Study Of Paulownia Witches’ Broom Based On Transcriptome And Methylome

Paulownia witches’ broom(Pa WB)is a devastating disease caused by phytoplasma,is one of the important problems affecting the industrialized production of paulownia.Over the past 40 years,researchers have conducted morphological,physicochemical studies on the pathogenesis of Pa WB.However,due to the fact that phytoplasma is difficult to be cultured in vitro and the genomes of Paulownia and phytoplasma are published,the pathogenesis of Pa WB remains unclear.Previous studies have shown that methyl methyl sulfonate(MMS)and rifampicin at appropriate concentrations can transform the morphology of disease seedlings into the healthy ones,in which,the 16 Sr RNA fragment of phytoplasma cannot be detected.Therefore,in this study,based on the Paulownia genome which drawn by your lab(version 3),the Illumina Hiseq 4000 platform was used to detect the changes of transcriptome and DNA methylation in the process of the invasion of phytoplasma and the process of the phytoplasma gradually disappear in paulownia body by 30mg·L-1(low concentration)and 100 mg·L-1(high concentration)rifampicin and 20mg·L-1(low concentration)and 60 mg·L-1(low concentration)MMS at different time points treated the witches’ broom seedlings,the main results as follows:1)To obtain the regulatory pathway of 20 differentially expressed genes which related to the occurrence of Pa WB.In the Paulownia fortunei witches’ broom seedling treated by MMS,a total of 33,302 genes were obtained,in the seedling treated by rifampicin,a total of 33,418 genes were obtinaed.Through WGCNA analysis,27 modules were obtained,in which,four specific module associated with Pa WB,including lightgreen,navajowhite2、darkmegenta and darkviolet.containing323,503,363 and 992 genes,respectively.Through analysis of the function of these genes,20 genes specifically related to the occurrence of Pa WB were obtained.2)A single base resolution methylation map was drawn.This study built a single-base resolution methylation map of the the sampls in the process of phytoplasma infected paulownia Pa WB,and analyzed the base preferences,the results showed that each sample m CG methylation level is the highest,followed by m CHG,finally is m CHH.The similar results were observed in different seedlings treated with rifampicin.In additional,the base preference of paulownia seedlings treated with two reagents at high and low concentrations was calculated based on the site numbers of three methylation types.The results showed that the DNA methylation event in the seedlings treated with the two reagents was preference for m CG type,followed by m CHG,and finally m CHH.3)The occurrence of Pa WB was closely related to the change of DNA methylation level.The DNA methylation levels anlaysis result showed that the methylation level of healthy seedlings after phytoplasma infection was 20.43% and that of diseased ones was 23.42%.In the treatment of paulownia seedlings by two reagents in high concentration,the the total DNA methylation level is gradually reduced.In the treatment of paulownia seedlings by two reagents in low concentration,the total DNA methylation level is also gradually reduced,while the DNA methylation level increased again in the revcoved seedling.In the paulownia seedlings treatment by MMS in high concentration.The DNA methylation level decreased from 24.85% to 20.87% with the extension of treatment time,in the paulownia seedlings treatment by MMS in low concentration and the recovery ones,the DNA methylation level decreased from 21.25% to 19.83% then increased to25.57%.In the paulownia seedlings treatment by rifampicin in high concentration.The DNA methylation level decreased from 21.65% to18.89% with the extension of treatment time,in the paulownia seedlings treatment by rifampicin in low concentration and the recovery ones,the DNA methylation level decreased from22.72% to16.72% then increased to 29.04%,showing that the phytoplasma infection increased the DNA methylation level.4)Changes of DNA methylation pattern was the other reason of the occurrence of Pa WB.all of the samples at the same time point showed that m CHH methylation pattern was the most frequent,followed by m CG,and m CHG was the least frequent in phytoplasma infection samples.The changes of DNA methylation pattern between different samples was dominated by m CHH and m CG,but no significant changes in m CHG.Compared to healthy seedlings,m CHH type increased from 50.64% to53.00% in the phytoplasma infected paulownia seedlings.In the seedlings treated with high concentration of MMS,the methylation ratio of m CHH type gradually decreased,from 54.17% to 47.53% with the extension of treatment time.The change trend of m CG type was opposite to that of m CHH.In the seedlings treated with low concentration of MMS,the methylation ratio of m CHH type gradually decreased,from 48.48% to 53.14% with the extension of treatment time.The change trend of m CG type was opposite to that of m CHH,the methylation ratio of this type decreased from 29.00%% to 26.22% with the extension of treatment time In the seedlings treated with high concentration of rifampicin,the methylation ratio of m CHH type gradually decreased,from 55.02% to 51.21% with the extension of treatment time.The change trend of m CG type was opposite to that of m CHH.In the seedlings treated with low concentration of rifampicin,the methylation ratio of m CHH type gradually increased,from 49.21% to51.72% with the extension of treatment time.The change trend of m CG type was opposite to that of m CHH.These results indicated that the occurrence of Pa WB may be more closely related to changes in m CHH and m CG types.5)Identifition of Pa WB related to genes and their regulatory pathways.Through correlation analysis of transcriptome and genome-wide methylation,a total of 404 common methylated genes with differentially expressed.GO analysis showed these methylated genes were mainly enriched to 50 GO terms,mainly in the REDOX process,regulating transcription,transcription,calcium ion correlation and protein phosphorylation.KEGG pathway analysis indicated that methylated genes were mainly involved in carbohydrate metabolism,lipid metabolism,amino acid metabolism,cofactors,vitamins and other metabolic pathways.Furthermore,a total of12 methylated genes associated with Pa WB were obtained by comparing the 404 methylated genes with those selected 20 genes by transcriptome,and the function analysis showed they mainly involved in photosynthesis,plant defense,and cell signal transduction.