Isolation And Identification Of Nucleoproteins In Verticillium Dahliae-infected Plants Via Proximity-dependent Biotinylation

Verticillium dahliae has a broad host range,such as eggplant,cotton,tomato and so on,and caused leaf wilting,vascular brown and,withered severe symptoms in infected plants,seriously harm the yield of cash crops in China.Like other plant pathogenic fungi,V.dahliae is a successful invader that interferes with the plant immune responses by secreting effectors.Plant nucleus is not only the main regulatory target of pathogen invasion,but also the regulatory center of the defense responses against fungal attack.Thus,understanding the changes of plant nuclear proteome during fungal invasion is of great significance for us to understand the infection mechanism of pathogenic fungi and the defense response mechanism of plants.In order to investigate the changes of nuclear protein components in plants during fungal infection,it is necessary to isolate and identify specific organelle(e.g.nuclear)protein proteomics.The APEX(Ascorbate peroxidase)is one of the enzymes used in the proximity labeling technique,which produces phenoxy radical during the labeling process.The existence time of phenoxy radical is short(<1 ms),and the labeling range is small(<20 n M).The phenoxy radical can be covalently linked with electron-rich amino acids such as Tyr,Trp,His and Cys to complete in situ biotin labeling.APEX provides a new method for proximity labeling,which can capture proteins in situ in biochemical environment and provide spatiotemporal information of target proteins for us.But APEX proximity labeling technique has not yet been used in plants.In this study,Arabidopsis thaliana and cotton were used as plant receptors to explore the application of APEX in plants and to isolate and identify nucleoprotein in plant-V.dahliae interactions.The main findings are as follows:1.Verification of subcellular localization of the NLS-APEXPlant expression vectors pLGN-35S-NLS-APEX-eGFP was constructed.By using of GV3101 mediated transient expression in tobacco,the subcellular localization of the NLS-APEX was observed in nuclear.After the tobacco leaves were stained by DAPI,the eGFP signal was co-located with the DAPI fluorescence signal.The results showed that NLS-APEX was targeted to plant nucleus,and the results were consistent in A.thaliana plants transgenic with pLGN-35S-NLS-APEX-eGFP.2.Determination and optimization of APEX labeling conditions in A.thaliana and cottonIn order to reduce the interference of endogenous biotin and enzyme,the variables,such as H₂O₂ processing time(0 S,3 S,10 S,30 S,60 S),reaction substrate Biotin phenol concentrations(0 n M,0.5 n M,5 n M,10 n M,25 n M,50 n M,75 n M),reaction generator-different parts of A.thaliana(leaf,root)and growth stage(seeding leaves,mature leaves),termination mode were investigated.The results showed that in mature leaves of APEX transgenic A.thaliana,the optimal labeling conditions were as follows:After the treatment with biotinolphenol at 50 n M,the initial reaction with H₂O₂ was carried out at 3 S.Immediately after the reaction,liquid nitrogen was rapidly frozen,and leaf proteins were extracted with Buffer containing the termination solution.Under these conditions,the adjacent biotin labeling could be better completed,and the WT background was lower.Indirect immunofluorescence verified that biotin proteins were located in the nucleus,that is,APEX in A.thaliana could function as adjacent markers.Similarly,the proximity labeling test was successfully completed in APEX transgenic cotton.3.Isolation and identification of nucleoprotein in plant-V.dahliae interactionsIsolation and identification of nuclear proteins were treated 4 d,2 d of transgenic APEX in A.thaliana and cotton leaves,near the biotin-labeled by treatment using streptavidin coupled to avidin beads for biotinylated protein purification,protein of sample was detected by LC-MS/MS mass spectrometry.Biotin-labeled protein samples of wild type A.thaliana was named AtWT,samples of APEX transgenic plants was named AtAPEX,samples of APEX transgenic plants inoculated with V.dahliae was named AtAPEX?Vd.APEX transgenic cotton treated identically,three sets of samples were named GhWT,GhAPEX and GhAPEX?Vd.Compared with wild AtWT of A.thaliana,228 proteins were detected at AtAPEX with the detected peptide number of>2 as the calibration line.Uni Prot KB and NCBI databases were used to conduct subcellular localization analysis of the proteins,among which 48(21.05%)had nuclear localization information.Four days after V.dahliae inoculation,152 proteins significant changes in the expression levels in AtAPEX?Vd compared with AtAPEX,of which 77 were up-regulated and 75 were down-regulated.The differential proteins are mainly involved in ribosome,TCA cycle,catalysis and so on.Similarly,328 proteins with significantly different expression levels were obtained,among which 198 were down-regulated and 130 were up-regulated,GhAPEX?Vd compared with GhAPEX.After screening out the captured proteins in GhWT of the control group,GhAPEX was compared with the 280 proteins measured in GhWT of the control group.The Uni Prot KB database was used to carry out the subcellular localization analysis of the proteins,and 35 proteins were predicted to be in the nucleus.4.Screening and validation of candidate nucleoprotein between A.thaliana and V.dahliae interactionsThrough proteomics analysis,three differentially expressed candidate nuclear proteins were screened,namely At1g07000,At5g22060 and At3g54360.After inoculation with V.dahliae for 0-6 d,differential expression was also detected at the transcriptional level.The use of Agrobacterium GV3101 mediated transient expression in tobacco indicated that the three candidate proteins were located in the plant nucleus.The transient expression of At1g07000 protein in tobacco caused the accumulation of ROS and increased the resistance of tobacco to V.dahliae.In our study,we obtained the biotin marker reaction system optimized by APEX in A.thaliana and cotton.After the separation and purification of biotin marker nucleoprotein treated by V.dahliae,proteomics analysis was conducted,and candidate nucleoproteins that might participate in defense regulation of A.thaliana were screened out.