Functional Analysis Of Metallophosphoesterase MaMPPED2 In Metarhizium Acridum

Filamentous fungus Metarhizium acridum(M.acridum)is used for the control of insect pests.As an environmental friendly insectcide with the advantage of safety and strong pathogenicity,M.acridum has a broad application prospect.However,the high environmental sensitivity,slow insecticidal speed and unstable effectivenesss have restricted its large-scale application in the field.Therefore,an in-depth study of the mechanisms that regulate the growth,development,pathogenicity and stress resistance of M.acridum will provide a theoretical basis to improve its effect of biological control.This study focused on M.acridum CQMa102,and identified the gene MAC＿09064 as MaMPPED2(XP＿007815404.1)which encodes a metaphosphatase by analyzing its sequence through bioinformatics.Next,through comparing and analyzing the biological characteristics(e.g.growth,development,stress resistance and virulence)among the wild-type strain(WT),gene-knockout strain(ΔMaMPPED2)and complementary strain(CP),this study clarified the biological function and possible mechanisms that regulate the function of MaMPPED2.The main results are as follows:1.Bioinformatical analyses of MaMPPED2 geneThe MaMPPED2 gene does not contain any introns,and its c DNA is 906 bp,which encodes a protein of 301 amino acids.The molecular mass of the protein is 33.0 k Da and the isoelectric point is 6.42.Through homology comparison analysis,it is highly conserved in fungi.2.Construction of MaMPPED2 disrupted and complemented vectorsDisrupted and complement vectors of the MaMPPED2 gene were successfully constructed through Agrobacterium tumefaciens mediated homologous recombination.MaMPPED2 disrupted and complemented strains were correctly identified by PCR and southern blot.3.The function of MaMPPED2 on the growth and sporulation abilityBy analyzing the conidia germination rate,sporulation mode and conidia yield,the study found that the conidia germination of the ΔMaMPPED2 strain was earlier than WT or CP strain.On the 1/4 SDAY medium,the germination time of the ΔMaMPPED2 strain was earlier and the hypha became shorter.In addition,when the MaMPPED2 gene was knocked out,the conidia yield on 1/4 SDAY medium was significantly reduced.However,on the SYA medium,the ΔMaMPPED2 strain did not change the conidiation pattern shift.4.Disruption of MaMPPED2 affects the stress toleranceKnocking out MaMPPED2 significantly reduced the conidial tolerance to UV-B irradiation and heat-shock,and was more sensitive to CFW and SOR compared to the WT or CP strain on 1/4 SDAY medium.5.Disruption of MaMPPED2 has no effect on virulenceThere is no significant differences in virulence among the WT,ΔMaMPPED2,and CP strains through bioassay of topical inoculation.5.Disruption of MaMPPED2 decreases the accumulation of melanin pigmentsPigment assays showed that the melanin accumulation decreased 62.7% inΔMaMPPED2.The transcript levels of DHN–melanin and DOPA–melanin synthesis-related genes were assessed via q RT-PCR,and the results showed that these genes were down-regulated significantly in ΔMaMPPED2 versus the WT and CP strains.MaMPPED2 played an important role in the synthesis of melanin pigments.6.RNA-seq analysisThe RNA-sequencing results of the conidia of WT and ΔMaMPPED2 strains cultured on 1/4 SDAY medium for 3 days found 204 up-regulated and 348down-regulated genes in the mutant compared with the WT strain.These genes are mainly related to metabolism,growth,cell wall components and adversity stress.In conclusion,MaMPPED2 regulate the morphogenesis,sporulation,stress tolerance and virulence of M.acridum by affecting the expression of signal transduction and metabolism-related genes.