| Wheat powdery mildew and Fusarium head blight(FHB)are the two devastating fungal diseases in wheat,which conferred severe losses in grain yield and quality every year.Compared to the chemical control,breeding of disease resistance wheat cultivars is the most effective method to control the losses.So for the wheat improvement,cloning of genes related to disease-resistance from wild relatives not only provide new genetic resources to wheat genetic engineering,but also help us to understand the molecular mechanism of wheat disease resistance.Haynaldia villosa L.(Dasypyrum villosum,2n=14,VV),an annual diploid relative of wheat from tertiary gene pool,possesses high level of resistance to several wheat diseases,such as powdery mildew,rusts,wheat yellow mosaic virus(WYMV)and full rot.In the previous work,a full length cDNA sequence of the CMPG1-V gene,which is rapidly induced by Blumeria graminis tritici(Bgt)inoculation,was isolated from H.villosa using GeneChip microarray analysis and cDNA library screening.Over-expressing CMPG1-V transgenic wheat showed improved broad-spectrum resistance to Bgt at seedling and adult stages which was found to be associated with an increasing expression of salicylic acid-responsive genes,H2O2 accumulation and cell-wall protein cross-linking at the Bgt infection sites.To further characterize the mechanism underlying CMPG1-V signaling pathway,we performed yeast two-hybrid(Y2H)and identified the protein ToxABP1-V(Toxin A Binding proteinl).The main results obtained in this research project are as follows:1.Cloning of ToxABPl-V from H.villosa:The cDNA sequence of ToxABP1-V obtained from H.villosa comprised of 1095 bp,which contained an ORF of 861 bp,encoded a protein of 286 amino acid residues,with a pI of 9 and a predicted molecular weight of 32.3kDa.Sequence analysis showed that ToxABP1-V contains a chloroplast transit peptide(cTP),with cleavage occurring after Arg48.Moreover,a coiled-coil motif from amino acid 231 to 266 was also found in this protein.Six genomic DNA sequences of ToxABP1s,which contained five exons and four introns,were also isolated from common wheat,Triticum monococcum,Aegilops tauschii and H.villosa.ToxABP1-V was mapped to the short arm of chromosome 2V of H.villosa.ToxABP1-V orthologue in barley,is located on the chromosome 2HS.Three ToxABP1s from wheat were assigned to short arm of homeologous group 2 chromosomes.ToxABP1-V orthologues in B.distachyon and rice,are located on the chromosome Bdl and chromosome R7,respectively.Both chromosome Bdl and rice chromosome R7 are syntenic to the short arm of homeologous group 2 chromosomes of wheat.2.Subcellular localization and expression pattern of ToxABP1-V:ToxABP1-V encoded a chloroplast localized protein which was predicted to have function in thylakoid formation and the biogenesis of PSII.ToxABP1-V exhibited tissue specific expression pattern,as high expression of ToxABP1-V was found in leaves than culm and spikelet,in addition,low ToxABP1-V transcript level was detected in callus and root.In H.villosa,expression of the ToxABP1-V sharply reduced to 0.02 folds after 24 h of Bgt inoculation.No significant change in expression level of ToxABP1-V was observed after chitin and flg22 treatments.3.Function analysis of ToxABP1-V in response to wheat powdery mildew:At first,single-cell transient silencing assay was used to elucidate the function of ToxABP1-V in response to wheat powdery mildew.It was observed that the haustorial index(HI)was 50.16%in moderately susceptible variety Yangmai158,when inoculated by E26.However,the HI was significantly decreased to 18.74-19.24%,when ToxABP1 was transiently silenced in Yangmai158.Silencing of ToxABP1 using VIGS(Virus Induced Gene Silencing)assay in Yangmai158 and CMPG1-V transgenic wheat plants enhanced resistance to powdery mildew,by triggering the expression of ROS generating/scavenging genes,SA pathway genes and JA pathway genes.We generated transgenic silenced ToxABP1 wheat plants with particle bombardment method.Three positive RNAi transgenic wheats exhibiting 3-8%ToxABP1 expression level showed enhanced resistance to powdery mildew.The resistance to susceptibility segregation ratio of their derived T1 lines:RNAi-ToxABP1-T1-1,RNAi-ToxABP1-T1-2 and RNAi-ToxABP1-T1-4 were 