The MiRNA Expression Profile Of Sugarcane Challenged By Sorghum Mosaic Virus And Mining Of Disease Resistant-related Genes

Sugarcane?Saccharum spp.hybrid?is the most important sugar crop.Sugarcane mosaic disease?SMD?is one of the major worldwide sugarcane diseases as well as an important virus disease that affecting sugarcane yield and sugar content.Among the several viruses causing this disease,Sorghum mosaic virus?SrMV?is a dominant pathogen in sugarcane cultivation regions in mainland China in recent more than ten years.The researches on this disease mainly concentrated in pathogen attribution,category and proportion of pathogen in different sugarcane producing regions,pathogenic mechanism,detection technology and genetic improvement by silence of virus protein genes.While the related signaling pathways,differentially expressed genes and proteins,interacting genes and proteins in the process of SrMV infection have not been studied in depth.Therefore,in this study,high-throughput sequencing and bioinformatics technology were used to construct the miRNA enrichment library in sugarcane resistance and disease genotypes after SrMV infection.The differentially expressed miRNA which was induced by SrMV and its target genes were identified according to the change of the miRNA expression abundance.The function of differentially expressed miRNA and its regulatory molecular mechanism were verified by fluorescence quantitative PCR?qRT-PCR?and transient over expression in Nicotiana tabacum.Meanwhile,the sugarcane resistant-related gene was identified and characterized so as to provide gene resource for improvement of suagracne mosaic disease resistance by genetic engineering.The main results are as follows:1.Virus-free and SrMV-infected leaves of sugarcane genotypes ROC22?susceptible?and FN39?resistant?with mosaic symptom were used as plant materials.Based on high-throughput sequencing by Illumina HiSeq2500 platform,the miRNAs in sugarcane challenged by SrMV were analyzed.Taking our previous transcriptome of sugarcane challenged by SrMV as the reference data,the target genes of miRNA were predicted.The main results and conclusions are as folows:?1?There was 174.71 M amount of data containing 161,145,158 clean reads,which were obtained by miRNA sequencing in 12 samples including from virus-free and virus-infected leaves from both resistant-and susceptible genotypes with three biological replicates.After comparing with the reference of sugarcane transcriptome data by miRDeep2 software,132 mature miRNAs were predicted in total,including 55 known miRNAs and 77novel miRNAs.In ROC22,19 significantly differential expressed miRNAs?P<0.01,|Log2?FC?>1?were identified,including 8 known miRNAs and 11 novel miRNAs,and 16 of them were up-regulated,while three were down-regulated.In FN39,30 significantly differential expressed miRNAs?P<0.01,|Log2?FC?|?1?were identified,including 12 known miRNAs and 18 novel miRNAs,and 17 of them were up-regulated,while 13 were down-regulated.?2?132 mature miRNAs were predicted to target to 1,037 target genes,653 of these target genes were annotated.There were 29 and 39 annotated target genes among the significantly differential expressed miRNAs were found in ROC22 and FN39,respectively.GO analysis indicated that there were 516 target genes involved in 40 GO classifications in two varieties.There were 25 and 33 target genes corresponding to the significantly differential expressed miRNAs observed in ROC22 and FN39,which involved in 26 and 28 GO classifications,respectively.And these GO classifications mainly concentrated in cellular component,catalytic activity,metabolic process,cellular process and single-organism process.KEGG enrichment analysis showed that there were seven target genes targeted by eight significantly differential expressed miRNAs involved in nine metabolic pathways,including mRNA surveillance pathway,valine,leucine and isoleucine degradation,plant-pathogen interaction,aminoacyl-tRNA biosynthesis,regulation of autophagy,glycerophospholipid metabolism,sulfur metabolism,selenocompound metabolism and purine metabolism.2.The