Paulownia Witches' Broom Research Articles

Novel Insight into the Prevention and Therapeutic Treatment of Paulownia Witches’ Broom: A Study on the Effect of Salicylic Acid on Disease Control and the Changes in the Paulownia Transcriptome and Proteome

Paulownia species not only have significant economic benefits but also show great potential in ecological conservation. However, they are highly susceptible to phytoplasma infections, causing Paulownia witches’ broom (PaWB), which severely restricts the development of the Paulownia industry. Salicylic acid (SA) plays a crucial role in plant disease resistance. However, there have been no reports on the effect of SA on PaWB. Due to the properties of SA, it may have potential in controlling PaWB. Based on the above speculation, the prevention and therapeutic effect of SA on PaWB and its effect on the PaWB-infected Paulownia transcriptome and proteome were studied in this work. The results indicated that 0.1 mmol/L was the optimal SA concentration for inhibiting the germination of Paulownia axillary buds. In terms of resistance physiological indicators, SA treatment significantly affected both Paulownia tomentosa infected (PTI) seedlings and Paulownia fortunei infected (PFI) seedlings, where the activities of peroxidase (POD) and superoxide dismutase (SOD) were enhanced. Malondialdehyde (MDA), O2−, and H2O2, however, were significantly reduced. Specifically, after SA treatment, SOD activity increased by 28% in PFI and 25% in PTI, and POD activity significantly increased by 61% in PFI and 58% in PTI. Moreover, the MDA content decreased by 30% in PFI and 23% in PTI, the H2O2 content decreased by 26% in PFI and 19% in PTI, and the O2− content decreased by 21% in PFI and 19% in PTI. Transcriptomic analysis showed that there were significant upregulations of MYB, NAC, and bHLH and other transcription factors after SA treatment. Moreover, genes involved in PaWB-related defense responses such as RAX2 also showed significant differences. Furthermore, proteomic analysis indicated that after SA treatment, proteins involved in signal transduction, protein synthesis modification, and disease defense were differentially expressed. This work provides a research foundation for the prevention and treatment of PaWB and offers references for exploring anti-PaWB methods.

Paulownia Witches' Broom Disease: A Comprehensive Review.

Phytoplasmas are insect-transmitted bacterial pathogens associated with diseases in a wide range of host plants, resulting in significant economic and ecological losses. Perennial deciduous trees in the genus Paulownia are widely planted for wood harvesting and ornamental purposes. Paulownia witches' broom (PaWB) disease, associated with a 16SrI-D subgroup phytoplasma, is a destructive disease of paulownia in East Asia. The PaWB phytoplasmas are mainly transmitted by insect vectors in the Pentatomidae (stink bugs), Miridae (mirid bugs) and Cicadellidae (leafhoppers) families. Diseased trees show typical symptoms, such as branch and shoot proliferation, which together are referred to as witches' broom. The phytoplasma presence affects the physiological and anatomical structures of paulownia. Gene expression in paulownia responding to phytoplasma presence have been studied at the transcriptional, post-transcriptional, translational and post-translational levels by high throughput sequencing techniques. A PaWB pathogenic mechanism frame diagram on molecular level is summarized. Studies on the interactions among the phytoplasma, the insect vectors and the plant host, including the mechanisms underlying how paulownia effectors modify processes of gene expression, will lead to a deeper understanding of the pathogenic mechanisms and to the development of efficient control measures.

Comprehensive Analysis of the GRAS Gene Family in Paulownia fortunei and the Response of DELLA Proteins to Paulownia Witches' Broom.

