Tobacco Mosaic Research Articles

Intranasal administration of a two-dose adjuvanted multi-antigen TMV-subunit conjugate vaccine fully protects mice against Francisella tularensis LVS challenge.

Tularemia is a fatal human disease caused by Francisella tularensis, a Gram-negative encapsulated coccobacillus bacterium. Due to its low infectious dose, ease of aerosolized transmission, and lethal effects, the CDC lists F. tularensis as a Category A pathogen, the highest level for a potential biothreat agent. Previous vaccine studies have been conducted with live attenuated, inactivated, and subunit vaccines, which have achieved partial or full protection from F. tularensis live vaccine strain (LVS) challenge, but no vaccine has been approved for human use. We demonstrate the improved efficacy of a multi-antigen subunit vaccine by using Tobacco Mosaic virus (TMV) as an antigen carrier for the F. tularensis SchuS4 proteins DnaK, OmpA, SucB and Tul4 (DOST). The magnitude and quality of immune responses were compared after mice were immunized by subcutaneous or intranasal routes of administration with a TMV-DOST mixture, with or without four different adjuvants. Immune responses varied in magnitude and isotype profile, by antigen, by route of administration, and by protection in an F. tularensis LVS challenge model of disease. Interestingly, our analysis demonstrates an overwhelming IgG2 response to SucB after intranasal dosing, as well as a robust cellular response, which may account for the improved two-dose survival imparted by the tetravalent vaccine, compared to a previous study that tested efficacy of TMV-DOT. Our study provides evidence that potent humoral, cellular and mucosal immunity can be achieved by optimal antigen combination, delivery, adjuvant and appropriate route of administration, to improve vaccine potency and provide protection from pathogen challenge.

Electrochemically-Driven Insertion of Biological Nanodiscs into Solid State Membrane Pores as a Basis for "Pore-In-Pore" Membranes.

Nanoporous membranes are of increasing interest for many applications, such as molecular filters, biosensors, nanofluidic logic and energy conversion devices. To meet high-quality standards, e.g., in molecular separation processes, membranes with well-defined pores in terms of pore diameter and chemical properties are required. However, the preparation of membranes with narrow pore diameter distributions is still challenging. In the work presented here, we demonstrate a strategy, a “pore-in-pore” approach, where the conical pores of a solid state membrane produced by a multi-step top-down lithography procedure are used as a template to insert precisely-formed biomolecular nanodiscs with exactly defined inner and outer diameters. These nanodiscs, which are the building blocks of tobacco mosaic virus-deduced particles, consist of coat proteins, which self-assemble under defined experimental conditions with a stabilizing short RNA. We demonstrate that the insertion of the nanodiscs can be driven either by diffusion due to a concentration gradient or by applying an electric field along the cross-section of the solid state membrane. It is found that the electrophoresis-driven insertion is significantly more effective than the insertion via the concentration gradient.

Speciation of Phenanthriplatin and Its Analogs in the Core of Tobacco Mosaic Virus.

Efficient loading of drugs in novel delivery agents has the potential to substantially improve therapy by targeting the diseased tissue while avoiding unwanted side effects. Here we report the first systematic study of the loading mechanism of phenanthriplatin and its analogs into tobacco mosaic virus (TMV), previously used by our group as an efficient carrier for anticancer drug delivery. A detailed investigation of the preferential uptake of phenanthriplatin in its aquated form (∼2000 molecules per TMV particle versus ∼1000 for the chlorido form) is provided. Whereas the net charge of phenanthriplatin analogs and their ionic mobilities have no effect on loading, the reactivity of aqua phenanthriplatin with the glutamates, lining the interior walls of the channel of TMV, has a pronounced effect on its loading. MALDI-MS analysis along with NMR spectroscopic studies of a model reaction of hydroxy-phenanthriplatin with acetate establish the formation of stable covalent adducts. The increased number of heteroaromatic rings on the platinum ligand appears to enhance loading, possibly by stabilizing hydrophobic stacking interactions with TMV core components, specifically Pro102 and Thr103 residues neighboring Glu97 and Glu106 in the channel. Electron transfer dissociation MS/MS fragmentation, a technique that can prevent mass-condition-vulnerable modification of proteins, reveals that Glu97 preferentially participates over Glu106 in covalent bond formation to the platinum center.

