Generation of dengue 3 envelope domain III using tobacco mosaic virus-based vector system and its immunological response mouse model by generating anti-dengue virus antibodies.

Producing recombinant proteins in plants has become a valuable alternative to traditional microbial or mammalian systems due to its cost-effectiveness, scalability, and ability to perform post-translational modifications. This study investigates the use of the Tobacco Mosaic Virus (TMV)-based vector system for producing the Dengue virus serotype 3 (DENV-3) envelope domain III (EDIII) protein in plants.. A fragment of the gene that encodes domain III of the dengue 3 envelope protein (D3EIII, comprising 300-420 amino acids), was effectively expressed within Nicotiana tabacum plants utilizing a transient expression system based on tobacco mosaic virus (TMV). The N-terminal 5' UTR region upstream of D3EIII notably enhanced protein yield in infected tissues. The produced recombinant protein exhibited reactivity with both (anti) D3EIII polyclonal antibodies and antibodies of anti-His tag. Upon injection of EDIII in mice, it stimulated the generation of antibodies against the dengue-specific virus. The induced antibodies demonstrated neutralizing activity against dengue virus type 3. These findings indicate that the TMV expression system is effective for producing dengue virus antigens in plants, resulting in antigens with appropriate properties and strong immunogenic potential.

Genome-wide analysis of tobacco NtTOM1/TOM3 gene family and identification of NtTOM1a/ NtTOM3a response to tobacco mosaic virus.

BACKGROUND: TOBAMOVIRUS MULTIPLICATION 1 (TOM1) and its homolog TOBAMOVIRUS MULTIPLICATION 3 (TOM3) play a prominent role in the multiplication of tobacco mosaic virus (TMV) in higher plants. Although homologs of NtTOM1/TOM3 genes have been identified in several plant species, little is known about the characteristics and functions of NtTOM1/TOM3 at the genome-wide level in tobacco (Nicotiana tabacum L.). RESULTS: In this study, we performed genome-wide identification and expression pattern analysis of the tobacco NtTOM1/TOM3 gene family. Twelve NtTOM1/TOM3 genes were identified and classified into four groups based on phylogenetic analysis. Sequence and conserved domain analyses showed that all these genes contained a specific DUF1084 domain. Expression pattern analysis showed that NtTOM1a, NtTOM1b, NtTOM1d, NtTOM3a, NtTOM3b, and NtTOM3d were induced by TMV at 1-, 3-, and 9 dpi, whereas the expression of other genes was not responsive to TMV at the early infection stage. TMV virion accumulation showed no obvious difference in either nttom1a or nttom3a mutants compared with the wild type. However, the virus propagation was significantly, but not completely, inhibited in the nttom1atom3a double mutant, indicating that other gene family members may function redundantly, such as NtTOM1b and NtTOM1d. In addition, overexpression of NtTOM1a or NtTOM3a also inhibited the TMV replication to some extent. CONCLUSIONS: The present study performed genome-wide analysis of the NtTOM1/TOM3 gene family in tobacco, and identified NtTOM1a and NtTOM3a as important genes involved in TMV multiplication based on functional analysis. These results provide a theoretical basis for further improving TMV resistance in tobacco.

Nanomaterial inactivates environmental virus and enhances plant immunity for controlling tobacco mosaic virus disease.

Tobacco mosaic virus (TMV) is extremely pathogenic and resistant to stress There are great needs to develop methods to reduce the virus in the environment and induce plant immunity simultaneously. Here, we report a multifunctional nano-protectant to reduce the virus in the environment and induce plant immunity simultaneously. The star polycation (SPc) nanocarrier can act as an active ingredient to interact with virus coat protein via electrostatic interaction, which reduces the proportion of TMV particles to 2.9% and leads to a reduction of the amount of virus in the environment by half. SPc can act as an adjuvant to spontaneously assemble with an immune inducer lentinan (LNT) through hydrogen bonding into nanoscale (142 nm diameter) LNT/SPc complex, which improves the physicochemical property of LNT for better wetting performance on leaves and cellular uptake, and further activates plant immune responses. Finally, the LNT/SPc complex displays preventive and curative effects on TMV disease, reducing TMV-GFP relative expression by 26% in the laboratory and achieving 82% control efficacy in the field We hope the strategy reported here would be useful for control of crop virus disease.

