Multi-omics Analysis Of Nicotiana Benthamiana In Response To Rice Stripe Virus Stress

Rice stripe virus(RSV)can infect many plants such as rice,barley,wheat and maize.RSV is the pathogen of rice stripe disease,which is mainly propagated through the planthopper and can overwinter in the ovary of the rice planthopper.Subsequent to RSV infection,the typical symptom of many host plants is the appearance of longitudinal yellow-green discontinuous stripes in the leaf.This sometimes also appears as a symptom of “dead heart”,namely if severe chlorosis occurred in the heart leaves,which curls and drops with the arcuation until heart leaves are withered.Nicotiana benthamiana can be easily inoculated with RSV indoors,so it is an important model plant for studying the interaction between plants and pathogens.In this study,a comprehensive systematic biology study,including transcriptome,proteome,phosphoproteome and metabolome,was applied to directly study the expression levels of the differential genes,proteins,phosphorylated proteins and metabolites in mock and RSV-infected N.benthamiana leaves.On the basis of the multi-omics research results,we tried to propose the possible plant defense mechanism against RSV.The main conclusions are as follows:Firstly,we carried out a differential transcriptomic study based on RNA-seq.We identified a total of 560 genes with a significant variation of over 1.5-fold change,among which 350 genes were up-regulated and 210 genes were down-regulated.Gene ontology(GO)analysis showed that the differentially expressed genes were mainly related to macromolecular complexes,membrane proteins and organelle proteins,and had molecular-binding activity.Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis showed that RSV infection significantly affected N.benthamiana three metabolic pathways including porphyrin and chlorophyll metabolism,photosynthesis and glycolysis.By designing specific primers,the qPCR results showed that 14 of the 17 genes were consistent with the overall average change trend of RNA-seq results.Secondly,we conducted a label-free quantitative differential proteomic study of N.benthamiana leaves.After strict screening,we identified a total of 963 proteins varying by more than 1.5-fold change,of which 249 proteins disappeared and 316 proteins were induced after RSV infection.We carried out GO and KEGG analysis of these 963 proteins,and found that these differential proteins mostly located in the chloroplasts and proteasomes,indicating that the RSV infection had the greatest influence on these two types of proteins.The quantitative result of the protein Niben101Scf04007g02007.1 which is related to chlorophyll metabolism was also validated by a semi-quantitative immunoblotting experiment.Thirdly,by a subsequent differential phosphoproteomic study,we found out that a total of 554 phosphorylation sites of the 392 proteins showed more than 1.5-fold change after RSV infection.KEGG analysis showed that these phosphorylated proteins had been widely involved in many important biological processes,such as pyruvate metabolism,photosynthesis,biosynthesis of secondary metabolites,oxidative phosphorylation and plant pathogen interaction.In addition,the sequence motifs of total phosphorylation sites were significantly enriched in SP,P*S and S*P.Fourthly,we also carried out a metabolomic analysis of N.benthamiana leaves based on liquid chromatography mass spectrometry.Multivariate statistical analysis showed that there existed a clear clustering and distinct differences between the mock and RSV-infected leaves.A total of 87 different metabolites were identified in the metabolomic analysis,of which 53 metabolites changed more than 1.5 times.Many important small molecular metabolites in the biosynthetic pathway of nicotinic acid,alkaloid and terpenoid metabolites,photosynthesis and pyruvate metabolism were found to be significantly changed.Finally,by combining the results of differential transcriptome,proteome,phosphoproteome and metabolome,we found out that RSV infection collectively affected the pyruvate metabolism,photosynthesis Calvin cycle,secondary metabolite biosynthesis,glycolytic / glycolysis pathway and alanine,aspartate and glutamate metabolism.The most significant impact on the photosynthesis Calvin cycle revealed that the serious obstruction of CO2 fixed reaction was the landmark event during the N.benthamiana-RSV interaction process.