| 21st century is coming for systems biology. Transcriptome and peoteome analysis are fundamental parts in this new research field. On the basis of genome sequencing, many biological questions of model monocotyledon plant, rice (Oryza sativa), would be deciphered by transcriptome and peoteome analysis using new high-throughput technologies. Rice stripe disease, caused by rice stripe virus (RSV), is one of the most economically important rice virus diseases prevailing in China in recent years. Although many studies have been conducted on the epidemic, molecular biology and molecular variation of RSV, little was known about the mechanisms underlying the development of rice stripe disease. To uncover this mystery and establish experimental basis for the ultimate goal of disease control, rice whole genome microarray and IPG-SDS-PAGE two dimensional electrophoresis combined with mass spectrum protein identification technologies were adopted in this study and changes in gene expression profiles in response to RSV in two japonica rice cultivars with similar genetic background but distinct resistant levels, WuYun3 and KT95-418, were analyzed in transcriptional and translational levels.Aerial parts of stripe diseased rice plants in the 7th day after symptoms’ initial appearance (treatments) and corresponding parts of non-inoculated ones growing in the same conditions (controls) of WuYun3 or KT95-418 were collected and total RNA were extracted from these materials and hybridized to Affymetrix rice whole genome array for transcriptome analysis. Three replicates were used in every treatment and materials from one single plant were regarded as one biological replicate. Gene expression profiles of treatments and controls were compared respectively in each cultivar. A total of 1931 differently expressed genes were picked out with thresholds of FDR close to 5% and fold change>=2 using SAM. Among them, 1666 differentially expressed genes, including 819 up-regulated and 847 down-regulated ones, were found in WuYun3 treatments comparing to WuYun3 controls. While in the comparison of KT95-418 treatments and KT95-418 controls, 561 genes were differentially expressed with 153 up-regulated and 408 down-regulated ones. 296 transcripts, including 95 up-regulated and 201 down-regulated ones, showed common change patterns in the transcriptome of both cultivars.Using the 18152 probe sets detected as "present" in all 12 chips in this study as background and a cutoff of at least 2 genes in one cluster and P<=1, functional annotations of differentially expressed genes in each comparison were clustered and annotation groups or key words were enriched by significance. Results showed that genes related to chloroplast, ribosome and electron transport were up-regulated in both cultivars. Genes annotated with metal-combining, oxidoreductase activity or abiotic stress response-related were down-regulated in both cultivars. And annotation keywords such as chloroplast, protein complex, heat shock proteins, photosynthesis, DNA-dependant RNA polymerase, plastid, metabolism, ribo-nucleoprotein complex, cation transporter activity etc. were clustered for genes up-regulated in WuYun3 only. Functional annotations in 646 repressed genes in WuYun3 treatments were effectively clustered for the P values of 8 clusters were up to the levels of significance. And the most significantly clustered annotation keywords were in the following 5 groups: photosynthesis, signal transduction, protein synthesis/localization or modification, enzymes in metabolism or transcriptional factors. Among them, plastid, thylakoid, photosystem II, ion binding or signal transduction were the most significantly enriched annotation keywords. Besides, protease inhibitors, protein biosynthesis, ribo-nucleoprotein or DNA-dependent RNA polymerase were also frequently annotated for these genes. Functional annotations of genes specifically regulated in KT95-418 (58 up-regulated and 207 down-regulated ones) could not be clustered significantly for lack of information.To clarify the connection of these differentially expressed genes in metabolism, twopathway databases KEGG (release 45.0) and RiceCyc (version 1.3) were combinedand genes differentially expressed in each comparison were analyzed. Results showedthat cytokinin biosynthesis pathway was significantly regulated by RSV in WuYun3 for13 differently expressed genes involved in this pathway (10 up-regulated and 3down-regulated ones). Photosynthesis pathways were disturbed obviously, which was incorrespondence with the chlorotic symptoms of stripe disease. But the change patternsof these photosynthesis related genes were complex (some were induced while otherswere repressed). Another possible RSV modulated pathway was fatty acid metabolism,while differentially expressed genes related to this pathway in WuYun3 and KT95-418were different. 