Self-Assembly of Protein Crystals with Different Crystal Structures Using Tobacco Mosaic Virus Coat Protein as a Building Block.

In this work, a typical cylinder-shaped tobacco mosaic virus coat protein (TMVCP) is employed as an anisotropic building block to assemble into triclinic and hexagonal close-packed (HCP) protein crystals by introducing cysteine residues at the 1 and 3 sites and four histidine residues at the C-terminal, respectively. The engineered functional groups of cysteine and histidine in the TMVCP and the self-assembly conditions determine the thermodynamics and kinetics in the self-assembly process for forming different crystal structures. The results show that the TMVCPs are thermodynamically driven to form triclinic crystals due to the formation of disulfide bonds between neighboring TMVCPs. On the other hand, the self-assembly of HCP crystals is kinetically directed by the strong metal-histidine chelation. This work not only greatly expands TMVCP for fabricating promising nanomaterials but also represents an approach to adjusting the protein crystal structures by tuning the thermodynamics and kinetics during crystallization.

Tobacco Mosaic Virus with Peroxidase-Like Activity for Cancer Cell Detection through Colorimetric Assay.

Cell-based ELISA (CELLISA) has been widely used in disease diagnosis due to its simplicity and low cost. Recently, peroxidase-like nanomaterials have emerged as promising systems for CELLISA applications. In this work, tobacco mosaic virus (TMV) was simultaneously tailored with peroxidase-like inorganic nanoparticles (platinum nanoparticles) and cancer cell target groups (folic acid, FA) to obtain TMV-FA-Pt nanoparticles for cancer cell detection. Induced by the uniformly distributed reactive groups and well-defined structure of the TMV particle, platinum nanoparticles could be grown in situ on the exterior surface of TMV with excellent monodispersity and uniform spatial distribution. Meanwhile, FA with a PEG1000 linker was successfully conjugated to the coat proteins of TMV through the Cu(I)-catalyzed alkyne-azide cycloaddition reaction, an efficient "click" chemistry. Our study demonstrated that the resultant TMV-FA-Pt had specific affinity to cancer cells and was successfully used to detect cancer cells through CELLISA. Less than 1.0 × 104 cells/mL of cancer cells could be readily detected.

