Microscopic Observation And Transcriptome Analysis Of Grapevine Microtubular Marker Line To Botryosphearia Dieback

The Botryosphaeria dieback of grapevine is mainly caused by Botryosphaeriaceae.The infection of this disease will lead to dry panicle axis,rotten fruit and grain drop of grapevine.In recent years,this disease has spread rapidly around the world,causing huge economic losses to more than 20 countries,including China,Spain,France,Italy and so on.It occurred in all of the 20 main grapevine growing provinces in China,resulting in a reduction more than 50% in grapevine yield.From reported in Portugal in 1998,the frequency of this disease has increased significantly.A variety of methods and production techniques have been tried to solve this problem at home and abroad,however,studies from the view of molecular cell biology in host is not sufficient.Microtubule(MT),as an important sensor adjacent to the cell membrane,has the function of sensing and conducting signals at the initial stage of pathogens invasion.It is of great significance to enhance the disease resistance of grapevine in production.In this study,the suspected pathogens of grapevine Botryosphaeria dieback were isolated and identified from susceptible branches collected in Penglai,Shandong province in March and July 2018.Extracellular secretions of pathogens were used to treat the suspension cells of Vitis rupestris expressing GFP-AtTUB6.Microscopic observation and transcriptome sequencing analysis were carried out.A suspension cell line marked with GFP-AtTUB6 in susceptible European grapevine was aimed to generate.The early endogenous immunity of grapevine cells mediated by microtubule was studied in order to provide cellular and molecular basis for the study of the mechanism of resistance to Botryosphaeria dieback in grapevine.These main findings are as follows:(1)The suspected pathogens of Botryosphaeria dieback were isolated from grapevine branches infected in the first year by tissue isolation.Six fungi,Botryosphaeria dothidea,Alternaria alternata,Alternaria tenuissima,Cladosporium tenuissimum,Fusarium equiseti and Penicillium brevicompactum were isolated and identified by biological characteristics observation and ITS-rDNA sequencing.They belong to 5 genera,4 families,3 classes and 2 subphyla,namely Botryosphaeria,Alternaria,Cladosporium,Fusarium and Penicillium.The ITS sequences of these isolated and identified suspected pathogens were uploaded to the NCBI database which obtained corresponding accession numbers.Compared with other existing ITS sequences to obtain the genetic relationship and genetic evolution tree of these suspected pathogens.(2)The calli of susceptible species Vitis vinifera L.cv.Cabernet Gernischet were induced from the petioles and stems.Of them,the suspension cell lines were established further.The Arabidopsis thaliana β tubulin 6 fusion green fluorescent protein(GFPAtTUB6)was tested to be transiently expressed in grapevine leaves as an initial step for stable transformation.The expression of GFP in grapevine leaves was observed by inverted fluorescence microscope.After these grapevine cells were transformed via Agrobacterium tumefaciens mediated transformation,the expression of GFP-At TUB6 was observed by stereoscopic fluorescence microscope in positively transformed calli.The stable expression of GFP-AtTUB6 around nuclear membrane were observed by inverted fluorescence microscope.(3)The response of Vitis rupestris expressing GFP-AtTUB6 cells to the extracellular secretion of Botryosphaeria dothidea and Diplodia seriata were studied.MTs in grapevine cells treated with extracellular secretion of D.seriata were significantly depolymerized at 3 dpi(days post inoculation),in contrast MTs were completely disappeared in cells treated with extracellular secretion of B.dothidea.In order to observe the response of MTs to extracellular secretion of pathogens at the molecular level,the cells of V.rupestris expressing GFP-AtTUB6 treated with extracellular secretion of D.seriata were deeply sequenced.After the clean reads were assembled and compared with the reference grapevine genome,the gene expression levels were quantified.Compared with the mock-control,1365 differentially expressed genes(DEGs)were obtained.The functional annotation of DEGs was carried out by using GO and KEGG databases.Pathways related to resistance,such as flavonoid synthesis pathway,plant-pathogen interaction pathway and plant hormone signal transduction pathway were annotated.Among the DEGs,53 were defined as transcription factors.Real-time quantitative PCR(qRT-PCR)was used to verify the expression of 12 genes in the transcriptome.The quantitative results were consistent with the transcriptome,which confirmed the reliability of transcriptome results.