CBCVd
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Viroids are smallest known pathogen that consist of non-capsidated, single-stranded non-coding RNA replicons and they exploits host factors for their replication and propagation. The severe stunting disease caused by Citrus bark cracking viroid (CBCVd) is a serious threat, which spreads rapidly within hop gardens. In this study, we employed comprehensive transcriptome analyses to dissect host-viroid interactions and identify gene expression changes that are associated with disease development in hop. Our analysis revealed that CBCVd-infection resulted in the massive modulation of activity of over 2000 genes. Expression of genes associated with plant immune responses (protein kinase and mitogen-activated protein kinase), hypersensitive responses, phytohormone signaling pathways, photosynthesis, pigment metabolism, protein metabolism, sugar metabolism, and modification, and others were altered, which could be attributed to systemic symptom development upon CBCVd-infection in hop. In addition, genes encoding RNA-dependent RNA polymerase, pathogenesis-related protein, chitinase, as well as those related to basal defense responses were up-regulated. The expression levels of several genes identified from RNA sequencing analysis were confirmed by qRT-PCR. Our systematic comprehensive CBCVd-responsive transcriptome analysis provides a better understanding and insights into complex viroid-hop plant interaction. This information will assist further in the development of future measures for the prevention of CBCVd spread in hop fields.
- MeSH
- Humulus genetika metabolismus virologie MeSH
- listy rostlin genetika metabolismus virologie MeSH
- nemoci rostlin genetika virologie MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné proteiny genetika metabolismus MeSH
- rostlinné viry genetika izolace a purifikace fyziologie MeSH
- stanovení celkové genové exprese MeSH
- viroidy klasifikace genetika izolace a purifikace fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Hop (Humulus lupulus L.) plants are grown primarily for the brewing industry and have been used as a traditional medicinal herb for a long time. Severe hop stunt disease caused by the recently discovered Citrus bark cracking viroid (CBCVd) is one of the most devastating diseases among other viroid infections in hop. MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in gene expression regulation. To identify miRNAs in hop and their response to CBCVd-infection, two small RNA (sRNA) libraries were prepared from healthy and CBCVd-infected hop plants and were investigated by high throughput sequencing. RESULTS: A total of 67 conserved and 49 novel miRNAs were identified. Among them, 36 conserved and 37 novel miRNAs were found to be differentially recovered in response to CBCVd-infection. A total of 311 potential targets was predicted for conserved and novel miRNAs based on a sequence homology search using hop transcriptome data. The majority of predicted targets significantly belonged to transcriptional factors that may regulate hop leaf, root and cone growth and development. In addition, the identified miRNAs might also play an important roles in other cellular and metabolic processes, such as signal transduction, stress response and other physiological processes, including prenylflavonoid biosynthesis pathways. Quantitative real time PCR analysis of selected targets revealed their negative correlation with their corresponding CBCVd-responsive miRNAs. CONCLUSIONS: Based on the results, we concluded that CBCVd-responsive miRNAs modulate several hormone pathways and transcriptional factors that play important roles in the regulation of metabolism, growth and development. These results provide a framework for further analysis of regulatory roles of sRNAs in plant defense mechanism including other hop infecting viroids in particular.
