Chrysanthemum CmWRKY15-1 Promotes Resistance To Chrysanthemum White Rust By Regulating CmNPR1 Expression

Chrysanthemum(Chrysanthemum morifolium)is a precious ornamental flower of compositae and chrysanthemum.Plants always face many kinds of stresses that lead to death throughout their life.Chrysanthemum white rust(CWR)is caused by the fungus Puccinia horiana Henn.which has become main disease affecting the quality of chrysanthemum.Through RNA-seq,screening the key genes of transcription factor CmWRKY15-1 in downstream salicylic acid(SA)signal transduction pathway,exploring the regulation of CmWRKY15-1 on CmNPR1 and clarifying the molecular mechanism of CmWRKY15-1 and CmNPR1 will help provide basis for improve the disease resistance of chrysanthemum and breeding excellent new resistant cultivars by using WRKY transcription factors.In this study,the resistant chrysanthemum ‘C029’ leaves of wild-type(WT)and CmWRKY15-1 silencd(CmWRKY15-1-RNAi)lines after treatment with P.horiana were used as materials to perform RNA-seq and obtain differentially expressed gene CmNPR1 to analyze the function.The regulatory mechanism of transcription factor CmWRKY15-1 and its target gene CmNPR1 in resistance to chrysanthemum white rust were analyzed by GUS staining technology、Y1H technology.The research results are as follows:(1)Total 12 biological samples(six from before inoculation and six from after inoculation)were prepared to perform RNA-seq and approximately 100.8 GB Clean Reads were accquired.The 326,062 transcripts with full length of 165,667,774 nt were obtained after De novo assembly.After further splicing analysis of transcripts,272,669 unigenes with full length of 152,620,397 nt were obtained.Finally,the function of unigenes were annotated and these unigenes were annotated in ’Posttranslational modification’,’Signal transduction mechanisms’ and so on.(2)Total 5449 and 6128 differentially expressed genes were identified from W0 vs WJ and R0 vs RJ.These differentially expressed genes are mainly relate to ’metabolic pathway’、’biosynthesis of secondary metabolites’ 、 ’Phenylpanoid biosynthesis’ 、 ’ ’Ribosome’.The expression levels of 8 disease resistance-related differentially expressed genes in SA signaling pathway were analyzed via q RT-PCR and our result were related to the transcriptome sequencing data.Finally,the key gene CmNPR1 in SA signaling pathway was chose for further research.(3)Based on RNA-seq database,the 1670 bp CDS sequence of CmNPR1 was cloned from ‘C029’.The CmNPR1-silenced lines of ‘C029’ were obtained by virus-induced gene silencing technology.The q RT-PCR result shows that CmNPR1 silencing lead to the decrease of pathogenesis-related(PR)genes expression in the SA pathway,indicating that CmNPR1 can respond to P.horiana by regulating PR genes.(4)The CmNPR1 1685 bp promoter was isolated by hi TAIL-PCR and numerous defense response cis-acting elements were discovered in the promoter,including pathogen-inducible element W-box which can bound to WRKY transcription factors.Full-length CmNPR1 promoter sequence and 5’-end fragments were obtained by PCR to construct three expression vectors with the GUS gene driven by CmNPR1 promoters and recombinant vectors were transiently transformed into ’C029’ to perform GUS staining.The result shows that the CmNPR1 promoter was active and the most active at 48 h,indicating that the promoter can respond to chrysanthemum white rust infection.(5)The CmNPR1 promoter was divided into two short truncation regions.Yeast one-hybrid assay shows that CmWRKY15-1 transcription factor could bound to CmNPR1 promoter region which has W-boxs,indicating that CmNPR1 is the downstream target gene of transcription factor CmWRKY15-1 and it can interact with the CmNPR1 promoter to regulate CmNPR1 expression to participate in the resistance to chrysanthemum white rust.