Transcriptome, microRNA, and degradome analyses of the gene expression of Paulownia with phytoplamsa

Primers of P. tomentosa miRNAs for qRT-PCR analysis. (DOCX 20.7 kb

Genome-Wide Analysis of Gene and microRNA Expression in Diploid and Autotetraploid Paulownia fortunei (Seem) Hemsl. under Drought Stress by Transcriptome, microRNA, and Degradome Sequencing

Drought is a common and recurring climatic condition in many parts of the world, and it can have disastrous impacts on plant growth and development. Many genes involved in the drought response of plants have been identified. Transcriptome, microRNA (miRNA), and degradome analyses are rapid ways of identifying drought-responsive genes. The reference genome sequence of Paulownia fortunei (Seem) Hemsl. is now available, which makes it easier to explore gene expression, transcriptional regulation, and post-transcriptional in this species. In this study, four transcriptome, small RNA, and degradome libraries were sequenced by Illumina sequencing, respectively. A total of 258 genes and 11 miRNAs were identified for drought-responsive genes and miRNAs in P. fortunei. Degradome sequencing detected 28 miRNA target genes that were cleaved by members of nine conserved miRNA families and 12 novel miRNAs. The results here will contribute toward enriching our understanding of the response of Paulownia fortunei trees to drought stress and may provide new direction for further experimental studies related the development of molecular markers, the genetic map construction, and other genomic research projects in Paulownia

Plant-Pathogen Interaction, Circadian Rhythm, and Hormone-Related Gene Expression Provide Indicators of Phytoplasma Infection in Paulownia fortunei

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

Identification and Functional Analysis of MicroRNAs and Their Targets in Platanus acerifolia under Lead (Pb) Stress

MicroRNAs (miRNAs) play important regulatory roles in development and stress responses in plants. Lead (Pb) is a non-essential element that is highly toxic to living organisms. Platanus acerifolia is grown as a street tree in cities throughout temperate regions for its importance in improving the urban ecological environment. MiRNAs that respond to abiotic stresses have been identified in plants; however, until now, the influence of Pb stress on P. acerifolia miRNAs has not been reported. To identify miRNAs and predict their target genes under Pb stress, two small RNA and two degradome libraries were constructed from Pb-treated and Pb-free leaves of P. acerifolia seedlings. After sequencing, 55 known miRNAs and 129 novel miRNAs were obtained, and 104 target genes for the miRNAs were identified by degradome sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to predict the functions of the targets. The expressions of eight differentially expressed miRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). This is the first report about P. acerifolia miRNAs and their target genes under Pb stress. This study has provided data for further research into molecular mechanisms involved in resistance of P. acerifolia to Pb stress

Discovery of genes related to witches broom disease in Paulownia tomentosa × Paulownia fortunei by a De Novo assembled transcriptome.

In spite of its economic importance, very little molecular genetics and genomic research has been targeted at the family Paulownia spp. The little genetic information on this plant is a big obstacle to studying the mechanisms of its ability to resist Paulownia Witches' Broom (PaWB) disease. Analysis of the Paulownia transcriptome and its expression profile data are essential to extending the genetic resources on this species, thus will greatly improves our studies on Paulownia. In the current study, we performed the de novo assembly of a transcriptome on P. tomentosa × P. fortunei using the short-read sequencing technology (Illumina). 203,664 unigenes with a mean length of 1,328 bp was obtained. Of these unigenes, 32,976 (30% of all unigenes) containing complete structures were chosen. Eukaryotic clusters of orthologous groups, gene orthology, and the Kyoto Encyclopedia of Genes and Genomes annotations were performed of these unigenes. Genes related to PaWB disease resistance were analyzed in detail. To our knowledge, this is the first study to elucidate the genetic makeup of Paulownia. This transcriptome provides a quick way to understanding Paulownia, increases the number of gene sequences available for further functional genomics studies and provides clues to the identification of potential PaWB disease resistance genes. This study has provided a comprehensive insight into gene expression profiles at different states, which facilitates the study of each gene's roles in the developmental process and in PaWB disease resistance

Expression of Pinellia pedatisecta Lectin Gene in Transgenic Wheat Enhances Resistance to Wheat Aphids

