Map-based Cloning And Functional Analysis Of CC-NB-LRR OsRLR1 In Rice (Oryza Sativa L.)

Rice?Oryza sativa L.?is the model plant for monocots and one of the main crops in the world wide as well.During the development,rice suffers kinds of biological and abiotic stresses,especially pathogens and insect pest diseases that can cause serious drop in rice yield and quality.At the same time,the extensive use of agriculture chemicals in the field has been causing a lot pollution to the environment and improved the tolerance of chemicals in pathogens and pests.Moreover,the usage of chemicals on crops planting cause serious concern in public about food safety.All together,the study of disease resistance mechanism in rice is of great theoretical significance and application value.The rice blast caused by agent fungus Pyricularia oryzae?P.oryzae?and bacterial blight caused by agent Xanthomonas oryzae pv.oryzae?Xoo?are two main diseases in rice,the beak out of which can both cause great damage to rice production.Thus,the research on P.oryzae and Xoo resistance study is of great importance to rice planting and environment protection.In this study,a new lesion mimic mutant,rlr1,was identified from EMS treated mutant bank from indica restorer line,Jinhui 10.Further study cloned candidate gene that yet unidentified,RPM1 like gene 1?OsRLR1?in rice.Besides gene expression study,OsRLR1 protein structure and defense response mechanism was analyzed.The main results are as follows:1.Gene mapping and cloning of OsRLR1HR-like lesions and early senescence phenotype was identified in rlr1 from early seedling stage and the appearance of the phenotype was induced by sunlight.Compared with the wild type,defence-related genes OsNPR1,OsWRKY45,PAL1,OsPR1a and OsPR10 were activated in mutant rlr1,which indicated the activation of defence response in plant.Disease inoculation study showed that rlr1 demonstrated enhanced resistance to rice blast and bacterial blight.Genetic analysis showed that the phenotype of rlr1 was controlled by a recessive nuclear gene that is located in a 66 kb region on chromosome 10 by map-based cloning using SSR and InDel molecular markers.Genome DNA and cDNA sequencing showed that only one base at 953 site changed?A to T?at the coding sequence of LOC_Os10g07978 was identified,causing amino acid change from Glu to Val.Therefore,LOC_Os10g07978 is regarded as the target gene that controls the phenotype in rlr1.2.the verification analysis of OsRLR1To verify whether LOC_Os10g07978 is the target gene,the coding sequence of LOC_Os10g07978 from the wild type was verified and overexpressed in mutant rlr1.No HR-like cell death and early senescence phenotype was observed in transgenic plants and the agronomy traits were recovered as well.Thus,the phenotype of rlr1 was recovered by overexpressing LOC_Os10g07978 from the wild type.At the same time,the disease resistance ability was reduced in transgenic plants:the transgenic plants showed reduced resistance to rice blast and bacterial blight.All toghether,LOC_Os10g07978 is OsRLR1,mutation of which is responsible for the phenotype in rlr1.3.Protein structure analysis of OsRLR1OsRLR1 encoded a typical CC-NBS-LRR resistance protein.The multiple sequences alignment and phylogenetic tree analysis showed that OsRLR1 was clustered well with other 19 CC-NB-LRR proteins from 10 species,in which OsRLR1 was most close to AtRPM1,ZmRPM1 and a blast resistance protein OsPid3 from rice.Sequence alignment showed that the mutation happened at the NB domain close to RNBS-B/Se motif.The 3D protein structure of ZAR1 was used to build the modeling 3D structure of OsRLR1.The result demonstrated that OsRLR1?E318V?is the same as that active ZAR1 structure binding ATP,which indicated that the mutation in OsRLR1?E318V?caused the failure of ATP hydrolysis or ADP binding to keep the protein in a sustained active state.Moreover,like most NLR protein in plant,OsRLR1 could form homodimers in yeast by its CC domain.OsRLR1 and OsRLR1?E318V?were both located in nucleus of rice and N.bencimiana cells.4.Gene expression analysis of OsRLR1At seedling,tillering and booting stage,different tissues were collected and used for RNA extraction to conduct qRT-PCR analysis.The results showed that:OsRLR1was mainly expressed in leaf blades at all three different stages.HR-like cell death and early senescence phenotype in leaf blade was induced by sunlight and the expression level of OsRLR1 was proportional to the severity of HR-like phenotype on leaf blade.Moreover,the promoter of OsRLR1 was cloned to express GUS gene and GUS staining of transgenic plant leaves showed that OsRLR1 was mainly expressed in vascular bundles of leaf blade.The expression of OsRLR1 was upregulated after rice blast and bacterial blight inoculation.All together,OsRLR1 is mainly expressed in vascular bundles in leaf blade and induced by sunlight and pathogen as well.5.Overexpression study of OsRLR1 geneUbiquitin promoter from maize was used to express OsRLR1 in the wild type,JH10.Compared with the wild type,T₂ OsRLR1 overexpression transgenic lines demonstrated enhanced resistance to rice blast and bacterial blight.At the same time,higher expression of disease resistance genes:OsNPR1,OsPR1a and OsPR10 were detected in OsRLR1 overexpression lines after pathogen inoculation.It was noticed that the elevated resistance level in OsRLR1 overexpression lines was considerable lower than that in rlr1.However,no HR-like cell death or deficient phenotype in agronomy traits such as,plant height and seed set rate were observed in OsRLR1 overexpression plants,which indicates its potencial use of in rice breeding.6.The selection of associated protein of OsRLR1The interaction between transcription factor OsWRKY19 and OsRLR1 CC in Y2H system was identified.The interaction was further verified by using truncated CC-NB,CC-NB-ARC and full length protein of OsRLR1 and OsRLR1?E318V?with OsWRKY19 in yeast.Moreover,fusion OsWRKY19-GFP proteins was observed in nucleus of N.bencimiana cells and rice protoplast.At the same time,BiFC technique was conducted to confirm the interaction of OsRLR1 and OsRLR1?E318V?with OsWRKY19 in planta,respectively.YFP signal was observed in nucleus in N.bencimiana cells that co-expressed OsWRKY19 with OsRLR1 or OsRLR1?E318V?,respectively.7.RNA interference analysis of OsWRKY19 in rlr1 mutantRNAi technique was used to silence the expression of OsWRKY19 in rlr1.All T₀transgenic plants showed reduced expression of OsWRKY19 compared with the wild type.The T₂ plants were generated and used for further study.Compared with mutant rlr1,the HR-like cell death,senescence phenotype and agronomic traits in rlr1OsWRKY19_RNAi plants were partially restored.At the same time,the expression of OsNPR1,OsPR1a and OsPR10 in T₂ plants were all reduced compared with rlr1 mutant after pathogen inoculation.Thus,blast and bacterial blight resistance response was compromised in rlr1 OsWRKY19_RNAi plants.8.The function study of transcription factor OsWRKY19OsWRKY19 demonstrated transcriptive activation activity in rice protoplast after transient expression of Pro35S:OsWRKY19-GAL.OsWRKY19 could facilitate the expression of OsPR10 when co-expression of Pro35S:OsWRKY19 and-1000bp promoter sequence of OsPR10.Moreover,OsRLR1 and OsRLR1?E318V?facilitated the transcriptive activity of OsWRKY19 in N.benthamiana leaves,and the effect of OsRLR1?E318V? was stronger than OsRLR1.