Tobacco Mosaic Research Articles

DNA technologies (molecular marking) in tomato breeding for resistance to Tobacco Mosaic Virus

Relevance. The purpose of this work is to create new discrete competitive tomato genotypes based on modern biotechnological approaches with increased resistance to the tobacco mosaic virus, economically valuable traits and adapted to the soil and climatic conditions of cultivation in the southern regions of the Russian Federation.Methods. At the first stage of the study, SSR markers taken from literature sources were tested [1;2] and PCR conditions were optimized for the identification of target Tm genes (Tm2a, Tm22, providing resistance to tobacco mosaic virus at the tissue level) in the breeding material available in the department of vegetable and potato growing.Results. Two informative molecular markers Tms 37 and UMD 2060 were selected, which reveal the allelic difference between resistant and susceptible samples. Hybridization of FMS tomato lines with samples having the desired genes of interest in the genotype for obtaining tomato lines resistant to TMV was carried out. Seeds of paternal forms - donors of target genes were obtained for their further use in breeding work. The scientific novelty lies in the study of the discrete genetic material of Solanumlycopersicum, used in the breeding process, and preserving its functions in a new genetic environment.

Nanoscale Wetting of Single Viruses.

The epidemic spread of many viral infections is mediated by the environmental conditions and influenced by the ambient humidity. Single virus particles have been mainly visualized by atomic force microscopy (AFM) in liquid conditions, where the effect of the relative humidity on virus topography and surface cannot be systematically assessed. In this work, we employed multi-frequency AFM, simultaneously with standard topography imaging, to study the nanoscale wetting of individual Tobacco Mosaic virions (TMV) from ambient relative humidity to water condensation (RH > 100%). We recorded amplitude and phase vs. distance curves (APD curves) on top of single virions at various RH and converted them into force vs. distance curves. The high sensitivity of multifrequency AFM to visualize condensed water and sub-micrometer droplets, filling gaps between individual TMV particles at RH > 100%, is demonstrated. Dynamic force spectroscopy allows detecting a thin water layer of thickness ~1 nm, adsorbed on the outer surface of single TMV particles at RH < 60%.

Analysis of Engineered Tobacco Mosaic Virus and Potato Virus X Nanoparticles as Carriers for Biocatalysts.

Plant virus nanoparticles are promising candidates for the development of novel materials, including nanocomposites and scaffolds/carriers for functional molecules such as enzymes. Their advantages for enzyme immobilization include a modular organization, a robust and programmable structure, and a simple, cost-effective production. However, the activity of many enzymes relies on posttranslational modification and most plant viruses replicate in the cytoplasm, so functional enzymes cannot be displayed on the virus surface by direct coat protein fusions. An alternative display system to present the Trichoderma reesei endoglucanase Cel12A on potato virus X (PVX) using SpyTag/SpyCatcher (ST/SC) technology was recently developed by the authors, which allows the carrier and enzyme to be produced separately before isopeptide conjugation. Although kinetic analysis clearly indicated efficient biocatalyst activity, the PVX carrier interfered with substrate binding. To overcome this, the suitability of tobacco mosaic virus (TMV) was tested, which can also accommodate a larger number of ST peptides. We produced TMV particles displaying ST as a new platform for the immobilization of enzymes such as Cel12A, and compared its performance to the established PVX-ST platform in terms of catalytic efficiency. Although more enzyme molecules were immobilized on the TMV-ST particles, we found that the rigid scaffold and helical spacing significantly affected enzyme activity.

Production of active human FGF21 using tobacco mosaic virus-based transient expression system

Fibroblast growth factor (FGF) family has a wide range of metabolic processes. FGF21 exerts critical physiological functions in clinical application. This study aimed to explore a convenient and highly efficient approach for rhFGF21 expression using TMV-TES. Firstly, the vector pTTEV-GFP was constructed, followed by optimisation of the expression parameters in Nicotiana benthamiana. Then, the rhFGF21 encoding gene harbouring vector pTTEV-rhFGF21 was constructed. Agrobacterium-mediated vacuum infiltration was performed with the optimised parameters and the expression of rhFGF21 was confirmed by the immunoblotting analysis. ELISA revealed that the protein accumulation of rhFGF21 accounts for 0.11% of total soluble proteins. The biological activity was evaluated and the results suggested that tobacco-expressed rhFGF21 could stimulate the glucose uptake in swiss 3T3-L1 adipocytes, which was similar to the activity of commercial products, suggesting its native biological activity. Therefore, using TMV-TES to express rhFGF21 will be a feasible approach for the mass production of rhFGF21.

