Phytoplasmas are mycoplasma-like pathogens of witches’ broom disease, and are responsible for serious yield losses of Paulownia trees worldwide. The molecular mechanisms of disease development in Paulownia are of considerable interest, but still poorly understood. Here, we have applied transcriptome sequencing technology and a de novo assembly approach to analyze gene expression profiles in Paulownia fortunei infected by phytoplasmas. Our previous researches suggested that methyl methane sulfonated (MMS) could reverse the effects of the infection. In this study, leaf samples from healthy, infected, and both infected and methyl methane sulfonate treated plants were analyzed. The results showed that the gene expression profile of P. fortunei underwent dramatic changes after Paulownia witches’ broom (PaWB) phytoplasma infection. Genes that encoded key enzymes in plant-pathogen interaction processes were significantly up-regulated in the PaWB-infected Paulownia. Genes involved in circadian rhythm and hormone-related genes were also altered in Paulownia after PaWB infection. However, after the PaWB-infected plants were treated with MMS, the expression profiles of these genes returned to the levels in the healthy controls. The data will help identify potential PaWB disease-resistance genes that could be targeted to inhibit the growth and reproduction of the pathogen and to increase plant resistance.

Paulownia witches’ broom (PaWB) is a fatal disease of Paulownia caused by a phytoplasma. In previous studies, we found that plants with PaWB symptoms would revert to a healthy morphology after methyl methane sulfonate (MMS) treatment. To completely understand the gene expression profiles of the Paulownia-phytoplasma interaction, three high-throughput sequencing technologies were used to investigate changes of gene expression and microRNAs (miRNAs) in healthy Paulownia tomentosa plantlets, PaWB-infected plantlets, and PaWB-infected plantlets treated with 60 mg · L−1 MMS. Transcriptome, miRNAs and degradome sequencing were performed to explore the global gene expression profiles in the process of Paulownia tomentosa with phytoplasma infection. A total of 98,714 all-unigenes, 62 conserved miRNAs, and 35 novel miRNAs were obtained, among which 902 differentially expressed genes (DEGs) and 24 miRNAs were found to be associated with PaWB disease. Subsequently, the target genes of these miRNAs were predicted by degradome sequencing. Interestingly, we found that 19 target genes of these differentially expressed miRNAs were among the 902 DEGs. The targets of pau-miR156g, pau-miR403, and pau-miR166c were significantly up-regulated in the P. tomentosa plantlets infected with phytoplasma. Interaction of miRNA -target genes mediated gene expression related to PaWB were identified. The results elucidated the possible roles of the regulation of genes and miRNAs in the Paulownia-phytoplasma interaction, which will enrich our understanding of the mechanisms of PaWB disease in this plant.

The response and adaptation to drought remains poorly understood for Paulownia australis. To investigate this issue, transcriptome profiling of four P. australis accessions (two diploid and the other two autotetraploid) under water stress condition were studied using Illumina Genome Analyzer IIx analysis. The current study aimed to identify genes of P. australis metabolism pathways that might be involved in this plant’s response to water deficit. Potted seedlings were subjected to well-watered conditions and drought stress, respectively. More than 290 million raw transcript reads were assembled into 111,660 unigenes, with a mean length of 1013 bp. Clusters of orthologous groups, gene ontology and the Kyoto Encyclopedia of Genes and Genomes annotations analyses were performed on the unigenes. Many differentially expressed genes and several metabolic pathways were identified. Quantitative real-time polymerase chain reaction was used to verify the expression patterns of 14 genes. Our study identified altered gene expression in P. australis induced by drought stress and provided a comprehensive map of drought-responsive genes and pathways in this species. To our knowledge, this is the first publicly available global transcriptome study of P. australis. This study provides a valuable genetic resource for this species.

DNA methylation is believed to play important roles in regulating gene expression in plant growth and development. Paulownia witches’ broom (PaWB) infection has been reported to be related to gene expression changes in paulownia plantlets. To determine whether DNA methylation is associated with gene expression changes in response to phytoplasma, we investigated variations in genomic DNA sequence and methylation in PaWB plantlets treated with methyl methane sulfonate (MMS) using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) techniques, respectively. The results indicated that PaWB seedings recovered a normal morphology after treatment with more than 15 mg·L⁻¹ MMS. PaWB infection did not cause changes of the paulownia DNA sequence at the AFLP level; However, DNA methylation levels and patterns were altered. Quantitative real-time PCR (qRT-PCR) showed that three of the methylated genes were up-regulated and three were down-regulated in the MMS-treated PaWB plantlets that had regained healthy morphology. These six genes might be involved in transcriptional regulation, plant defense, signal transduction and energy. The possible roles of these genes in PaWB are discussed. The results showed that changes of DNA methylation altered gene expression levels, and that MSAP might help identify genes related to PaWB.

Paulownia witches’ broom (PaWB) disease caused by phytoplasmas is a fatal disease that leads to considerable economic losses. Long non-coding RNAs (lncRNAs) have been demonstrated to play critical regulatory roles in posttranscriptional and transcriptional regulation. However, lncRNAs and their functional roles remain poorly characterized in Paulownia. To identify lncRNAs and investigate their roles in the response to PaWB phytoplasmas, RNA sequencing was performed for healthy Paulownia tomentosa, PaWB-infected P. tomentosa, and for healthy and PaWB-infected P. tomentosa treated with 100 mg L−1 rifampicin. A total of 28,614 unique mRNAs and 3693 potential lncRNAs were identified. Comparisons between lncRNAs and coding genes indicated that lncRNAs tended to have shorter transcripts and fewer exon numbers, and displayed significant expression specificity. Based on our comparison scheme, 1063 PaWB-related mRNAs and 110 PaWB-related lncRNAs were identified; among them, 12 PaWB-related candidate target genes that were regulated by nine PaWB-related lncRNAs were characterized. This study provides the first catalog of lncRNAs expressed in Paulownia and gives a revealing insight into the molecular mechanism responsible for PaWB.