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Virus-induced gene silencing as a tool for functional genomics in weeds: Challenges and future directions.

Virus-induced gene silencing (VIGS) has evolved from a conceptual demonstration of antiviral defense into a pivotal reverse-genetics platform for plant functional genomics. By exploiting engineered DNA- or RNA-based viral vectors, VIGS enables rapid, sequence-specific transcript knockdown through RNA-mediated degradation of target transcripts. Recent refinements in vector design, inoculation strategies, and viral species selection, such as TRV, BSMV, and FoMV, have expanded its application to previously recalcitrant plants, including major crops and emerging weed models. In weeds, functional genomics remains particularly challenging due to high genetic variability, limited genomic resources, and incompatibility with conventional viral vectors and transformation systems. In this context, VIGS provides a tractable approach to investigate genes associated with herbicide resistance, metabolic adaptation, and stress tolerance. Beyond weed biology, its application to studies of immune signaling, hormonal crosstalk, and secondary metabolism highlights VIGS as a versatile biotechnology for elucidating gene function and supporting next-generation strategies in plant improvement and integrated pest management.

Journal Article

Smallpox vaccination reactions, prophylaxis, and therapy of complications.

Smallpox vaccination in the United States is a routine public health measure which has been under intensive review during the last decade. The most frequently occurring adverse reactions to vaccination are benign and require little or no systemic therapy. These reactions include accidental infection, erythematous and urticarial rash, and generalized vaccinia. Chickenpox occurring concurrently with vaccination presents no problem unless vaccinia has widely superinfected the chickenpox lesions. There is no risk to the pregnant woman who is vaccinated, but there is a slight risk that the fetus will develop fetal vaccinia. The vaccinia does not cause congenital malformations. Vaccinia hyperimmune globulin (VIG) in prophylactic dosage may be given to a pregnant woman who is traveling to a smallpox infected or endemic area in order to prevent fetal vaccinia. Vaccinia necrosum and eczema vaccinatum require vigorous systemic therapy with VIG, and often thiosemicarbazone. Post-vaccinial encephalitis, while frequently serious, has not been shown to be ameliorated by VIG therapy, although there are data which suggest VIG has some value in prophylaxis for encephalitis. Prophylaxis, prompt recognition, and proper therapy may reduce the fatality rates of these complications. Revaccination of patients who have suffered a complication is a frequent clinical problem. Revaccination of an individual who has had post-vaccinial encephalitis or vaccinia necrosum is contraindicated unless the risk of contracting smallpox outweighs the risk of the above two diseases. Revaccination of children who have had eczema vaccinatum is not contraindicated. Revaccination of children with a history of accidental infection or erythematous or urticarial rash presents no known or theoretically increased risk.

Eczema

Genome-wide AP2/ERF analysis identifies HmaERF87 as a positive regulator of Hydrangea macrophylla leaf spot resistance.

A total of 164 APETALA2/ethylene-responsive factor (AP2/ERF) genes were identified in Hydrangea macrophylla, and HmaERF87 positively contributes to leaf spot resistance. The APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor family plays important roles in plant stress responses, but its contribution to disease resistance in Hydrangea macrophylla (hydrangea) remains poorly understood. In this study, 164 AP2/ERF genes were identified in the H. macrophylla genome and classified into APETALA2 (AP2), ethylene-responsive factor (ERF), dehydration-responsive element-binding (DREB), and related to ABI3/VP1 (RAV) subfamilies. Their chromosomal distribution, conserved motifs, gene structures, and duplication patterns were analyzed. A total of 46 pathogen-responsive H. macrophylla AP2/ERF (HmaERF) genes were identified from the RNA sequencing (RNA-seq) dataset of resistant and susceptible cultivar leaves collected before and after Corynespora cassiicola inoculation. Promoter analysis revealed that the HmaERF genes with upregulated expression post-C. cassiicola infection showed a higher frequency and copy number of jasmonate-responsive cis-regulatory elements, suggesting their possible involvement in hormone-mediated defense responses. Three infection-induced candidate genes, including HmaERF56, HmaERF87, and HmaERF129, were selected for functional validation using virus-induced gene silencing (VIGS) in hydrangea leaf discs. Silencing of HmaERF87 expression via VIGS significantly increased lesion development after C. cassiicola inoculation, whereas the transient overexpression of HmaERF87 reduced the area of leaf disc lesions. Subcellular localization showed that the HmaERF87 protein was localized in the nucleus, and yeast assays indicated that its transcriptional activation activity was mainly associated with the C-terminal region of the protein. These results support a role for HmaERF87 as a positive regulator of H. macrophylla resistance to leaf spot disease and provide a candidate gene for further studies of disease resistance in hydrangea.

