| Powdery mildew,caused by Blumeria graminis f.sp.tritici,is one of the most important cereal fungal diseases resulting in serious yield loss in wheat(Triticum aestivum).Breeding for disease-resistant cultivars is a major measure to control this disease.The discovery of disease resistance genes and understanding of the interaction mechanism between wheat plants and the fungus is the theoretical basis and source of resistance genes for improving powdery mildew resistance.To this end,we carried out the following three parts of studies.(1)A pair of residue heterozygous lines H962R(highly resistant to powdery mildew)and H962S(highly susceptible to powdery mildew)with similar genomic compositions were used to construct a RIL population for mapping the powdery mildew-resistance gene in H962 R.An array of 103 Bgt isolates collected from different wheat producing areas were used to compare the reaction patterns between the H962 lines with 21 known powdery mildew resistance genes.Line H962 R produced similar reaction patterns as those of the differential cultivars carrying Pm5 e alleles,such as Fuzhuang 30(Pm5e),Hongquanmang(Pm H)and Xiaobaidong(Mlxbd).RNA-Seq analysis was used to reveal the early interaction events between the two H962 lines and isolate Bgt1.A total of 155 genes that were differentially expressed were selected.Primers were designed based on these gene sequences,and two pairs of primers Xics-NL2 and Xics-X5 were polymorphic between the parents,and X5 was co-segregated with the phenotype.Since Xics-X5 is designed based on the sequence of Traes CS7B01G441700.1,the functional marker of Pm5 e,Pm5e-KASP,was used to genotype the RIL population,and it was cosegregated with the phenotype.The gene-specific primers were used to amplify the full length of Pm5 e coding sequences from H962 R and H962 S.The Pm5 e sequence of H962 R was exactly identical with that of Fuzhuang 30(Pm5e),confirming the disease resistance gene in H962 R is Pm5 e.(2)The 2-DE and i TRAQ combined mass spectrometry approaches were used to study the proteomic changes of the early interaction between the two H962 lines with isolate Bgt1.Through GO and KEGG enrichment,disease resistance gene annotation,transcription factor annotation and protein-protein interaction network analyses,we studied the process from pathogen recognition to signal transduction and further to defense responses.Five candidate genes CDPK6,Hsp90,HCHIB,Glb3,and CAT3 were functionally verified by VIGS.It was found that transient silencing of these genes can result in the susceptibility of H962 R,indicating that these genes participate the Pm5e-mediated defense reactions in the H962 lines.(3)The compatible and incompatible interaction between Liangxing 99(carrying Pm52)and the Bgt isolates(A21 and Bgt1,respectively)were studied using transcriptome,proteomic and metabolomic techniques.Transcriptome studies showed that the compatible and incompatible interactions between Liangxing 99 and Bgt were most different at 48 h after inoculation(hai).The DEGs were mainly involved in cinnamic acid biosynthetic process,phenylpropanoid biosynthesis,phenylalanine metabolism,glutathione metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,recognition of pollen,calcium ion transmembrane transport,regulation of systemic acquired resistance,plant-pathogen interaction,and MAPK signaling pathway.Proteomic and metabolomic studies found that most proteins and metabolites were also different at 48 hai,and picked at 72 hai.These proteins and metabolites were mainly involved in defense response to fungus,protein phosphorylation,hydrogen peroxide metabolic process,phenylpropanoid biosynthesis,flavonoid biosynthesis,alpha-Linolenic acid metabolism,tryptophan metabolism,tyrosine metabolism,glutathione metabolism,stilbenoid,diarylheptanoid,and gingerol biosynthesis.Results of multi-omics combined analyses found that α-linolenic acid,tryptophan,cysteine,methionine and glutathione metabolism,as well as the biosynthesis of zeatin,penylpropanoid,favonoid,silbenoid,diarylheptanoid and gingerol,were involved in the interaction between Liangxing 99 and Bgt isoaltes,both in the compatible and incompatible interactions.In addition,the metabolites OPDA,methyl jasmonate,serotonin,tryptophan,and indole may be associated with powdery mildew resistance. |