- MeSH
- datové kurátorství MeSH
- fenotyp MeSH
- Humulus genetika virologie MeSH
- messenger RNA genetika MeSH
- mikro RNA genetika MeSH
- nemoci rostlin genetika virologie MeSH
- regulace genové exprese u rostlin MeSH
- reprodukovatelnost výsledků MeSH
- RNA interference MeSH
- RNA rostlin MeSH
- stanovení celkové genové exprese MeSH
- výpočetní biologie metody MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
The hop metabolome important for the brewing industry and for medical purposes is endangered worldwide due to multiple viroid infections affecting hop physiology. Combinatorial biolistic hop inoculation with Citrus bark cracking viroid (CBCVd), Apple fruit crinkle viroid (AFCVd), Hop latent viroid, and Hop stunt viroid (HSVd) showed a low CBCVd compatibility with HSVd, while all other viroid combinations were highly compatible. Unlike to other viroids, single CBCVd propagation showed a significant excess of (-) over (+) strands in hop, tomato, and Nicotiana benthamiana, but not in citruses. Inoculation of hop with all viroids led to multiple infections with unstable viroid levels in individual plants in the pre- and post-dormancy periods, and to high plant mortality and morphological disorders. Hop isolates of CBCVd and AFCVd were highly stable, only minor quasispecies were detected. CBCVd caused a strong suppression of some crucial mRNAs related to the hop prenylflavonoid biosynthesis pathway, while AFCVd-caused effects were moderate. According to mRNA degradome analysis, this suppression was not caused by a direct viroid-specific small RNA-mediated degradation. CBCVd infection led to a strong induction of two hop transcription factors from WRKY family and to a disbalance of WRKY/WDR1 complexes important for activation of lupulin genes.
- MeSH
- Citrus genetika virologie MeSH
- Humulus genetika virologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- Malus genetika virologie MeSH
- messenger RNA genetika MeSH
- ovoce genetika virologie MeSH
- tabák genetika virologie MeSH
- viroidy genetika patogenita MeSH
- Publikační typ
- časopisecké články MeSH
Viroids are small non-capsidated, single-stranded, covalently-closed circular noncoding RNA replicons of 239-401 nucleotides that exploit host factors for their replication, and some cause disease in several economically important crop plants, while others appear to be benign. The proposed mechanisms of viroid pathogenesis include direct interaction of the genomic viroid RNA with host factors and post-transcriptional or transcriptional gene silencing via viroid-derived small RNAs (vd-sRNAs) generated by the host defensive machinery. Humulus lupulus (hop) plants are hosts to several viroids among which Hop latent viroid (HLVd) and Citrus bark cracking viroid (CBCVd) are attractive model systems for the study of viroid-host interactions due to the symptomless infection of the former and severe symptoms induced by the latter in this indicator host. To better understand their interactions with hop plant, a comparative transcriptomic analysis based on RNA sequencing (RNA-seq) was performed to reveal the transcriptional alterations induced as a result of single HLVd and CBCVd infection in hop. Additionally, the effect of HLVd on the aggressiveness of CBCVd that underlies severe stunting in hop in a mixed infection was studied by transcriptomic analysis. Our analysis revealed that CBCVd infection resulted in dynamic changes in the activity of genes as compared to single HLVd infection and their mixed infection. The differentially expressed genes that are involved in defense, phytohormone signaling, photosynthesis and chloroplasts, RNA regulation, processing and binding; protein metabolism and modification; and other mechanisms were more modulated in the CBCVd infection of hop. Nevertheless, Gene Ontology (GO) classification and pathway enrichment analysis showed that the expression of genes involved in the proteolysis mechanism is more active in a mixed infection as compared to a single one, suggesting co-infecting viroids may result in interference with host factors more prominently. Collectively, our results provide a deep transcriptome of hop and insight into complex single HLVd, CBCVd, and their coinfection in hop-plant interactions.
- MeSH
- Humulus genetika virologie MeSH
- nemoci rostlin genetika virologie MeSH
- transkriptom * MeSH
- viroidy patogenita MeSH
- Publikační typ
- časopisecké články MeSH
Transcription factors (TFs) play a major role in controlling gene expression by intricately regulating diverse biological processes such as growth and development, the response to external stimuli and the activation of defense responses. The systematic identification and classification of TF genes are essential to gain insight into their evolutionary history, biological roles, and regulatory networks. In this study, we performed a global mining and characterization of hop TFs and their involvement in Citrus bark cracking viroid CBCVd infection by employing a digital gene expression analysis. Our systematic analysis resulted in the identification of a total of 3,818 putative hop TFs that were classified into 99 families based on their conserved domains. A phylogenetic analysis classified the hop TFs into several subgroups based on a phylogenetic comparison with reference TF proteins from Arabidopsis thaliana providing glimpses of their evolutionary history. Members of the same subfamily and subgroup shared conserved motif compositions. The putative functions of the CBCVd-responsive hop TFs were predicted using their orthologous counterparts in A. thaliana. The analysis of the expression profiling of the CBCVd-responsive hop TFs revealed a massive differential modulation, and the expression of the selected TFs was validated using qRT-PCR. Together, the comprehensive integrated analysis in this study provides better insights into the TF regulatory networks associated with CBCVd infections in the hop, and also offers candidate TF genes for improving the resistance in hop against viroids.
