Functional Analysis Of BrSWEET15 Which Involved In Infection Process Of Plasmodiophora Brassicae

SWEET(sugars will eventually be exported transporters)is one kind of sugar transporter which was widely found in eukaryotes and prokaryotics.It contains seven transmembrane helixes to form two MtN3/saliva domains.They play an important role in many aspects such as plant reproductive development,response to stress,senescence,and plant-microbe interactions and so on.Results showed there are 34 members in the BrSWEET gene family of Brassica campestris L.syn B.rapa L.,which are located on chromosomes 1 to 10 and participate in multiple physiological and biochemical processes.Moreover,the infection of Plasmodiophora brassicae can induce the expression of SWEETs in B.rapa,and improve the transportation and regulation of sugar in host tissues during the occurrence of disease.Currently,Clubroot and Sclerotinia sclerotiorum have always been two major diseases threatening the product of crucifer crops around the world.Both can be fastly spreaded through soil,agricultural activities,flowing water,etc.Once the soil infected,it was difficult to eradicate.Meanwhile,P.brassicae is a species with a life cycle of protoplast stage,thus it is classified into the Protozoa,Rhizobium,Rhizobium,Rhizobium.In addition,S.sclerotiorum is a facultative parasitic fungus.Both parasitic processes involve the grabbing of sugar between the pathogen and the host.In this study,the bioinformatics and molecular experimental techniques have been utilized,and the BrSWEET15 has been employed as the research gene in Chinese cabbage,and exploring the functions of its paralogous members in the pathogenesis of and S.sclerotiorum.Aim to clarify the molecular mechanism of BrSWEET15 a,15b and 15 c during the infection process.The study results obtained as follows:(1)RNA-seq analysis of the infected roots by P.brassicae after 24 and 48 hours.Clubroot experiment was performed on B.rapa ECD05 by spore injection method.Infected roots have been sampled after 24 and 48 hours,and transcriptome sequencing was performed subsequently.There were three groups in the comparison group:24hCKvs24hI,48hCKvs48 hI,and 24hIvs48 hI.The numbers of differentially expressed genes(DEGs)were 4406,1412,and 1253,respectively.The enrichment analysis results of KEGG pathway showed that three comparison groups contained pathways with significant enrichment levels such as starch metabolism,sucrose metabolism and pathogen interaction.There are 19 main terms of DEGs in the GO classification.The differences in enrichment trends include immune system processes,stimulus responses,detox pathways,growth,signals,multi-organism processes,and cell death.Among them,detoxification pathway,stimulus response,and biological adhesion are the terms with obvious differences among the three groups.The results of qRT-PCR showed that the expression trends of 22 BrSWEET genes were consistent with the expression trends obtained by transcriptome sequencing.(2)BrSWEET15a is the preferential expression gene among the three paralogous genes of BrSWEET15.Combining the transcriptome sequencing results and qRT-PCR quantitative results,we employed BrSWEET15a/15b/15 c as the main research genes for the further expression character analysis.By constructing BrSWEET15a/15b/15 c subcellular localization vectors and promoter fusion GUS expression vectors,the subcellular localization results of BrSWEET15a/15 b and the expression signals of BrSWEET15 a promoter were obtained.In addition,the online promoter analysis software PlantCARE was used to analyze the structure and function of the promoter sequences.BrSWEET15 a and 15 b were confirmed to be expressed and located on the cell membrane.The promoters of the three genes all have core promoter elements such as TATA-box and CAAT-box.Only the expression activity of BrSWEET15 a promoter has been found.Thus,BrSWEET15 a is the preferential expression gene among the three paralogous genes of BrSWEET15.(3)Functional analysis of BrSWEET15 a.Construction of ectopic expression vector BrSWEET15 a and AtSWEET15 overexpression vector,and transformation with Arabidopsis floral dipping method had been carried out.At the same time,after the homozygous detection of the T-DNA insertion mutant of AtSWEET15,we observed the phenotype of the plants which is infected.The results showed that during the infection of P.brassicae,although the over-expressed BrSWEET15 and AtSWEET15 were not significantly different from the control group,but the T-DNA-inserted Arabidopsis plants showed stronger disease susceptibility than wild-type plants significantly.Therefore,SWEET15 is probably a resistant gene for the infection of P.brassicae.During the infection of S.sclerotiorum,no significant difference was found between the over-expressed BrSWEET15 and the control group,but the T-DNAinserted arabidopsis plants showed more resistant than wild-type.It seems that SWEET15 is a susceptible gene for the infection of S.sclerotiorum.In summary,theinfection process and molecular mechanism are significantly different between the P.brassicae and S.sclerotiorum.