Transcriptome Analysis Of Sugarcane Response To Sorghum Mosaic Virus Infection And Mining Of Pathogenesis Related Genes

Sugarcane mosaic disease isone of the most widely spread virus disease causing serious loss in eco-important crop of sugarcane(Saccharum spp.).Breeding and cultivation of resistant cultivars have been identified as the most efficient way to control the prevalence of mosaic disease.The abundant germplasms for mosaic disease resistance were found in Saccarum spontaneum and Saccharum robustum,while Saccharum officinarum is high sensitive to sugarcane mosaic disease.Unfortunately,the process of modern sugarcane breeding is known as "the nobliztion",which taking S.officinarum as the crossing female,and thus the genes related to cane yield,suerose and sensitive to sugarcane mosaic disease are coexisted in modem varieties which containing more than 80%of S.officinarum and showed to be adverse linkage of the above genes.This reason,combined with a selection method which based on phenotypic traits,result in an unsatisfactory improvement of the mosaic disease resistance.The investigation of the mosaic disease in guangxi suggests Sorghum mosaic virus(SrMV)was the absolutely predominant strain in sugarcane,which taking nearly 30%of the samples collected from the major progduction region of Guangxi.Therefore,sugarcane cultivar ROC22,which takes more than 60%of the total cultivated area in China in recent more than ten years,is selected as the investigated genotype,and three types of plants were used for RNA sequencing.The methods of transcriptomeanalyzed by bioinformatics,gene cloning,microarray and qRT-PCR were used in this study.For the first time,the interaction in the biosystem of sugarcane-SrMV was recognized on the transcriptional level,and the interactional unigene library was established,together with investigation of the changes of the related metabolisms and pathogenesis-related genes in sugarcane response to SrMV infection,and mining of the sugarcane-SrMV interactor and genes related to resistance or defense.The target is focused on understanding of molecular mechanism of sugarcane-SrMV interaction,and providing virus resistance gene resources,target genes of molecular improvement and potential functional marker genes for mosaic disease resistant breeding.Therefore,this study has important scientific significance and potential practical value.The main research contents,research results and conclusion are as follows.1.Validation of reference genes.With the increasing interest in sugarcane gene mining,the demands for qRT-PCR stable expressed reference genes in sugarcane were urgent.Due to the greatly effect of a reference gene on the quantifying of a target gene expression,a systematic validation of reference genes work was conducted using geNorm,NormFinder,BestKeeper and deltCt.The total of 33 genes and 12 miRNAs were selected as the candidate reference genes in three sugarcane genotypes infected by SrMV,as well as 13 candidates were used in selection of tissue-specific reference gene in five tissue organs of five genotypes,and the selection of reference genes based on the same 13 candidates in four genotype under 8 different abiotic stresses,were validated.Here,in this study we found that PP2A and miR159 can be a suitable reference control for gene transcript quantification in sugarcane under mosaic disease infection,as well as GAPDH in sugarcane tissue-specific experiments,eIF-4a in sugarcane unde 8 kinds of enviroumental stresses(NaCl,PEG,H2O2,CuCl2,CdCl2,ABA,SA and JA),together with eEF-1a in sugarcane under the treatment of hormone only and APRT in sugarcane under heavy metal(CuCl2 and CdCl2)treatment.CAC and CUL were suitable for using as sugarcane reference genes together under virus-infection,abiotic stress,drought/ostimatic and heavy metal stresses when reference genes combination is required in gene expression quantification,the same as eEF-1a and GAPDHin hormone treatment.Similarly,miR171 and miR1520 can use for the calibration of miRNA expression data in sugarcane mosaic virus-infection sample.2.RNA-seq and transcriptomic analysis.In this study,IluminaHiseq2000 was used to generate the sugarcane-SrMV interaction transcriptome database based on the threee different types of leaf samples,non-infected and SrMV-infected,as well as the non-infected transgenic ROC22 plants that derived from RNAi lines targeting at the CP gene of SrMV.The RNA-seq produced totally 88,414 unigene sequences with complete open reading frame(ORF),and 70,309 of them possess functional unique annotation from NCBI_Nr,NCBI_Nt,SwissProt,TrEMBL,GO,COG or KEGG public databases.The bioinformatics analysis of differential expressed sequences indicated that several hormone signaling