- MeSH
- fylogeneze MeSH
- Humulus klasifikace genetika imunologie virologie MeSH
- kůra rostlin imunologie virologie MeSH
- nemoci rostlin genetika imunologie virologie MeSH
- rostlinné proteiny genetika imunologie MeSH
- stanovení celkové genové exprese MeSH
- transkripční faktory genetika imunologie MeSH
- viroidy genetika fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Some viroids-single-stranded, non-coding, circular RNA parasites of plants-are not transmissible through pollen to seeds and to next generation. We analyzed the cause for the elimination of apple fruit crinkle viroid (AFCVd) and citrus bark cracking viroid (CBCVd) from male gametophyte cells of Nicotiana tabacum by RNA deep sequencing and molecular methods using infected and transformed tobacco pollen tissues at different developmental stages. AFCVd was not transferable from pollen to seeds in reciprocal pollinations, due to a complete viroid eradication during the last steps of pollen development and fertilization. In pollen, the viroid replication pathway proceeds with detectable replication intermediates, but is dramatically depressed in comparison to leaves. Specific and unspecific viroid degradation with some preference for (-) chains occurred in pollen, as detected by analysis of viroid-derived small RNAs, by quantification of viroid levels and by detection of viroid degradation products forming "comets" on Northern blots. The decrease of viroid levels during pollen development correlated with mRNA accumulation of several RNA-degrading factors, such as AGO5 nuclease, DICER-like and TUDOR S-like nuclease. In addition, the functional status of pollen, as a tissue with high ribosome content, could play a role during suppression of AFCVd replication involving transcription factors IIIA and ribosomal protein L5.
The mediator (MED) represents a large, conserved, multi-subunit protein complex that regulates gene expression through interactions with RNA polymerase II and enhancer-bound transcription factors. Expanding research accomplishments suggest the predominant role of plant MED subunits in the regulation of various physiological and developmental processes, including the biotic stress response against bacterial and fungal pathogens. However, the involvement of MED subunits in virus/viroid pathogenesis remains elusive. In this study, we investigated for the first time the gene expression modulation of selected MED subunits in response to five viroid species (Apple fruit crinkle viroid (AFCVd), Citrus bark cracking viroid (CBCVd), Hop latent viroid (HLVd), Hop stunt viroid (HSVd), and Potato spindle tuber viroid (PSTVd)) in two model plant species (Nicotiana tabacum and N. benthamiana) and a commercially important hop (Humulus lupulus) cultivar. Our results showed a differential expression pattern of MED subunits in response to a viroid infection. The individual plant MED subunits displayed a differential and tailored expression pattern in response to different viroid species, suggesting that the MED expression is viroid- and plant species-dependent. The explicit evidence obtained from our results warrants further investigation into the association of the MED subunit with symptom development. Together, we provide a comprehensive portrait of MED subunit expression in response to viroid infection and a plausible involvement of MED subunits in fine-tuning transcriptional reprogramming in response to viroid infection, suggesting them as a potential candidate for rewiring the defense response network in plants against pathogens.
- MeSH
- druhová specificita MeSH
- Humulus genetika virologie MeSH
- listy rostlin genetika mikrobiologie MeSH
- mediátorový komplex genetika MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné proteiny genetika MeSH
- rostlinné viry MeSH
- stanovení celkové genové exprese MeSH
- tabák genetika virologie MeSH
- viroidy genetika patogenita MeSH
- Publikační typ
- časopisecké články MeSH
