The Structural And Functional Analysis Of StSLT2 Gene In The CWI-MAPK Cascade Pathway In Setosphaeria Turcica

Previous studies showed that the cell wall integrity mitogen activated protein kinase(CWI-MAPK)pathway was one of the important MAPK signal transduction pathways in eukaryotic cells.CWI-MAPK pathway was mainly involved in the regulation of cell wall component biosynthesis and cell cycle.Up to date,SLT2 homologous gene is an important MAPK gene in the CWI-MAPK pathway in fungi,but the function of this gene has not been reported in Setosphaeria turcica.In this study,StSLT2 was identified in the whole genome of S.turcica by the bioinformatics technology and the structural characteristics of the gene were analyzed.Furthermore,the gene knockout mutants were obtained using target gene knockout technology and the function of the gene was clarified by comparing the differences between the mutant and the wild type(WT)strain in growth,cell wall morphogenesis and pathogenicity.This study will not only clarify the function of StSLT2,but also lay a foundation for further elucidation of the regulatory mechanism of CWI-MAPK pathway.The main research results are as follows:1.In this study,the SLT2 homologous gene was identified in the whole genome database of S.turcica,and it was designated as StSLT2.The full length of the gene was 5842 bp,and the cDNA was 3105 bp in size.The gene contained 13 exons and 12 introns,encoded 1034 amino acids.Conserved domain analysis showed that the gene contained a conserved serine/threonine protein kinase domain,which contained the typical activation motif(TEY)of MAPK protein.Phylogenetic analysis showed that S.turcica was closer evolutionary relationship with Bipolaris maydis and Alternaria brassicicola.2.StSLT2 gene knockout recombinant plasmid was constructed by means of pBluescriptⅡSK(-)vector.Based on the principle of homologous recombination,two mutants(ΔStSLT2-1,ΔStSLT2-2)with resistance to glufosinate were obtained by PEG mediated genetic transformation method.These strains were further determined through BAR PCR,specific PCR,RT-PCR technique.3.Compared with WT strain,the growth rates of mutant ΔStSLT2-1,ΔStSLT2-2 colony were slowed down.The colony morphology color was gray and became lighter,aerial hyphae was denser,no conidia were found,melanin content decreased significantly.The results showed that StSLT2 gene was involved in regulating the growth and development of S.turcica.4.Microscopic observation showed that compared with WT,the mycelial branches of the mutants increased significantly,and the tips expanded.Chitin staining was performed on mycelial cells,and it was found that the chitin synthesis of the mutant hyphal tips were vigorous.The contents of chitin and β-glucan in StSLT2 knockout mutants were significantly increased and decreased,respectively.By comparing the tolerance of mutant and WT strain to cell wall sensitive substances,it was found that the tolerance of gene knockout mutant increased at 100 g/mL Congo red,20 g/mLCFW and 0.01% SDS.The results showed that StSLT2 gene was involved in the regulation of cell wall development of S.turcica.5.By observing the appressorium development state,it found that StSLT2 knockout mutants formed germ tube at 24 h,germ tube tip enlarged at 36 h,formed many mature multiple appressoria at 48 h,some appressoria formed the infection hypha at 60 h,and penetrated cellophane.Compared with the WT strain,the appressoria development of the mutants was delayed for 24 h,and the formation rate of infection hypha was significantly reduced.Pathogenicity analysis showed that both WT strain and StSLT2 gene knockout mutant strains could infect and colonize B73 maize leaves,but disease spot formational time of mutant was delayed and the disease spot area was smaller than that of WT strain.The results showed that StSLT2 was involved in the regulating appressorium development and pathogenicity.6.The expression pattern of StSLT2 gene at the the five development stages(hypha,conidium,germ tube,appressorium and penetration peg)was analyzed by RNA-Seq technology.It was found that the gene had different level expression at the five development stages and was the highest expression level at the conidia development stage and the lowest expression level at the germ tube forming stage.