Kumar Mishra, Ajay* Dotaz Zobrazit nápovědu
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
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
Molecular cloning is based on isolation of a DNA sequence of interest to obtain multiple copies of it in vitro. Application of this technique has become an increasingly important tool in clinical microbiology due to its simplicity, cost effectiveness, rapidity, and reliability. This review entails the recent advances in molecular cloning and its application in the clinical microbiology in the context of polymicrobial infections, recombinant antigens, recombinant vaccines, diagnostic probes, antimicrobial peptides, and recombinant cytokines. Culture-based methods in polymicrobial infection have many limitation, which has been overcome by cloning techniques and provide gold standard technique. Recombinant antigens produced by cloning technique are now being used for screening of HIV, HCV, HBV, CMV, Treponema pallidum, and other clinical infectious agents. Recombinant vaccines for hepatitis B, cholera, influenza A, and other diseases also use recombinant antigens which have replaced the use of live vaccines and thus reduce the risk for adverse effects. Gene probes developed by gene cloning have many applications including in early diagnosis of hereditary diseases, forensic investigations, and routine diagnosis. Industrial application of this technology produces new antibiotics in the form of antimicrobial peptides and recombinant cytokines that can be used as therapeutic agents.
Among computationally predicted and experimentally validated plant miRNAs, several are conserved across species boundaries in the plant kingdom. In this study, a combined experimental-in silico computational based approach was adopted for the identification and characterization of miRNAs in Humulus lupulus (hop), which is widely cultivated for use by the brewing industry and apart from, used as a medicinal herb. A total of 22 miRNAs belonging to 17 miRNA families were identified in hop following comparative computational approach and EST-based homology search according to a series of filtering criteria. Selected miRNAs were validated by end-point PCR and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), confirmed the existence of conserved miRNAs in hop. Based on the characteristic that miRNAs exhibit perfect or nearly perfect complementarity with their targeted mRNA sequences, a total of 47 potential miRNA targets were identified in hop. Strikingly, the majority of predicted targets were belong to transcriptional factors which could regulate hop growth and development, including leaf, root and even cone development. Moreover, the identified miRNAs may also be involved in other cellular and metabolic processes, such as stress response, signal transduction, and other physiological processes. The cis-regulatory elements relevant to biotic and abiotic stress, plant hormone response, flavonoid biosynthesis were identified in the promoter regions of those miRNA genes. Overall, findings from this study will accelerate the way for further researches of miRNAs, their functions in hop and shows a path for the prediction and analysis of miRNAs to those species whose genomes are not available.
- MeSH
- databáze genetické MeSH
- exprimované sekvenční adresy * MeSH
- genové regulační sítě genetika MeSH
- Humulus genetika MeSH
- mikro RNA genetika MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- regulace genové exprese u rostlin genetika MeSH
- rostlinné geny genetika MeSH
- software * MeSH
- stanovení celkové genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Molecular and biochemical studies have shown that gene contains single or combination of different cis-acting regulatory elements are actively controlling the transcriptional regulation of associated genes, downstream effects of these result in the modulation of various biological pathways such as biotic/abiotic stress responses, hormonal responses to growth and development processes and secondary metabolite production. Therefore, the identification of promoters and their cis-regulatory elements is one of intriguing area to study the dynamic complex regulatory network of genes activities by integrating computational, comparative, structural and functional genomics. Several bioinformatics servers or database have been established to predict the cis-acting elements present in the promoter region of target gene and their association with the expression profiles in the TFs. The aim of this study is to predict possible cis-acting regulatory elements that have putative role in the transcriptional regulation of a dynamic network of metabolite gene activities controlling prenylflavonoid and bitter acids biosynthesis in hop (Humulus lupulus). Recent release of hop draft genome enabled us to predict the possible cis-acting regulatory elements by extracting 2kbp of 5' regulatory regions of genes important for lupulin metabolome biosynthesis, using Plant CARE, PLACE and Genomatix Matinspector professional databases. The result reveals the plausible role of cis-acting regulatory elements in the regulation of gene expression primarily involved in lupulin metabolome biosynthesis including under various stress conditions.
