Mechanism Of NcRNAs Response To Plasmodiophora Brassicae Infection In Brassica Rapa

NoncodingRNA is a new class of regulators with multiple biological functions.In recent years,it has been found that many noncodingRNAs can respond to biotic and abiotic stress in plant.In order to explore the potential immune defense mechanism in plants,how noncodingRNA responds to the infection of pathogenic has become a hot topic in plants disease defense.In this study,the whole transcriptome analysis was performed on the roots of Chinese cabbage‘BJN 222’ containing clubroot resistance gene CRa in response to pathogenic and nonpathogenic P.brassicae,to explore the important role of noncodingRNA participate in stress response mechanism of B.rapa.The results are as follows:1.The roots of Chinese cabbage ‘BJN 222’ inoculated with non-pathogenic P.brassicae(Pb4)or pathogenic P.brassicae(Pb E)did not change significantly after 8 days.However,the roots inoculated with pathogenic pathotype(Pb E)displayed obvious disease symptoms at 23 dpi,while no changes were observed in roots inoculated with the non-pathogenic pathotype(Pb4).In addition,the root cell morphology of ‘BJN 222’ infected by P.brassicae was observed by paraffin section.It was found that no P.brassicae spore was observed in the root tissue of the control group,and the cell morphology was normal.The root cells have been enlarged at 23 dpi,which is full of P.brassicae spores,thus leading to the formation of clubroot.Thess results indicate that the ‘BJN 222’ was resistance to Pb4 and susceptible to Pb E.2.In this study,high-throughput sequencing was used to analyze the whole transcriptome data of non-pathogenic P.brassicae(Pb4)and pathogenic P.brassicae(Pb E)-infected roots of Chinese cabbage ‘BJN 222’.A total of 12971 novel lncRNAs with high confidence were identified,of which 1217 were significantly differentially expressed;a total of 1636 novel circRNAs were detected of which 231 differentially expressed,indicating that ncRNAs played a crucial role in the infection of P.brassicae.A total of 17466 differentially expressed mRNAs were screened.In the group of ck-0d-vsPb E-8d,up-regulated genes were significantly enriched in the “Brassinosteroid biosynthesis”pathway,while in the group of ck-0d-vs-Pb E-8d,down-regulated genes were enriched in the“Plant-pathogen interaction” pathway.In addition,“plant hormone signal transduction”pathway in this study were up-regulated at 23 dpi inoculated with Pb E;and downregulated at 8dpi with inoculated with Pb4.Some metabolic pathways were significantly enriched in the late stage,especially the “Glucosinolate biosynthesis” pathway,which was significantly enriched in the comparison between the eight groups.The results revealed the important role of noncodingRNAs in response to P.brassicae infection,and laid a foundation for studying the disease resistance mechanisms of Brassica crops and other important economic crops.3.Using PCR and Sanger sequencing technology,the cycliczation formation mechanism of circRNAs were identified.Some circRNAs contained several miRNA binding sites,and multiple circRNAs could also share the same miRNA binding site.Based on differentially expressed circRNAs,circRNA-miRNA-mRNA network was constructed using target genes directly or indirectly related to stress-associated.Upregulated novel＿circ＿000495 supressed the expression of mi R5656-y,leading to the up regulation of Bra026508,whose function was cytochrome P450 705A5-like.P450 s perform two types of biosynthetic and metabolic detoxification functions in plants,some of which have essential roles in plant defense responses.Our study will give a new sight into the exploration of the relationship between the CYP450 family and ncRNAs and lay a foundation for further studies exploring complex gene regulation networks in B.rapa.4.A total of 36 pairs of lncRNA with antisense function were identified.631 pairs of differential lncRNA-mRNA co-localization;812 pairs of lncRNA with trans-acting were identified.Further analysis showed that most of the predicted DE lncRNA-DE mRNA pairs in the co-expression network were up-regulated,indicating that P.brassicae infection could strongly activate the expression of many genes in B.rapa.In order to explore the network regulation relationship between protein-codingRNA and non-codingRNA related to P.brassicae,the interaction between DE lncRNA,DE miRNA and DE mRNA were screened.Protein-coding genes annotated with plant-pathogen interaction and disease resistance were selected to construct the lncRNA-miRNA-mRNA regulation network.Among them,the sequence of Bra019410 annotated as resistance protein TAO1 showed 73.1 % sequence identity with CRa.Pearson correlation analysis showed that TCONS＿0025646 was positively correlated with the expression level of CRa(r = 0.981)and negatively correlated with the expression level of novel-m0732-3p(r =-0.644).The TCONS＿0025646-novel-m0732-3p-CRa regulatory pair was selected to further explore the mechanism of clubroot disease resistance.5.To explore the function of CRa in the interaction between B.rapa and P.brassicae inoculation,CRa was overexpressed in B.rapa plants.Seven overexpressing transgenic lines were identified via selective media and PCR identification in conjunction with the whole-genespecific primers.Results showed that the disease grade and disease index of the three transgenic lines were lower than those of the wild type ‘GT-24’,regardless of the non-pathogenic P.brassicae(Pb4)or pathogenic P.brassicae(Pb E).The activities of CAT,SOD and POD enzyme contents in ‘OE-CRa’ plants were apparently higher than those in wild-type plants under P.brassicae conditions,while the MDA contents in ‘OE-CRa’ roots were significantly lower than those of wild-type.These results showed that overexpressing CRa in B.rapa significantly increasely clubroot resistance.Pearson correlation analysis showed that TCONS＿0025646 was positively correlated with the expression level of CRa(r = 0.917)and negatively correlated with the expression level of novel-m0732-3p(r =-0.488).Therefore,the TCONS＿0025646-novel-m0732-3p-CRa regulatory network plays an important role in the disease resistance in B.rapa.6.In order to explore the resistance mechanism of CRa in B.rapa,the hormone content of three CRa overexpressing lines and Chinese cabbage ‘BJN 222’ was determined after inoculation with different pathogenic P.brassicae.Results showed that ABAs,AUX,CKs,GAs,JAs and SAs changed significantly.Under P.brassicae conditions,the content of SAs in CRa overexpressed plants reached 2-3 times that of the ‘BJN 222’;the content of JAs also increased significantly;the content of AUX was significantly reduced.Studies have shown that CRa overexpressing activates plant hormone signal transduction pathways to affect plant disease resistance.In summary,this study demonstrated that ‘BJN 222’ was resistant to P.brassicae Pb4 and susceptible to Pb E by observing phenotypic and cytological morphology of root tissue.Using whole transcriptome sequencing to explore the ceRNA regulatory module in B.rapa,combined with the mechanism of plant hormone signal regulating clubroot resistance,the molecular mechanism of B.rapa response to clubroot infection was explored from the perspective of noncodingRNA,which laid a foundation for further exploration of plant disease resistance mechanism.