Expression And Functional Characterization Of Wheat Antimicrobial Peptides Genes Induced By Deoxynivalenol

Fusarium head blight（FHB）,caused by Fusarium graminearum（Fg）,is a devasting fungal disease in creal crop worldwide.In addition to reducing crop yields,Fg can also produce a variety of mycotoxins in infected grains,especially Deoxynivalenol,posing a grave threat to the food safety.Despite of its cytotoxic effect on plant and mammalian cells,deoxynivalenol also acts as an important virulence factor of Fg,and play a vital role in Fusarium-wheat interaction,which can promote symptom development after initial wheat infection.Previously,a DON induced Zhengmai 9023 wheat embryogenic suspension cell line c NDA suppression subtractive hybridization library was constructed.Four DON induced up-regulated full-length genes TaAn,Ta Un,Ta221 and Ta C7 were cloned by Rapid amplification of c DNA ends（RACE）.TaAn encoding a ankyrin-like protein,while Ta Un has no annotation among the existing database,and their biological functions have not yet been well elucidated.On the basis,this study carried out the following contents:1. Identification an novel antimicrobial peptides TaAn,which inhibited the germination and development of multiple pathogenic fungal species.TaAn,coding for a 73 amino acids short peptide,was located in wheat 3A,3B and 3D chromosome,with a secondary structure comprised of twoα-helixes and oneβ-sheets.TaAn exhibited DON binding affinity with a K_d value of 91μM measured by Microscale thermophoresis assay.GST Fusion protein of TaAn gene was expressed in E.coli expression system and purified via affinity chromatography.Germination and development Fg conidia was significantly inhibited by TaAn-GST fusion protein or chemical synthetic N-terminal FITC-Ahx labled TaAn peptide.The mechanisms of action of TaAn was demonstrated by flow cytometric analysis and fluorescent microscopic assay,that TaAn inhibited Fg conidia gemination by directly interacting with the cell membrane components of fungus spores.The antifungal activity of TaAn against Fg conidia showing a minimal inhibitory concentration of 96μM.Furthermore,TaAn also exhibited a broad spectrum resistance against both Fusarium graminearum and Alternaria alternate.TaAn can improve wheat resistance against Fg,we determinated the lesion length of coleoptile and relative biomass of fungi three days after coleoptile inoculation with mixture of TaAn peptide and Fg conidia.The results showed that TaAn can significantly reduced the relative biomass of fungi and the lesion length of the coleoptile by 70%and 72%,respectively.Overall,TaAn can improve wheat resistance against Fg 5035.2. Identification another novel antimicrobial peptides Ta Un,which inhibited the germination and development of multiple pathogenic fungal species.Ta Un,coding for a 55 amino acids short peptide,was located in wheat 7A,7B and 7D chromosome,shared a similar secondary structure consisted of twoα-helixes and twoβ-sheets.GST Fusion protein of Ta Un gene was expressed in E.coli expression system and purified via affinity chromatography.Germination and development Fg conidia was significantly inhibited by Ta Un-GST fusion protein or chemical synthetic N-terminal FITC-Ahx labled Ta Un peptide.The mechanisms of action of Ta Un was demonstrated by flow cytometric analysis and fluorescent microscopic assay,that Ta Un can influence the prolification of pathogen in a manner of targeting both the cell membrane and cytoplasm compotents of fungus spores.The antifungal activity of Ta Un against Fg conidia showing a minimal inhibitory concentration of 154μM.Furthermore,Ta Un also exhibited a broad spectrum resistance against both Fusarium graminearum and Alternaria alternate.Ta Un can improve wheat resistance against Fg,we determinated the lesion length of coleoptile and relative biomass of fungi three days after coleoptile inoculation with mixture of Ta Un peptide and Fg conidia.The results showed that Ta Un can also significantly reduced the relative biomass of fungi and the lesion length of the coleoptile by 59%and 51%,respectively.Overall,Ta Un can improve wheat resistance against Fg 5035.3. DON induced genes TaAn,Ta Un,Ta221 and Ta C7 increased FHB resistance of transgenic wheat.DON induced genes TaAn,Ta Un,Ta221 and Ta C7 were constructed into gene gun expression vector and Agrobacterium vector,separately,then transformed into susceptible wheat cultivar Xiangmai 76,seedling inoculation indicated that the transgenic lines significantly improve the FHB resistance by 40%-60%as compared with Xiangmai 76.4. Identification of five candidate wheat genes FS2,FS3,FS7,FS12 and FS15,encoding PAMP recognition receptors proteins based on RNA-Seq analysis of different expressed genes in wheat spikes after deoxynivalenol treatment.They shared a canonical structure consist of extracellular leucine rich repeat,transmembrane domain and intracellular Ser/Thr kinase domain.GST Fusion protein of Ser/Thr kinase domain of FS2,FS3,FS7,FS12 and FS15 genes were expressed in E.coli expression system and purified via affinity chromatography,then their kinase activity was assessed by phosphorylation assay.All FSK proteins showed exogenic substrate phosphorylation activity against Myelin basic protein.LRR domains of FS2,FS3,FS7,FS12 and FS15were predicted to had capacity of DON binding affinity by SWISS-MODEL homologous modeling and molecular docking,but Far Western Blot assay failed to prove the intact interactions between candidate wheat LRR domains and DON.Additionaly,a Arabidopsis ethyl methanesulfonate mutants library was conducted.250,000 M₂ seeds were subjected to screen DON-resistant mutants,no desirable DON-resistant seedling was obtained.