| Plasmodiophora brassicae Woron.,the causal agent of clubroot,is an obligate biotrophic plant pathogenic protist in the Rhizarian kingdom.Clubroot is a disastrous cultivated cruciferous crops disease which responses for huge economical losses on Brassica crops every year.The genome of P.brassicae strain e3 and Pbe3 has been sequenced,which provides a new opportunity for the intensive study on the growth,development and pathogenic mechanism of P.brassicae.Regarding the diversity pathotype of this pathogen,strains with different pathotype or regional distribution will share the variousness in the genome content.In this study,we conducted denove genome sequencing on the single spore strain ZJ-1（Pathotype 1）,which was derived from the clubroot of rapeseed plant from Zhijiang in Hubei Province.The genome characteristics of strain ZJ-1 were compared with strain e3 and Pbe3.We studied this pathogen by combinating the de nove genome sequencing and transcriptomic analysis,explained the phylogenetic relationship and the bases for molecular mechanisms of dormancy,growth,development and pathogenesis.The signal transduction pathways and pathogenic factors were verified.Here we present the main progresses as follows:We conducted the de nove genome sequencing of strain ZJ-1.The genome final assembly was 24.1 Mb with N50 sizes of the assembly scaffolds and contigs were 510.27 kb and 73.856 kb,respectively.The integrity and continuity of ZJ-1 genome is similar or slightly better than strain e3 and Pbe3.The genome size of ZJ-1 is similar to strain e3（24 M）and Pb3（24.2 M）.10,951 genes were predicted in ZJ-1 genome,which was 1221 and 100 more than in strain e3 and Pb3,respectively.The genome of strain ZJ-1 and e3 shared a high collinearity,there were 104 local collinearity blocks between them.However,there also existed significant genome rearrangement events.Whole-genome dot-plot comparison revealed obvious differences between the two genomes.The total length of repetitive sequences in ZJ-1 genome was 495,571 bp,comprised 2.01% of the assembly genome.The P.brassicae genome,with a low proportion of repetitive sequences and high gene density（454.40 gene/Mb）,has a smaller size than species from Rhizaria and SAR supergroup.The phylogenetic comparison was conducted between P.brassicae and 18 sequenced representative species from the eukaryotic evolutionary node.The result proved amoebozoa and chromalveolata from protista formed an independent branch with fungi,plant and animal kingdom,and P.brassicae has a closer evolution relationship with other Rhizaria species（R.filosa,B.natans）among all other model species.The Rhizaria group was an independent branch in the phylogenetic tree with a unique evolutionary status.From the perspective of strain ZJ-1,there are 4,197 members from the P.brassicae species-special gene family.Enrichment analysis of GO terms on the species-special genes and 611 genes from 117 expanded gene families revealed their function in G-protein coupled receptor（GPCRs）signaling pathway,chitin biosynthesis and DNA integration.These processes were presumed to be critical for the development of P.brassicae.What is more,the treatment of antagonists of GPCRs could delay the development process of P.brassicae and relieve the clubroot symptom of the host significantly,which confirmed the roles of GPCRs related genes were involved in growth,development and pathogenesis of P.brassicae.We applied multi-omics method to explore the molecular mechanism of gene regulation of P.brassicae ZJ-1 at three different growth state（resting spores stage,resting spores germinating stage and the secondary plasmodia development stage）by RNA-Seq analysis.During the resting spore stage,the expression level of genes involved in steroid metabolism,flavonoid biosynthesis,linolenic acid metabolism,phospholipid metabolism and ether lipid metabolism pathway were significantly up-regulated,revealing the important roles in the maintenance of the dormancy state of resting spore.During the germinating stage of resting spores,the pathogen activated more genes in carbohydrate metabolism pathway.Compared to other stages,the development stage of multinucleate secondary plasmodia showed a different pattern of gene expression.There were 458 genes up-regulated,involving in cell division,growth,DNA replication and protein translation processes.During the germinating stage of resting spores and the secondary plasmodia development stage,Ankyrin repeat-containing domain genes,P-loop containing nucleoside triphosphate hydrolase and serine/threonine-protein kinase coding gene were activated.Resting spores are critical for the survival and spread of P.brassicae.The roles of lipid droplets