- MeSH
- Humulus genetika MeSH
- promotorové oblasti (genetika) * MeSH
- regulace genové exprese u rostlin genetika MeSH
- regulační elementy transkripční genetika MeSH
- regulační oblasti nukleových kyselin genetika MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
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 plant (Humulus lupulus L.) produces several valuable secondary metabolites, such as prenylflavonoid, bitter acids, and essential oils. These compounds are biosynthesized in glandular trichomes (lupulin glands) endowed with pharmacological properties and widely implicated in the beer brewing industry. The present study is an attempt to generate exhaustive information of transcriptome dynamics and gene regulatory mechanisms involved in biosynthesis and regulation of these compounds, developmental changes including trichome development at three development stages, namely leaf, bract, and mature lupulin glands. Using high-throughput RNA-Seq technology, a total of 61.13, 50.01, and 20.18 Mb clean reads in the leaf, bract, and lupulin gland libraries, respectively, were obtained and assembled into 43,550 unigenes. The putative functions were assigned to 30,996 transcripts (71.17%) based on basic local alignment search tool similarity searches against public sequence databases, including GO, KEGG, NR, and COG families, which indicated that genes are principally involved in fundamental cellular and molecular functions, and biosynthesis of secondary metabolites. The expression levels of all unigenes were analyzed in leaf, bract, and lupulin glands tissues of hop. The expression profile of transcript encoding enzymes of BCAA metabolism, MEP, and shikimate pathway was most up-regulated in lupulin glands compared with leaves and bracts. Similarly, the expression levels of the transcription factors and structural genes that directly encode enzymes involved in xanthohumol, bitter acids, and terpenoids biosynthesis pathway were found to be significantly enhanced in lupulin glands, suggesting that production of these metabolites increases after the leaf development. In addition, numerous genes involved in primary metabolism, lipid metabolism, photosynthesis, generation of precursor metabolites/energy, protein modification, transporter activity, and cell wall component biogenesis were differentially regulated in three developmental stages, suggesting their involvement in the dynamics of the lupulin gland development. The identification of differentially regulated trichome-related genes provided a new foundation for molecular research on trichome development and differentiation in hop. In conclusion, the reported results provide directions for future functional genomics studies for genetic engineering or molecular breeding for augmentation of secondary metabolite content in hop.
- MeSH
- flavonoidy biosyntéza chemie metabolismus MeSH
- genová ontologie MeSH
- Humulus chemie metabolismus MeSH
- listy rostlin genetika metabolismus MeSH
- propiofenony chemie metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné proteiny genetika metabolismus MeSH
- sekvenování transkriptomu MeSH
- terpeny chemie metabolismus MeSH
- transkripční faktory metabolismus MeSH
- transkriptom genetika MeSH
- trichomy genetika metabolismus ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
Micronutrient deficiency conditions, such as anemia, are the most prevalent global health problem due to inadequate iron and folate in dietary sources. Biofortification advancements can propel the rapid amelioration of nutritionally beneficial components in crops that are required to combat the adverse effects of micronutrient deficiencies on human health. To date, several strategies have been proposed to increase micronutrients in plants to improve food quality, but very few approaches have intrigued `clustered regularly interspaced short palindromic repeats' (CRISPR) modules for the enhancement of iron and folate concentration in the edible parts of plants. In this review, we discuss two important approaches to simultaneously enhance the bioavailability of iron and folate concentrations in rice endosperms by utilizing advanced CRISPR-Cas9-based technology. This includes the 'tuning of cis-elements' and 'enhancer re-shuffling' in the regulatory components of genes that play a vital role in iron and folate biosynthesis/transportation pathways. In particular, base-editing and enhancer re-installation in native promoters of selected genes can lead to enhanced accumulation of iron and folate levels in the rice endosperm. The re-distribution of micronutrients in specific plant organs can be made possible using the above-mentioned contemporary approaches. Overall, the present review discusses the possible approaches for synchronized iron and folate biofortification through modification in regulatory gene circuits employing CRISPR-Cas9 technology.
- MeSH
- biofortifikace * MeSH
- CRISPR-Cas systémy * MeSH
- editace genu metody MeSH
- geneticky modifikované rostliny * metabolismus genetika MeSH
- kyselina listová * metabolismus MeSH
- lidé MeSH
- rýže (rod) metabolismus genetika MeSH
- železo * metabolismus MeSH
- zemědělské plodiny * metabolismus genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy 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.
