Quantitative Proteomics Of Simultaneous Resistant Wheat N9134 Response To Blumeria Graminis F.sp.tritici And Puccinia Striiformis F.sp.tritici Infection

Powdery mildew(caused by Blumeria graminis f.sp.tritici,Bgt)and stripe rust caused by(Puccinia striiformis f.sp.tritici,Pst)are two serious fungal diseases of wheat worldwide and perennial occurred in the wheat growing areas of China,which greatly threatened food security of England.Deployment of resistant cultivars provides an effective approach for disease control to eliminate the use of fungicides and minimize crop losses.At present,the research on the interaction between wheat and these two fungi at the molecular level focuses on developing functional markers,the location and cloning of resistance-related genes,rare on the proteomic level.In this paper,the simultaneous resistant winter wheat line N9134 that shows high resistance to Bgt and Pst,were taken as plant materials to analyze the proteome change in seedling leaves after Bgt and Pst infection with a quantitative proteomic technique.Then,the correlation analysis between protein and previous RNA-seq transcriptomics data were conducted,and compared the two correlation results.The aim of this study was to reveal the molecular mechanism related to metabolism and signaling network of wheat N9134 response to Bgt and Pst stress on the whole level,and to identify the key protein and gene,which can provide theoretical foundation for understanding the resistance mechanism and wheat breeding resistant to Bgt and Pst.The main results are included as follows:1.An iTRAQ-based quantitative proteomic technique was used to analyze the proteome change in N9134 leaves after Bgt E09 infection,a total of 280,698 mass spectra were generated.After data filtering to exclude low-scoring spectra,40,604 unique spectra that matched to special peptides were obtained.Through searching using Mascot search engine(version 2.3.02),15,446 matched peptides,13,036 matched unique peptides,and the final 2182 proteins were quantified in the samples,394 proteins showed a significant difference.These differentially expressed proteins(DEPs)mainly included pathogenesis-related(PR)polypeptides(PR-1,chitinase and glucanase),oxidative stress responsive proteins(peroxidase,superoxide dismutase and ascorbate peroxidase)and proteins involved in defense-related metabolic pathways(cinnamyl alcohol dehydrogenase,Hsp90 and SGTl).KEGG enrichment analysis showed that biosynthesis of secondary metabolites,phenylpropanoid biosynthesis,phenylalanine metabolism and photosynthesis antenna proteins pathways showed significant differential enrichment at 24,48 and 72 hpi.The results indicated that these activated metabolic pathways played vital roles in resistant wheat in response to Bgt attack.According to the analysis of Self Organizing Map(SOM),the protein profile was built in different time points on the interaction between wheat and Bgt,and revealed the diversity and complexity of proteomic regulatory networks.2.An iTRAQ technique was used to conduct differential proteomics study for N9134 leaves after Pst race CYR31 infection,a total of 372,848 mass spectra were generated.After data filtering to exclude low-scoring spectra,56,544 unique spectra that matched to special peptides were obtained.Through searching using Mascot search engine(version 2.3.02),14,217 matched peptides,13,685 matched unique peptides,and the final 2292 proteins were quantified in the samples,489 proteins showed a significant difference.These DEPs mainly included defense-related proteins(thaumatin-like proteins,lipoxygenase and 14-3-3 protein),oxidative stress responsive proteins(glutathione transferase,thioredoxin and superoxide dismutase)and proteins involved in oxidative phosphorylation pathways(NADH dehydrogenase,cytochrome c1 precursor,cytochrome c oxidase subunit 6b-1,F-type ATPase,V-type proton ATPase subunit B2 and soluble inorganic pyrophosphatase).KEGG enrichment analysis showed that ribosome,oxidative phosphorylation and photosynthesis pathways showed significant differential enrichment at 24,48 and 72 hpi.Based on SOM analysis,DEPs were generally classed into seven clusters,and the protein profile was built in different time points on the interaction between wheat and Pst.Addtionally,we performed a comparison between wheat in reponse to Bgt and Pst infection,and inferred that DEPs were specifically induced in response to two different fungi infection.3.We performed a correlation analysis that couples quantitative proteomics and previous RNA-seq transcriptomics of wheat N9134 in response to Bgt and Pst.The results showed as follows:(1)The correlation analysises showed that after infection with Bgt for 24,48 and 72 h,the number of correlated DEPs is 50,59 and 69 respectively;the correlation coefficients(r)of the responsive protein and RNA pairs were 0.637,0.525 and 0.379 respectively.(2)The correlation analysises showed that after infection with Pst for 24,48 and 72 h,the number of correlated DEPs is 29,15 and 26 respectively;while r were 0.020,-0.316 and-0.265 respectively.(3)The correlation between the proteome and transcriptome after Bgt infection was high,while the Pst was lower.The cause might be: various genes were influenced in transcriptional and post-transcriptional regulation after different fungi infection.Hence,there was a difference in the correlation of proteome and transcriptome.