Plant Pathology and the Greenhouse Tomato Industry in Ohio, 1937–82

Plant Pathology and the Greenhouse Tomato Industry in Ohio, 1937–82 Christopher Cumo (bio) During the twentieth century, the tomato emerged as the leading crop grown in greenhouses in Ohio and the United States in general. During the first half of the century, Fusarium wilt, a fungal disease, and a virus related to Tobacco Mosaic Virus (TMV) or Tomato Mosaic Virus (ToMV), if not identical to one of these viruses, threatened the livelihood of tomato growers, particularly those who managed greenhouses. To combat these diseases in Ohio, Leonard Jay Alexander bred tomatoes resistant to Fusarium wilt after 1939 and to both the fungus and the virus after 1942. Success in these endeavors earned him national as well as international recognition. greenhouse tomato culture in the united states During the twentieth century, the U.S. tomato crop consisted of tomatoes grown in the field and the greenhouse. Our focus is on the rise of the greenhouse industry and its problems because these issues shaped agricultural research, notably at the Ohio Agricultural Experiment Station (today the Ohio Agricultural Research and Development Center) in Wooster. Before examining these issues, a general account of the development of tomato culture is in order. The tomato is indigenous to South and Central America, where a pre–Mexican people in the Monte Alban valley in southern Mexico first cultivated it around 200 bce.1 Europeans became aware of the tomato only in the [End Page 95] early sixteenth century, when the Spanish introduced it to Europe as a curiosity. Accordingly, the tomato was part of the Columbian Exchange, which was the transfer of plants, animals, and pathogens both ways across the Atlantic Ocean in the years following the European discovery of the Americas. In Europe, true to the stereotype, the Italians embraced it as the staple ingredient in sauces. After Europeans established the relationship between the tomato and the poisonous belladonna, its popularity diminished, especially in northern Europe. Gardeners, thinking it toxic, grew the tomato as an ornamental rather than as food. In the seventeenth century, the English carried this misapprehension to the New World. Not until 1710 does literary evidence mention the tomato in colonial America, and then only as an ornamental.2 It was, however, being grown as food in New Orleans in 1812 and marketed in Philadelphia in 1829.3 Nevertheless, belief in its toxicity remained strong enough that in the latter year one man achieved notoriety by brazenly eating several tomatoes at the courthouse in Salem, New Jersey, without becoming ill.4 Commercial tomato culture grew slowly during the nineteenth century. The fresh, ripe tomato bruised and cracked easily and spoiled after being stored less than a week without refrigeration. Also, before the discovery of vitamins, beginning in 1913, nutritionists conceived of human dietary requirements in terms of protein, carbohydrates, lipids, and minerals. The tomato, which was understood to contain little more than carbohydrates and water, was regarded as nutrient poor.5 It was, however, suitable for cultivation as fresh produce near urban markets. Rapid urbanization in the United States after the Civil War—the 1920 census revealed for the first time that more than half of the population inhabited cities—created concentrated centers of demand for fresh produce, including tomatoes.6 Large cities in the northeast and near the Great Lakes, including Cleveland, Grand Rapids, Indianapolis, and Boston, manifested sufficient demand for fresh tomatoes that growers established greenhouses on their outskirts during the 1910s.7 In general, then, the development of the U.S. green-house [End Page 96] tomato industry has been inextricably bound to urban population size and consumption patterns. This development, however, is difficult to measure with precision. The U.S. Department of Agriculture's Agricultural Statistics has divided tomato production into the categories of fresh and processed tomatoes.8 Greenhouses have supplied tomatoes for only a portion of fresh produce, but not for processing into juice, sauce, catsup, paste, soup, or canned goods. Field tomato growers supply the rest. Joan Stemner, secretary-treasurer of the Cleveland Greenhouse Growers' Association, has estimated that greenhouses have traditionally supplied, with minor perturbations, roughly 35 percent of fresh tomatoes in the United States.9 If she is correct, one can obtain a...

RNA-Directed Assembly of Tobacco Mosaic Virus (TMV)-Like Carriers with Tunable Fractions of Differently Addressable Coat Proteins.

Taking advantage of the ability for in vitro assembly of the plant-infecting virus tobacco mosaic virus (TMV), rod-shaped nanoscale scaffolds presenting different addressable groups can be obtained. We have established procedures resulting in virus-like particles with randomly distributed functional groups, with different groups arranged in striped but randomized structures, and even with distinct groups clustered in adjacent, better-defined domains. The TMV coat protein (CP) variants combined in these approaches can either originate all from TMV mutants propagated in planta, or be mixed with CP expressed in E. coli (CPEc). Protocols for expression and purification of a CPEc-His6 mutant in E. coli as well as the different methods for in vitro assembly and the visualization by decoration of one CP type are explained in detail.

烟草苗床期普通花叶病(TMV)组织印迹检测技术研究

烟草苗床期普通花叶病(TMV)组织印迹检测技术研究

Propagation and Isolation of Tobacco Mosaic Virus That Surface Displays Metal Binding and Reducing Peptides for Generation of Gold Nanoparticles.

In this chapter we describe an approach for propagating, isolating and characterizing tobacco mosaic virus (TMV) genetically modified to surface display a metal binding and reducing peptide, and its utilization for the production of free gold metal nanoparticles from metal salt precursors.

Virological Quality of Irrigation Water Sources and Pepper Mild Mottle Virus and Tobacco Mosaic Virus as Index of Pathogenic Virus Contamination Level.

