Transcription Profile Analysis Of Interaction Mechanism Between Sugarcane And Sporisorium Scitamineum Using Microarray

Sugarcane smut is a fungal disease caused by S.scitamineum,which leads to serious yield loss and sucrose content reduction.Sugarcane production is seriously affected by smut disease in the world.Although some excellent resistant breeding materials have been created,the utilization of sugarcane germplasm and the breeding of resistant varieties are still greatly restricted for the complex genetic background of sugarcane and the differentiation of smut race.To solve this problem,much effort should be devoted to underlying the mechanism of sugarcane smut resistance and S.scitamineum virulence,mining resistance associated genes and markers,improve the application of resistant germplasm and thus shorten the conventional breeding process of resistant varieties.According to our previous RNA-seq data,the gene expression chip was customized.The transcription profile of gene set,which referred to 2,0392 from sugarcane and the whole CDS sequence(6,621)from smut genome,was identified on smut spore injected samples.These samples were from sugarcane smut-susceptible genotype YC71-374 and smut-resistant genotype NCo376.After that,the differential expressed genes from sugarcane and smut were selected and used to construct the weighted gene co-expression network of sugarcane-smut interaction.Finally,two smutresistance related genes and four putative smut effector genes were cloned and function characterized.Based on the technologies like customized gene expression chip,gene cloning,real-time quantitative PCR,BiFC and yeast two hybrid,this study aims to understand the molecular mechanism of sugarcane-smut interaction by the characterization of gene transcription and gene function,as well as to identify gene related to the proliferation of smut fungus,mine pathogenesis-related genes and smut effectors.It should help to pave the way to the conventional breeding for smut resistance in sugarcane.The main results and conclusions of this study are as follows:1.The design,customization and hybridization of the sugarcane-smut gene expression chip.According to the key words,the gene set,including oxidation-reduction,RNA silence,disease resistance,phytohormone,phosphorylation,dephosphorylation,transcription factor,receptor,kinase,ubiquitinationand RNA binding,etc.from sugarcane and the whole CDS sequence from smut genome,was achieved,and the chip was designed and spotted by company.Based on smut infection samples from smutsusceptible sugarcane genotype YC71-374 and smut-resistant sugarcane genotype NCo376,the hybridization was conducted and validated by realtime fluorescent quantitative PCR,and the result showed that the data achieved from chip was reliable.It would provide a basic data for the study on sugarcane-smut interaction mechanism,which referred to sugarcane smut resistance and smut pathogenesis.2.Sugarcane and smut fungus reference gene.Due to its excellent expression stability,ACAD,SARMp1 and their combination(ACAD+SARMp1)could be used for sugarcane gene expression normalization in the sugarcane bud when the smut infected,as well as S10,S11 and their combination(S10+S11)for smut gene expression normalization in the interaction process.3.Sugarcane gene expression profile.During smut fungus infestation in sugarcane bud,6,842 sugarcane genes were differentially expressed.According to the GO and KEGG enrichment analysis,the differential expressed genes involved in primary metabolism pathway for material and energy supplement,MAPK pathway for smut infection signaling,plant hormone signal transduction pathway for regulation of defense response and ROS response,as well as phenylalanine biosynthesis and cell wall organization or biosynthesis.4.Smut gene expression profile.During smut fungus infestation in sugarcane bud,3,569 smut genes were differentially expressed,including 94 sequence of candidate smut effector genes.In the process of smut colonization,the up-regulated genes from smut fungus mostly located in vesicles,and have activities like transportation,signal transduction and redox.These genes also involved in the anabolism pathway,which referred to carbohydrate,protein and aromatic compounds.5.Gene co-expression network.The weight gene co-express networks(WGCNA)of susceptible,resistant or the consensus of both genotypes were constructed based on the differential expressed genes from sugarcane and smut.The gene modules from the network,which were specific to susceptible or resistant genotype,and those significantly related to smut infection,were obtained.6.Gene selection and functional characterization.Based on the smut infection significant related gene module,two sugarcane genes(vacuolarsorting receptor,ScVSR;leucine-rich repeat receptor-like protein kinase,ScLRR-RLK)and four putative smut effector genes:SsPep1(Protein essential during penetration 1),SsE4(hypothetical cysteine-protease inhibitor),SsE11(conserved hypothetical protein)and SsE14(hypothetical protein),were selected from the same network for their high co-expression weight value(weight>0.15)and cloned from the smut infected sugarcane bud sample.Bioinformatics analysiswas performed with these cloned genes,and their expression patterns in S.scitamineum infected sugarcane buds for 0 to 7 dpi were analyzed by qRT-PCR.The results of qRT-PCR showed that two sugarcane genes continued to down-regulate expression in different sugarcane genotypes.In the susceptible genotype,the four S.scitamineum effector genes were all up-regulated from 0 dpi to 5 dpi,and then decreased after 5 dpi.In the resistant genotype,the expression pattern of SsPepl in the resistant genotype was the same as that in the susceptible one,while SsE4,SsE11 and SsE4 were up-regulated in all the resistant genotype.Subsequently,the interaction between ScVSR and ScLRR-RLK,SsPep1,SsE11 or SsE4 were validated by bimolecular fluorescence complementation and yeast two hybrid.The trials indicated that the Sc VSR gene interacted with ScLRR-RLK,SsE4 and SsE14,however Sc VSR did not interact with SsPep1 and SsE11.These results will provide a basis for the interaction research and a reference for the role elucidation of vesicle transport in the interaction between sugarcane and smut fungus.