The Role Of MiRNA1885b-CRa Regulatory Ele Ments In The Immune Response Of Brasscia Napus And Arabidopsis

Brassica napus is one of the main economical crops in China,but in recent years,clubroot has become one of the main diseases that harm rapeseed production.Therefore,it is of great significance to excavate disease resistance genes and analyze the resistance mechanism,to provide a theoretical basis for the improvement of rapeseed resistance,yield,and quality.CRa is the clubroot resistance gene,but the regulatory mechanism of the CRa gene and its relationship with other genes are still unclear.In recent years,micro RNAs have attracted much attention as gene regulators in plant biotic stress response.Previous work in our lab showed that miR1885 b was involved in CRa gene regulation.The purpose of this project is to analyze the CRa-mediated regulatory pathways of hypersensitivity response(HR)in Arabidopsis thaliana,and the regulation of CRa by bra-miR1885 b.The research results are as follows:1.Transcriptome analysis of inducible::CRa in A.thaliana.An inducible promoter was used to drive the CRa gene expression in A.thaliana wild type(Columbia).Upon inducible expression,the inducible::CRa transgenic material showed phenotypes of programmed cell death(PCD).At later stage of growth,the inducible::CRa transgenic materials also showed premature senescence.The accumulation of ROS in the transgenic materials was higher than that of WT.In addition,the relative expression of EDS1 and PAD4 gene,critical genes for ETI and PTI pathways,were higher in the transgenic material at the third day after induction.In order to explore the mechanism of PCD in inducible::CRa materials,RNA-seq analysis was performed from samples taken from different induction time.Transcriptome data showed that CRa expression had a strong response in the early stage.GO and KEGG enrichment on DEGs(differential expressed genes)showed that CRa-induced expression resulted in changes on transcript level of genes involved in chloroplasts,thylakoids,mitochondria,and outer membrane components,as well as responses to peroxides and nitrogen-containing compounds.This indicated that CRa can affect plant redox reactions and electron transport,which might lead to an increase in the content of ROS(reactive oxygen species)and causing cell damage.In addition,plant hormones such as JA and IAA,and MAPK pathway are also involved in this process.2.Bra-miR1885 b negatively regulates CRa expressionThrough the prediction analysis of the RNA target website,we found that bra-miR1885 b can target CRa as target gene.Combined with the degradome results from 409 R with or without clubroot inoculation,we found that bra-miR1885 b could cleave CRa gene exon1 at 292 nt,and the cleavage was not dependant on Plasmodiophora infection.In addition,when CRa gene was driven by its native promoter and introduced into A.thaliana,it can lead to PCD;however when35S::bra-miR1885 b and Native::CRa were introduced,it did not result in PCD,indicating that bra-miR1885 b can negatively regulate the expression of CRa.3.Functional identification of miR1885b-CRaThe bra-miR1885 b overexpression vector,the bra-miR1885 b interference vector,and the CRa target-site-mutation vector were constructed and transgenic materials were generated.bra-miR1885 b OE,bra-miR1885 b RNAi were transformed into 409 R where CRa is present,CRa-target-site-mutation vector was transformed into J9707 which did not contain CRa gene.The positive transgenic rapeseed materials were inoculated with clubroot pathogen from Zhijiang,both the bra-miR1885 b overexpression and bra-miR1885 b interference in 409 R background showed complete resistance,while the CRa target-site-mutation in J9707 showed susceptible phenotypes.By analysis of the expression level of CRa,we found that CRa was indeed negatively regulated by bra-miR1885 b.We explained the result as: CRa might not recognize the mixed population of clubroot pathogen collected from zhijiang,therefore although the CRa transcript level is high in transgenic materials with target-site-mutations,the plants were susceptible.In conclusion,our functional study of the miR1885-CRa regulatory module contributes to the understanding of the disease resistance mechanism of small RNA-mediated disease.The HR response and transcriptome analysis induced by CRa in Arabidopsis thaliana are helpful for understanding the pathogenesis of plant immune response and HR process in rapeseed and Arabidopsis,and the specific role played by the disease resistance gene CRa.