Conservation of molecular responses upon viral infection in the non-vascular plant Marchantia polymorpha.

After plants transitioned from water to land around 450 million years ago, they faced novel pathogenic microbes. Their colonization of diverse habitats was driven by anatomical innovations like roots, stomata, and vascular tissue, which became central to plant-microbe interactions. However, the impact of these innovations on plant immunity and pathogen infection strategies remains poorly understood. Here, we explore plant-virus interactions in the bryophyte Marchantia polymorpha to gain insights into the evolution of these relationships. Virome analysis reveals that Marchantia is predominantly associated with RNA viruses. Comparative studies with tobacco mosaic virus (TMV) show that Marchantia shares core defense responses with vascular plants but also exhibits unique features, such as a sustained wound response preventing viral spread. Additionally, general defense responses in Marchantia are equivalent to those restricted to vascular tissues in Nicotiana, suggesting that evolutionary acquisition of developmental innovations results in re-routing of defense responses in vascular plants.

Design, Synthesis, Anti-TMV Activity, and Structure-Activity Relationships of Seco-pregnane C21 Steroids and Their Derivatives.

seco-pregnane C21 steroids exhibit high antiviral activity against the tobacco mosaic virus (TMV). However, the structural modification of seco-pregnane C21 steroids and the structure-activity relationship (SAR) of the modified compounds remain unevaluated. Hence, the present study investigated how variations in the original skeletons of natural seco-pregnane C21 steroids affect their antiviral activity. A series of glaucogenin C and A derivatives were designed and synthesized for the first time, and their anti-TMV activity was evaluated. Bioassay results showed that most of the newly designed derivatives exhibited good to excellent antiviral activity; among these derivatives, 5g, 5j, and 5l with higher antiviral activity than that of ningnanmycin emerged as new antiviral candidates. Reverse transcription-polymerase chain reaction and Western blotting assay revealed reduced levels of TMV coat protein (TMV-CP) gene transcription and TMV-CP protein expression, which confirmed the antiviral activity of these derivatives. These compounds also downregulated the expression of NtHsp70-1 and NtHsp70-061. Computational simulations indicated that 5l displayed strong van der Waals energy and electrostatic with the TMV coat protein, affording a lower binding energy (ΔGbind = -56.2 kcal/mol) compared with Ribavirin (ΔGbind = -47.6 kcal/mol). The SAR of these compounds was also evaluated, which demonstrated for the first time that substitutions at C-3 and double bonds of C-5/C-6 and C-13/C-18 are crucial for maintaining high anti-TMV activity.

Synthesis, Anti-TMV Activities, and Action Mechanisms of a Novel Cytidine Peptide Compound.

Cytidine has a broad range of applications in the pharmaceutical field as an intermediate of antitumor or antiviral agent. Here, a series of new cytidine peptide compounds were synthesized using cytidine and Boc group-protected amino acids and analyzed for their antiviral activities against tobacco mosaic virus (TMV). Among these compounds, the structure of an effective antiviral cytidine peptide SN11 was characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometer. The compound SN11 has a molecular formula of C15H22N6O8 and is named 2-amino-N-(2- ((1- (3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl) -2-oxo-1,2-dihydropyrimidin-4-yl) amino) -2-oxyethyl) amino). The protection, inactivation, and curation activities of SN11 at a concentration of 500 µg/mL against TMV in Nicotiana glutinosa were 82.6%, 84.2%, and 72.8%, respectively. SN11 also effectively suppressed the systemic transportation of a recombinant TMV carrying GFP reporter gene (p35S-30B:GFP) in Nicotiana benthamiana by reducing viral accumulation to 71.3% in the upper uninoculated leaves and inhibited the systemic infection of TMV in Nicotiana tabacum plants. Furthermore, the results of RNA-seq showed that compound SN11 induced differential expression of genes involved in the biogenesis and function of ribosome, plant hormone signal transduction, plant pathogen interaction, and chromatin. These results validate the antiviral mechanisms of the cytidine peptide compound and provide a theoretical basis for their potential application in the management of plant virus diseases.