Plant Proteins

Genome-Wide Identification of the R2R3-MYB Gene Family in Solanum americanum and Functional Analysis of Its Role in Fruit Coloration.

Anthocyanins are key secondary metabolites responsible for fruit coloration in plants, and their biosynthesis is largely regulated by R2R3-MYB transcription factors. However, the R2R3-MYB regulators controlling fruit anthocyanin accumulation in wild Solanum species remain poorly understood. Here, Solanum americanum was used to identify candidate R2R3-MYB genes associated with fruit coloration through genome-wide identification, phylogenetic analysis, synteny analysis, expression profiling, and virus-induced gene silencing (VIGS). A total of 122 SaMYB genes were identified, and phylogenetic analysis revealed that SaMYB proteins clustered with Arabidopsis thaliana R2R3-MYB members in conserved subgroups, suggesting evolutionary conservation of this family. Synteny analysis identified 37 syntenic gene pairs among SaMYB genes, and the Ka/Ks values of all analyzable gene pairs were below 1, indicating that these duplicated genes are subject to functional constraint. Integrated analysis of phylogenetic relationships, protein structures, promoter cis-elements, and fruit developmental expression patterns identified SaMYB59 and SaMYB106 as candidate regulators of anthocyanin accumulation. VIGS analysis demonstrated that silencing SaMYB106 reduced purple coloration, decreased anthocyanin content, and downregulated the expression of the structural gene DFR. These results indicate that SaMYB106 functions as a positive regulator of fruit anthocyanin accumulation in S. americanum. This study provides insights into the molecular basis of fruit coloration in wild Solanum species.

Solanum americanum

Genome-wide identification and analysis of paclobutrazol-resistance gene family in cotton and the positive role of GhPRE3 in salt stress and drought stress resistance.

Compared with other transcription factors, much less studies have been performed on paclobutrazol-resistance (PRE), a subgroup of the extensive bHLH transcription factor gene family, and the research in cotton was also limited. By utilizing the PRE genes and their conserved domains identified in Arabidopsis, a total of 23, 22, 11, and 12 PRE genes were identified from two major cultivated cotton species and their two ancestors, respectively. The cotton PRE gene family was categorized into three subgroups based on evolutionary tree analysis. Motif and intron analyses indicated that the PRE gene has remained highly conserved throughout evolution. Collinearity analysis indicated that gene duplication, particularly through fragment replication, has significantly contributed to the expansion of the cotton PRE family. An exploration of the conserved elements within the PRE gene family uncovered numerous elements associated with plant stress resistance. Additionally, cotton transcriptome and qRT-PCR analysis showed that PRE genes were associated with a variety of abiotic stresses, including salt, drought, and cold treatments. Subcellular localization experiments indicated that the GhPRE3 gene is associated with membrane proteins. Finally, we selected the GhPRE3 gene for a VIGS experiment, which revealed that under salt stress and drought stress conditions, the wilting of leaves in the GhPRE3-silenced plants was significantly more severe than that observed in the control group, with T-AOC levels notably lower and MDA levels significantly higher. Overexpression of GhPRE3 enhanced seed germination and root development in transgenic Arabidopsis thaliana under salt stress and drought stresses. This suggests that GhPRE3 plays a positive regulatory role in cotton tolerance to salt and drought stressed, providing a reference for molecular genetic breeding of cotton with salt and drought tolerance.

Gossypium

Genome-Wide Characterization of the ScLOR Gene Family in Wild Tomato Solanum lycopersicoides Reveals ScLOR16 as a Negative Regulator of Cold-and Drought-Stress Tolerance.

