Functional Analysis Of TaMCA5 Involved In Resistance To Fusarium Head Blight And Identification Of Mutants Of Sumai 3 Susceptible To Fusarium Head Blight

Wheat Fusarium head blight（FHB）is a fungal disease that is widespread worldwide.It is mainly caused by Fusarium graminearium Schwabe（F.g）in humid regions which adversely affects yield and quality of wheat.Cultivation of wheat varieties resistant to FHB is the most economical,effective and environmentally friendly measure for the control and prevention FHB.Exploration of the resistance mechanism is important for breeding of FHB resistance verieties.Due to the diverse types of wheat FHB resistance,and the infection process of F.g involves both biotrophic infection stage and necrotrophic expansion stage,the mechanism of wheat FHB resistance is very complicated.Programmed cell death is a critical mechanism of disease resistance produced by plants to limit pathogen infecton and developemt,however,nechtrophic pathogen can induce plant cell death in host for successful infection.The later stage of F.g infection involves necrotrophic expansion stage.Therefore,mining and functional research on genes involved in the regulation of cell death at this stage will help to understand the regulatory mechanism of wheat FHB resistance.Mutants are important materials for mining important genes and revealing molecular networks in plants.The creation and in-depth study of FHB-susceptible mutants can be better used for analyzing the molecular network of FHB resistance.In the early stage of the laboratory,it was found that the wheat cysteine protease gene TaMCA5 participated in the resistance pathway in the FHB resistant variety 06Y86,and silencing of this gene can effectively compromise the FHB resistance of 06Y86.In this study,the yeast two-hybrid technique was used to screen the interaction protein of TaMCA5.The interaction protein gene Ta EIN3 was cloned and functionally studied,and the molecular mechanism of TaMCA5 involved in the regulation of FHB resistance was analyzed.In addition,the phenotypic and molecular identification of the susceptible mutant produced by the EMS mutagenesis of Sumai 3 was carried out.The differentially expressed genes that respond to F.g between wild Sumai 3 and the mutant were screened,and then the network invoved the differential expressed genes were constructed.The main findings are as follows:1.Functional analysis of TaMCA5 participated in the FHB resistanceTaMCA5 is a cysteine protease gene in wheat whcih plays an important role in the process of PCD.In the previous research,it was found that TaMCA5 played a positive role in FHB.In this study,in order to analyze the molecular mechanism of TaMCA5-mediated resistance to FHB,the interaction proteins of TaMCA5 were screened and functionally studied.Using TaMCA5 as the bait protein,the protein fragments that interacting with TaMCA5 were screened by yeast two-hybrid technology,and a key protein Ta EIN3 in the ethylene signaling pathway was discovered.Using VIGS system to make Ta EIN3 silence,it was found that Ta EIN3 the resistance to FHB was decreased in the gene silenced plants.It was indicated that Ta EIN3 may play a positive regulatory role in synergy with TaMCA5 in FHB resistance,but its synergistic mechanism still needs to be further explored.2.Phenotypic identification and genetic analysis of Sumai 3 susceptible mutantSumai 3 is a wheat variety with high resistance to FHB,and it carries multiple head blight resistance QTLs.In the previous study,about 20 FHB susceptible mutants were produced by EMS mutagenesis through evaluation by single flower instillation.This study continued to evaluate the resistance of these putative mutants in multiple locations and multiple years,and at the same time the agronomic traits of these mutant were also investigated.It was found that some susceptible mutants had significant changes in plant height,ear length and other traits.Among them,plant height and resistance displayed a certain degree of correlation in SFM-12.In order to geneticly analyze of the mutation site of the mutant,the mutant was crossed with the wild-type Sumai 3 and the Fhb1-carrying resistant variety 06Y86.Then,the produced F₁generation plants were tested for resistance to FHB resistance,and it was found that all the F₁ individual plants showed similar level of resistance to FHB as that of whet wild type,indicating that recessive mutations occurred in some disease resistance positively regulated genes.3.Transcriptome analysis of mutants induced by F.gIn order to study the disease resistance mechanism of Sumai 3 using the developed susceptible mutants,the differentially expressed genes in response to FHB were screened and analyzed.The wild type and the mutant plants were inoculatated with Fg0609,then RNA-seq data were performed in the samples inoculated at 0h,24h,and72h,and the gene expression profiles of the two materials at three time periods were obtained.Using GO,KEGG and other databases to analyze the differentially expressed genes between the two materials at different time periods,it was revealed that phenylpropane biosynthesis,plant MAPK signal transduction,flavonoid biosynthesis etc responded to pathogen in both materials,but genes enriched in the MAPK cascade pathway were found upregulated in the susceptible mutants.