Irrigation water is a doorway for the pathogen contamination of fresh produce. We quantified pathogenic viruses [human adenoviruses, noroviruses of genogroups I and II, group A rotaviruses, Aichi virus 1 (AiV-1), enteroviruses (EnVs), and salivirus (SaliV)] and examined potential index viruses [JC and BK polyomaviruses (JCPyVs and BKPyVs), pepper mild mottle virus (PMMoV), and tobacco mosaic virus (TMV)] in irrigation water sources in the Kathmandu Valley, Nepal. River, sewage, wastewater treatment plant (WWTP) effluent, pond, canal, and groundwater samples were collected in September 2014, and in April and August 2015. Viruses were concentrated using an electronegative membrane-vortex method and quantified using TaqMan (MGB)-based quantitative PCR (qPCR) assays with murine norovirus as a molecular process control to determine extraction-reverse transcription-qPCR efficiency. Tested pathogenic viruses were prevalent with maximum concentrations of 5.5-8.8 log10 copies/L, and there was a greater abundance of EnVs, SaliV, and AiV-1. Virus concentrations in river water were equivalent to those in sewage. Canal, pond, and groundwater samples were found to be less contaminated than river, sewage, and WWTP effluent. Seasonal dependency was clearly evident for most of the viruses, with peak concentrations in the dry season. JCPyVs and BKPyVs had a poor detection ratio and correspondence with pathogenic viruses. Instead, the frequently proposed PMMoV and the newly proposed TMV were strongly predictive of the pathogen contamination level, particularly in the dry season. We recommend utilizing canal, pond, and groundwater for irrigation to minimize deleterious health effects and propose PMMoV and TMV as indexes to elucidate pathogenic virus levels in environmental samples.

Dysprosium-Modified Tobacco Mosaic Virus Nanoparticles for Ultra-High-Field Magnetic Resonance and Near-Infrared Fluorescence Imaging of Prostate Cancer

The increasing prevalence of ultra-high-field magnetic resonance imaging (UHFMRI) in biomedical research and clinical settings will improve the resolution and diagnostic accuracy of MRI scans. However, better contrast agents are needed to achieve a satisfactory signal-to-noise ratio. Here, we report the synthesis of a bimodal contrast agent prepared by loading the internal cavity of tobacco mosaic virus (TMV) nanoparticles with a dysprosium (Dy3+) complex and the near-infrared fluorescence (NIRF) dye Cy7.5. The external surface of TMV was conjugated with an Asp-Gly-Glu-Ala (DGEA) peptide via a polyethylene glycol linker to target integrin α2β1. The resulting nanoparticle (Dy-Cy7.5-TMV-DGEA) was stable and achieved a high transverse relaxivity in ultra-high-strength magnetic fields (326 and 399 mM-1 s-1 at 7 and 9.4 T, respectively). The contrast agent was also biocompatible (low cytotoxicity) and targeted PC-3 prostate cancer cells and tumors in vitro and in vivo as confirmed by bimodal NIRF imaging and T2-mapping UHFMRI. Our results show that Dy-Cy7.5-TMV-DGEA is suitable for multiscale MRI scanning from the cellular level to the whole body, particularly in the context of UHFMRI applications.

Programming Self-Assembly of Tobacco Mosaic Virus Coat Proteins at Pickering Emulsion Interfaces for Nanorod-Constructed Capsules.

Pickering emulsion constructions on nanorods with high aspect ratio are a great challenge because of the geometry restrictions. On the basis of the theory that the stability of Pickering emulsion is highly dependent on the size and amphiphilicity of the nanoparticle at fluid interfaces, we report a novel strategy to fabricate long-time stable Pickering emulsion consisting of tobacco mosaic virus (TMV)-like nanorods through the programming self-assembly of TMV coat protein (TMVCP). The first step is the self-assembly of amphiphilic TMVCP at Pickering emulsion interfaces, and the second step is the in situ interfacial self-assembly of TMVCP into nanorods with increased particle size. The robust capsules can be further fabricated through cross-linking of the proteins. By taking advantage of both the amphiphilicity of TMVCP and the subsequent size growth induced by TMVCP self-assembly, this work provides a powerful strategy for constructing robust capsules consisting of nanorods with high aspect ratio, which may show potential applications for drug delivery and virus recognition.

Calculation of the cross-sectional shape of a fibril from equatorial scattering.