Identification and physical characterization of a spontaneous mutation of the tobacco mosaic virus in the laboratory environment

Virus-like particles are an emerging class of nano-biotechnology with the Tobacco Mosaic Virus (TMV) having found a wide range of applications in imaging, drug delivery, and vaccine development. TMV is typically produced in planta, and, as an RNA virus, is highly susceptible to natural mutation that may impact its properties. Over the course of 2 years, from 2018 until 2020, our laboratory followed a spontaneous point mutation in the TMV coat protein—first observed as a 30 Da difference in electrospray ionization mass spectrometry (ESI–MS). The mutation would have been difficult to notice by electrophoretic mobility in agarose or SDS-PAGE and does not alter viral morphology as assessed by transmission electron microscopy. The mutation responsible for the 30 Da difference between the wild-type (wTMV) and mutant (mTMV) coat proteins was identified by a bottom-up proteomic approach as a change from glycine to serine at position 155 based on collision-induced dissociation data. Since residue 155 is located on the outer surface of the TMV rod, it is feasible that the mutation alters TMV surface chemistry. However, enzyme-linked immunosorbent assays found no difference in binding between mTMV and wTMV. Functionalization of a nearby residue, tyrosine 139, with diazonium salt, also appears unaffected. Overall, this study highlights the necessity of standard workflows to quality-control viral stocks. We suggest that ESI–MS is a straightforward and low-cost way to identify emerging mutants in coat proteins.

Green Synthesis of Silver Nanoparticles with the Tobacco Mosaic Virus

Green Synthesis of Silver Nanoparticles with the Tobacco Mosaic Virus

Mode Switching of the Natural Low-Frequency Oscillations of the Tobacco Mosaic Virus when the Temperature of Its Aqueous Suspension Changes

Mode Switching of the Natural Low-Frequency Oscillations of the Tobacco Mosaic Virus when the Temperature of Its Aqueous Suspension Changes

Hybrid Nanoassemblies from Viruses and DNA Nanostructures.

Viruses are among the most intriguing nanostructures found in nature. Their atomically precise shapes and unique biological properties, especially in protecting and transferring genetic information, have enabled a plethora of biomedical applications. On the other hand, structural DNA nanotechnology has recently emerged as a highly useful tool to create programmable nanoscale structures. They can be extended to user defined devices to exhibit a wide range of static, as well as dynamic functions. In this review, we feature the recent development of virus-DNA hybrid materials. Such structures exhibit the best features of both worlds by combining the biological properties of viruses with the highly controlled assembly properties of DNA. We present how the DNA shapes can act as “structured” genomic material and direct the formation of virus capsid proteins or be encapsulated inside symmetrical capsids. Tobacco mosaic virus-DNA hybrids are discussed as the examples of dynamic systems and directed formation of conjugates. Finally, we highlight virus-mimicking approaches based on lipid- and protein-coated DNA structures that may elicit enhanced stability, immunocompatibility and delivery properties. This development also paves the way for DNA-based vaccines as the programmable nano-objects can be used for controlling immune cell activation.

DsRNA Molecules From the Tobacco Mosaic Virus p126 Gene Counteract TMV-Induced Proteome Changes at an Early Stage of Infection.