and their components in the resting spore are still undiscovered.From the perspective of multiple omics,we analyzed lipid droplets and related metabolism pathways of P.brassicae.295 lipid droplet-associated proteins were identified from P.brassicae ZJ-1 lipid droplets and167 genes involved in lipid metabolism were identified on the basis of genome analysis of P.brassicae ZJ-1.Fatty acid biosynthesis,fatty acid elongation and fatty acid degradation were highly abundant in the secondary plasmodia development stage,indicating a high conversion of lipids in this active life-stage.Arachidonic acid metabolism,Ether lipid metabolism and Ketone synthesis and degradation pathway were highly abundant in the resting spore stages.P.brassicae accumulated the largest amounts of TAG among related species.The genome contained a complete pathway for TAG synthesis,storage,and degradation.Gene expression pattern analysis showed P.brassicae utilized the stored energy in lipid droplets by activating the degradation pathways of TAG and fatty acid during the germinating stage of resting spores.The proportion of saturated fatty acids（C16:0 and C18:0）and unsaturated fatty acids was 76.6: 23.4.Approximately half of the unsaturated fatty acids were C20:4（arachidonic acid,ARA）,which accounted for 50% of the total unsaturated fatty acid content.As an obligate biotrophic intracellular parasite,P.brassicae shows special living-style compared with other eukaryotic plant pathogens.It maintains the vitality of the host cells and suppresses the host defense response meanwhile.We analyzed the unique obligate biotrophic life style of P.brassicae with the comparative genomics,transcriptomics,and secretomics and found a significant reduction in the genes devoted to metabolic pathways and enzymes,such as primary and secondary metabolites,and CAZymes families.The genes encoding ABC transporters were expanded in the genome of strain ZJ-1,which showed a unique way to obtain nutrients from plant cells,and the ABC transporter related genes were up regulated during the secondary plasmodia development stage.P.brassicae could regulate hormone level of host plant by at least 4 genes.Two proteins PBHK1 and PBHK2 in the genome of strain ZJ-1 might involve in phytohormone signal transduction pathway,genes PBHK1 and PBHK2 also existed in the genome of e3 strain.We identified 739 candidate secreted proteins（SP）,the expression level of 111 secreted protein coding genes was highly inducible during the germinating stage of resting spores or secondary plasmodia development stage.Finally,20 candidate effectors were mined from the P.brassicae genome,of which 8 are were exclusive in ZJ-1 strain.Function of 6 selected secreted proteins of P.brassicae was validated.Transient expression systems of TRV were used to test on N.benthamiana.The EC4（PlasB07397）caused serious hypersensitive cell death on the cell of host plant.What’s more,the apoplastic localization of EC4 was required for the cell death of tobacco leaves caused by EC4.Cell death reaction in N.benthamiana induced by Bax protein was suppressed by EC1（PlasB06695）.The secondary zoospore of P.brassicae can penetrate host cortical cell,and then develop into secondary plasmodia.The nuclei of the secondary plasmodia keep continuous division and finally the plasmodia split into resting spores,which occupy the whole host cell.For the differential expression genes from secondary plasmodia development stage,the top four significant enriched KEGG pathway classification were “translation”（Genetic information processing）,“Cell growth and death”,“Cell communication”（Cellular Processes）,and “Cancers”（Human Diseases）.Taking this as a clue,we identified 171 genes from the genome of strain ZJ-1 involving in cancer related pathways,of which three predicted proto-oncogenes Pb-Raf1、Pb-Raf2 and Pb-MYB,showed homology to human proto oncogenes Raf and MYB,were specifically activated during the secondary plasmodia development stage.Gene networks involved in the tumorigenic related signaling transduction pathways were identified,in which 12 core components were activated.What is more,the treatment of antagonists of PI3 K could relieve the clubroot symptom and the development process of secondary plasmodia was blocked significantly,this provides further evidence that PI3 K signal transduction pathway plays an important role in the course of secondary plasmodia development stage of P.brassicae.In summary,we performed the genome sequening of strain ZJ-1,and analyzed the life activities of dormancy,growth,development and pathogenesis of P.brassicae.The research results shed new light on the establishment of nuisanceless techniques on management of the disease. |
