Dynamic expression of novel and conserved microRNAs and their targets in diploid and tetraploid of Paulownia tomentosa

AbstractMicroRNAs (miRNAs) play profound roles in plant growth and development by regulating gene expression. Tetraploid plants often have better physical characteristics and stress tolerance than their diploid progenitors, but the role of miRNAs in this superiority is unclear. Paulownia tomentosa, (Paulowniaceae) is attracting research attention in China because of its rapid development, wide distribution, and potential economic uses. To identify miRNAs at the transcriptional level in P. tomentosa, Illumina sequencing was used to sequence the libraries of diploid and tetraploid plants. Sequence analysis identified 37 conserved miRNAs belonging to 14 miRNA families and 14 novel miRNAs belonging to seven miRNA families. Among the miRNAs, 16 conserved miRNAs from 11 families and five novel miRNAs were differentially expressed in the tetraploid and diploid; most were more strongly expressed in the former. The miRNA target genes and their functions were identified and discussed. The results showed that several P. tomentosa miRNAs may play important roles in the improved traits seen in tetraploids. This study provides a foundation for understanding the regulatory mechanisms of miRNAs in tetraploid trees

ceRNA Cross-Talk in Paulownia Witches’ Broom Disease

Long noncoding RNA (lncRNA), circular RNA (circRNA), and microRNA (miRNA) are important in the regulation of life activities. However, their function is unclear in Paulownia fortunei. To identify lncRNAs, circRNAs, and miRNA, and investigate their roles in the infection progress of Paulownia witches’ broom (PaWB) disease, we performed RNA sequencing of healthy and infected P. fortunei. A total of 3126 lncRNAs, 1634 circRNAs, and 550 miRNAs were identified. Among them, 229 lncRNAs, 65 circRNAs, and 65 miRNAs were differentially expressed in a significant manner. We constructed a competing endogenous RNA (ceRNA) network, which contains 5 miRNAs, 4 circRNAs, 5 lncRNAs, and 15 mRNAs, all of which were differentially expressed between healthy and infected P. fortunei. This study provides the first catalog of candidate ceRNAs in Paulownia and gives a revealing insight into the molecular mechanism responsible for PaWB

Combined Analysis of mRNAs and miRNAs to Identify Genes Related to Biological Characteristics of Autotetraploid Paulownia

Autopolyploid plants and their organs are larger than their corresponding diploid ancestors, and they attract considerable attention for plant breeding. Paulownia is a fast-growing tree. To identify genes related to the biological characteristics of tetraploid Paulownia, transcriptome and small RNA sequencing were used to identify the key gene expression regulation in tetraploid Paulownia fortunei and tetraploid P. tomentosa and their corresponding diploids. A total of 1977 common differentially expressed genes (DEGs) and 89 differentially expressed miRNAs (DEMs) (38 conserved and 51 novel) were obtained in tetraploid vs. diploid comparisons of the two Paulownia species, and 18 target genes were identified by target prediction. Finally, by analyzing the expression profiles of the DEGs and DEMs and their target genes, we discovered that Pau-miR169, Pau-miR408 and Pau-miR156 interacted with their target gene nuclear transcription factor Y subunit A-9 (NF-YA9), serine/threonine protein phosphatase (PP1) and s-adenosyl-methionine-sterol-c-methyltransfera—se (SAM:SMT) to regulate the abiotic stress tolerance and the timber quality of the tetraploid Paulownia. This study lays a molecular biology foundation for understanding autotetraploid Paulownia and will benefit future breeding work

Long Non-Coding RNAs Responsive to Witches’ Broom Disease in Paulownia tomentosa

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

Genome-Wide Characterization of Calmodulin and Calmodulin-like Protein Gene Families in <i>Paulownia fortunei</i> and Identification of Their Potential Involvement in <i>Paulownia</i> Witches’ Broom

As significant Ca2+ sensors, calmodulin (CaM) and calmodulin-like proteins (CML), have been associated with a variety of environmental conditions in plants. However, whether CaMs/CMLs are related to the stress of phytoplasma infection has not been reported in Paulownia fortunei. In the current study, 5 PfCaMs and 58 PfCMLs were detected through a genome-wide investigation. The number of EF-hand motifs in all PfCaMs/CMLs varied. Bioinformatics analyses, including protein characteristics, conserved domain, gene structure, cis-elements, evolutionary relationship, collinearity, chromosomal location, post-translation modification site, subcellular localization and expression pattern analyses, represented the conservation and divergence of PfCaMs/CMLs. Furthermore, some PfCaMs/CMLs might be involved in plants’ reaction to phytoplasma infection and exogenous calcium therapy, indicating these genes may play a role in abiotic as well as biotic stress responses. In addition, subcellular localization analysis showed that PfCML10 was located in the cell membrane and nucleus. In summary, these findings establish a stronger platform for their subsequent functional investigation in trees and further characterize their roles in Paulownia witches’ broom (PaWB) occurrence

Comparative Analysis of MicroRNA Expression in Three Paulownia Species with Phytoplasma Infection

