| Brassicaceae family includes a variety of oil crops,vegetables and feed.Clubroot disease,caused by Plasmodiophora brassicae Woron,threatens the cultivation of Cruciferous plant and leads to economic loses eventually.To date,scientist have cloned incompletely dominant clubroot resistant(CR)gene Crrla and dominant CR gene CRa from Brassicae.However,little is known about the mechanism of plant resistance to clubroot disease.This study identified the function of CRa by the tissue localization,subcellular localization and expression verification in A.thaliana.Then screened the metabolic pathways involved in the interaction between Chinese cabbages and P.brassicae,and downstream signaling receptor of CRa by mRNA-seq and miRNA-seq in CR 3-2.Moreover,confirmed the downstream signaling receptors of CRa using yeast two hybrid,bimolecular fluorescence complementation,luciferase complementation,hypersensitive response and ROS detection assay.Finally,analyzed expression profiles of MKK and MPK genes in Chinese cabbage under P.brassicae infection in a pair of Chinese cabbage near-isogenic lines(NILs)CR 3-2 and CS 3-2.1.Functional analysis of CRa genewe tested the trans-breeding clubroot-resistant chinese cabbage CR3-2 line,which harbors the resistant gene CRa,with 9 different P.brassica pathotypes.CR 3-2 showed resistance to 5 different P.brassica pathotypes.Subsequently,we created stable CRa transgenic Arabidopsis lines,which also exhibited resistance to the infection of P.brassica.Summarily,CRa functions as a dominant resistant gene conferring the plant with clubroot resistance.By applying GUS histochemical assay and subcellular localization assay,we conclude that CRa is mainly expressed in the root hair cells of maturation zone,and the CRa protein localized in the cytosol and at the plasma membrane.These results are also compatible with the fact that P.brassica infects plants root at root hair cells.2.mRNA-seq and miRNA-seq analysis in CR 3-2 after inoculation of pb4 and pbE.mRNA and miRNA transcriptomes of Chinese cabbage line CR 3-2 were sequenced on 0,8 and 23 days after inoculating the roots with pb4 and pbE,respectively.In pbE-8d-vs-pb4-8d,129 differentially expressed genes(DEGs)and 49 differentially expressed miRNAs(DEMs)were found,and the DEGs were significantly enriched in the linoleic acid metabolism and alpha-linolenic acid metabolism pathway of KEGG.In pbE-23d-vs-pb4-23d,830 DEGs and 49 DEMs were found,and the DEGs were significantly enriched in the glucosinolate biosynthesis,linoleic acid metabolism and alpha-linolenic acid metabolism pathway of KEGG.Bra012655 was not expressed in pbE-23d group,but highly induced in pb4-23d group.Moreover,by aligning the transcriptomic data of miRNA and mRNA,we also screen out 5 pairs of negatively correlated miRNA-mRNA in pbE-8d-vs-pb4-8d and pbE-23d-vs-pb4-23d.Then,19 DEGs and 3 DEMs were validated by qRT-PCR.3.Calcium signaling pathway is involved in CRa-mediated immune response through AtCBL1-AtCIPK9The amino acid sequence alignment reveals Bra012655 is a Ca2+-sensor encoding gene,which contains four EF-hand domains.Thereafter,we aligned the AtCBL1 homologous fromA.thaliana and B.rapa,and performed phylogenic trees.Strikingly,BraCBL1s shares highly conserved amino acid sequences with AtCBL1.Furthermore,by applying yeast two hybrid assay,bimolecular fluorescence complementation assay,and luciferase complementation assay,we succeeded in finding out the downstream AtCBL1 interacting protein kinases,AtCIPK6,AtCIPK9,and AtCIPK13.The GUS histochemical staining and subcellular localization assay indicate that AtCBL1,AtCIPK9,and AtCIPK13 share similar spatial-temporal expression pattern like CRa.In PCD assay,we observed that co-expression of AtCIPK6 or AtCIPK9 with CRa and AtCBLl in tobacco leaves could accelerate the PCD,in comparison with expressing CRa and AtCBL1.And disease index in pCRa::CRa in cbl1/cipk9 line is higher than pCRa::CRa in Col-0 line.4.AtRBOHD and AtRBOHF are involved in CRa-mediated plant immunityThe results of ROS detection in Arabidopsis leaf protoplasts showed that both Col-0 and rbohd mutant generate CRa-triggered ROS.Comparing with Col-0,the second ROS peak triggered by CRa was significantly reduced and the duration also shortened in rbohd mutant.And disease index in pCRa::CRa in rbohd/f line is higher than pCRa::CRa in Col-0 line.5.MAPK cascades is involved in the early immune response mediated by CRaIn present study,a total of 11 BraMKK and 30 BraMPK genes were identified and bioinformatic analysis was performed.Expression profiles of BraMPK and BraMKK genes in different tissues in Chinese cabbage were analyzed by semi-quantitative RT-PCR.Additionally,expression pattern of BraMKKs and BraMPKs genes in the roots of CR 3-2and CS 3-2 was analyzed by qRT-PCR at 0,10,12,13,14 and 20 hours after pb4 treatment,then screen out differentially expressed genes. |
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