| Paulownia witches’ broom(Pa WB),caused by phytoplasma,is a devastating disease of Paulownia trees,and the incidence of it is as high as 85% in some plantation regions,which can result in significant economic losses.Thus,it’s important to dissect the molecular mechanism of Phytoplasma-Paulownia interaction.In this research,the SMRT sequencing and metabomic methods had been used to analysis the transcripts and metabolites changes in Pa WB-infected Paulownia fortunei plants.The main results are as follows:(1)A total of 1,132,801 polymerase reads and 18,344,143 subreads were obtained from four libraries by using the SMRT sequencing.1,304,487 ROIs with full passes ≥ 0 and sequence accuracy > 0.75 were obtained after filtering.After cluster analysis,422,332 transcripts were identified,among which,263,662 were high quality reads.(2)By aligning the transcripts to the reference genome of Paulownia,10,901,16,176;16,749;16,193 alternative splices were identified PF,PFI,PF-60 and PFI-60,respectively.The results showed that the alternative splices events in PF-60 were most,while those in PF were least,and the most frequent alternative splice event in these four samples was intron retention.The results of SSRs analysis suggested that 13978,18236,18180 and 18726 SSRs existed in PF,PFI,PF-60 and PFI-60,respectively.Further analysis showed that the SSRs in PFI-60 were most,while those in PF were least,and the number of Mono-nucleotide was highest and that of Penta-nucleotide was lowest.(3)The coding potential prediction of the transcripts identified in this study were performed by CPC,CNCI,pfam and CPAT analysis.EMBOSS was used to predict the ORFs of candidate transcripts,those with ORF length >100 were selected as lnc RNAs.Finally,1019 lnc RNAs were obtained,after comparative analysis,494 lnc RNAs associated with Pa WB were obtained.(4)A total of 637,382,580 clean reads were obtained from 12 libraries by using illumina sequencing,and FPKM were calculated by aligning these reads to genome.Differential expression analysis of the identified transcripts was performed in different comparison.A total of 3848,3240 and 2528 differentially expressed transcripts,and 18715 non-differentially expressed transcripts were obtained in PFI/PF,PFI-60/PFI,PF-60/PF and PFI-60/PF,respectively.(5)According to the different comparisons results above,1423 transcripts were considered to be related to Pa WB.GO terms and KEGG pathways analysis of these transcripts showed that transcripts were mainly involved in plant-pathogen interaction,phenylpropanoid biosynthesis and plant hormone signal transduction.Furthermore,q RT-PCR was used to verified the expression level of 9 transcripts,and the results showed that the data generated from RNA-Seq in this study were sufficient to be used to assess the transcriptomic changes of Pa WB-infected plants.These results provide a new perspective about the research on the interaction of the phytoplasma-Paulownia.(6)In the present study,645 metabolites were identified in four Paulownia samples by using UPLC-MS/MS.Differential expression analysis of the identified metabolites was performed in different comparison.A total of 109,94 and 102 differentially expressed secondary metabolites,and 297 non-differentially expressed secondary metabolites were obtained in PFI/PF,PFI-60/PFI,PF-60/PF and PFI-60/PF,respectively.Finally,99 metabolites were selected as Pa WB-related metabolites,these metabolites were mainly related to flavonoid,phytohormone and phenols.Combined with the results obtained from transcriptome sequencing,the molecular mechanisms of Pa WB were discussed.These results provide a new perspective about the research on the interaction of the phytoplasma-Paulownia. |
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