The LOR (LURP-one related) gene family encodes proteins containing conserved LOR domains; however, its functions in plant abiotic stress responses remain largely unexplored. In this study, we systematically identified and characterized the LOR gene family in the stress-tolerant wild tomato Solanum lycopersicoides using comprehensive bioinformatic analyses and conducted functional validation of the candidate gene ScLOR16. A total of 19 ScLOR members were identified and classified into eight phylogenetic subgroups. Numerous cis-acting elements associated with responses to abscisic acid (ABA), cold, and drought, including ABRE, LTR, and MBS, were detected in the promoter regions, suggesting that the ScLOR family may be broadly involved in ABA-mediated stress signaling pathways. RT-qPCR analysis revealed that ScLOR16 expression was significantly induced by both cold and drought treatments. Subcellular localization assays demonstrated that ScLOR16 is localized in both the nucleus and cytoplasm. Virus-induced gene silencing (VIGS) was subsequently employed to generate ScLOR16-silenced plants. Following 24 h of cold treatment at 4 °C and four days of drought stress, ScLOR16-silenced seedlings exhibited significantly less severe wilting symptoms than empty-vector controls. Physiological analyses showed that silenced plants exhibited enhanced superoxide dismutase (SOD) and peroxidase (POD) activities, increased proline accumulation, and decreased thiobarbituric acid-reactive substances (TBARS) content. Collectively, these results indicate that reduced ScLOR16 transcript levels are associated with enhanced cold and drought tolerance, accompanied by alterations in antioxidant defense and osmoprotection-related physiological markers. This study yields new insights into the evolution and stress-related functions of the ScLOR family.

LOR gene family

Mutagens in a river heavily polluted with paper recycling wastes: results of field and laboratory mutagen assays.

Paper recycling industries generate considerable quantities of waterborne wastes, and thus water pollution constitutes the greatest environmental problem associated with this industrial activity [Hunt and Franklin, 1973]. Generally the impact of this water pollution is considered in terms of aesthetic blight and deterioration of water quality. We present data that document another aspect of this pollution, the environmental contamination of an aquatic ecosystem with mutagenic materials. A natural population of the fern Osmunda regalis growing in a river heavily polluted with paper recycling wastes had a high incidence of chromosome mutations. This population was monitored for four years for the frequency of two-break chromosome mutations. These mutations were postzygotic in origin and suggested the presence of mutagens in the river water. The fern population is downstream from the outfalls of a paper recycling mill, which was discharging 13.3 X 10(6) liters of untreated paper recycling waste per day. In 1977 a waste-water-treatment facility was constructed to remove the solid waste previously discharged into the river. This facility generates 69,300 kg of solid waste daily, which is taken to a landfill. Periodic samples of this solid waste were collected from the waste-treatment facility in the summer of 1978, extracted with various solvents, and the extracts tested for mutagenic activity with the Salmonella/mammalian microsome mutagenicity test [Ames et al, 1975]. A majority of the solid waste samples contained mutagenic materials, but in all cases S-9 activation was required for mutagenic activity. The samples also were assayed with the soybean mitotic crossing-over assay [Vig, 1975]. Four out of six samples were positive. These results document the presence of mutagens in the solid waste generated by a paper recycling industry and the genetic impact of these mutagens on the local biota.

Chromosome Aberrations

Transforming Plant Viruses into Vectors for Next-Generation Agriculture-A Review.

Plant viral vectors have evolved from tools for transient gene expression into a versatile platform for precise genetic intervention, offering a rapid, transgene-free alternative to conventional crop transformation. This review critically assesses their engineering for scalable field application, moving beyond foundational techniques like virus-induced gene silencing (VIGS). We highlight how advanced vector design, including deconstructed genomes and synthetic regulatory circuits enhances cargo capacity, specificity, and biosafety. The integration of viral delivery with CRISPR-Cas systems has unlocked virus-induced genome editing (VIGE), base editing, and prime editing, enabling heritable trait modification without tissue culture. However, the transition from proof-of-concept in model plants to robust field technology hinges on overcoming critical bottlenecks: expanding host range through chimeric vectors, ensuring environmental containment, and developing scalable delivery methods such as nano-formulations or adjusted agroinfiltration protocols. We evaluate these delivery routes and emerging synergies with nanobiotechnology for targeted and efficient applications. While challenges in regulation, public perception, and large-scale production persist, the strategic engineering of viral vectors for stability, specificity, and safety positions them as a transformative, next-generation biotechnological input for achieving sustainable crop improvement and protection under changing climatic conditions.