3:0,8:4 and 9:2,respectively.We had also developed ToxABP1-V over-expressed transgenic wheat plants.18 positive plants,which exhibited highly susceptible to powdery mildew,were identified by western blot.ToxABP1-V-T1-9 derived from ToxABP1-V-T0-172 showed high susceptibility to Bgt mixture in field conditions.All these results clearly indicates that ToxABP1s negatively regulates wheat powdery mildew resistance.4.Mechanism analysis of ToxABP1-V meadiated disease resistance:After H2O2,methyl jasmonate(MeJA),and ethylene(ET)treatments,ToxABP1-V transcripts reduced at 1-12 h post treatment(p.t.)and then up-regulated at 24h p.t.The expression of ROS generating/scavenging genes,SA pathway genes and JA pathway genes were high in ToxABP1 RNAi positive plant after the inoculation of Bgt mixture.In addition,hypersensitive response(HR)and ROS accumulation triggerred by E26 in CMPG1-V transgenic plant were light dependent.Compared to Yangmail58,ToxABP1-V RNAi plant exhibit higher ROS accumulation and stonger cell death after Bgt inoculation using DAB and trypan staining.Yangmai158 exhibits high susceptibility(infection grades 4)to E26,and ToxABP1 was slightly down-regulated after 24h E26 inoculation in Yangmail58.CMPG1-V transgenic wheat(in the background of Yangmail58)exhibits high resistance(infection grades 0)to E26,and ToxABP1 transcripts significantly down-regulated after E26 inoculation in CMPG1-V transgenic wheat.However,Both Yangmai158 and CMPG1-V transgenic wheat exhibit high susceptibility(infection grades 4)to E31,then the ToxABP1 expression level in both Yangmai158 and CMPG1-V transgenic wheat slightly down-regulated after E31 inoculation.Yeast two hybrid showed that the strong interaction was detected between CMPG1-V or ARM domain and the ToxABP1-V C-terminal 233aa.Pull-down assay showed ToxABPl-V directly interacted with CMPG1-V.An in vitro ubiquitination assay showed that ToxABP1-V can be polyubiquitinated by CMPG1-V.In addition,cell-free degradation assay showed that ToxABP1-V can be degraded by CMPG1-V.Compared to coexpreession with pCambia 1305.1-GFP or 35S:GFP-CMPG1CW(lost ubiquitination activity),ToxABPl-GFP was lower accumulation when coexpressed with 35S:GFP-CMPG1,moreover,MG 132 treatment increased the protein level of ToxABP1-GFP under same experimental conditions.It indicated that ToxABP1 was a substrate of CMPG1-V.5.Function analysis of ToxABP1s in wheat FHB resistance in Sumai3 using VIGS:The results showed that compared to BSMV:?,Sumai3 exhibits enhanced FHB susceptibility after inoculation with BSMV:ToxABP1.qRT-PCR assay showed that ToxABP1s expression level was significantly decreased after inoculation with BSMV:ToxABP1.Compared to BSMV:?,the expression of JA pathway genes,TaCOI1,TaPR3 and SA pathway genes,TaPR1,TaPR2 were decreased in BSMV:ToxABP1 plants.However,the expression of ROS generating/scavenging genes,TaNOX,TaGST were increased in BSMV:ToxABP1 plant.6.Phylogenetic analysis of LecRK-V and its response to wheat powdery mildew disease:In our previous work,we isolated LecRK-V from H.villosa.The expression of LecRK-V was rapidly up-regulated by Bgt inoculation and chitin treatment.Single-cell transient over-expression of LecRK-V led to decreased haustorial index in wheat variety Yangmai158.Stable transformation LecRK-V into Yangmai158 significantly enhanced the powdery mildew resistance at both seedling and adult stages.At seedling stage,the transgenic line was highly resistance to 18 of the tested 23 Bgt isolates,and hypersensitive responses(HR)were observed for 22 Bgt isolates,and more ROS was accumulated at the Bgt infection sites.The main results obtained were as follows:LecRK-V was mapped to the chromosome 5V of H.villosa.By searching databases,a total of 276 LecRKs were identified,and phylogenetic analysis showed 276 LecRKs could be classified into 11 different types.Southern blot analysis detected two additional copies of LecRK-V in genome of LecRK-V-T3-2 line.Dynamic changes were detected for the expression levels of ROS generating/scavenging genes and marker genes of the SA pathway,indicating that ROS and SA pathways may contribute to ROS accumulation and associated with the powdery mildew resistance mediated by LecRK-V. |
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