Study On The Regulation Of Potato Virus Y Infection On The Ubiquitination Of Tobacco MLP-like Protein

In the co-evolution of host and virus,host plants used multiple strategies to deal with viral infections,involving complex but strictly regulated networks of interactions between different types of cells,cell receptors,and signaling pathways.Specificity and complexity also depended on post-translational modifications of proteins involved in the initiation,maintenance,and termination of immune responses.Among them,the ubiquitin / 26 S proteasome system(UPS)was involved in almost every step of the plant defense mechanism,not only the weapon that plants used to defend themselves,but also the target of certain pathogens,which have evolved mechanisms to inhibit and / or use the system for its own purposes.In this study,Potato Virus Y(PVY),which is a serious damage to economic crops,and Nicotiana benthamiana were the main research materials.Based on the comprehensive analysis of proteomics and ubiquitination proteomics,the related differential proteins and their ubiquitination proteins in host plants in response to PVY infection stress were determined.Using ubiquitination site mutation and pull-down technology,it has been verified that PVY infection promoted the ubiquitination of MLP-like protein 43(Nb MLP43)and was conducive to self-infection,which was hoped to provide theoretical basis for studying the pathogenic mechanism of plant virus and the mechanism of plant antiviral.The results of this study are as follows:1.Analysis of proteomics and ubiquitination proteomics after PVY infection to N.benthamianaCombining with lysine ubiquitinated peptide enrichment technology and LC-MS / MS technology,differential analysis of proteins and proteins ubiquitination levels,GO enrichment analysis,KEGG and subcellular localization analysis were performed using N.benthamiana after PVY infection.Proteomics data showed that 4 days after PVY infection,67 proteins were up-regulated and 50 proteins were down-regulated.Through GO enrichment and KEGG enrichment analysis,it was found that in the interaction between virus and host,proteins in response to stress and hormone signal transduction pathway were up-regulated.Among them,MLP-like protein 28 was up-regulated at the protein level.The ubiquitin-omics results showed that the ubiquitination levels of 12 proteins were up-regulated,while the ubiquitination levels of 19 proteins were down-regulated.MLP-like protein 43 was down-regulated at the protein level,and its ubiquitination level was up-regulated.Validation tests showed that PVY infection caused up-regulation of host proteins ubiquitination,and the ubiquitination level was highest at 3-5 dpi.The results of treatment with the protease inhibitor MG132 showed that PVY was significantly reduced at both the RNA and protein levels,and it was concluded that the ubiquitination of the host was conducive to virus infection.2.Response of MLP-like protein 28(Nb MLP28)to PVY infection stressMLP-like protein 28(denoted Nb MLP28)was first identified and functionally analyzed in N.benthamiana.In this study,the Nb MLP28 CDS sequence(Gen Bank accession number MK780769)was obtained from N.benthamiana.Nb MLP28 was localized in the plasma membrane and nucleus,and was the highest in roots.Nb MLP28 gene was presumed to be triggered by PVY infection and was highly expressed in the jasmonic acid(JA)signaling pathway.Nb MLP28 reached its peak expression 2 days after PVY infection.Virus-induced gene silencing(VIGS)and transient overexpression experiments verified the function of Nb MLP28 in response to PVY infection stress.The promoter of this gene was cloned and analyzed.After analysis,it contained cis-acting elements in response to Me JA,light,drought,plant hormones,etc.,in order to obtain stress-induced transcription factors of specific expression.3.Response analysis of MLP-like protein 43(Nb MLP43)to PVY infection stressThe Nb MLP43 CDS sequence(Gen Bank accession number MK780770)was obtained from N.benthamiana by homologous cloning.Sequence alignment showed that the N.benthamiana Nb MLP43 had the closest relationship to MLP28 in Gossypium hirsutum.The promoter sequence of Nb MLP43 on tobacco was also amplified,and bioinformatics analysis showed that the promoter sequence contained cis-acting elements in response to SA,light,temperature,stress and so on.By constructing an RFP tag and laser confocal technology,we observed that Nb MLP43 was mainly localized in the cytoplasm and nucleus.Nb MLP43 was the highest expressed in tobacco leaves.The expression pattern analyzed the response of the gene to PVY stress,Nb MLP43 reached its peak expression 3 days after PVY infection. CRISPR and constitutive overexpression further verified the function of the gene in response to PVY stress,and the results showed that knocking out Nb MLP43 promoted PVY infection.The results of reactive oxygen staining and enzyme activity measurement indicated that Nb MLP43 may be related to CAT activity and downstream related to the elimination of reactive oxygen species.Nb MLP43 overexpression inhibited PVY infection and replication in N.benthamiana.Through hormone spraying and silencing of key genes in the hormone pathway,a pathway that regulated the expression of Nb MLP43 in N.benthamiana was identified,and Nb MLP43 responded to the SA signal transduction pathway.In addition,the transcriptomics analysis of the over-expressed Nb MLP43 transgenic N.benthamiana also enriched the expression of SA pathway genes.4.Study the effects of Nb MLP43 ubiquitination to PVY infectionBased on proteomic and ubiquitin proteomic analysis,we identified that Nb MLP43 was down-regulated at the protein level,while its ubiquitination level was up-regulated.The ubiquitinated peptide was identified as SNPHQTSTMSPDK(1)IK.In order to further identify the ubiquitination site of Nb MLP43,the transgenic tobacco carrying the RFP tag was constructed,and the Nb MLP43 carrying the RFP tag was purified using pull-down technology.The LC-MS / MS technology was also used to identify the ubiquitination site of Nb MLP43 after PVY infection,which was also K38.Ubiquitinated antibodies were used to verify that the ubiquitination level of Nb MLP43 was increased after PVY infection.By point mutation technology,K38 was mutated to R38.The PVY infection test after point mutation showed that the ubiquitination level of Nb MLP43 was down-regulated,and the virus expression was down-regulated at both the gene and protein levels.Therefore,it was verified that PVY infection promoted the ubiquitination of Nb MLP43,which was beneficial to self-infection.However,detailed molecular mechanisms need further study.