An alternate formulation of helical diffraction theory is used to generate cross-sectional shapes of fibrous structures from equatorial scattering. We demonstrate this approach with computationally generated scattering intensities and then apply it to scattering data from Tobacco Mosaic Virus (TMV) and in vitro assembled fibrils of Aβ40 peptides. Refining the cross-sectional shape of TMV from SAXS data collected on a 26mg/ml solution resulted in a circular shape with outer diameter of ∼180Å and inner diameter of ∼40Å consistent with the known structure of TMV. We also utilized this method to analyze the equatorial scattering from TMV collected by Don Caspar from a concentrated (24% ∼295mg/ml) gel of TMV as reported in his Ph.D. thesis in 1955. This data differs from the SAXS data in having a sharp interference peak at ∼250Å spacing, indicative of strong interparticle interactions in the gel. Analysis of this data required consideration of interatomic vectors as long as 2000Å and resulted in generation of images that were interpreted as representative of local organization of TMV particles in the sample. Peaks in the images were separated, on average by about 250Å with a density consistent with Caspar's original measurements. Analysis of SAXS data from Aβ fibrils resulted in a cross-sectional shape that could be interpreted in terms of structural models that have been constructed from ssNMR and cryoEM. These results demonstrate an unexpected use of the small-angle region of fiber diffraction patterns to derive fundamental structural properties of scattering objects.

Development of a Multivalent Subunit Vaccine against Respiratory Tularemia

Abstract Francisella tularensis (Ft); the causative agent of a fatal human disease tularemia is classified as a Category A Select Agent. No licensed vaccine is available for prevention of tularemia in the U.S.A. In this study, we used a novel Tobacco Mosaic Virus (TMV) based delivery platform for development of a fully protective multi-antigen subunit tularemia vaccine. Previously we have published that a trivalent TMV-conjugate vaccine confers 50% protection in immunized mice against respiratory Ft LVS challenge. In this study, we refined TMV-conjugate vaccine formulation to improve the level of protection in immunized C57BL/6 mice against respiratory tularemia. We developed a tetravalent vaccine by conjugating OmpA, DnaK, Tul4 and SucB proteins of Francisella to TMV. CpG adjuvant was also included in the vaccine formulation. C57BL/6 mice were immunized intranasally (i.n.) on days 0, 14 and 28 and challenged with 10LD100 of Ft LVS on day 49 post-primary immunization (PPI). Mice were monitored for survival, weight loss, antibody and recall responses, and duration of immune protection. 100% of immunized mice were protected against a 10LD100 i.n. challenge dose of Ft LVS. Mice vaccinated with TMV-tetravalent vaccine showed high levels of TH1 antibodies than those with trivalent vaccine formulation. The challenged mice exhibited significantly reduced bacterial burden in lungs, liver and spleen. Strong ex-vivo recall responses were observed in immunized mice as late as day 84 PPI and 80% of mice survived when challenged 163 days PPI. Collectively, this study demonstrates that tetravalent vaccine formulation provides complete protection, induces strong protective and memory immune responses against respiratory challenge with F. tularensis.

Detection of Viral-like Particles by Electron Microscopy of Negatively Stained Extracts from Grapevines

Viral-like particles were detected by electron microscopy of negatively stained extracts from young leaves and roots of grapevines infected with fanleaf virus and purported to be infected with the graft-transmissible agents of Ieafroll, corky bark, fleck, and stem grooving. Preparations were extracted in 0.01 M phosphate buffer (pH 7.0) containing 2.5% nicotine and negatively stained with 2% ammonium molydate. Membrane associated spherical particles were detected in extracts from grapevines that tested positive for fanleaf virus by ELISA and bioassay. Similar membrane associated particles were detected in herbaceous plants inoculated with grapevine extracts. Rigid rod tobacco mosaic virus-like particles were detected in extracts from some grapevines but they were not disease associated. Flexuous rod viral-like particles about 11x800 nm with cross-banding helical substructure similar to closteroviruses were detected in extracts from leafroll infected grapevines.

A novel hairpin library-based approach to identify NBS\u2013LRR genes required for effector-triggered hypersensitive response in Nicotiana benthamiana