3 genes involved in epicuticular wax biosynthesis and 2 genes injasmonic acid biosynthesis were specifically up-regulated in WuYun3 treatmentscomparing to WuYun3 controls. And genes in response to circadian rhythm werespecifically down-regulated in WuYun3. Flavonoid biosynthesis pathway was repressedin both WuYun3 and KT95-418 while genes involved in in the two cultivars were notthe same. Amino acid metabolism was disordered with genes involved in valinebiosynthesis or arginine, praline metabolism down-regulated in both cultivars and genesinvolved in tyrosine, tryptophan, cysteine or threonine biosynthesis up-regulated inWuYun3 only, while alanine, arginine, valine, leucine, isoleucine or lysine metabolismrelated genes were down-regulated in WuYun3 only. Functional annotations fordown-regulated genes in both cultivars were amino acid (alanine) biosynthesis, sucrosedegradation, fatty acid metabolism, circadian rhythm, nicotinamide adeninedinucleotide (NAD) salvage pathway II, oxidative branch of the pentose phosphatepathway, galactose metabolism, glutathione-mediated detoxification, methylglyoxaldegradation trehalose biosynthesis I and trehalose biosynthesis III.In the proteomic analysis, ImageMaster was adopted to compare the two dimensional electrophoresis maps (2DE maps) of proteins from the treatments and controls in WuYun3 and KT95-418. In gel regions of relative molecular wight about 20 kDa～25kDa and isoelectric point about 4.5～5.5, RSV disease specific protein (RSV SP) accumulated markedly in the protein 2DE maps of treatments in both cultivars. Moreover, another 44 protein spots with different expression patterns in treatments and controls of the two cultivars were picked out for the following analysis. Among them, 33 were picked out in the comparison of 2DE maps of WuYun3 treatments and WuYun3 controls, including 25 decreased spots and 8 newly appeared ones. 15 spots were picked out in the comparison of 2DE maps of KT95-418 treatments and KT95-418 controls, including 7 decreased ones, 5 disappeared ones and 3 induced ones. In the two comparisons, signals of 4 protein spots in the 2DE maps decreased in treatments of both cultivars, but less reduction were found in KT95-418 than in WuYun3.25 out of 44 protein spots were identified by MALDI-TOF-TOF/MS and NCBInr database search using Mascot. The 25 proteins were encoded by 22 non-redundant genes, of which 16 records were from rice, 1 from RSV and 5 from other plants. Successfully identified proteins included ChainS in crystal structure of activated rice Rubisco complexed with 2-carboxyarabinitol-l,5-bisphosphate, Rubisco large subunit form Ypsilandra thibetica, Bentinckia nicobarica or Utricularia pubescens, chloroplast precursor of plastocyanin, chloroplast precursor of thylakoid lumenal 17.4 kDa protein, Cytochrome B6-F complex Fe-S subunit, 23 kDa polypeptide of photosystem II oxygen-evolving complex protein precursor, chloroplast precursor of Thioredoxin M-type, chloroplast precursor of putative superoxide dismutase [Cu-Zn], Os02g0328300(Oxidoreductase NAD-binding domain. Xanthine dehydrogenases, that also bind FAD/NAD), Os02g0192700(Thioredoxin peroxidase), Os08g0116500(60s Acidic ribosomal protein), chloroplast precursor of 50S ribosomal protein L12, adenosine diphosphate glucose pyrophosphatase, putative ribose-5-phosphate isomerase, glycine cleavage system protein H, putative small ubiquitin-like modifier (SUMO) protein, RSV NS2 protein and two conserved hypothetical proteins (Os06g0705100 and Os10g0330000).By combining the results of transcriptome and proteome analysis, conclusion could be made in this study that photosynthesis, dynamic balance of cellular redox state or ions, protein translation/translocation or modification, carbohydrate, amino acids or fatty acids metabolism were the most significantly disturbed pathways by RSV infection. The possible connections between these metabolism disorders and the formation of rice stripe disease and the important roles these pathways might serve in host-virus interaction were discussed and mass experimental data for exploring rice systemic pathology was supplied in this study. |
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