Discovery of microRNAs and transcript targets related to witches’ broom disease in Paulownia fortunei by high-throughput sequencing and degradome approach

Abstract

Paulownia witches' broom (PaWB) caused by the phytoplasma is a devastating disease of Paulownia trees. It has caused heavy yield losses to Paulownia production worldwide. However, knowledge of the transcriptional and post-transcriptional regulation of gene expression by microRNAs (miRNAs), especially miRNAs responsive to PaWB disease stress, is still rudimentary. In this study, to identify miRNAs and their transcript targets that are responsive to PaWB disease stress, six sequencing libraries were constructed from healthy (PF), PaWB-infected (PFI), and PaWB-infected, 20 mg L(-1) methyl methane sulfonate-treated (PFI20) P. fortunei seedlings. As a result, 95 conserved miRNAs belonging to 18 miRNA families, as well as 122 potential novel miRNAs, were identified. Most of them were found to be a response to PaWB disease-induced stress, and the expression levels of these miRNAs were validated by quantitative real-time PCR analysis. The study simultaneously identified 109 target genes from the P. fortunei for 14 conserved miRNA families and 24 novel miRNAs by degradome sequencing. Furthermore, the functions of the miRNA targets were annotated based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The results presented here provide the groundwork for further analysis of miRNAs and target genes responsive to the PaWB disease stress, and could be also useful for addressing new questions to better understand the mechanisms of plant infection by phytoplasma in the future.

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

“…These results, together with the results of previous studies, indicate that an integrated co-regulatory network exists in the pathological process of phytoplasma infection [23]. Based on an integrated analysis of the functions of the target genes of the DEMs in this study and the previous ones [23,[51][52][53], we constructed a network that describes the regulation relationship of the DEM-mediated target genes in the manifestation of PaWB symptoms (Figure 3). …”

“…When the same miRNAs were detected in seedlings treated with both reagents, the miRNAs were identified as key miRNAs related to PaWB. Thence, we compared the common DEMs in the three paulownia species with the DEMs detected in our previous studies [23,[51][52][53][54], and found that miR156, miR398, miR408, and miR2118 were common DEMs in the healthy vs. phytoplasma-infected seedling comparisons. Thirdly, to confirm the key miRNAs related to witches' broom, we compared the four identified miRNAs with the miRNAs detected in phytoplasma-infected Mexican lime, Ziziphus jujuba, and mulberry [7, [14][15][16], and found that miR156 was the only common miRNA among the phytoplasma-responsive miRNAs in all these studies.…”

“…Although some progress has been made in understanding paulownia-phytoplasma interactions, various aspects still need to be verified [23,[51][52][53]. Firstly, the accuracy of annotation information needs to be improved.…”

ceRNA Cross-Talk in Paulownia Witches’ Broom Disease

“…PaWB is caused by phytoplasmas that belong to the Aster Yellows group Candidatus Phytoplasma asteris (16SrI-D) and are spread by insect vectors [4]. PaWB has been the focus of many studies, and some PaWB-related genes [5,6,7,8,9,10], microRNAs (miRNAs) [11,12,13,14], proteins [15,16,17], metabolites [18], and long noncoding RNAs (lncRNAs) [19] have been revealed. However, the underlying mechanisms of PaWB are still not known.…”

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

“…Rifampicin treatment can make PaWB-infected Paulownia recover to normal morphology [13]. Furthermore, the rapid development of 'omics' has allowed for the investigation of the transcriptomes [3,[14][15][16][17][18], microRNAs (miRNAs), degradomes [19][20][21][22], and proteomes [4,23] of Paulownia species, and the results have revealed changes after PaWB infection at transcriptional, post-transcriptional, and translational levels.…”