| Soybean[Glycine max(L.)Merr]is widely cultivated around the world,and is one of the important crops,as well as the important source of plant protein and edible oil.In soybean cultivation the loss of yield and quality is often caused by diseases,which leads to great economic loss.Phytophthora root rot(PRR),caused by soil spread oomycetes Phytophthora sojae(P.sojae),is one of the most serious and widely distributed diseases in soybean-growing regions throughout the world and showed a trend of expansion,leaded to a lot of economic losses every year.Application of resistant cultivars is the most effective and environment protection method to control PRR.Thus,researchers have carried out a lot of studies to screening resistance germplasms and discover resistant genes.As a result,a certain number of resistance genes have been mapped.To date,however,there are few studies on the isolation of the mapped Rps gene,Rps-mediated defense resistance mechanism or breeding applications of Rps gene.Understanding the Rps-mediated resistance mechanism can point the way for subsequent functional research and provide new ideas for breeding to speed up the breeding process.It is also beneficial to overcome the limitations of the full resistance gene,such as short effective time,possible reduction of production,so as to breeding varieties with more lasting,more broader spectrum resistance and stable yield.Using RNA-Seq and GC-MS technology platforms,,we investigated in both transcriptional and metabolic changes in hypocotyl of two soybean lines during the P.sojae infection:Nannong 10-1(Resistance line,R)and 06-070583(Susceptibility line,S).We previously characterized RpsJS,a novel resistance gene to P.sojae in Nannong 10-1.Expression analysis of candidate genes in the RpsJS loci by RNA-Seq and qRT-PCR was performed to narrow down the candidate genes.Transcriptome and metabolome analysis may uncover new resistance-associated genes and potential resistance materials,and at the same time enable us to have a preliminary understanding of RpsJS-mediated defense resistance mechanisms.The main results of this study are listed as follows:1.RNA-Seq sequencing,screening of resistance-related differentially expressed genes and expression analysis of RpsJS candidate genesExpression profiling of R and S soybean lines which inoculated by P.sojae in a time course were conducted by RNA-Seq.By comparison,9,809 significantly differentially expressed genes(DEGs)were identified after P.sojae infection.Individually,822 DEGs in S12(343 up-regulated and 479 down-regulated),9218 DEGs in S36(3816 up-regulated and 5402 down-regulated),69 DEGs in R12,(45 up-regulated and 24 down-regulated),1489 DEGs in R36,(576 up-regulated and 913down-regulated).When the S line and R line libraries were compared,2,077 genes showed different expressed.Individually,822 DEGs in RS12,(343 up-regulated and 479 down-regulated);9218 DEGs in S36,(3816 up-regulated and 5402 down-regulated).GO and KEGG pathway enrichment analysis of DEGs revealed that the difference in the response between R and S soybean lines to P.sojae infection at the transcriptional level was mainly reflected in the phytohormone signal transduction,plant-pathogen interaction(involving transcription factors,disease-resistance-related genes,etc.),physical defenses,and metabolism of resistance metabolites.Meanwhile,plant-pathogen interactions,physical defenses,and metabolism of resistance metabolites are also major natural differences between R and S soybean lines.Thus,these parts may be major components of RpsJS-mediated defense resistance mechanismsRNA-Seq analysis showed that 10 of 14 candidate genes in the RpsJS loci with expression levels.The expression of the 10 genes that have RNA-Seq data were confirmed by qRT-PCR,results showed that three nucleotide-binding site and a leucine-rich repeat(NBS-LRR)type genes(Glyma18g51930,Glyma18g51950 and Glyma18g51960)were not significantly changed after P.sojae infection in both R and S lines while their expression level in R line were naturally much higher than S line with a fold change more than 5 times.The other 7 candidate genes did not present significant differentially expression in any comparison group.These three genes all showed high similarity with AtRPP13,an NBS-LRR type gene.Thus,we have reason to believe that RpsJS is likely to be an NBS-LRR type gene.2.GC-MS and screening of potential resistant substancesMetabolic profiling of the S line and R line has been carried out by GC-MS,and totally 311 metabolites of samples were identified.All of the differentially accumulated metabolites(DAMs)were selected by using multidimensional analysis(O)PLS-DA and single-dimensional analysis(t-test).As a result,a total of 118 DAMs were identified.Relative to the levels in mock-inoculated hypocotyls,the levels of 96 metabolites were significantly differentially accumulated in P.sojae infected hypocotyls.Individually,21 DAMs in S12(14 up-regulated and 7 down-regulated),58 DAMs in S36(25 up-regulated and 33 down-regulated),24 DAMs in R12(11 up-regulated and 13 down-regulated),21 DAMs in R36(11 up-regulated and 1 down-regulated).Relative to the levels in S line,the levels of 50 metabolites were significantly differentially accumulated in R line.Individually,37 DAMs in RS12(21 up-regulated and 16 down-regulated),22 DAMs in RS36(13 up-regulated and 9 down-regulated).28 metabolites showed differentially accumulated between R line and S line;meanwhile,these metabolites also differentially accumulated after P.sojae infection.Based on the response patterns of these metabolites to P.sojae infection and the differences in the content of S line and R line,we screened out some potential resistant metabolites,including sugars such as melezitose,levoglucosan,erythrose,trehalose,isomaltose,and 1-kestose;organic acids such as cumic acid,oxalic acid,and 2-methylfumaric acid;amino acid derivatives such as saccharopine,tyramide,N-formyl-L-methionine,N-?-acetyl-L-ornithine,phenylacetaldehyde indole-3-acetamide,4-hydroxybenzoic acid,trans-4-hydroxy-L-proline,treo-beta-hydroxy aspartate and S-carboxymethylcysteine;secondary metabolites such as octanal and daidzein.These potential resistant metabolites may also be part of RpsJS-mediated defense resistance mechanisms,but this hypothesis and its specific regulatory mechanisms need further research to confirm.We also attempted to integrate transcriptomics and metabolomics data using the KEGG pathway as an intermediary.However,we found that the two omics data were not highly correlated that due to technical limitations of GC-MS,the quantity of DAMs cannot be match with the the quantity of DEGs;on the other hand,the metabolic responded more sensitively lead to the adjustment was faster,and the undetectable differences in gene expression could be amplified and detected at the metabolic level.3.A potential RpsJS-mediated defense resistance mechanismsBased on the screened resistance-related DEGs and potential resistant metabolites,we deduced a potential RpsJS-mediated disease resistance mechanisms.First of all,expression analysis of candidate genes in the RpsJS loci points to great possibility of RpsJS is an NBS-LRR type gene.Plant can recognize pathogens and activate downstream defense responses though the action of R gene.The downstream defense responses involved in phytohormone signaling,transcription factors,PRs,plant cell wall modification and cuticle reinforcement,and accumulation of resistant metabolites.It is important to emphasize that plant cell wall modification and cuticle reinforcement may play an indispensable role in the defense response mechanism.Not only because of the related gene expression patterns revealed this fact,but also the up-regulation of PR9 and PR14 genes as well as oligosaccharides accumulation contribute to plant cell wall modification. |
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