The GRAS (GAI\RGA\SCL) gene family encodes plant-specific transcription factors that play crucial roles in plant growth and development, stress tolerance, and hormone network regulation. Plant dwarfing symptom is mainly regulated by DELLA proteins of the GRAS gene subfamily. In this study, the association between the GRAS gene family and Paulownia witches' broom (PaWB) was investigated. A total of 79 PfGRAS genes were identified using bioinformatics methods and categorized into 11 groups based on amino acid sequences. Tandem duplication and fragment duplication were found to be the main modes of amplification of the PfGRAS gene family. Gene structure analysis showed that more than 72.1% of the PfGRASs had no introns. The genes PfGRAS12/18/58 also contained unique DELLA structural domains; only PfGRAS12, which showed significant response to PaWB phytoplasma infection in stems, showed significant tissue specificity and responded to gibberellin (GA3) in PaWB-infected plants. We found that the internodes were significantly elongated under 100 µmol·L-1 GA3 treatment for 30 days. The subcellular localization analysis indicated that PfGRAS12 is located in the nucleus and cell membrane. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays confirmed that PfGRAS12 interacted with PfJAZ3 in the nucleus. Our results will lay a foundation for further research on the functions of the PfGRAS gene family and for genetic improvement and breeding of PaWB-resistant trees.

Bioinformatic Analysis of the BTB Gene Family in Paulownia fortunei and Functional Characterization in Response to Abiotic and Biotic Stresses.

To learn about the gene structure, phylogenetic evolution, and function under biotic and abiotic stresses of BTB (Bric-a-Brac/Tramtrack/Broad Complex) genes in Paulownia fortunei, a whole-genome sequence evaluation was carried out, and a total of 62 PfBTB genes were identified. The phylogenetic analysis showed that PfBTB proteins are divided into eight groups, and these proteins are highly conserved. PfBTB genes were unevenly distributed on 17 chromosomes. The colinearity analysis found that fragment replication and tandem replication are the main modes of gene amplification in the PfBTB family. The analysis of cis-acting elements suggests that PfBTB genes may be involved in a variety of biological processes. The transcriptomic analysis results showed that PfBTB3/12/14/16/19/36/44 responded to Paulownia witches' broom (PaWB), while PfBTB1/4/17/43 responded to drought stress, and the RT-qPCR results further support the reliability of transcriptome data. In addition, the association analysis between miRNA and transcriptome revealed a 91-pair targeting relationship between miRNAs and PfBTBs. In conclusion, the BTB genes in Paulownia are systematically identified in this research. This work provides useful knowledge to more fully appreciate the potential functions of these genes and their possible roles in the occurrence of PaWB and in response to stress.

Genome-Wide Characterization of Calmodulin and Calmodulin-like Protein Gene Families in Paulownia fortunei and Identification of Their Potential Involvement in Paulownia 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.

N6‐methyladenosine modification changes during the recovery processes for Paulownia witches' broom disease under the methyl methanesulfonate treatment

AbstractPhytoplasmas induce diseases in more than 1000 plant species and cause substantial ecological damage and economic losses, but the specific pathogenesis of phytoplasma has not yet been clarified. N6‐methyladenosine (m6A) is the most common internal modification of the eukaryotic Messenger RNA (mRNA). As one of the species susceptible to phytoplasma infection, the pathogenesis and mechanism of Paulownia has been extensively studied by scholars, but the m6A transcriptome map of Paulownia fortunei (P. fortunei) has not been reported. Therefore, this study aimed to explore the effect of phytoplasma infection on m6A modification of P. fortunei and obtained the whole transcriptome m6A map in P. fortunei by m6A‐seq. The m6A‐seq results of Paulownia witches' broom (PaWB) disease and healthy samples indicate that PaWB infection increased the degree of m6A modification of P. fortunei. 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. Moreover, the functions of PaWB‐related genes 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). 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 methyl methanesulfonate, and m6A modification was found in m6A‐seq results. Moreover, Reverse Transcription–Polymerase Chain Reaction (RT‐PCR) verified that the alternative splicing of these two genes was associated with m6A modification. This comprehensive map provides a solid foundation for revealing the potential function of the mRNA m6A modification in the process of PaWB. In future studies, we plan to verify genes directly related to PaWB and methylation‐related enzymes in Paulownia to elucidate the pathogenic mechanism of PaWB caused by phytoplasma invasion.