Exogenous application of double-stranded RNA (dsRNA) in the tobacco–Tobacco mosaic virus (TMV) pathosystem was shown previously to induce resistance against TMV providing an alternative approach to transgenesis. In the present study, we employed proteomics technology to elucidate the effect of TMV on tobacco as well as the effect of exogenous application of TMV p126 dsRNA molecules (dsRNAp126) at an early stage of the tobacco–TMV interaction. The proteome of tobacco leaf at 15 min post inoculation (mpi) in the presence or absence of dsRNAp126 molecules was studied. Thirty-six tobacco proteins were differentially accumulated in TMV-infected vs. healthy tobacco leaf tissue. The identified main differential TMV-responsive proteins were found to be involved in photosynthesis, energy metabolism, stress, and defense responses. Most of the virus-induced changes in the tobacco leaf proteome were not observed in the leaves treated with dsRNAp126 + TMV. The results indicated that the protein changes induced by TMV infection were counteracted by the exogenous application of dsRNAp126 molecules. Moreover, using small RNA sequencing, we showed that the exogenously applied dsRNAp126 was efficiently processed in tobacco as early as 15 min post application (mpa) to produce small interfering RNAs (siRNAs); the dicing pattern was not affected by the presence of TMV. The presence of dsRNAp126 reduced TMV p126 RNA abundance suggesting virus titer reduction via a sequence-specific mechanism, since a non-homologous dsRNA did not protect from TMV infection nor affect TMV accumulation.

Negative modulation of SA signaling components by the capsid protein of tobacco mosaic virus is required for viral long-distance movement.

An important aspect of plant-virus interaction is the way viruses dynamically move over long distances and how plant immunity modulates viral systemic movement. Salicylic acid (SA), a well-characterized hormone responsible for immune responses against virus, is activated through different transcription factors including TGA and WRKY. In tobamoviruses, evidence suggests that capsid protein (CP) is required for long-distance movement, although its precise role has not been fully characterized yet. Previously, we showed that the CP of Tobacco Mosaic Virus (TMV)-Cg negatively modulates the SA-mediated defense. In this study, we analyzed the impact of SA-defense mechanism on the long-distance transport of a truncated version of TMV (TMV ∆CP virus) that cannot move to systemic tissues. The study showed that the negative modulation of NPR1 and TGA10 factors allows the long-distance transport of TMV ∆CP virus. Moreover, we observed that the stabilization of DELLA proteins promotes TMV ∆CP systemic movement. We also characterized a group of genes, part of a network modulated by CP, involved in TMV ∆CP long-distance transport. Altogether, our results indicate that CP-mediated downregulation of SA signaling pathway is required for the virus systemic movement, and this role of CP may be linked to its ability to stabilize DELLA proteins.

The Role of 3'UTR of RNA Viruses on mRNA Stability and Translation Enhancement.

The central dogma of molecular biology explains the flow of genetic information from DNA to functional products such as proteins. In most cases, a linear relationship with a high correlation coefficient exists between the concentration of mRNA, the middle man, and the functional product. Untranslated regions (UTRs) of RNA form a considerable base pairing that contributes to the secondary and tertiary structures of mRNA. The interaction between the mRNA secondary structures (cis-elements), RNA-binding proteins (RBP) and miRs (trans-element) are critical determinants of mRNAs' fate and stability. Among different viral families, the positive sense (+) RNA viruses use the simplest possible strategy of replication and expression, as the same molecule functions both as a genome and mRNA. Additionally, nucleotide composition and codon usage of +RNA viruses are the closest to human codon adaptation index (CAI). Since the origin of replication of viral intermediate RNA molecules is at the 3'-end of the genome, the 3'UTR plays a role in viral RNA replication. Moreover, the messenger role of RNA likely places functional demands on the 3'UTR to serve a role typical of cellular mRNA. This article reviews the effect of 3'UTR of RNA viruses with positive sense and genomes on mRNA stability and translation improvement. A range of animal (e.g., Dengue, Sindbis, Corona and Polio) and plant (Barley yellow dwarf, Brome mosaic, Turnip crinkle, Tobacco mosaic, Cowpea mosaic and Alfalfa mosaic) viruses are examined to highlight the role of 3'UTR in viral survival and as a potential target for pharmaceutical applications.

Transcriptomic and Functional Analyses Indicate Novel Anti-viral Mode of Actions on Tobacco Mosaic Virus of a Microbial Natural Product ε-Poly-l-lysine.

