Resistance Mechanism Study Of BrRPP1 Gene In Chinese Cabbage Responsed To Plasmodiophora Bacteriae

Clubroot caused by Plasmodiophora brassicae is a serious threat to the economic value of cruciferous crops,and it is an urgent problem to be solved all over the world.Many resistance genes of Plasmodiophora brassicae have been mapped,such as Rcr1 and Rcr2 were located to the same candidate gene Bra A03g049860.3C,but the resistance machenism against clubroot has not been reported.In this study,it was found that Bra A03g049860.3C was homologous with the recognition of peronospora parasitica 1(RPP1)of Arabidopsis,so it was named BrRPP1.the cDNA sequence of this gene was different in resistant / susceptible materials.In order to furtherly verify function and mechanism of BrRPP1 against the clubroot disease,we designed experiments as following: Through the infection observation between the Arabidopsis thaliana deletion mutant of the BrRPP1 homologous gene and wild type,we verified the disease resistance function of BrRPP1;The promoter region of BrRPP1 was cloned and their promoter elements were analyzed to predict the regulatory mechanism of BrRPP1;The subcellular localization of BrRPP1 was analyzed to determine the expression pattern of BrRPP1 in cells;The Yeast two hybrid vector was constructed and interaction proteins of the target gene were screened;The results showed:1.The coding sequence of BrRPP1 in LRR domain was different between resistant and susceptible materials.The bioinformatics analysis showed that BrRPP1 had the same properties in the resistant and susceptible materials of Chinese cabbage,and both of them contained TIR and LRR domains.BrRPP1 belonged to TIR-NBS-LRR(TIR-NBS-LRR)protein,which was a R protein in plant ETI reaction and a non secretory hydrophilic protein without transmembrane structure.2.The Arabidopsis thaliana homologous deletion mutant ’Salk＿065253’ of BrRPP1 gene was used to identify its resistant function against clubroot.The roots of Arabidopsis thaliana were observed under light microscope at 24 h,48 h and 72 h after inoculation with P.brassicae.It was found a faster infection in ’Salk＿065253’,which verified that BrRPP1 deletion accelerated the infection process of P.brassicae.3.The GFP fusion expression vector pGPTVII.GFP-BrRPP1 was constructed and transformed into tobacco by Agrobacterium tumefaciens,and the empty vector was used as control experiment.The GFP fluorescence signal of the control empty vector and pGPTVII.GFP-BrRPP1 was observed by laser confocal microscope,and it was found that the GFP fluorescence signal of the control empty vector existed in the nucleus and cell membrane,but the GFP fluorescence signal of the pGPTVII.GFP-BrRPP1 vector existed only in the nucleus,which make it localized in the nucleus.4.The bait vector PGBKT7-BrRPP1 of BrRPP1 was constructed and hybridized with yeast double heterozygous cDNA library.Eleven interacting proteins were screened,including plant galacturonosyltransferase involved in cell wall modification,phospholipase A1 involved in jasmonic acid synthesis and cytochrome C involved in programmed cell death.The functions of the other proteins are unknown.5.In order to further understand the upstream regulation mechanism of BrRPP1,the1167 bp upstream promoter region of BrRPP1 was amplified and sequenced.The sequencing results were analyzed by Plantcare online software.Finally,transcription factors binding sites of WRKY,MYB,MYC and abscisic acid and anti oxidative response site were found,but promoter related regulatory mechanisms need to be furtherly studied.