Functional Analysis Of Macwh Gene In Metarhizium Acridum

In fungi,the cell wall as the first physiological barrier involves a variety of physiological functions,such as cell attachment,nutrient acquisition,response to the destruction of the external environment and the formation.The Calcofluor white hypersensitive protein Cwh has the function of maintaining the the cell wall integrity.This phenomenon is only reported in Saccharomyces cerevisiae,and there is no research and report on such genes in pathogenic fungi.Due to the unique body wall infection of biocontrol,the cell wall plays an important role in the pathogenesis of pathogenic fungi and the pathogenesis of infection.In this study,we analyzed the function of cwh genes by gene knockout in Metarhizium acridum.The main results are as follows:1.Bioinformatics analysis of Macwh1 and Macwh2The Macwh1 and Macwh2 genes were cloned based on the genomic DNA sequence of M.acridum.The open reading frame?ORF?of Macwh1?XP₀07815818.1?was 2874 bp.The genomic sequence of Macwh1 contains seven introns,encodes a protein of 957 amino acids with an isoelectric point of 7.02 and molecular weight of 107.02 KDa.The open reading frame?ORF?of Macwh2?XP₀07808921.1?was 402 bp.The genome of Macwh2 contains two introns,encodes a protein of 133 amino acids with with an isoelectric point of 9.48 and molecular weight of 13.85 KDa.Bioinformatics analysis showed that there was no homology between Macwh1 and Macwh2.2.Macwh deletion and complementationIn order to study the function of Macwh1 and Macwh2,the genes were knocked out by homologous recombination method.Transformation was performed mediated by Agrobacterium tumefaciens was and transformants were obtained by resistance screening and confirmed by Southen hybridization.To complement deletion mutants,the complete Macwh genes were reserted into the deletion mutant to get the complementation strains.3.Virulence is attenuated in the ?MacwhThe virulence was assayed using the fifth instar nymphs of L.migratoria manilensis?Meyen?by larvale.The pathogenicity of ?Macwh mutant was significantly reducted compared with WT by the topical inoculation.The hyphal bodies in insect haemolyph decreased in ?Macwh compared to WT and CP.However,compared with WT and CP,?Macwh mutant had similar virulence in injection assays.There was no significant difference in the number of hyphal bodies in the in insect haemolyph.The pathogenicity factors of M.acridum contain appressoria formation rate,appressorial turgor pressure and the enzyme involved in degradation of the insect wall.All the result indicated that Macwh affected the appressoria formation,appressorial pressure turgor and the expression of genes related to penetration process.5.Macwh were involoved in the cell wall integrity and resistanceThemain cell wall components of conidium were analyzed.Results indicated that compared to the WT strain,the content of chitin,mannose glycoprotein were reduced significantly.However,the content of ?-1,3-glucan has no significant difference.Analysis of conidial surface hydrophobicity revealed that ?Macwh mutant conidia had significantly decreased indexes of hydrophobicity compared to wild type conidia.Genes expression of chitin synthase and hydrophobic protein,which have been reported to involve in the cell wall integrity,were analyzed by quantitative RT-PCR.Results showed that chsI-chsVII,hyd were signigficantly downregulated.Colony diameter of ?Macwh mutant was significant smaller than WT,when the fungi were grown on the 1/4SDAY added with CFW.Furthermore,?Macwh mutant had significant weakened tolerance to UV irradiation compared with WT.Expression of genes photolyase phr1,UV-endonuclease uve-1,which have been reported involved in UV irradiation,were analyzed by qRT-PCR.The results indicated that phr1 and uve-1 genes were all downregulated.All the results indicated that Macwh maintained the cell wall integrity and involoved in UV irradiation.The results showed that Macwh1 and Macwh2 had very important roles in the pathogenicity,especially the penetration process and cell wall integrity of M.acridum.