Novel anti-viral natural product ε-poly-l-lysine (ε-PL) produced by Streptomyces is a homopolymer of l-lysine, of which the underlying molecular mode of action remains to be further elucidated. In this study, ε-PL induced significant fragmentation of tobacco mosaic virus (TMV) virions and delayed the systemic infection of TMV-GFP as well as wild-type TMV in plants. ε-PL treatment also markedly inhibited RNA accumulation of TMV in tobacco BY-2 protoplasts. The results of RNA-seq indicated that the agent induced significantly differential expression of genes that are associated with defense response, stress response, autophagy, and ubiquitination. Among them, 15 critical differential expressed genes were selected for real-time quantitative PCR validation. We further demonstrated that ε-PL can induce host defense responses by assessing the activity of several defense-related enzymes in plants. Our results provided valuable insights into molecular anti-viral mode of action for ε-PL, which is expected to be applied as a novel microbial natural product against plant virus diseases.

Single-dose intranasal subunit vaccine rapidly clears secondary sepsis in a high-dose pneumonic plague infection

Yersinia pestis, the causative agent of plague, has killed millions throughout human history. Though public health initiatives have reduced the number of plague cases, it remains endemic in many areas of the world. It also remains a significant threat for use as a biological weapon. Naturally occurring multi-drug antibiotic resistance has been observed in Y. pestis, and resistant strains have been engineered for use as a biological weapon. Vaccines represent our best means of protection against the threat of antibiotic resistant plague. We have developed a vaccine consisting of two Y. pestis virulence factors, LcrV (V) and F1, conjugated to Tobacco Mosaic Virus (TMV), a safe, non-replicating plant virus that can be administered mucosally, providing complete protection against pneumonic plague, the deadliest form of the disease and the one most likely to be seen in a biological attack. A single intranasal (i.n.) dose of TMV-F1 + TMV-V (TMV-F1/V) protected 88% of mice against lethal challenge with 100 LD50 of Y. pestis CO92pgm-, while immunization with rF1 + rV without TMV was not protective. Serum and tissues were collected at various timepoints after challenge to assess bacterial clearance, histopathology, cytokine production, and antibody production. Overall, TMV-F1/V immunized mice showed a significant reduction in histopathology, bacterial burden, and inflammatory cytokine production following challenge compared to rF1 + rV vaccinated and unvaccinated mice. Pneumonic challenge resulted in systemic dissemination of the bacteria in all groups, but only TMV-F1/V immunized mice rapidly cleared bacteria from the spleen and liver. There was a direct correlation between pre-challenge serum F1 titers and recovery in all immunized mice, strongly suggesting a role for antibody in the neutralization and/or opsonization of Y. pestis in this model. Mucosal administration of a single dose of a Y. pestis TMV-based subunit vaccine, without any additional adjuvant, can effectively protect mice from lethal infection.

Synthesis and cytotoxicity evaluation of gemcitabine-tobacco mosaic virus conjugates

Gemcitabine is one of the nucleoside analogue drugs that acts against a wide range of solid tumors such as pancreatic, non-small lung, breast, and ovarian cancers. Unfortunately, gemcitabine possesses rapid body clearance that limits its efficacy. This drawback is due to kidney excretion and metabolism by plasmatic enzyme cytidine-deaminase, which yields an inactive metabolite. Plant virus nanoparticles are one of the biggest groups of natural nanoparticles, which are less likely to interact with mammalian system to make adverse effects and can be used in drug delivery. In this study, chemical conjugation of gemcitabine and Tobacco Mosaic Virus (TMV); as one of the mostly used plant viruses, has been investigated through the creation of amide bond and the percentage of conjugation was calculated using UV spectroscopy. The drug release from conjugate was pH-dependent and, at acidic pH, the drug release was almost complete. The hemolysis percentage caused by the virus and the conjugate was also proved to be very small. Cytotoxicity studies at 24 and 48-h showed that there was no significant difference between the free drug and drug conjugate in HepG2 and MCF7 cell lines, but the results of the toxicity study were better on HepG2 cell line. Also in both cell lines, the observed cytotoxicity of the conjugate was more acceptable after 48 h which is due to enough time for drug release. It is expected that these viral nanoparticles make better in vivo drug delivery, pharmacokinetics improvement and reduced dose related toxicity of chemotherapeutic agents.