pathways,calcium signaling pathway,photosynthetic pathway,pigment metabolism,plant-pathogen interaction pathway,carbon fixation pathway and other 51 pathway were positive response to virus infection.A total of 68 differential expressed genes were annotated to these pathways,including MAPKK5,PP2C,MYB,WRKY,NPR1 and others.The sugarcane sequences,which are homologous to the potyvirus-host interactors,can mostly be found in the present unigene database.However,only eEF1A,PCaP1,rgs-CaM and SGS3 varied in transcriptional level when ROC22 plants infected by SrMV.3.A global analysis of the RNA-seq and microarray.The result indicated that,the four cultivars with different resistant levels exhibited different transcript profiles in response to SrMV infection,and the susceptible one can accumulate more virus in its leaf tissue.Comparing with the resistant genotypes,the susceptibleone possessed more down-regulated genes.The results of differential expressed analysis showed that the basal metabolism in susceptible genotypesinfected by SrMV,such as photosynthesis,carbon fixation and pigment biosynthesis,varied more seriously due to the virus infection and accumulation..The sugarcane differential transcript profile revealed by qRT-PCR in sugarcane-SrMV showed that several auxin-induced protein,regulatory factor in cytokinin signaling system,ABA responsive elements binding factor,ethylene-responsive transcription factors,jasmonate O-methyltransferase and two-component response regulator encoded genes exhibited a different expression pattern in different genotypes after SrMV infection.It indicated that the sugarcane hormone signaling pathways,which referred to auxin,cytokinin,ethylene,abscisic acid,salicylic acid and jasmonic acid,were activated.The infection of SrMV also affected chloroplast,and the following Ca2+ leakage from chloroplast and activation of Ca2+signaling pathway,which would lead to the differential expression of CRT,CIPK and some other Ca2+related genes and chloroplastic located genes.These primarily Ca2+ and hormone signal activated a serial of plant defense responses.Some regulators varied in transcript profiles,including LRR-RLKs,Lec-RLKs,WRKY,bHLH,MAPKs,X1,SGS3,RH8,SGS3,G3 and G26,and some of them had significantly correlation in transcription.It indicated that these genes likely participate in the regulatory network of sugarcane-SrMV interaction.All these provide a global and new insight to the interaction of sugarcane-SrMV biosystem.It is worth to stress,the difference of the statistical methods used for calculation of a relative expression and for gene expression normalization in microarray and qRT-PCR,which may leading to a deviation of some gene expression,while the above two methods still shared high correlation of gene expression data.It indicates that the customized microarray,employingthe unigenes obtained from RNA-seq as probe targets,is a reliable,economical and effective way to generate huge of transcriptomic data in sugarcane.4.The functional charaterization of sugarcane genes.The potyvirus host interactor could be categorized based on its function and subcellular localization reported by the previous studies.ScSCE1 was up-regulated in sugarcane infected by SrMV.The expression pattern of ScSNARE,ScSGS3 and ScX1 across four sugarcane genotypes exhibited a similar trend and shared significantly positive correlation.At the same time,these three genes appeared to be down-regulated in susceptible genotypes and up-regulated in the resistant one.SNARE(Syntaxin-71)is potyvirus replication related factor,which localizes with 6K2 vesicle on endoplasmic reticulum.Finally,the further function characterization of ScSCE1,ScSNARE,ScSGS3 and ScX1 were conducted subsequently.Based on comparision of the three-dimensional models of the protein,it indicated that ScSNARE protein from sugarcane shared a highly identity of the three-dimensional model with AtSNARE from Arabidosis,as well as ScSGS3 and ScX1 with StSGS3a/b from Solanum tuberosum.In addition,the interaction between ScSCE1 and SrMV NIb in yeast two-hybrid(Y2H)was observed,which was in accordant with the similarity of their three-dimensional structure.The expression of ScSCE1 was suppressed in sugarcane treated by SA and JA,while induced by ABA.The expression of ScSCE1 in different sugarcane tissues was nearly on the same level.The mRNA level of ScSNARE tends to be more abundant in the more active tissues,such as stem apex,bud and intemode.The qRT-PCR results suggested that hormone signaling,especially ABA,had obviously impact on the expression of ScSNARE.Both ScSGS3 and ScX1 shared a VPg-interaction conserved domain and the expression patterns of these two genes under hormone treatment or in different tissues were similar.