Genome-Wide Identification and Expression of the Paulownia fortunei MADS-Box Gene Family in Response to Phytoplasma Infection.

Paulownia witches' broom (PaWB), caused by phytoplasmas, is the most devastating infectious disease of Paulownia. Although a few MADS-box transcription factors have been reported to be involved in the formation of PaWB, there has been little investigation into all of the MADS-box gene family in Paulownia. The objective of this study is to identify the MADS-box gene family in Paulownia fortunei on a genome-wide scale and explore their response to PaWB infection. Bioinformatics software were used for identification, characterization, subcellular localization, phylogenetic analysis, the prediction of conserved motifs, gene structures, cis-elements, and protein-protein interaction network construction. The tissue expression profiling of PfMADS-box genes was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Transcriptome data and the protein interaction network prediction were combined to screen the genes associated with PaWB formation. We identified 89 MADS-box genes in the P. fortunei genome and categorized them into 14 subfamilies. The comprehensive analysis showed that segment duplication events had significant effects on the evolution of the PfMADS-box gene family; the motif distribution of proteins in the same subfamily are similar; development-related, phytohormone-responsive, and stress-related cis-elements were enriched in the promoter regions. The tissue expression pattern of PfMADS-box genes suggested that they underwent subfunctional differentiation. Three genes, PfMADS3, PfMADS57, and PfMADS87, might be related to the occurrence of PaWB. These results will provide a valuable resource to explore the potential functions of PfMADS-box genes and lay a solid foundation for understanding the roles of PfMADS-box genes in paulownia-phytoplasma interactions.

Genome-Wide Analysis of Specific PfR2R3-MYB 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.

Molecular Diversity and Evolutionary Relatedness of Paulownia Witches’-Broom Phytoplasma in Different Geographical Distributions in China

To reveal the distribution and transmission pathway of Paulownia witches'-broom (PaWB) disease, which is caused by phytoplasmas related to genetic variation, and the adaptability to the hosts and environments of the pathogenic population in different geographical regions in China, in this study, we used ten housekeeping gene fragments, including rp, fusA, secY, tuf, secA, dnaK, rpoB, pyrG, gyrB, and ipt, for multilocus sequence typing (MLST). A total of 142 PaWB phytoplasma strains were collected from 18 provinces or municipalities. The results showed that the genetic diversity was comparatively higher among the PaWB phytoplasma strains, and substantially different from that of the other 16SrI subgroup strains. The number of gene variation sites for different housekeeping genes in the PaWB phytoplasma strains ranged from 1 to 14 SNPs. Among them, rpoB (1.47%) and dnaK (1.12%) had higher genetic variation, and rp (0.20%) had the least genetic variation. The tuf and rpoB genes showed the fixation of positively selected beneficial mutations in the PaWB phytoplasma populations, and all housekeeping genes except tuf followed the neutral evolutionary model. We found an absence of recombination among PaWB phytoplasma sequence types (STs) for each housekeeping gene except dnaK, and no evidence for such recombination events for concatenated sequences of PaWB phytoplasma strains. The 22 sequence types were identified among the concatenated sequences of seven housekeeping genes (rp, fusA, secY, secA, tuf, dnaK, and rpoB) from 105 representative strains. We analyzed all 22 STs by goeBURST algorithm, forming two clonal complexes (CCs) and three singletons. Among them, ST1, as the primary founder of CC1, had the widest geographical distribution, accounting for 72.38% of all strains, with a high frequency of shared sequence type. The results of phylogenetic analysis of the concatenated sequences further revealed that the 105 strains were clustered into two representative lineages of PaWB phytoplasma, with obvious geographical differentiation. The ST1 strains of highly homogeneous lineage-1 were a widespread and predominant population in diseased areas. Lineage-2 contained strains from Jiangxi, Fujian, and Shaanxi provinces, highlighting the close genetic relatedness of the strains in these regions, which was also consistent with the results of most single-gene phylogenetic analysis of each gene. We also found that the variability in the northwest China population was higher than in other geographical populations; the range of genetic differentiation between the south of the Yangtze River population and the Huang-huai-hai Plain (or southwest China) population was relatively large. The achieved diversity and evolution data, as well as the MLST technique, are helpful for epidemiological studies and guiding PaWB disease control decisions.