Top Ten Viruses Reported on Chilly in Southeast Asia

Chilly is a common spice crop grown in large quantities in South Asia. India produces 75% of the total chilly crop followed by Nepal at 8% and Bangladesh at 6%, where it is grown primarily by large and small-scale farmers. It can also be found in the kitchen garden. Disease and insect pests are the most significant factors affecting the production of the chilly crop. The viral disease represents a significant threat to chilly production. Recently, 68 viruses have been reported influencing the chilly crop all over the world. Chilly Leaf Curl Virus Disease, Cucumber Mosaic Virus, Tobacco Mosaic Virus, Tomato Spotted Wilt Virus, Potato Virus Y, Alfalfa Mosaic Virus, Pepper Vein Mottle Virus, Beet Curl Top Virus, and Pepper Mild Mottle Virus are among the 68 viruses belonging to seven genera namely, Begomovirus, Cucumovirus, Tobamovirus, Tospovirus, Potyvirus, Alfamovirus and Curtovirus. In chilly cultivation, yield losses vary from 15 to 100%, depending on the virus. This review article is an analysis providing of top ten viruses reported in Chilly, including their geographic range, yield losses, symptomatology, transmission and management in South Asian Countries.

Exploration on the expression and assembly of virus-like particles

Virus-like particles (VLPs) have a great application prospect in vaccines and molecule delivery carriers. In this study, in order to solve the problem of low expression and low assembly efficiency of VLPs, the conditions for the assembly and purification of eight representative VLPs (hepatitis B virus core antigen protein particles, Qbeta phage, MS2 phage, P22 phage, cowpea chlorotic mottle virus, tobacco Mosaic virus, ferritin and encapsulin) expressed in Escherichia coli were optimized. The VLPs with high expression, easy assembly and good purification properties were selected as the preferred objects for potential biological applications.

Enhancing Tomato Plant Resistance Against Tobacco Mosaic Virus Using Riboflavin

Plant systemic resistance induced by natural product is an alternative technique of disease management. Riboflavin (vitamin B2) usually used as abiotic elicitor to improve the plant immunity against different pathogens. This work aimed to explore the efficiency of three riboflavin concentrations (0.5, 2.5, and 5 mM) to enhance resistance toward Tobacco Mosaic Virus (TMV) in tomato plants. Our results showed that exogenous application of 2.5mM riboflavin, 5 days before virus challenge was the most effective concentration, which provided a reduction in disease incidence and disease severity by 80% and 75%, respectively. Furthermore, obtained results were confirmed by using DAS-ELISA test, which showed that concentration 2.5 mM of riboflavin decreased the virus concentrations by 46.4% in treated- inoculated plants. It was remarked that exogenous application of 2.5 mM riboflavin showed a positive effect on some plant growth parameters either in presence or absence of TMV challenge. The plant height and number of leaves per plant were significantly improved in treated- inoculated plants about 30% and in compared to the infected control 78%. In time course investigation, 2.5mM riboflavin treatment reduced the virus symptoms particularly at 9th day, where after the symptoms become evident. In addition, expression of phenylalanine ammonia-lyase (PAL) and pathogenesis-related protein (PR10), which are the markers of systemic acquired resistance (SAR), were rapidly increased in inoculated-treated tomato from 1-3 days after treatment. Moreover, the results of the biochemical changes analysis revealed that, the levels of the defense markers including peroxidase (PO), and polyphenol oxidase (PPO) were increased four days after of treatment and reached maximum levels at 8 days in the treated- inoculated plants. In conclusion, it could be suggesting that riboflavin exerted a great influence on TMV disease, as indicated by reduction of disease symptoms as well as enhancement of biochemical changes in plant defense against infection with TMV.

Coronavirus rotational diffusivity.

