Characteristics And Functional Analysis Of The Dual-specificity Phosphatase Gene MaCdc14 In Metarhizium Acridum

Entomopathogenic fungi such as Metarhizium spp.have been widely studied because of its safety and eco-friendly.Metarhizium acridum,a locust-specific pathogen,is used as an excellent model organism to explore a broad array of questions in ecology,evolution and the mechanisms of speciation,and it has great potential in biological control.Such biological agents have been widely used in Australia,West Africa and Asia.However,Vulnerable to environmental impacts and poor efficacy are serious obstacles for the application of biological agents in agriculture.Thus,it is of great significance to study the entomopathogenic fungi on molecular level for arthropod pest control.Reversible phosphorylation of protein kinases and phosphatases plays a pivotal role in multiple biological events in eukaryotes.In this study,a dual-specificity cell division cycle 14 phosphatase gene,MaCdc14,was functionally characterized in Metarhizium acridum.According to bioinformatics analysis,this gene has a total length of 1963 bp with an intron of 103 bp,and it encodes a protein of 619 amino acids with a molecular weight of 69.11 kDa and an isoelectric point of 9.29,and this protein has no signal peptide.To determine the functions of Cdc14 in M.acridum,the MaCdc14-deletion mutant(?MaCdc14)and complemented strain(CP)were generated.Deletion of MaCdc14 decreased the branch numbers,affected the septum formation and caused multiple nuclei in each hyphal compartment.The conidial yield of ?MaCdc14 was significantly decreased with delayed conidiation,and the transcription levels of the conidiation related genes were significantly changed in ?MaCdc14.The morphology of conidia was uneven in size and the germination rate of conidia was increased and the conidial tolerance to UV irradiation was significantly enhanced after deleted MaCdc14,while the thermotolerance,resistance to chemical disrupting agents and virulence of ?MaCdc14 were not affected compared to WT and CP strains.Furthermore,the increased pigmentation resulted from the upregulated expression of melanin synthesis related genes may contribute to the enhanced UV tolerance.In addition,it was found that MaCdc14 regulated the conidiation pattern shift in SYA medium in M.acridum.The conidiation pattern was shift from microcycle conidiation to normal conidiation and the color of the colony was changed from green to white after deleted MaCdc14,and the spore production capacity of ?MaCdc14 was seriously affected with decreased conidial yield.In order to explore the mechanism of conidiation pattern shift in SYA medium,protein phosphorylation level of WT and ?MaCdc14 were conducted.The results showed that the phosphorylation levels of proteins of growth and development,cell membrane,septum,sporulation,cell cycle,cell differentiation,metabolism and some important signaling pathways were changed.Taken together,our study indicated that MaCdc14 affected cytokinesis,morphogenesis and conidiation,and it possibly that MaCdc14 affected the anti-ultraviolet ability of spores by regulating melanin synthesis.In addition,MaCdc14 regulated the conidiztion pattern shift of M.acridum jointly by affecting the the phosphorylation levels of proteins of growth and development,conidiation and cell cycle to regulate spore production capacity.