Integrated full-length transcriptome and metabolome analysis of the early defense mechanisms in Paulownia witches’ broom disease

ABSTRACT Paulownia witches’ broom (PaWB) is a devastating disease of Paulownia spp. caused by phytoplasma infection. To reveal the reason for the absence of morphological changes at the early stage of phytoplasma infection, in this study, a combination of second-and third-generation sequencing (single-molecule real-time long-read isoform) and liquid chromatography tandem mass spectrometry were performed to analyse the changes of full-length transcripts and metabolites at the early stage of phytoplasma infection. A total of 140,528 non-redundant full-length transcripts, 597 transcription factors, 1,658 long non-coding RNAs, and 645 metabolites were identified. The third-generation sequencing improved the annotation of the reference Paulownia fortunei (Seem.) Hemsl. genome by 8.86%. Combination analysis of the full-length transcriptome and metabolome revealed that phenolics and phenylalanine ammonia-lyase synthesis related genes were significantly up-regulated in the early stage of phytoplasma-infected P. fortunei. Based on the results of full-length transcriptome and metabolome integrative analysis, we constructed an early defence model diagram of PaWB disease. Our results provide powerful insights into the reason for absence of morphological at the early stage of phytoplasma infection.

Conformational Changes in Three-Dimensional Chromatin Structure in Paulownia fortunei After Phytoplasma Infection.

Higher-order chromatin structures play important roles in regulating multiple biological processes such as growth and development as well as biotic and abiotic stress response. However, little is known about three-dimensional chromatin structures in Paulownia or about whole-genome chromatin conformational changes that occur in response to Paulownia witches' broom (PaWB) disease. We used high-throughput chromosome conformation capture (Hi-C) to obtain genome-wide profiles of chromatin conformation in both healthy and phytoplasma-infected Paulownia fortunei genome. The heat map results indicated that the strongest interactions between chromosomes were in the telomeres. We confirmed that the main structural characteristics of A/B compartments, topologically associated domains, and chromatin loops were prominent in the Paulownia genome and were clearly altered in phytoplasma-infected plants. By combining chromatin immunoprecipitation sequencing, Hi-C signals, and RNA sequencing data, we inferred that the chromatin structure changed and the modification levels of three histones (H3K4me3/K9ac/K36me3) increased in phytoplasma-infected P. fortunei, which was associated with changes of transcriptional activity. We concluded that for epigenetic modifications, transcriptional activity might function in combination to shape chromatin packing in healthy and phytoplasm-infected Paulownia. Finally, 11 genes (e.g., RPN6, Sec61 subunit-α) that were commonly located at specific topologically associated domain boundaries, A/B compartment switching and specific loops, and had been associated with histone marks were identified and considered as closely related to PaWB stress. Our results provide new insights into the nexus between gene regulation and chromatin conformational alterations in nonmodel plants upon phytopathogen infection and plant disease resistance.

Genomic insights into the fast growth of paulownias and the formation of Paulownia witches' broom