Just 11 weeks after the confirmation of first infection, one team had already discovered and published [D. Wrapp et al., “Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation,” Science 367(6483), 1260–1263 (2020)] in exquisite detail about the new coronavirus, along with how it differs from previous viruses. We call the virus particle causing the COVID-19 disease SARS-CoV-2, a spherical capsid covered with spikes termed peplomers. Since the virus is not motile, it relies on its own random thermal motion, specifically the rotational component of this thermal motion, to align its peplomers with targets. The governing transport property for the virus to attack successfully is thus the rotational diffusivity. Too little rotational diffusivity and too few alignments are produced to properly infect. Too much, and the alignment intervals will be too short to properly infect, and the peplomer is wasted. In this paper, we calculate the rotational diffusivity along with the complex viscosity of four classes of virus particles of ascending geometric complexity: tobacco mosaic, gemini, adeno, and corona. The gemini and adeno viruses share icosahedral bead arrangements, and for the corona virus, we use polyhedral solutions to the Thomson problem to arrange its peplomers. We employ general rigid bead–rod theory to calculate complex viscosities and rotational diffusivities, from first principles, of the virus suspensions. We find that our ab initio calculations agree with the observed complex viscosity of the tobacco mosaic virus suspension. From our analysis of the gemini virus suspension, we learn that the fine detail of the virus structure governs its rotational diffusivity. We find the characteristic time for the adenovirus from general rigid bead–rod theory. Finally, from our analysis of the coronavirus suspension, we learn that its rotational diffusivity descends monotonically with its number of peplomers.

Induction of Plant Resistance against Tobacco Mosaic Virus Using the Biocontrol Agent Streptomyces cellulosae Isolate Actino 48

Viral plant diseases represent a serious problem in agricultural production, causing large shortages in the production of food crops. Eco-friendly approaches are used in controlling viral plant infections, such as biocontrol agents. In the current study, Streptomyces cellulosae isolate Actino 48 is tested as a biocontrol agent for the management of tobacco mosaic virus (TMV) and inducing tomato plant systemic resistance under greenhouse conditions. Foliar application of a cell pellet suspension of Actino 48 (2 × 107 cfu. mL−1) is performed at 48 h before inoculation with TMV. Peroxidase activity, chitinase activity, protein content, and the total phenolic compounds are measured in tomato leaves at 21 dpi. On the other hand, the TMV accumulation level and the transcriptional changes of five tomato defense-related genes (PAL, PR-1, CHS, PR-3, and PR-2) are studied. Treatment with Actino 48 before TMV inoculation (48 h) induced tomato plants to increase their levels of peroxidase and chitinase enzymes. Furthermore, a significant increase in the concentration of total phenolic compounds was observed in Actino 48 and TMV-treated tomato plants compared to TMV-treated tomato plants alone. Treatment with Actino 48 reduced the TMV accumulation level (53.8%) compared to treatment with the virus alone. Actino 48 induced plant growth, where the fresh and dry weights of tomato plants increased. Additionally, significant increases of the PAL, PR-1, CHS, and PR-3 transcripts were observed. On the other hand, a higher induction of PR-2 was only observed in TMV-treated tomato plants. In conclusion, S. cellulosae isolate Actino 48 can be used as a biocontrol agent for the reduction of symptoms and severity of TMV.

Identification of Proteins of Tobacco Mosaic Virus by Using a Method of Feature Extraction.

Tobacco mosaic virus, TMV for short, is widely distributed in the global tobacco industry and has a significant impact on tobacco production. It can reduce the amount of tobacco grown by 50–70%. In this research of study, we aimed to identify tobacco mosaic virus proteins and healthy tobacco leaf proteins by using machine learning approaches. The experiment's results showed that the support vector machine algorithm achieved high accuracy in different feature extraction methods. And 188-dimensions feature extraction method improved the classification accuracy. In that the support vector machine algorithm and 188-dimensions feature extraction method were finally selected as the final experimental methods. In the 10-fold cross-validation processes, the SVM combined with 188-dimensions achieved 93.5% accuracy on the training set and 92.7% accuracy on the independent validation set. Besides, the evaluation index of the results of experiments indicate that the method developed by us is valid and robust.