BackgroundPTI and ETI are the two major defence mechanisms in plants. ETI is triggered by the detection of pathogen effectors, or their activity, in the plant cell and most of the time involves internal receptors known as resistance (R) genes. An increasing number of R genes responsible for recognition of specific effectors have been characterised over the years; however, methods to identify R genes are often challenging and cannot always be translated to crop plants.ResultsWe present a novel method to identify R genes responsible for the recognition of specific effectors that trigger a hypersensitive response (HR) in Nicotiana benthamiana. This method is based on the genome-wide identification of most of the potential R genes of N. benthamiana and a systematic silencing of these potential R genes in a simple transient expression assay. A hairpin-RNAi library was constructed covering 345 R gene candidates of N. benthamiana. This library was then validated using several previously described R genes. Our approach indeed confirmed that Prf, NRC2a/b and NRC3 are required for the HR that is mediated in N. benthamiana by Pto/avrPto (prf, NRC2a/b and NRC3) and by Cf4/avr4 (NRC2a/b and NRC3). We also confirmed that NRG1, in association with N, is required for the Tobacco Mosaic Virus (TMV)-mediated HR in N. benthamiana.ConclusionWe present a novel approach combining bioinformatics, multiple-gene silencing and transient expression assay screening to rapidly identify one-to-one relationships between pathogen effectors and host R genes in N. benthamiana. This approach allowed the identification of previously described R genes responsible for detection of avirulence determinants from Pseudomonas, Cladosporium and TMV, demonstrating that the method could be applied to any effectors/proteins originating from a broad range of plant pathogens that trigger an HR in N. benthamiana. Moreover, with the increasing availability of genome sequences from model and crop plants and pathogens, this approach could be implemented in other plants, accelerating the process of identification and characterization of novel resistance genes.

Packaged and Free Satellite Tobacco Mosaic Virus (STMV) RNA Genomes Adopt Distinct Conformational States.

The RNA genomes of viruses likely undergo multiple functionally important conformational changes during their replication cycles, changes that are poorly understood at present. We used two complementary in-solution RNA structure probing strategies (SHAPE-MaP and RING-MaP) to examine the structure of the RNA genome of satellite tobacco mosaic virus inside authentic virions and in a capsid-free state. Both RNA states feature similar three-domain architectures in which each major replicative function-translation, capsid coding, and genome synthesis-fall into distinct domains. There are, however, large conformational differences between the in-virion and capsid-free states, primarily in one arm of the central T domain. These data support a model in which the packaged capsid-bound RNA is constrained in a local high-energy conformation by the native capsid shell. The removal of the viral capsid then allows the RNA genome to relax into a more thermodynamically stable conformation. The RNA architecture of the central T domain thus likely changes during capsid assembly and disassembly and may play a role in genome packaging.

Preparation and Characterization of Functional Tobacco Mosaic Virus Spherical Nanoparticles

Preparation and Characterization of Functional Tobacco Mosaic Virus Spherical Nanoparticles

The genome sequence of a novel RNA virus in Adelphocoris suturalis.

The complete genome of a novel virus found in Adelphocoris suturalis was determined by RNA-seq and named Adelphocoris suturalis-associated virus 1 (ASV1). ASV1 has a single-stranded RNA genome of 10,845 nucleotides in length and contains five putative open reading frames (ORFs). ORF1 encodes a polypeptide of 2592 amino acids (aa) and contains four conserved domains: a viral RNA methyltransferase domain, an FtsJ-like methyltransferase domain, a viral RNA helicase domain and an RNA-dependent RNA polymerase domain. ORF2, ORF3, ORF4 and ORF5 encode polypeptides of 190, 461, 103 and 159 aa, respectively, of which only ORF5 contains a conserved domain, the Tobacco mosaic virus-coat superfamily. Phylogenetic analysis with the deduced amino acid sequences indicated that ASV1 clusters with the Drosophila-related Boutonnet virus. The similar genomic structure and high bootstrap value identified in the maximum-likelihood tree suggest that ASV1 (possibly alongside Boutonnet virus) could be considered the prototype of a new taxon of unclassified insect viruses. The prevalence of ASV1 infection in wild populations of A. suturalis was at a low level (6.60%, 14 positives from 212 samples).

The Importance of Calcium Ions in Poly-A RNA Mediated Tobacco Mosaic Virus-Like Rod Formation

Due to its well-understood structural and chemical features, Tobacco mosaic virus (TMV) has attracted interest as a template for producing a variety of novel nanostructured materials and devices. However, for TMV based nanotechnology to reach its potential, controlling the size of TMV rods without using infectious native RNA is a key issue. Here, we re-examine the interactions of TMV coat protein (CP) with poly A RNA. Our results confirm that poly A RNA can induce virus-like rod formation through interactions with the coat protein. In addition, we show that calcium ions have an important role in stabilizing such rods, a previously unknown interaction that enhances the reliability of poly A induced assembly.