Interaction Among Plasimodiophora Brassicae,Host And Endo-Microbiome Of Root

Clubroot of cruciferous plants,caused by Plasmodiophora brassicae,severely occurs all over the world.In recent years,it has spread from the upper reaches of the Yangtze River and Yunnan-Guizhou Plateau to the middle and lower reaches of the Yangtze river region of rapeseed(Brassica napus),and become a serious threat to security of rapeseed industry in China.Therefore,it is necessary to seek safe,effective and green measures to control the clubroot disease.In this research,the early reaction mechanisms of Arabidopsis thaliana responding to the infection of P.brassicae were investigated,and the interactions of the rapeseed,P.brassicae and the root microbiome were explored.The main results obtained were as follows:To clarify the early reaction of host to the infection of P.brassicae,gene expression profile of Arabidopsis thaliana was detected using RNA-Seq technique,and differently expressed genes at 24 hours post inoculation(hpi)and 48 hpi were analyzed.The results showed that 536 genes were up-regulated and 1367 genes were down-regulated at 24 hpi,while 374 genes were up-regulated and 985 genes were down-regulated at 48 hpi.Through Gene Ontology and Pathway significance enrichment analysis,the function modules and channels of the differently expressed genes were displayed by the MAPMAN program.The results showed that Flavonoids and the lignin synthesis pathways were enhanced,glucosinolates,terpenoids,and proanthocyanidins accumulated and many hormonal-and receptor-kinase related genes were expressed,caused by P.brassicae infection during its early phase.In addition,some homologs of nodulation genes participating in leguminous plants and rhizobia symbiosis were identified to be up-regulated in Aarabidopsis after infection by P.brassicae.These responses suggest that P.brassicae and host have strong interaction at the root hair infection stage,which may set up foundation for late cortex infection and P.brassicae growth within host's cortical cells.AM fungi form symbiosis with more than 80% land plants and rhizobia only nodulate leguminous plants with very rare exception.AM fungi and N-fixing rhizobia share similar signaling pathways during the early stage of infection,and infect their host through root hair of hosts.Plant auxin,kinetin,and other hormones,and flavonoids involve in the symbiosis of both the symbiosis microbes.Most interestingly,both could not naturally infect cruciferous plants.Considering that P.brassicae can also activate hormones and flavonoids synthesis pathway,we presume that rhizobia or AM fungi may enter the P.brassicae-infected root of rapeseed.By PCR and in situ hybridization examination,rhizobia were detected in naturally occurred clubroot rapeseed collected in the field.The co-infection of M.huakuii,a kind of rhizobium on Chinese milk vetch with P.brassicae was confirmed with rDNA sequences fluorescence in situ hybridisation,direct observation of rhizobia based on GFP and scanning electron microscopy,and re-isolation of the inoculated bacteria.The rhizobium-infection was further found to alleviate some clubroot symptoms.More interestingly,the rhizobia in the roots may had a trace nitrogen fixation activity in the co-infection rapeseed roots based on the results of 15 N isotope dilution tests and nitrogenase activity assays.The P.brassicae-infected rapeseed roots could attract the attachment of M.huakuii,and root exudates of P.brassicae-infected plants contained higher-level of flavonoids that could activate the expression of rhizobial Nod D.At the same time,homologues of some legume genes required for symbiosis and early-nodulin genes appeared to be present in Arabidopsis,and expression of such genes were significantly upregulated in rapeseed during early infection by P.brassicae.Furthermore,the co-infection of AM fungi and P.brassicae were discovered and confirmed in the chamber by PCR detection,WGA staining observation and qRT-PCR analysis,and we found that AM fungi formed arbuscule-like structures in the rapeseed root cells.With the infection of P.brassicae,rhizobia and AM fungi can penetrate the rape root and live in the cortex cells.At the same time,we often find rapeseed plants infected with P.brassicae without any symptoms.Therefore,we presumed that the rapeseed root infected by P.brassicae may also be suitable for other microorganisms' growth and these microorganisms may have significant inhibitory effects on P.brassicae.Microbiome of P.brassicae-infected rapeseed root was investigated with 16S rRNA and ITS high throughput sequencing technologies,and a large number of microorganisms were detected in both the symptomatic samples and asymptomatic samples.The characteristic and content of microorganisms in asymptomatic samples were obvious higher than those in the symptomatic samples;in the asymptomatic samples,many microorganisms have the functions of biological control and promoting plant growth.At the same time,we identified some rhizobia and Glomeromycota in all the detected samples,and the Glomus genus was only existed in the asymptomatic samples.In this study,we found that P.brassicae could change the host hormone synthesis pathway and secondary metabolic pathways,and these changes could trigger the invasion of rhizobia and AM fungi,two kinds of symbiotic microbes,into their nonhost plants-rapeseed.Microbiome analysis also showed significant microorganism differences between the symptomatic and asymptomatic rapeseed root samples.These findings are expected to advance the study of the interactions between rapeseed,P.brassicae and rapeseed roots endophytes,and provide a foundation for clubroot biological control.The results provide a new clue for the investigation of the rhizobia and AM fungi symbiosis with non-host plants(such as rapeseed).