Identification And Biological Function Analysis Of The Methylation Differential Genes Involved In Phytoplasma Infection In Mulberry

As an important epigenetic modification,DNA methylation is one of the important molecular mechanisms of plant in response to stresses.It has been shown that phytoplasma infection affects the dynamic regulation of plant DNA methylation.However,the change and regulation mechanism of plant DNA methylation in mulberry response to phytoplasma infection is still unclear.Using transcriptome sequencing and methylation-dependent restriction-site associated DNA sequencing(methyl RAD-Seq)techniques,the changes of transcriptome and genome DNA methylation levels in response to phytoplasma infection in mulberrywere analyzed.DNA methylation regulated differentially expressed genes were screened and their functions were studied.The regulation of DNA methylation on gene expression in mulberry response to phytoplasma infection was preliminarily elucidated,which will lay a foundation for revealing the resistance mechanism of mulberry to yellow dwarf disease.The main results of this study are as follows:(1)Construction and sequencing analysis of the transcriptome sequencing libraries of phytoplasma-infected and healthy mulberry leaves.The transcriptome sequencing libraries of healthy and infected mulberry leaves was successfully constructed,respectively,and sequenced by Illumina Solexa sequencing technology.A total of 2722 differentially expressed genes were identified,of which 1 701 genes were up-regulated and 1021 genes were down-regulated in the infected leaves.The differentially expressed genes were enriched into 154 metabolic pathways,including metabolism,hormone synthesis,signal transduction and so on,by KEGG pathway analysis.(2)Genomic DNA methylation differential analysis of mulberry genome DNA in response to phytoplasma infection.Methyl RAD-Seq technology was used to analyze the genome DNA methylation level of infected and healthy mulberry leaves.A total of 34 595 methylation sites were identified,and there were 5 042 differential methylation sites in the infected and healthy mulberry genomes.Among them,3 676 CCGG differential methylation sites were identified.In the infected mulberry leaves,there were 190 methylation sites with elevated methylation level,and there were 1 686 sites with decreased methylation level,and 935 genes were detected to contain CCGG methylation differential sites.In addition,1 366 CCNGG methylation differential sites were identified,and there were 818 methylation differential sites with elevated methylation level and 548 sites with decreased methylation level in the infected mulberry trees.There were 935 genes which were detected to contain CCGG methylation differential sites.(3)Association analysis of the levels of DNA methylation and gene expression in response to phytoplasma infection in mulberry.Association analysis of the differentially expressed genes and the differential DNA methylation level genes showed that there were 55 genes which expression levels were significantly correlated with their methylation levels.The correlated DNA methylation sites included 45 CCGG methylation sites and 10 CCNGG methylation sites.There were 35 genes which methylation levels and gene expression levels showed the same trend,while 20 genes showed the opposite trend.Association analysis showed that there were 35 genes which DNA methylation and gene expression were both differential in the infected leaves.These genes included six resistance-related genes and some other genes which were involved in metabolism,signal transduction,transcriptional regulation,etc.qPCR and CHOP-PCR were used to verify the expression abundance and DNA methylation level of the differentially expressed genes,respectively,and the results showed that the results of high-throughput sequencing were reliability.(4)Functional identification of candidate genes related to plant disease resistance.According to the results of association analysis,two differentially expressed genes,disease resistance protein RPM1(named as Mul-RPM1)gene and serine-threonine protein kinase(Mul-STK)gene,were selected to analyze their biological functions.The two genes were successfully cloned by PCR,and subcellular localization analysis showed that Mul-RPM1 and Mul-STK threonine protein kinase were located in the plasma membrane.The results of quantitative PCR and CHOP-PCR analysis showed that the levels of gene expression of the two genes were induced by salicylic acid and pathogens.Moreover,it was showed that the DNA methylation levels of the two genes were affected by the treatments with pathogens but not affected by salicylic acid treatment.The plant expression vectors of Mul-RPM1 and Mul-STK were constructed,respectively.Moreover,the transgenic Arabidopsis plants of Mul-RPM1 and Mul-STK were obtained successfully.Overexpression of Mul-RPM1 and Mul-STK in Arabidopsis thaliana increased the resistance of transgenic plants to Pst.DC3000.Therefore,the change of methylation levels of Mul-RPM1 and Mul-STK in mulberry may affect the expression of the two genes and play an important role in regulating plant disease resistance.These results will contribute to the further study of the function and expression regulation mechanism of Mul-RPM1 and Mul-STK genes in mulberry,and lay a foundation for revealing the pathogenesis of mulberry yellow dwarf disease at the level of DNA methylation modification.