Paulownias are among the fastest growing trees in the world, but they often suffer tremendous loss of wood production due to infection by Paulownia witches' broom (PaWB) phytoplasmas. In this study, we have sequenced and assembled a high-quality nuclear genome of Paulownia fortunei, a commonly cultivated paulownia species. The assembled genome of P. fortunei is 511.6 Mb in size, with 93.2% of its sequences anchored to 20 pseudo-chromosomes, and it contains 31 985 protein-coding genes. Phylogenomic analyses show that the family Paulowniaceae is sister to a clade composed of Phrymaceae and Orobanchaceae. Higher photosynthetic efficiency is achieved by integrating C3 photosynthesis and the crassulacean acid metabolism pathway, which may contribute to the extremely fast growth habit of paulownia trees. Comparative transcriptome analyses reveal modules related to cambial growth and development, photosynthesis, and defense responses. Additional genome sequencing of PaWB phytoplasma, combined with functional analyses, indicates that the effector PaWB-SAP54 interacts directly with Paulownia PfSPLa, which in turn causes the degradation of PfSPLa by the ubiquitin-mediated pathway and leads to the formation of witches' broom. Taken together, these results provide significant insights into the biology of paulownias and the regulatory mechanism for the formation of PaWB.

Whole-genome landscape of H3K4me3, H3K36me3 and H3K9ac and their association with gene expression during Paulownia witches' broom disease infection and recovery processes.

Histone methylation and acetylation participate in the modulation of gene expression. Here, chromatin immunoprecipitation sequencing (ChIP-Seq) was used to determine genome-wide patterns of three histone modifications, H3K4me3, H3K36me3, and H3K9ac (associated with actively expressed genes) and their associations with gene expression in Paulownia fortunei following phytoplasma infection and recovery from Paulownia witches' broom (PaWB) disease after methyl methane sulfonate treatment. The three histone marks were preferentially deposited in genic regions, especially downstream of transcription start sites, and were highly concurrent with gene expression. Genes with all three histone marks exhibited the highest expression levels. Based on the comparison scheme, we detected 365, 2244, and 752 PaWB-associated genes with H3K4me3, H3K36me3, and H3K9ac marks, separately. KEGG pathway analysis showed that these genes were involved in plant-pathogen interaction, plant hormone signal transduction, and starch and sucrose metabolism. A small proportion of differentially modified genes showed changes in expression in response to phytoplasma infection, including genes involved in calcium ion signal transduction, abscisic acid signal transduction, and ethylene biosynthesis. This comprehensive analysis of genome-wide histone modifications and gene expression in Paulownia following phytoplasma infection provides new insights into the epigenetic responses to phytoplasma infection and will be useful for further studies on epigenetic regulation mechanisms in plants under biotic stress.

Correction: Phytoplasma-induced Changes in the Acetylome and Succinylome of Paulownia Tomentosa Provide Evidence for Involvement of Acetylated Proteins in Witches' Broom Disease.

Lysine acetylation and succinylation are post-translational modifications of proteins that have been shown to play roles in plants response to pathogen infection. Phytoplasma infection can directly alter multiple metabolic processes in the deciduous plant Paulownia and lead to Paulownia witches' broom (PaWB) disease, the major cause of Paulownia mortality worldwide. However, the extent and function of lysine aceylation and succinylation during phytoplasma infection have yet to be explored. Here, we investigated the changes in the proteome, acetylome, and succinylome of phytoplasma-infected Paulownia tomentosa seedlings using quantitative mass spectrometry. In total, we identified 8963 proteins, 2893 acetylated proteins (5558 acetylation sites), and 1271 succinylated proteins (1970 succinylation sites), with 425 (533 sites) simultaneously acetylated and succinylated. Comparative analysis revealed that 276 proteins, 546 acetylated proteins (741 acetylation sites) and 5 succinylated proteins (5 succinylation sites) were regulated in response to phytoplasma infection, suggesting that acetylation may be more important than succinylation in PaWB. Enzymatic assays showed that acetylation of specific sites in protochlorophyllide reductase and RuBisCO, key enzymes in chlorophyll and starch biosynthesis, respectively, modifies their activity in phytoplasma-infected seedlings. On the basis of these results, we propose a model to elucidate the molecular mechanism of responses to PaWB and offer a resource for functional studies on the effects of acetylation on protein function.

