Cloning And Functional Characterization Of Wheat Genes Induced By Deoxynivalenol Mycotoxin

Deoxynivalenol(DON)is one of the trichothecene mycotoxins produced by Fusarium species including F.graminearum,which can cause Fusarium Head Blight(FHB)in wheat and the most prevalent contaminents of grains and feeds around the world.DON is a potent inhibitor of protein translation that targets the 60S ribosomal protein L3 in eukaryotes.DON also acts as a virulence factor in plants,promoting the spread of fungi in plant tissues.DON is highly toxic to humans and domestic animals;consumption of DON-contaminated cereals by domestic animals or humans can cause several chronic effects such as immune dysregulation,anorexia,and growth retardation,as well as acute poisoning leading to emesis and diarrhea.The knowledge of mechanisms underlying the plant resistance to FHB pathogens is still limited.Consequently,identification and screening of the plant genes resistant to DON and FHB are crucial in wheat breeding.Besides,study on the interaction between DON and plant cells is significant to reveal the mechanism of the occurrence of wheat FHB.The objectives of this study were:i)Screening DON-induced genes from DON-treated wheat suspension cells by suppression subtractive hybridization(SSH);cloning the full length sequences of the DON-induced genes and characterization of the functions in DON tolerance and resistance to F.graminearum,which may provide new type resistance genes for wheat breeding.ii)In order to uncover the gene interaction network and signal transduction between DON and wheat cells,high-throughput RNA sequence technology(RNA-seq)was performed to analyze the transcriptome changes in wheat in response to DON.The main results are as follows:I)Cloning and characterization of the full length of a DON-induced gene TaAn.The results indicated that the full length of TaAn was 523 bp,containing a 222 bp ORF encoding 71 amino acids.The TaAn was localized in the cytomembrane and nucleus.The expression of TaAn was induced by DON up to 17,040-folds 12 h post inoculation(hpi)in wheat cultivar Z9.The promoter of TaAn was cloned by TAIL-PCR,which contained a number of cis-elements,and could be used in wheat expression.Constitutive expression of TaAn in Arabidopsis plants conferred significant and durable tolerance to DON and resistance to E.graminearum.II)Cloning and characterization of the full length of a DON-induced gene TaUn.The results indicated that the full length of TaUn was 563 bp,containing a 168 bp ORF encoding 55 amino acids.The TaUn was localized in the cytomembrane and karyotheca.The expression of TaUn was induced by DON up to 28,033-folds 24 hpi in wheat cv.Z9.The promoter of TaUn was cloned by TAIL-PCR,which contained different cis-elements,and could be further used in wheat expression.Constitutive expression of TaUn in Arabidopsis plants conferred significant and durable tolerance to DON and resistance to F.graminearum.III)Cloning and characterization of the full length of a DON-induced gene TaMetRS.The results indicated that the full length of TaMetRS was 3,905 bp,containing 7 introns and a 1,791 bp ORF encoding 596 amino acids.The TaMetRS was localized in the nucleus.The expression of TaMetRS was only induced by DON or the DON-producing F.graminearum strain in wheat spikes,while the non-DON producing Tri5-strain did not activate the TaMetRS expression.Constitutive expression of TaMetRS in Arabidopsis plants conferred significant and durable tolerance to DON and resistance to F.graminearum.IV)Cloning and characterization of the full length of a DON-induced gene TaMATE.The results indicated that the full length of TaMATE was 2,572 bp,containing 7 introns and a 1,449 bp ORF encoding 482 amino acids.The TaMATE was localized on the cytomembrane.The expression of TaMATE was induced by DON and F.graminearum strain in wheat spikes.Constitutive expression of TaMATE in Arabidopsis plants conferred significant and durable tolerance to DON and resistance to F.graminearum.V)RNA-seq was performed to analyze the transcriptome changes in wheat in response to DON.The results clarified the gene interaction network between DON and plant cells,and a model of the interaction between DON and plant cells was deduced from the results;i)DON could make many damages to plant cells,such as inducing the expression of pore-forming toxin-like protein Hfr-2(PFT),which disrupts the plasma membrane integrity;DON could bind to the ribosome to repress the protein biosynthesis,and activate retrotransposons,which would result in an overwhelmingly high level of mutation;ii)DON induced the pathways which facilitate F.graminearum spreading in the plant.Purtrescine biosynthesis pathway was activated by DON as this biosynthesis supplied nutrients for F.graminearum growth on wheat;iii)DON also induced the ribosome biosynthesis pathway to rescue the protein biosynthesis repression and induced UDP-glucosyltransferase and glutathione S-transferase and many transporters to reduce the damage caused by DON;iv)DON tiggered plant immunities PTI and ETI simultaneously as well as other defense responses and detoxification pathways to fight against F.graminearum.In addition we confirmed that DON tiggerred plant immunities PTI and ETI simultaneously in Arabidopsis,in which the epoxy structure plays a pivotal role.