Plant Viruses

CgMYC2 directly activates jasmonate-induced naringin biosynthesis in Citrus grandis 'Tomentosa'.

CgMYC2 links jasmonate signaling to naringin biosynthesis by binding G-box motifs and activating flavonoid-pathway promoters in Citrus grandis 'Tomentosa' Naringin, the predominant bitter-flavanone glycoside in Citrus grandis 'Tomentosa', has well-characterized biosynthetic enzymes, yet the transcriptional regulators coupling hormonal signals to pathway activation remain poorly understood. We demonstrate that CgMYC2, a jasmonate-responsive bHLH transcription factor, functions as a central activator of naringin biosynthesis. Exogenous methyl jasmonate (MeJA) treatment increased naringin content 3.45-fold in seedlings, coinciding with a rapid 6.6-fold induction of CgMYC2 that preceded the peak transcription of five core biosynthetic genes (CgPAL5, CgCHS, CgFNS, Cg7GlcT, and Cg1,2RhaT). Physical interaction between CgMYC2 and the JAZ protein CgJAZ3 was confirmed by pull-down and Co-IP assays, placing CgMYC2 within the canonical jasmonate signaling cascade. Y1H confirmed CgMYC2 binding to the Cg1,2RhaT promoter, EMSA demonstrated direct G-box-dependent binding to all five pathway promoters, and dual-luciferase assays showed transactivation of all five promoters, with the strongest activation for CgCHS. As complementary chromatin-level support, a single-sample CUT&Tag profile revealed G-box-enriched CgMYC2-associated chromatin regions across jasmonate-responsive and secondary-metabolic loci. Virus-induced gene silencing (VIGS) of CgMYC2 reduced naringin content by ~21% and suppressed biosynthetic gene expression, supporting its positive contribution. Furthermore, heterologous overexpression in tomato activated the flavonoid pathway and elevated 16 flavonoid compounds, consistent with evolutionary conservation of the MYC2-G-box regulatory logic. These findings establish CgMYC2 as a central, JA-responsive activator bridging jasmonate perception and naringin biosynthesis, providing a molecular framework for the targeted improvement of bitter-flavonoid traits in citrus.

Citrus

Identification of the BrSK gene family in flowering Chinese cabbage and functional characterization of BrSK2 subfamily involvement in heat stress.

Glycogen synthase kinase 3 (GSK3) kinases are evolutionarily conserved regulators of plant development and stress signaling, yet their contributions to thermotolerance in cool-adapted Brassica crops remain poorly understood. Here, we identified 16 BrSK genes in the Caixin (Brassica rapa ssp. chinensis var. parachinensis) genome, all harboring intact catalytic motifs indicative of functional kinase activity. Spatiotemporal expression profiling revealed preferential accumulation of BrSK transcripts in stem apices and floral organs during reproductive transition, while promoter analysis identified abundant heat- and abiotic stress-responsive cis-elements. Under heat stress, BrSK21, BrSK22, and BrSK23 displayed striking genotype-specific expression dynamics. BrSK21/22/23 transcripts were stably suppressed in the heat-tolerant cultivar '49-19' but transiently declined before rapidly rebounding in the heat-sensitive 'Liuye 50', mirroring RNA-seq profiles. Protein-protein interaction assays (Y2H, BiFC, and LCI) demonstrated specific associations between BrSK kinases and BrHSFA1. Functional validation via VIGS revealed that silencing of BrSK21 significantly enhanced thermotolerance, with triple silencing of BrSK21/22/23 conferring additive protection, indicating functional redundancy within the BrSK2 subfamily. Collectively, these findings establish the BrSK2 subfamily as negative regulators of heat tolerance in Caixin, likely via modulation of BrHSFA1 expression. This work identifies high-priority targets for molecular breeding of climate-resilient Brassica vegetables.