Phytoplasma-induced Changes in the Acetylome and Succinylome of Paulownia tomentosa Provide Evidence for Involvement of Acetylated Proteins in Witches' Broom Disease

Lysine acetylation and succinylation are post-translational modifications of proteins that have been shown to play roles in plants response to pathogen infection. Phytoplasma infection can directly alter multiple metabolic processes in the deciduous plant Paulownia and lead to Paulownia witches' broom (PaWB) disease, the major cause of Paulownia mortality worldwide. However, the extent and function of lysine aceylation and succinylation during phytoplasma infection have yet to be explored. Here, we investigated the changes in the proteome, acetylome, and succinylome of phytoplasma-infected Paulownia tomentosa seedlings using quantitative mass spectrometry. In total, we identified 8963 proteins, 2893 acetylated proteins (5558 acetylation sites), and 1271 succinylated proteins (1970 succinylation sites), with 425 (533 sites) simultaneously acetylated and succinylated. Comparative analysis revealed that 276 proteins, 546 acetylated proteins (741 acetylation sites) and 5 succinylated proteins (5 succinylation sites) were regulated in response to phytoplasma infection, suggesting that acetylation may be more important than succinylation in PaWB. Enzymatic assays showed that acetylation of specific sites in protochlorophyllide reductase and RuBisCO, key enzymes in chlorophyll and starch biosynthesis, respectively, modifies their activity in phytoplasma-infected seedlings. On the basis of these results, we propose a model to elucidate the molecular mechanism of responses to PaWB and offer a resource for functional studies on the effects of acetylation on protein function.

Multilocus sequence analysis for detection of finer genetic variation and phylogenetic interrelatedness in 16SrI group phytoplasma strains infecting different plants in China

The group 16SrI phytoplasmas are associated with severe diseases of many cash and ecological plants throughout the world. To date, the genetic variation and population structure of very closely related phytoplasma strains are still not fully understood in China. In this study, a multilocus sequence analysis (MLSA) scheme was developed using ten housekeeping genes (rp, tuf, secA, secY, ipt, dnaK, fusA, gyrB, pyrG and rpoB) fragments compared with 16S rDNA to analyze 18 phytoplasma strains infecting chinaberry, lettuce, mulberry, paulownia and periwinkle from ten provinces in China. The nucleotide site polymorphisms resolved all strains into 15 sequence types (STs), demonstrating extensive genetic diversity among the 16SrI group strain population. All the strains, classified in 16SrI-B and-D subgroup by 16S rDNA analysis, clustered into one clade and clearly differentiated into discrete subclades by phylogenetic analysis of the concatenated gene sequences. The 10 chinaberry witches’ broom (CWB) strains that were most closely related to two mulberry dwarf (MD) and hardly distinguished with 16S rDNA, were definitely split into four distinct clusters and 8 STs apparently congruent to their geographical locations. Two lettuce yellows (LY) strains in Sanming, Fujian province, China were more closely related to the onion yellows OY-M strain in Japan than the periwinkle virescence (PeV) and paulownia witches’ broom (PaWB) strains in China. The levels of variation in dnaK gene were higher than those in 16S rDNA and other genes tested. This MLSA is a promising approach for phytoplasma differentiation as well as for in depth examination of strain diversity and evolution of various 16Sr groups or subgroups.

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.

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

Paulownia witches’ 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.

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’ 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·L−1 (for Paulownia tomentosa (PT) and Paulownia fortunei (PF) or 15 mg·L−1 (for P. tomentosa × 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’ 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.

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

Paulownia witches’ 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–target regulation in healthy and diseased Paulownia tomentosa, Paulownia fortunei, and P. tomentosa × P. fortunei seedlings, and the corresponding diseased seedlings treated with 75 mg L−1 dimethyl sulfate. A total of 212, 111, and 197 differentially expressed miRNAs (DEMs) were obtained in P. tomentosa, P. fortunei, and P. tomentosa × P. fortunei, respectively. Degradome sequencing detected 559, 251, and 568 target genes of the DEMs in P. tomentosa, P. fortunei, and P. tomentosa × 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’ 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.