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 ΔMaPepDA mutant. Digital gene expression data revealed that the ΔMaPepDA 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.
Highlights
The harmful and irreversible impacts of toxic chemical insecticides on the environment are receiving widespread attention and have accelerated extensive research for alternatives, especially biological control agents such as fungi and bacteria (Aw and Hue, 2017)
The multiple sequence alignment analysis showed that the C69 domain of MaPepDA was homologous to C69 family peptidases of other fungal species (Figure 1A)
Fluorescent microscopic visualization showed that the fusion MaPepDA-Enhanced green fluorescence protein (EGFP) was evenly localized in cytoplasm (Figure 2A), which was consistent with the estimated results of the online program1
Summary
The harmful and irreversible impacts of toxic chemical insecticides on the environment are receiving widespread attention and have accelerated extensive research for alternatives, especially biological control agents such as fungi and bacteria (Aw and Hue, 2017). Metarhizium is regarded as reliable substitute for chemical pesticide because of its distinctive advantages, such as safety, environmental friendliness, and low insect resistance (Steven et al, 2003; Jesper et al, 2007; Guerrero-Guerra et al, 2013). Combined application of Metarhizium with Bacillus thuringiensis was more effective in controlling pests than single use of B. thuringiensis, and the host insects could hardly develop resistance (Tupe et al, 2017). Metarhizium spores were reported to be detected in soil or as endophytes in plants root and can persist over a long time (Greenfield et al, 2016). Many challenges, such as unstable tolerance to physical and natural conditions and low virulence, limited the efficiency and large-scale application of Metarhizium. Exploring the mechanism of heat shock and UV-B tolerance would help to improve the efficiency of fungal biocontrol agents by genetic engineering techniques (Zhao et al, 2016)
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