expression data revealed that the MaPepDA mutant had a higher UV-B and heat-shock tolerance compared to wild type by regulating transcription of sets of genes involved in cell surface component, cell growth, DNA repair, amino acid metabolism, sugar metabolism and some important signaling pathways of stimulation. Our results suggested that disruption of the MaPepDA could potentially improve the performance of fungal pesticides in the field application with no adverse effect on virulence and conidiation.

MaAts, an Alkylsulfatase, Contributes to Fungal Tolerances against UV-B Irradiation and Heat-Shock in Metarhizium acridum

“…During the applications of mycopesticides in the field, many natural abiotic factors, especially UV irradiation and heat-shock, directly affect the effectiveness of fungal biocontrol agents [ 15 , 18 ]. In addition, the conidial tolerances to UV-B irradiation and high temperature are also served as the important indicators of the conidial quality during the industrial production of mycopesticides [ 54 ].…”

“…Conidia are the asexual propagules in many entomopathogenic fungi and usually the infective unit of mycopesticides [ 12 , 13 ]. Thus, the conidial pathogenicity, conidial yield, as well as sensitivities of conidia to various adverse conditions are all critical for the production cost and the application efficiency of the mycopesticides [ 14 , 15 ]. Among the adverse factors, the high-temperature and the ultraviolet (UV) irradiation from sunlight can lead to the decrease of the conidial vitality and are closely related to the application efficiency of the mycopesticides [ 16 , 17 , 18 , 19 ].…”

Calcofluor white hypersensitive proteins contribute to stress tolerance and pathogenicity in entomopathogenic fungus, Metarhizium acridum

“…Inhibition of these chemicals on fungal growth was assessed as relative growth inhibition (RGI), which was computed with the formula [(control colony diameter −stressed colony diameter)/control colony diameter × 100]. The heat and UV radiation tolerance of the spores were tested according to the methods in the previous study 30 . The inhibition times of 50% germination (IT 50 ) under heat (45 °C) and UV radiation (1350 mW m −2 ) were calculated for WT, Δ Macwh1 , Δ Macwh43 and CP strains.…”

“…The heat and UV radiation tolerance of the spores were tested according to the methods in the previous study. 30 The inhibition times of 50% germination (IT 50 ) under heat (45°C) and UV radiation (1350 mW m −2 ) were calculated for WT, ΔMacwh1, ΔMacwh43 and CP strains. The assays were repeated three times.…”

The Deubiquitinating Enzyme MrUbp14 Is Involved in Conidiation, Stress Response, and Pathogenicity in Metarhizium robertsii

“…For conidial tolerance to heat stress, conidial suspensions (1 ml) in 1.5 ml Eppendorf tubes were placed in a water bath at 42 • C for different amounts of time. After incubation, 10 µl aliquots of the treated suspensions were added to PDA plates and incubated at 25 • C. The rates of conidial germination were measured after culturing on PDA for 20 h. The IT 50 (time for 50% inhibition of germination rate by heat treatment) for different strains was analyzed using GraphPad Prism software (Li et al, 2020;Wang Z. X. et al, 2021).…”