Multi-Omics Analysis Of Maize (Zea Mays L.) In Response To The Infection By Setosphaeria Turcica And The Mining Of Important Infection-Related Genes

Northern corn leaf blight(NCLB)is one of the devastating foliar disease of maize(Zea mays)in maize producing areas of the world.The disease injures or kills the leaf tissues,as a result reducing the area of green chlorophyll which in turn affects maize quality and yield.The pathogen which causes the disease is Setosphaeria turcica.Previous many studies have focused on the infection strategy and process of the pathogen,but the molecular mechanism of the interaction between the pathogen and host is still largely unknown.In this study,S.turcica was used as a research object.Further,the transcriptome data based the interaction between the pathogen and maize was analyzed to screen and identify the key genes related to the infection.On the other hand,a comprehensive systematic biology study,including transcriptome,proteome and phosphoproteome,was applied to directly study the expression levels of the response genes,proteins and phosphorylated proteins in S.turcica-infected maize B73 inbred line leaves during the infection.On the basis of the multi-omics research results,we tried to screen the possible function genes and metabolic pathways which were closely related to infection process.Moreover,we initially explored the possible molecular mechanism of the interaction between maize and Sturcica.The main conclusions are as follows:1.Screening and identification of the key genes related to infection process of S.tucica(1)By analyzing the maize transcriptome during the early stage of S.turcica infection,a total of 583 candidate genes which were closely related to infection process were obtained.These gens included 7 MAPK genes(such as StPBS2,StMKK1,StHOG1,and StKSS1),11 transcription factor genes(such as StSTE12),15 effect factor genes with functional notes(such as StSP1 and StSP2),19 genes related to genetic information transmission(such as StKU80).(2)Some important genes related to the infection process of the pathogen were verified by corresponding experiments.The results showed that the silence of SrPBS2 or the deletion of StHOG1 in HOG-MAPK pathway can lead to the pathogenic loss of the pathogen,and StHOG1 also effected HT-toxin activity.The deletion of transcription factor gene StSTE12 could make the pathogen lose the ability to penetrate and lose pathogenicity,but did not affect the virulence of the pathogen.The deletion of StKU80 related to genetic information transmission could make the pathogen lose the ability of infection.Effect factor genes StSP1 and StSP2 were transiently expressed in N.benthamiana leaves,and the results confirmed that the above genes were related to the infection process of the pathogen.2.Multi-omics analysis of maize reaction to S.turcica infection(1)The transcriptome of maize leaves which infected by S.turcica was performed at 24 and 72 hours post-inoculation(hpi).When compared with CK(0 hpi),a total of 7,196 differentially expressed genes(DEG)were identified to response the infection process of S.turcica.Functional annotation showed that these DEGs were enriched in multiple metabolic pathways.It’s worth noting that the genes related to pathways of "benzoxazinone biosynthesis","tetracyclic pyrrole biosynthesis" and "photosynthesis" were all down-regulated.While,the DEGs which related to "phenol metabolism" and "phenylpropane metabolism" pathways were up-regulated.In addition,some RLKs,WAKs,and NLRs receptor genes which related to plant immune response were also induced during the stages of S.turcica infection.Particularly,the gene ZmWAK-RLK1 that confers quantitative resistance to NCLB was specifically up-regulated at 72 hpi.Moreover,the genes which belong to NAC,MYB_related,HB and WRKY transcription factor family were also significantly enriched in the DEGs.Finally,the expression level of 6 random selected DEGs were further confirmed by the qRT-PCR results,indicating the reproducibility of the RNA-Seq results.(2)Quantitative proteome with TMT labeling technology was performed on the maize leaves which infected by S.turcica at 24 and 72 hpi.A total of 1,331 differentially expressed proteins(DEPs)were identified.Functional annotation shown that KEGG pathways"phenylpropionic acid biosynthesis","α-linolenic acid metabolism","fatty acid degradation","endoplasmic reticulum protein processing" and "metabolism of starch and sucrose" were significantly enriched in up-regulated DEPs.However,the pathways of "benzoxazinone biosynthesis" and "photosynthesis" were specifically enriched in 72 hpi down-regulated DEPs.In addition,11 proteins related to the jasmonic acid biosynthesis were differentially expressed,and most of them were up-regulated.Furthermore,the relative abundance of 14 selected DEPs were validated by using Parallel Reaction Monitoring(PRM),the results showed that the PRM and TMT quantification results were highly consistency,which lends confidence to the TMT data.(3)The phosphoproteome was performed on the above infected maize leaves.A total of 1.598 proteins and 3.416 phosphorylation sites were detected.These sites were enriched into 21 different types of phosphotylated conserved motifs,including 17 serine(S)phosphorylation motifs and 4 threonine(T)phosphorylation motifs,respectively.Compared with CK(0 hpi).183 DP proteins were identified in maize leaves during the stages of S.turcica infection.And the numbers of up-regulations DP proteins were more than the down-regulations.Functional annotation analysis showed that the DP proteins had a wide range of functions,mainly involving carbohydrate-related kinases,redox and transmembrane transporter activity.(4)The correlation between multiple omics were analyzed.There was a significant positive correlation(P<0.001)between the transcript level and protein abundance of the co-detected differential genes,but the correlation coefficient was relatively low(Pearson correlation coefficients of 24 hpi and 72 hpi were 0.26 and 0.43,respectively).However,no obvious correlation between the protein abundance and phosphorylation level was found.In summary,the interaction between maize and S.turcica is a complex dynamic biological process.On the one hand,pathogens require the participation of many important infection-related genes including MAPK signal transduction,transcription factors,and effectors during infection process.On the other hand,in order to response to S.turcica infections maize demand a large number of gene transcription rearrangements,protein abundance changes and changes in phosphotylation modification levels.It’s worth noting that the down regulation of genes which associated with the "photosynthesis" and"benzoxazinone biosynthesis" pathways may be a representative event in the maize response to the infection of S.turcica.This study will not only provide theoretical support for the early diagnosis and prevention of NCLB but also for breeding resistant maize varieties in the future.This research provides basic data for in-depth understanding of the molecular mechanism of the interaction between the S.turcica and maize.