Functional Analysis Of MaVeA In Metarhizium Acridum

Fungi are a kind of microorganisms which are the earliest concerned and can cause insect disease.Its special infection pattern and considerable host registration mode show strong vitality in pest control.In recent decades,entomopathogenic fungi have made great breakthroughs in the basic and applied research,and promoted the rapid development of its industry.So far,there are nearly 200 kinds of fungal pesticides have been registered in the international community,but the high cost of production,the effect of instability and other factors are restricting its wide range of applications.Conidia are the important units of pest control,so its yield and quality directly determine the production costs and control effect of the fungal pesticides.Metarhizium acridum is an important entomopathogenic fungus,which is widely used in the biological control of pests,and the study of the mechanism of sporulation and pathogenesis have a positive effect on its wide application.The sporulation of pathogenic fungi is regulated by multiple genes,including feedback roles in sporulation.VeA is an important gene that performs feedback function in the process of sporulation and regulates asexual and sexual conversion in Aspergillus nidulans.A gene had been found in Metarhizium acridum that is highly homologous to VeA and named MaVeA,while the function of which in entomopathogenic fungi is unclear.In this paper,locusta migratoria and Metarhizium acridum were selected as experimental materials to explore the following purposes: First,the biological function of MaVeA on the growth,sporulation and pathogenicity in Metarhizium acridum;Second,the mechanism of MaVeA in sporulation and pathogenesis in Metarhizium acridum.(1)MaVeA deletion and complementationUsing M.acridum complete genome sequence database and complete genomic library to cloned the full-length MaVeA cDNA sequence.To construct MaVeA deletion,MaVeA was replaced by the Bar as a selectable marker gene through homologous recombination.Deletion and recovery strains of MaVeA was confirmed by PCR and Southern blot.(2)MaVeA affected fungal growth and stress toleranceIn the 1/4 SDAY solid medium,the GT50 of MaVeA had significant increase compared with WT.However,deletion of MaVeA obviously decreased the colony,the mycelium and the production of sporulation.The resistance analysis showed that the loss of MaVeA did not affect the heat shock tolerance,but it reduced its ability to resist UV.(3)The relationship between MaVeA and conidiation regulatory pathway genesThe expression of MaflbA and MabrlA was significantly higher than that of wild-type WT at the 9 day,indicating that MaVeA may negatively regulate the expression of these two genes,and it may be related to the conversion of spore maturation.MaflbA,MaflbB,MaflbC,MabrlA,MafadA and MafluG in the knockout strain MaVeA were significantly higher than those in the wild type WT at the 15 day,indicating that the MaVeA post-feedback sporulation situation,and further promote spore maturation.(4)MaVeA leads to a change in pigmentWhen the strains were cultured on 1/4 SDAY solid medium,the spores of ?MaVeA strain showed yellow,while WT and CP showed green,indicating that ?MaVeA could lead to a change in the pigment.However,?MaVeA,WT and CP showed a consistent color on the CM medium,indicating that some or several elements may be related to the formation of the pigment.(5)Virulence is attenuated in the ?MaVeA mutantBioassay against locust indicated that pathogenicity of ?MaVeA was attenuated compared with WT and CP in topical inoculation.The LT50 values were significantly delayed in the topical inoculation assays,but no difference was found in LT50 between WT and ?MaVeA in injection assays.These results suggested that MaVeA may possibly affect the process of penetrating into insect cuticle.(6)Reasons for the decrease of virulence in M.acridumAfter the loss of MaVeA,the hydrophobicity of the strain was decreased,the rate of the formation of appressorium was decreased,the rate of the collapse of turgor pressure was increased,which all reduced pathogenicity in Metarhizium acridum.In a word,MaVeA plays an important role in sporulation and pathogenicity.This study provides a theoretical basis for exploring the mechanisms of sporulation and pathogenicity in entomopathogenic fungus,which is of great theoretical and practical value to fully exploit its potential biological control ability.