accuracy of the sequencing results were verified by qRT-PCR analysis on the gene expression of 12 selected significantly differential expressed miRNAs.The result indicated that nine of the 16 sequencing results?4 miRNAs were differentially expressed in the 2 varities?were consistent with the quantitative results,which exhibited the same up or down regulated expression.Meanwhile,the expression patterns of 12 evidently differential expressed miRNAs and 15 target genes in ROC22 and FN39 infected by SrMV were analyzed.The results showed that:?1?Among eight miRNAs which displayed the same expression patterns in both ROC22 and FN39,six miRNAs?miR396a-5p;miR812f;miR1510b-5p;nov₉492;nov₉377 and nov₂0472?were up-regulated,while the other two?nov₃741 and nov₄0875?were down-regulated in both resistant-and susceptible cultivars.In addition,five of them?miR396a-5p;miR812f;miR1510b-5p;nov₉377 and nov₂0472?and their target genes followed a negatively regulated mode,and two miRNAs?nov₃741 and nov₄0875?and their target genes followed a positively regulated mode in both cultivars.While the regulatory mode between nov₉492 and its target gene was positive in ROC22 and negative in FN39.?2?The remaining four miRNAs?miR165a-3p;nov₂2650;nov₂8432 and miR395b?were all up-regulated in ROC22,but down-regulated in FN39.miR165a-3p and miR395b negatively regulated their target genes in both cultivars,while nov₂2650 and nov₂8432 positively regulated their target genes in ROC22 and negatively in FN39.3.The precursor sequences of three novel significantly differential expressed miRNAs?nov₃741;nov₂2650 and nov₄0875?were identified from ROC22 leaf infected with SrMV.Based on Agrobacterium tumefaciens-mediated over expression technology,the function of the candidate miRNA was verified.The result showed that three miRNAs could all cause the accumulation of H2O2 in N.benthamiana leaves after transient over expression.Besides,the transcripts of the immunity associated marker genes?including hypersensitive response marker genes:NtHSR203 and NtHSR515;jasmonic acid pathway associated genes:NtPR-1?/c and NtPR2 and the ethylene synthesis depended genes:NtEFE26 and NtAccdeaminase?were all up-regulated in N.benthamiana leaves after over expression of nov₃741.And the over expression of both nov₂2650 and nov₄0875 caused the increase of HR marker genes?NtHSR203 and NtHSR515?and ET synthesis depended genes?NtEFE26 and NtAccdeaminase?.These results indicated that nov₃741,nov₂2650 and nov₄0875 may get involved in the early stage of plant HR reponse.In addition,based on the regulation mode between markedly differential expressed miRNA and their target genes,we outlined the regulatory network of miRNA and its target gene in response to SrMV infection.4.A pathogenesis-related gene 10,termed as ScPR10?GenBank Acc.No.KT887884?based on the SrMV infected sugarcane transcriptome data were identified.Bioinformatic analysis showed that ScPR10 containes a 'P-loop' motif and a Bet_v 1-like domain and sharing the highest homology?91.25%?with a PR10 protein from Erianthus arundinaceus.qRT-PCR analysis showed that ScPR10 was not constitutively expressed in sugarcane which showed the highest expression in sugarcane buds while was barely detected in sugarcane root tips.After SA and MeJA?Methyl Jasmonate?stimulation,ScPR10 reached a peak at 6 h and 12 h,respectively.And the expression of ScPR10 was increased in resistant varieties while decreasded in susceptible varieties after challenged by pathogenic fungus?Sporisorium scitamineum?and virus?SrMV?.Subcellular localization demonstrated that ScPR10 mainly located in the nucleus.The transient over expression of ScPR10 in the leaves of N.benthamiana indicated the accumulation of H2O2 and the increase expression of HR marker genes,NtHSR201,NtHSR203 and NtHSR515 and the JA pathway associated genes,NtPR-1a/c,NtPR2 and NtPR3.Moreover,the enhanced resistance of N.benthamiana leaves to the infection of Pseudomonas solanacearum and Fusarium solani var.coeruleum were found after infiltration.These results suggest that ScPR10 plays a potential role in sugarcane antiviral defense.