Plant Proteins

Genome-wide screening and functional analysis of protein glycosylation-related genes involved in tomato fruit ripening.

Protein glycosylation, an essential co- and post-translational modification, plays critical roles in plant growth, development, and stress responses. However, its functional role in tomato fruit ripening has not been extensively investigated. Here, key protein glycosylation-related genes involved in tomato fruit ripening were identified by genome-wide screen and subsequently functional characterization. First, a dataset comprising 242 glycosylation-related proteins was established based on Gene Ontology annotations in tomato, combined with sequence homology to protein glycosylation-related proteins from Arabidopsis thaliana and Homo sapiens. Then, Subsequently, 28 genes encoding highly expressed glycosylation-related proteins (RPKM > 30) at the breaker (BR) stage were selected for functional screening, and subsequently 6 genes were identified as regulators of fruit ripening by method of virus-induced gene silencing (VIGS). Among them, Solyc03g098600 (STT3B), Solyc01g109410 (OST48), Solyc04g082670 (RPN1), and Solyc08g076460 (DAD1) functioned as positive regulators of tomato fruit ripening, whereas Solyc04g005340 (UAM2) and Solyc08g075340 (XEG113), acted as negative regulators. The expression of these genes responded dynamically to multiple ripening-related cues, including temperature, light, ethylene, and transcription factors. Furthermore, silencing of these genes individually affected the expression of genes involved in fruit ripening, including ethylene biosynthesis genes (ACS2, ACS4, ACO1, and ACO3), ripening-associated transcription factors (RIN, NOR, NOR-LIKE1, FUL1, and FUL2), and the key gene (PSY1) of lycopene biosynthesis pathway. Collectively, these findings demonstrate that protein glycosylation plays an important role in tomato fruit ripening by modulating ethylene signaling, ripening-associated transcriptional regulation, and lycopene biosynthesis.

Fruit ripening

Identification and Catalytic Optimization of Pinene Oxidases in Paeoniflorin Biosynthetic Pathway.

Paeoniflorin is a pharmacologically important cage-like monoterpene glycoside characteristic of Paeonia plants, yet its biosynthetic pathway has remained largely unresolved, hindering sustainable production. Here, we confirmed that paeoniflorin biosynthesis originates from α-pinene and identified three novel cytochrome P450 enzymes that catalyze pinene oxidation. CYP71AN126 catalyzes the hydroxylation of α-pinene at positions C4 and C10, followed by further oxidation of the alcohol to a ketone at C4, whereas CYP76A225/226 exclusively catalyze C10 hydroxylation. Virus-induced gene silencing (VIGS) assays demonstrated that silencing CYP71AN126, but not CYP76A225 and CYP76A226, significantly reduced the paeoniflorin content, indicating that C4 hydroxylation plays an important role in paeoniflorin biosynthesis, whereas C10 hydroxylation is not. Through the analysis of natural sequence and activity divergence among CYP71AN126 and CYP76A225/226, combined with protein structure prediction and site-directed mutagenesis, we identified L493 as a critical residue involved in regulating catalytic site specificity and substrate specificity of CYP71AN126. Mutation of L493 reduced or eliminated the formation of undesired C10 hydroxylation side-product and enhanced substrate specificity. These findings establish C4 oxidation of α-pinene as the critical committed step in paeoniflorin biosynthesis. Our study lays a foundation for elucidating the complete biosynthetic pathway of paeoniflorin in Paeonia and provides a target for enzyme engineering of CYP71AN126 aimed at the efficient production of paeoniflorin via synthetic biology approaches.

Paeonia genus

A cooperative regulatory module between TAGL2 and JMJC1 activates specific defense genes against root-knot nematodes in tomato.

