N6-Methyladenosine Modification Changes in the Genome of Paulownia Fortunei Seedlings Infected With Phytoplasma
Abstract Background: Phytoplasmas induce diseases in more than 1,000 plant species and cause substantial ecological damage and economic losses, but the specific pathogenesis of phytoplasma has not yet been clarified. N6-methyladenosine sequencing (m6A-seq) has been applied mainly to model plants and not to woody plants. Results: In this study, we applied m6A-seq to study changes in m6A modification in the Paulownia fortunei genome after phytoplasma infection. We found that the m6A modification level in seedlings infected with the phytoplasma that causes Paulownia witches' broom (PaWB) was slightly higher than the m6A modification level in PaWB-infected seedlings treated with 60 mg·L−1 methyl methanesulfonate (MMS). MMS has been shown to restore PaWB-infected seedlings to their normal form and no phytoplasma can be detected in MMS-treated PaWB-infected seedlings. RNA sequencing (RNA-seq) and m6A-seq were used to analyze the expression of genes with m6A peaks and m6A motifs in genes, respectively. The correlation analysis between the RNA-seq and m6A-seq data detected that a total of 315 differentially methylated genes were predicted to be significantly differentially expressed at the transcriptome level. The functions of genes related to PaWB were predicted by functional enrichment analysis, and two genes related to maintenance of the basic mechanism of stem cells in shoot apical meristem were discovered. One of the genes encodes the receptor protein kinase CLV2 (Paulownia_LG2G000076), and the other gene encodes the homeobox transcription factor STM (Paulownia_LG15G000976). The m6A modification levels were higher in PaWB-infected seedlings than they were in MMS-treated seedlings. In addition, genes F-box (Paulownia_LG17G000760) and MSH5 (Paulownia_LG8G001160) had exon skipping and mutually exclusive exon types of alternative splicing in PaWB-infected seedling treated with MMS. RT-PCR verified that the alternative splicing of these two genes was related to m6A modification. Conclusions: In this study, we applied m6A-seq to determine methylation levels in phytoplasma-infected Paulownia, and combined m6A-seq with transcriptome analysis to screen differentially expressed genes associated with PaWB. Also analyzed the effect of m6A methylation on alternative splicing. In future studies, we plan to verify genes directly related to PaWB and methylation-related enzymes in Paulownia to elucidate the pathogenicity mechanism of PaWB caused by phytoplasma invasion.