Sequencing Of Transcriptome And Function Analysis Of Canadidate Gene SsCI68230 Of Sporisorium Scitamineum Isolates With Different Pathogenicity

Sugarcane smut disease caused by the basidiomycetous fungus Sporisorium scitamineum is one of the most severe diseases in world-wide sugarcane growing areas.Further mining pathogencity genes related to S.scitamineum wilt can provide valuable reference for explaining the pathogencity mechanism of S.scitamineum and facilitate the disease control.In this study,we explored the genome transcription of difference pathogenicity isolates Ss16(strongly pathogenic)and Ss47(weakly pathogenic)of S.scitamineum were analyzed using Illumina Sequencing technology.Differential expression analysis,gene ontology annotation(GO)and classification,GO enrichment analysis,Kyoto encyclopedia of genes and genomes(KEGG)pathway analysis were performed to screen candidate pathogencity genes.To further analysis these candidate pathogencity genes,we constructed the mutants by homologous disruption,the main results of this study are as follows:1 Screening of candidate pathogencity genes related to S.scitamineum.(1)Differential of expression analysis and GO annotation.A total of 649 significantly differentially expressed genes(DEGs)between isolates Ss16 and Ss47 of S.scitamineum,of which 299 were up-regulated and 350 were down-regulated.(2)GO and KEGG enrichment analysis.Enrichment analysis of the DEGs were performed,the statistical results showed that a total of 441 Molecular Functions,984Biological Processes,and 238 Cellular Components were generated.Deferentially expressed genes main enriched in intracellular,organelle and protein complex in Cellular Component.While in Biological Process,redox process were the most significant.And in Molecular Function,peptidase activity,GTPase activity and oxidoreductase activity were the most significant.KEGG pathway enrichment analysis showed that DEGs were enriched significantly in KEGG pathways such as starch and sucrose metabolism,ABC transporters,degradation of aromatic compounds,metabolic pathways,purine metabolism,and these pathways are mostly related to biosynthesis,transport and metabolism of substances.2.Functional analysis of candidate genes related to S.scitamineumBased on the transcriptome analysis,ATP-binding cassette transporter gene SsCI68230was choosen for functional analysis.The Split-marker method was applied to construct the deletion cassette of the SsCI68230 gene.The primers were designed according the sequence in the S.scitamineum genome database and the PCR products were transformed into the protoplast of S.scitamineum wild-type by the PEG-mediated transformation method.Subsequently,we employed phenotypic comparison tests such as sexual mating,pathogenicity determination,abiotic stress,and strain growth to identify differences between wild-type haploids(Ss16~+and Ss16~-)and mutants.The results demonstrated that the gene had no effects on abiotic stress,cell wall integrity or strain growth,but was related to sexual mating and pathogenicity.When compared with the wild types,the two mutants displayed a significant reduction in mating with each other or wild types,which also weakened their pathogenicity.We also found that mating ability could be restored by exogenous addition of small molecular signal substance(cAMP or tryptophol).