Plant-parasitic nematodes (PPNs) threaten global food security. Although epigenetic modifications are crucial for plant immunity, how histone modifiers contribute to root-knot nematodes (RKNs, Meloidogyne incognita) resistance remains unclear. Here, using genetic, molecular and biochemical approaches, we investigated the epigenetic and transcriptional mechanisms underlying RKN resistance mediated by the histone demethylase (HDM) JMJC1 and the MADS-box transcription factor TAGL2 in tomato (Solanum lycopersicum). We identified JMJC1 as an RKN-induced positive defense regulator targeting H3K9me3 and H3K27me3 histone marks. JMJC1 physically interacts with TAGL2, which also positively regulates RKN resistance. Transcriptomic analysis indicated that TAGL2 regulates multiple layers of the plant defense network, transcriptionally activating representative genes from distinct pathways (including PUB10, bHLH98, CCaMK, and SAUR3), which we validated as positive regulators of RKN resistance via virus-induced gene silencing (VIGS). At the chromatin level, TAGL2 and JMJC1 co-regulate these loci, associating with localized H3K9me3 and H3K27me3 reduction. Furthermore, TAGL2 directly activates JMJC1 transcription, establishing a positive feedback loop that amplifies immune signaling. Our findings reveal a cooperative model wherein a HDM and a transcription factor coordinate at specific loci to fine-tune multiple defense layers at both epigenetic and transcriptional levels, providing insights for breeding durable nematode-resistant plants.

Solanum lycopersicum

CsMYB219 and CsMYB196 influence epigallocatechin gallate biosynthesis in tea plant (Camellia sinensis) by regulating CsSCPL1A gene expression.

Epigallocatechin gallate (EGCG) is the most abundant and biologically active catechin in tea leaves and has been widely utilized in the development of functional foods. EGCG is catalyzed by serine carboxypeptidase-like 1A (CsSCPL1A) acyltransferases in tea plants. Although CsSCPL family genes are regulated by several transcription factors (TFs), systematic studies on their regulation by MYB TFs are lacking. This study integrates targeted metabolomics, transcriptomics, DNA-protein, and protein-protein interaction analyses to elucidate the transcriptional regulation of EGCG biosynthesis-related genes CsSCPL4 and CsSCPL5-1 by R2R3-MYB TFs. CsMYB219 and CsMYB196 can specifically bind to CsSCPL4 and CsSCPL5-1 promoters and activate their expression. CsMYB196 also interacted with CsTT8a and CsTTG1 to activate the transcription activity of CsSCPL4 and CsSCPL5-1 promoters by forming a MYB/bHLH/WD40 (MBW) complex. Promoter truncation assays delineated MYB-responsive cis-elements in CsSCPL4 (-613 to -1 bp with enhancers at -1967 to -1622) and CsSCPL5-1 (-503 to -296 bp). Silencing of CsMYB219 and CsMYB196 by virus-induced gene silencing (VIGS) assay significantly reduced the expression levels of CsSCPL4 and CsSCPL5-1 and EGCG content in tea leaves. Transient overexpression of CsMYB219 and CsMYB196 in tea leaves upregulated CsSCPL4 and CsSCPL5-1 expression and elevated EGCG content. These findings enhance our understanding of the regulatory network underlying EGCG biosynthesis in tea plants and provide a solid foundation for future genetic improvement of tea plant cultivars.

Catechin

Orthodontic attitudes toward national health insurance.

Data were obtained by surveying North Carolina orthodontists by means of a mailed questionnaire. Their responses were coded to make possible analysis by computer and were compared to responses from a national sample of dentists in the 1975 Survey of Dentists. Both North Carolina orthodontists and dentists nationwide agreed that some form of dental care should be provided if a national health insurance system were established. Compared to the dentists surveyed nationwide, the orthodontists favored a wider range of coverages but advocated providing a narrower scope of dental services. They indicated more strongly their general belief that a government health program would lead to regulation outside the private sector. The orthodontists disagreed more strongly with the claim that government health programs could provide more people with high-quality dental care. Both groups anticipated that comprehensive dental care in a national health insurance system would result in fixed fees set by the government. Only 62 percent of the orthodontists polled were familiar with HMOs. Of this group, nearly 90 percent chose not to contract with these organizations.

Attitude

Computed tomography: a three-dimensional study of the nasal airway.

1. Accurate volume measurement of the nasal airway based upon computed tomography images sequentially generated along the length of the airway is possible. 2. The cross-sectional area of the airway at any position along its length can be determined. 3. The most constricted part of the airway is not necessarily at the location of the turbinates. 4. Details of hard- and soft-tissue anatomy not otherwise discernible can be detected with computed tomography imaging.

Cephalometry