Wheat aphids are major pests during the seed filling stage of wheat. Plant lectins are toxic to sap-sucking pests such as wheat aphids. In this study, Pinellia pedatisecta agglutinin (ppa), a gene encoding mannose binding lectin, was cloned, and it shared 92.69% nucleotide similarity and 94% amino acid similarity with Pinellia ternata agglutinin (pta). The ppa gene, driven by the constitutive and phloem-specific ribulose bisphosphate carboxylase small subunit gene (rbcs) promoter in pBAC-rbcs-ppa expression vector, was transferred into the wheat cultivar Baofeng104 (BF104) by particle bombardment transformation. Fifty-four T0 transgenic plants were generated. The inheritance and expression of the ppa gene were confirmed by PCR and RT-PCR analysis respectively, and seven homozygous transgenic lines were obtained. An aphid bioassay on detached leaf segments revealed that seven ppa transgenic wheat lines had lower aphid growth rates and higher inhibition rates than BF104. Furthermore, two-year aphid bioassays in isolated fields showed that aphid numbers per tiller of transgenic lines were significantly decreased, compared with wild type BF104. Therefore, ppa could be a strong biotechnological candidate to produce aphid-resistant wheat

Transcriptome-Wide Profiling and Expression Analysis of Diploid and Autotetraploid <i>Paulownia tomentosa</i> × <i>Paulownia fortunei</i> under Drought Stress

<div><p>Paulownia is a fast-growing deciduous hardwood species native to China, which has high ecological and economic value. In an earlier study, we reported ploidy-dependent differences in Paulownia drought tolerance by the microscopic observations of the leaves. Autotetraploid Paulownia has a higher resistance to drought stress than their diploid relatives. In order to obtain genetic information on molecular mechanisms responses of Paulownia plants to drought, Illumina/Solexa Genome sequencing platform was used to <i>de novo</i> assemble the transcriptomes of leaves from diploid and autotetraploid <i>Paulownia tomentosa</i> × <i>Paulownia fortunei</i> seedlings (PTF2 and PTF4 respectively) grown under control conditions and under drought stress and obtained 98,671 nonredundant unigenes. A comparative transcriptome analysis revealed that hundreds of unigenes were predicted to be involved mainly in ROS-scavenging system, amino acid and carbohydrate metabolism, plant hormone biosynthesis and signal transduction, while these unigenes exhibited differential transcript alteration of the two accessions. This study provides a comprehensive map of how <i>P. tomentosa</i> × <i>P. fortunei</i> responds to drought stress at physiological and molecular levels, which may help in understanding the mechanisms involve in water-deficit response and will be useful for further study of drought tolerance in woody plants.</p></div

Transcriptome/Degradome-Wide Discovery of MicroRNAs and Transcript Targets in Two <i>Paulownia australis</i> Genotypes

<div><p>MicroRNAs (miRNAs) are involved in plant growth, development, and response to biotic and abiotic stresses. Most of the miRNAs that have been identified in model plants are well characterized, but till now, no reports have previously been published concerning miRNAs in <i>Paulownia australis</i>. In order to investigate miRNA-guided transcript target regulation in <i>P. australis</i>, small RNA libraries from two <i>P. australis</i> (diploids, PA2; and autotetraploids, PA4) genotypes were subjected to Solexa sequencing. As a result, 10,691,271 (PA2) and 10,712,733 (PA4) clean reads were obtained, and 45 conserved miRNAs belonging to 15 families, and 31 potential novel miRNAs candidates were identified. Compared with their expression levels in the PA2 plants, 26 miRNAs were up-regulated and 15 miRNAs were down-regulated in the PA4 plants. The relative expressions of 12 miRNAs were validated by quantitative real-time polymerase chain reaction. Using the degradome approach, 53 transcript targets were identified and annotated based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. These targets were associated with development, stimulus response, metabolism, signaling transduction and biological regulation. Among them, 11 targets, including TCP transcription factors, auxin response factors, squamosa promoter-binding-like proteins, scarecrow-like proteins, L-type lectin-domain containing receptor kinases and zinc finger CCCH domain-containing protein, cleaved by four known miRNA family and two potentially novel miRNAs were found to be involved in regulating plant development, biotic and abiotic stresses. The findings will be helpful to facilitate studies on the functions of miRNAs and their transcript targets in <i>Paulownia.</i></p></div