Paulownia witches&rsquo; broom (PaWB), caused by phytoplasma, is an important disease of Paulownia. To further identify the key miRNAs associated with the formation of PaWB symptoms, miRNA and degradome sequencing were performed to explore important miRNAs&ndash;target regulation in healthy and diseased Paulownia tomentosa, Paulownia fortunei, and P. tomentosa &times; P. fortunei seedlings, and the corresponding diseased seedlings treated with 75 mg L&minus;1 dimethyl sulfate. A total of 212, 111, and 197 differentially expressed miRNAs (DEMs) were obtained in P. tomentosa, P. fortunei, and P. tomentosa &times; P. fortunei, respectively. Degradome sequencing detected 559, 251, and 568 target genes of the DEMs in P. tomentosa, P. fortunei, and P. tomentosa &times; P. fortunei, respectively. The expression patterns of selected miRNAs and the target genes were verified be qRT-PCR. Through analysis of the expression level of the DEMs in this study, combined with the results in our previous studies, as well as with those reported in other phytoplasma-infected plants, we concluded that miR156 is an important miRNA related to witches&rsquo; broom. According to the functions of the target genes of DEMs, we constructed a co-regulatory network of the DEMs-target genes interaction. These results will help to advance the understanding of the mechanism of PaWB

Comparative Transcriptomics Analysis of Phytohormone-Related Genes and Alternative Splicing Events Related to Witches’ Broom in Paulownia

Paulownia is a native fast-growing tree in China that has been introduced into many countries. However, it is often infected by Paulownia witches&rsquo; broom (PaWB) disease, which can lead to large declines in yield. PaWB is caused by a phytoplasma that is an obligate biotrophic plant pathogen. Until now, the molecular mechanisms of interactions between the host plants and the phytoplasma have not been clear. In previous studies, it was reported that PaWB-infected Paulownia exhibited healthy morphology after being treated with methyl methane sulfonate (MMS) at the concentration of 20 mg&middot;L&minus;1 (for Paulownia tomentosa (PT) and Paulownia fortunei (PF) or 15 mg&middot;L&minus;1 (for P. tomentosa &times; P. fortunei) MMS. In this study, the whole transcriptome expression profile of PaWB-infected Paulownia was studied using high-throughput sequencing technology. In total, 74 significantly differentially expressed genes were detected among three species of healthy, PaWB-infected Paulownia, and the Paulownia treated with MMS. We identified and analyzed genes related to the roles of phytohormones and alternative splicing events involved in regulating plant growth. In response to phytoplasma infection, the concentrations of the plants&rsquo; phytohormones were altered, leading to morphology transformation. This research will provide valuable information to detect the molecular mechanisms involved in the Paulownia response to phytoplasma infection

Genome-Wide Analysis of Specific <i>PfR2R3-MYB</i> Genes Related to Paulownia Witches’ Broom

Paulownia witches’ broom (PaWB), caused by phytoplasmas, is the most devastating infectious disease of Paulownia. R2R3-MYB transcription factors (TF) have been reported to be involved in the plant’s response to infections caused by these pathogens, but a comprehensive study of the R2R3-MYB genes in Paulownia has not been reported. In this study, we identified 138 R2R3-MYB genes distributed on 20 chromosomes of Paulownia fortunei. These genes were classified into 27 subfamilies based on their gene structures and phylogenetic relationships, which indicated that they have various evolutionary relationships and have undergone rich segmental replication events. We determined the expression patterns of the 138 R2R3-MYB genes of P. fortunei by analyzing the RNA sequencing data and found that PfR2R3-MYB15 was significantly up-regulated in P. fortunei in response to phytoplasma infections. PfR2R3-MYB15 was cloned and overexpressed in Populus trichocarpa. The results show that its overexpression induced branching symptoms. Subsequently, the subcellular localization results showed that PfR2R3-MYB15 was located in the nucleus. Yeast two-hybrid and bimolecular fluorescence complementation experiments showed that PfR2R3-MYB15 interacted with PfTAB2. The analysis of the PfR2R3-MYB15 gene showed that it not only played an important role in plant branching, but also might participate in the biosynthesis of photosystem elements. Our results will provide a foundation for future studies of the R2R3-MYB TF family in Paulownia and other plants

Transcriptome and Small RNA Sequencing Analysis Revealed Roles of PaWB-Related miRNAs and Genes in Paulownia fortunei

Paulownia witches&rsquo; broom (PaWB) is an epidemic disease caused by phytoplasmas infection, which is responsible for large production and economic losses. The study of PaWB has made significant progress, but the specific molecular mechanisms associated with PaWB remain unclear. To clearly know the gene expression profiles of plantlets infected with phytoplasmas, in this study, we used high-throughput sequencing technology to generate an integrated analysis of the transcriptome and microRNAs (miRNAs) of Paulownia fortunei (seem.) Hemsl. plantlets, and to obtain a comprehensive resource for the relationship between vital miRNA-target gene pairs and PaWB. A total of 756 genes, and 45 conserved and 22 new miRNAs were identified associated with PaWB. In addition, 635 target genes were predicted for the 67 DERs (Differentially expressed miRNAs). An interaction network of these miRNAs and their target genes was constructed. Gene ontology (GO) and KEGG pathway analysis of these target genes indicated that genes encoding transcription factors (TFs), including auxin response factors (ARF), WRKY, NAC (NAM, ATAF1/2 and CUC2), and MYB (v-myb avian myeloblastosis viral oncogene homolog), and genes encoding superoxide dismutase (SOD), as well as alternative splicing were related directly or indirectly to PaWB. Our results shed light on the possible roles of genes and miRNAs in PaWB-infected plantlets, which will enhance the understanding of the PaWB mechanism in Paulownia plants