| Fusarium head blight(FHB)is a devastating disease caused by Fusarium graminearum Schwabe [teleomorph Gibberella zeae(Schw.)Perch],resulting in great yield and economic losses in global wheat production each year.It is urgent to prevent wheat FHB and reveal the mechanistic basis of host plant resistance to infection by F.graminearum.However,due to the scarcity of wheat FHB resistance sources and the complexity of its own resistance mechanism,little is known about wheat FHB so far.As an effective tool,'omics' analysis can accelerate our study on the mechanism of wheat FHB.In this study,the wheat cultivar Sumai 3 was used as the research material.Firstly,the Sumai 3 leaves inoculated with F.graminearum were analyzed by integrating metabolomics and transcriptomics.On this basis,the resistance mechanism of wheat FHB was analyzed.The FHB resistance-related pathways were explored,and the key genes of wheat FHB resistance were found.The key FHB resistance genes were verified by transgenic and other molecular biological techniques.The main research contents are as follows:1.In order to gain insight into metabolic changes following infection with F.graminearum,a metabolomics platform which integrates ultra-high-performance liquid chromatography(UHPLC)and tandem mass spectrometry(MS/MS)was applied.The leaf tissues of the wheat cultivar “Sumai 3”,as inoculated with four different F.graminearum races(R40,R64,S52,S66)were examined via metabolomic analyses.The differences were screened according to the criteria of fold change ? 2and fold change ? 0.5.The metabolic profiles were characterized at day 4 post F.graminearum inoculation.It was clear that Sumai 3 leaves were much more resistant to F.graminearum races R40 and R64 than to the S52 and S66 races.Our metabolomics data were detected 789 metabolites with known structures in wheat.There were 215,213,206,and 198 significantly differentially accumulated metabolites,respectively,in the comparisons of the mock inoculation control VS R40,R64,S52,and S66.Among the differentially accumulated metabolites,220 metabolites were the flavonoid class(e.g.,flavonols,flavones,flavanones,isoflavones,anthocyanins,catechins),28 metabolites were involved in plant hormone metabolism(e.g.,SA,JA,IAA)biosynthesis and signal transduction pathways,and 57 metabolites were related to the metabolism of tryptamine,phenolamides,alkaloids,or cholines.2.Transcriptome analysis were performed by Illumina HiSeq 2000 sequencing platform.We used chinese spring gene annotation file as reference genomic information and Pval < 0.05 as significant differential expression evaluation criteria to screen for differential genes.The results showed that the expression levels of a large number of differential genes were changed.In order to better understand the influence of F.graminearum infection on the relationship between the metabolite levels and the genes of the biosynthesis pathway,an analysis of the metabolome alongside the RNAseq based transcriptome data for inoculated leaves was performed based on Pearson's correlation coefficient values.All of the metabolites and biosynthesis pathway genes were combined to establish a network to illustrate relationships.This analysis examined that a total of 16 genes were found in the flavonoid biosynthesis pathway:when considering the data for each of the F.graminearum races together,PAL,PTAL,CL,CYP73 A,CYP75B1,ANS,and FLS were up-regulated compared to the control,while the CHS and ANR genes were down-regulated.15 genes were enriched in the salicylic acid pathway,among which PAL,PTAL,PR1,and TGA were significantly up-regulated in F.graminearum-inoculated leaves compared to mock control leaves,whereas NPR1 was significantly down-regulated.Fifty-five auxin-related genes were enriched upon F.graminearum infection,among which AROA,AROC,TRPD,TRP1,TRPA,TRP3,AROKTAA1,AAO,YUCCA,and GH3 were up-regulated compared to controls,while ALDH,AUX1,TIR1,AUX/IAA,and ARF were down-regulated.Twenty-two jasmonic-acid-related genes were enriched upon F.graminearum infection: LOX,OPR,AOS,AOC,and JAZ1 were up-regulated by F.graminearum infection compared to controls,while JAR1,COI1,and MYC2 were down-regulated.An integrative analysis of the metabolome and transcriptome data revealed significant differences in the expression of many phenolamide and tryptamine pathway genes,among which three genes(ARD,ODC1,TYNA)were up-regulated in response to F.graminearum infection.In contrast,the ARG,PAO,and SPE genes were significantly down-regulated upon F.graminearum infection.3.Our metabolome and transcriptome analyses revealed that the phytohormone auxin was responsive to F.graminearum infection.These results showed that exogenous auxin treatment could apparently aggravate the incidence of wheat FHB.Itsuggested that auxin pathway played a certain role in wheat resistance to FHB.In addition,we confirmed that TaTIR1 expression was responsive to treatment with F.graminearum,SA,JA,IAA and ABA.We selected auxin receptor gene TaTIR1 as a candidate gene to verify the role of TaTIR1 in wheat FHB resistance.Compared with wild type,TaTIR1 RNAi transgenic lines were more resistant to FHB.The stereo fluorescence microscope observation results showed that there was no significant difference in the infection mode of F.graminearum in panicle between TaTIR1 RNAi transgenic lines and wild-type plants.Moreover,our scanning electron microscopy analysis showed that there was a reduced amount of F.graminearum hyphae in the rachis of TaTIR1 knockdown compared to WT plants.To further characterize the mechanism through which TaTIR1 contributes to FHB resistance,we used qPCR to analyze the expression of known TaTIR1 downstream genes.This showed that upon F.graminearum infection TaTIR1 regulates the expression of genes including AUX,AUX/IAA,ARF,GH3,and SAUR;the levels of transcripts for all of these genes were significantly decreased(by ~2-3 fold)in TaTIR1 knockdown plants compared with WT plants.Our study therefore provides new insights about how wheat responds to F.graminearum infection and about mechanisms of FHB resistance,while also illustrating a promising application of TaTIR1 knockdown to increase FHB resistance in crop improvement programs.4.The resistance of wheat to FHB is mainly controlled by type II resistance(inhibition of the spread of F.graminearum through wheat rachis).As a new monocotyledon model plant,it has been shown that Brachypodium distachyon 21(Bd21)could interact with F.graminearum,while further study is needed to evaluate the disease development in spike in detail.Here,we found that F.graminearum spores mainly germinate on the pistil of Bd21,and then the hyphae rapidly extend to the bottom of the florets and enter the spike rachis,which was similar to the infection process of F.graminearum in wheat.However,there were differences in the spike rachis structure between Brachypodium distachyon and wheat.In our study,we found that the spread of the fungus through the rachis node of inoculated spikelets could be used as an important index to evaluate the resistance of FHB type II in Brachypodium under optimal conditions at 28 °C and 50%–70% humidity.5.In order to verify the strategy feasibility of the Brachypodium distachyon in F.graminearum infection,we constructed overexpressed transgenic plants oftranscription factor TaTGA2,and found that TaTGA2 transgenic plants could enhance resistance to F.graminearum in both spikes and detached leaves.In order to further verify the resistance of TaTGA2 to FHB in wheat,we found that TaTGA2 silencing plants could increase FHB severity through the barley stripe mosaic virus induced gene silencing(BMSV-VIGS)(about 20%).The identification results of FHB resistance in wheat were consistent with Brachypodium distachyon.It was showed that the study on the resistance of wheat FHB was feasible by using the Brachypodium distachyon as the model plant.In addition,we also screened and identified another 49 Brachypodium germplasms with FHB resistance,and found some materials with different resistance to FHB,such as Bd21,Bd3-1. |
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