Molecular Mechanism Of Aspartic Protease Gene TiAP1 Against Wheat Powdery Mildew

The biotrophic fungal pathogen Blumeria graminis f.sp.tritici(Bgt)is a crucial factor causing reduction of global wheat production.Cultivating new wheat varieties resistant to powdery mildew and revealing its resistance mechanism has become an urgent problem to be solved.Wild wheat relatives are an important source of disease resistance genes.Among them,Thinopyrum intermedium has become one of the wild parents commonly used in the genetic improvement of wheat.Aspartic proteinase is widely distributed in eukaryotes and microorganisms,and participates in the body's metabolism and biological regulation,such as protein processing and degradation,biotic and abiotic stress,senescence and programmed cell death.The laboratory obtained an aspartic protease gene TiAP1 from T.intermedium in the early stage.After gene function verification,it was found that the TiAP1 gene can resist the invasion of pathogenic.To better understand its disease resistance mechanism,this study identified the resistance to Bgt in the overexpression of TiAP1 gene in wheat,cloned and analyzed the promoter of TiAP1 gene.Subsequently,the subcellular localization and accumulation of TiAP1were analyzed and the yeast two-hybrid(Y2H)experiment was used to screen the proteins that interact with TiAP1,and the interaction were verified by bimolecular luciferase(LUC)complementation imaging(LCI)and bimolecular florescent complimentary(BiFC)to verify the interaction.In addition,this study also used HIGS and VIGS combined with transcriptome analysis to preliminarily analyze the disease resistance molecular mechanism of TiAP1 gene.The main research results were as follows:1.Analysis of resistance to Bgt in wheat of the overexpression of TiAP1 gene.In the identification of resistance to Bgt in the seedling stage of the overexpression of TiAP1 gene in Bobwhite(OE1 and OE2)and the hybrid F₃between homozygous overexpression of TiAP1gene in Bobwhite and YN15(Line1-Line10),this study found the overexpression of the TiAP1gene can enhance resistance to Bgt of the recipients wheat.Through histological observation and statistics,it was found that the conidia development of Bgt mainly stayed in the appressorium(App)stage in the overexpressing TiAP1 gene wheat lines at 2 day post-inoculation(dpi)with Bgt,and the haustor index(HI)was significantly lower than that of the control.At the same time,this study also found that overexpression of the TiAP1 gene can enhance the resistance of the recipients wheat to Bgt at the adult stage,and the development of Bgt for 2 dpi at the adult stage are similar to those at the seedling stage.The above results indicate that the overexpression of TiAP1 gene in wheat can inhibit the formation of Bgt haustorium to enhance the resistance of the recipient wheat Bobwhite to Bgt.2.Analysis of ROS burst in adult stage of the overexpressing TiAP1 gene in wheat.By measuring the hydrogen peroxide content of the overexpression of TiAP1 gene in wheat inoculated with Bgt at different time points,we found that the hydrogen peroxide content of the overexpression of TiAP1 gene in wheat at the adult stage was significantly higher than that of the control Bobwhite,and the enzyme activities of SOD,POD and CAT related to hydrogen peroxide were also different in the transgenic lines and control.3.Cloning and analysis of the promoter of TiAP1 gene.In this study,a 2460 bp TiAP1gene promoter was cloned,and the structure of the promoter was analyzed.It was found that the promoter contains cis-acting elements that are related to signal transduction,drought,salt stress,and pathogen induction.The analysis of a series of promoter truncation experiments showed that in the P4G and P7G promoter segments,the promoter activity disappeared.In the P4 and P7 promoter segments,the promoter activity disappeared.It is speculated that the cis-acting elements WRKY71OS and GT-1 lacking in the P4G and P7G promoter segments may activate the promoter activity after being induced by pathogen.Therefore,this study constructed p ABi-WRKY71OS and p Ab Ai-GT-1 bait vectors,screened the yeast library,and obtained some candidate gene sequences that may interact with WRKY71OS and GT-1.4.Subcellular localization analysis found that TiAP1 was secreted protein.In Blumeria graminis f.sp.hordei(Bgh)-infected barley leaves transfected with the TiAP1 gene,a large number of TiAP1 protein was observed to accumulate in the primary germination tube(PGT)and appressorium(App)of Bgh,as well as the intercellular of barley leaves.5.Screening and verification of TiAP1 interacting proteins.In order to verify that the interaction of TiAP1 and wheat powdery mildew could cause the overexpression of TiAP1 gene in wheat to resist the invasion of powdery mildew.Utilization the constructed yeast library,the TiAP1 gene was recombined into the bait vector,and the Y2H mating SOP method was used,after sequencing and analysis,this study obtained three Bgt protein which were the ribosomal60s large subunit(Bgt60S),glycosyl hydrolase family 61(Bgt GHF61)and named Chitin deacetylase 1(BgtCDA1)that containing"polysaccharide deacetylation"domain,belongs to the class of chitin deacetylases(EC 3.5.1.41).Simultaneously,utilization the LCI and BiFC experimental systems,this study determined that TiAP1 interacts with Bgt60S,Bgt GHF61 and BgtCDA1 in tobacco cells,respectively.6.BgtCDA1 gene silencing can slow down the infection of Bgt.HIGS analysis found that1 dpi with Bgt,the HI in YN15 leaves silenced by BgtCDA1 gene decreased significantly compared with the control;BSMV-VIGS analysis showed the diseased area of BSMV:BgtCDA1as wheat leaves infected with Bgt for 5 days was significantly lower than that of control BSMV:00 wheat leaves,and the hypha density in wheat leaves of BSMV:BgtCDA1as for 3dpi with Bgt was also significantly lower than the hypha density in BSMV:00 wheat leaves.7.Transcriptome analysis of OE2 and Bobwhite wheat before inoculation with Bgt(Bob-0 and OE2-0)and 2 dpi with Bgt(Bob-2 and OE2-2).Through analysis,this study found that the gene Traes CS1A02G410500,Traes CS2B02G187500,Traes CS2A02G161500,Traes CS3B02G379200 and Traes CS1B02G440300 encoding WRKY transcription factors;PIL13,TIFY family genes and ERF transcription factors related to plant hormone signal transduction;at the same time,it was found a gene that is crucial in lignin biosynthesis,PAL encoded by Traes CS2B02G224300 and XTH30,a gene involved in xylan metabolism were up-regulated in OE2-2 relative to Bob-2.Based on the above results,this study speculate that there is a double-layer defense system in the overexpression of TiAP1 gene in wheat.First,the overexpression of TiAP1 gene in wheat induced by Bgt,its activates the expression of genes related to cell wall synthesis and metabolism,thereby blocking the penetration of Bgt conidia and enhancing the tolerance of the overexpression of TiAP1 gene in wheat to pathogens;Second,the interaction between TiAP1and BgtCDA1 inhibits the deacetylation activity of BgtCDA1 and stimulates the immune response triggered by chitin.The host's genes related to hormone signal transduction are also activated,and their expression is up-regulated after 2 d of induction by Bgt of the overexpressing TiAP1 gene in wheat.These two mechanisms in wheat enhance the resistance of wheat to powdery mildew,and provide a reference for plant genetic improvement.