Identification And Functional Analysis Of Susceptibility Candidate Genes To The Powdery Mildew In Triticum Aestivum

In recent years,wheat powdery mildew caused by Blurmeria graminis f.sp.tritici occurred fluently and widely in China.It adversely affected the yield and quality of wheat,therefore,it is particularly important to develop and cultivate disease-resistant wheat varieties in production.At present,researches about resistance to powdery mildew mostly focus on exploring resistance genes and the pathogenesis-related genes,but there are few reports about susceptibility genes or susceptibility mechanisms in wheat.Susceptibility gene is a kind of plant protein that is required for host susceptibility to a certain pathogen.One type is susceptibility gene that negatively regulates disease resistance reactions;the other type is susceptibility factor which fulfills growth and reproduction of pathogen.The resistance caused by loss of susceptibility gene function was always persistent and broad-spectrum.In barley,the mlo mutant results in complete and race-nonspecific resistance to otherwise virulent barley powdery mildew fungi,and it had been applied in production for more than 30 years.In this study,the gene expression profiles of two groups of resistant and susceptible near isogenic lines were sequenced by next-generation sequencing technology.The gene expression data were compared between two resistant materials and the corresponding susceptible near isogenic lines,and 14 susceptibility candidate genes(SCG)that only up-regulated in susceptible materials after infection of Bgt were identified,including TaHBP encoding heme binding protein,TaFBX containing the conserved F-box domain and TaABC encoding an adenosine triphosphate-binding cassette transporter protein.Evolutions and expressions of these three SCG were further studied and their functions were analysed based on RNAi technology.The results showed that these three SCG played negative roles in resistant reactions to wheat powdery mildew.1.Screening susceptibility candidate genes to powdery mildew in wheat by the next-generation sequencing technologyTwo near-isogenic lines were inoculated by Bgt,including resistant Tstpk-V transgenic wheat vs susceptible recipient Yangmai158 and resistant Nannong 9918 vs susceptible mutant NM14.The RNA was extracted from leaves of non-inoculated samples and Bgt inoculated samples after 24 hours,then the gene expression profiles were sequenced by the next-generation sequencing technology.The number of distinct tag in each material was more than 130,000,and specific tags ranging from 26,000 to 34,000 could be matched with the wheat unigene data.The gene expression data were compared between two resistant materials and the corresponding susceptible near isogenic lines,and the result showed only 14 genes were up-regulated in susceptible materials but down-regulated or no response to Bgt in resistant materials,so they were identified as susceptibility candidate gene(SCG).2.Bioinformatics analysis of susceptibility candidate geneAmong these 14 susceptibility candidate genes,three genes which had putative functions were studied by bioinformatics analysis,including TaHBP gene encoding a heme binding protein,TaFBX gene encoding an F-box domain containg protein,and TaABC gene encodeing an adenosine triphosphate-binding cassette transporter protein.Bioinformatics analysis located TaHBP to the chromosome 7AS,TaFBX to chromosome 5AL,and TaABC to homoelogpus group 6 chromosomes.3.The expression pattern analysis of susceptibility candidate genesThese susceptibility candidate genes were differentially expressed in the two resistant materials which showed broad-spectrum resistance to powdery mildew,for example,Nannong 9918 contained the Pm21 gene and Stpk-Ⅴ transgenic plants contained the key gene from the Pm21 locus,so the expression pattern of the SCG were analysed by QRT-PCR in the different varieties from different ecology districts containing the Pm21 gene after Bgt inoculation,including Shimai 14,Neimai 8 and Lantian 17.The result showed that TaHBP expression level was significantly decresed in all these materials after Bgt inoculation for 24 hours,the expression level of TaFBX was decresed in Shimai 14 and Neimai 8,but not changed in Lantian 17,and the expression level of TaABC had a significant decline in all these materials.It was proposed that the TaHBP and TaABC mightplay negative roles in defense response mediated by Pm2l.At the same time,the expression patterns of these three genes were also studied in other Bgt resistance pathway mediated by Pm2 or Pm4a.The result showed that the expression of TaHBP and TaABC were down-regulated in Pm2 and Pm4a containing materials after Bgt inoculation for 24 hours,while TaFBX was significantly down-regulated in Pm4a,but slightly down-regulated in Pm2.Taken together,functions of TaHBP,TaFBX and TaABC were all conserved in powdery mildew resistance pathway mediated by Pm2 or Pm4a.4.Functional analysis of susceptibility candidate genesTo study the functions of TaHBP,TaFBX and TaABC in powdery mildew resistance pathway,several approaches were carried out including knockdown of genes by TIGS(transient induced gene silencing)at the single-cell level and using RNAi transgenic technology to silence these three genes at the plant level.The parameter of ’haustorium index’ was used to evaluate functions of the genes at single-cell level,and the ’resistance scale’ was used to evaluate function of the genes at the plant level.The results showed that silencing of these three genes in Yangmai158 leaves leading to decresing of haustorium index,for example,haustorium index was decreased from 51.1 percent to 40.2 percent in the TaHBP silenced cells,from 51.8 percent to 43.8 percent in the TaFBX silenced cells,and from 52.4 percent to 43.5 percent in the TaABC silenced cells.Stable transgenic wheat plants expressing RNAi cassette in recipient Yangmail58 were produced by gene gun bombdment.The TaFBX RNAi T0 generation plants were tested with Bgt inoculation for resistant evaluation,and three plants were identified for a consistently decreased colonization of Bgt.The resistance levels of the T1 generation plants of the corresponding three T0 plants were segregated at seedling or adult stage.QRT-PCR indicated that TaFBX was silenced successfully in the resistance improved plants.So,TaFBX played a negative role in the resistance pathway to Bgt.For TaABC,four segregating lines were identified in the T1 generation,and the corresponding T0 generation plants of these lines also showed an enhanced resistance level,indicating that TaABC played a negative role in Bgt resistance pathway.For TaHBP,three positive plants were identified by PCR amplification from 48 regenerated transgenic plants of T0 generation in the field,and the resistance needs to be evaluated using the T1 generation.In summary,it is a feasible strategy to screen susceptibility candidate genes by using the near isogenic lines containing resistant and susceptible materials,and high consistency of their genetic backgrounds could improve the efficiency of screening.Silencing of susceptibility candidate genes in susceptible materials would enhance their resistance to Bgt,and provide new gene resources and strategies for resistance improvement in wheat breeding.