Biosynthesized silver nanoparticles mediated by Ammi visnaga extract enhanced systemic resistance and triggered multiple defense-related genes, including SbWRKY transcription factors, against tobacco mosaic virus infection.

BACKGROUND: Tobacco mosaic virus (TMV) is a highly infectious plant virus that affects a wide variety of plants and reduces crop yields around the world. Here, we assessed the effectiveness of using Ammi visnaga aqueous seed extract to synthesize silver nanoparticles (Ag-NPs) and their potential to combat TMV. Different techniques were used to characterize Ag-NPs, such as scanning and transmission electron microscopy (SEM, TEM), energy-dispersive X-ray spectroscopy (EDS), fourier transform infrared spectroscopy (FTIR), and dynamic light scattering (DLS). RESULTS: TEM demonstrated that the synthesized Ag-NPs had a spherical form with an average size of 23-30 nm and a zeta potential value of -15.9 mV, while FTIR revealed various functional groups involved in Ag-NP stability and capping. Interestingly, the Pre-treatment of tobacco plants (protective treatment) with Ag-NPs at 100-500 µg/mL significantly suppressed viral symptoms, while the Post-treatment (curative treatment) delayed their appearance. Furthermore, protective and curative treatments significantly increased chlorophyll a and b, total flavonoids, total soluble carbohydrates, and antioxidant enzymes activity (PPO, POX and CAT). Simultaneously, the application of Ag-NPs resulted in a decrease in levels of oxidative stress markers (H2O2 and MDA). The RT-qPCR results and volcano plot analysis showed that the Ag-NPs treatments trigger and regulate the transcription of ten defense-related genes (SbWRKY-1, SbWRKY-2, JERF-3, GST-1, POD, PR-1, PR-2, PR-12, PAL-1, and HQT-1). The heatmap revealed that GST-1, the primary gene involved in anthocyanidin production, was consistently the most expressed gene across all treatments throughout the study. Analysis of the gene co-expression network revealed that SbWRKY-19 was the most central gene among the studied genes, followed by PR-12 and PR-2. CONCLUSIONS: Overall, the reported antiviral properties (protective and/or curative) of biosynthesized Ag-NPs against TMV lead us to recommend using Ag-NPs as a simple, stable, and eco-friendly agent in developing pest management programs against plant viral infections.

Strigolactones Negatively Regulate Tobacco Mosaic Virus Resistance in Nicotiana benthamiana.

Strigolactones (SLs) are plant hormones that regulate diverse developmental processes and environmental responses in plants. It has been discovered that SLs play an important role in regulating plant immune resistance to pathogens but there are currently no reports on their role in the interaction between Nicotiana benthamiana and the tobacco mosaic virus (TMV). In this study, the exogenous application of SLs weakened the resistance of N. benthamiana to TMV, promoting TMV infection, whereas the exogenous application of Tis108, a SL inhibitor, resulted in the opposite effect. Virus-induced gene silencing (VIGS) inhibition of two key SL synthesis enzyme genes, NtCCD7 and NtCCD8, enhanced the resistance of N. benthamiana to TMV. Additionally, we conducted a screening of N. benthamiana related to TMV infection. TMV-infected plants treated with SLs were compared to the control by using RNA-seq. The KEGG enrichment analysis and weighted gene co-expression network analysis (WGCNA) of differentially expressed genes (DEGs) suggested that plant hormone signaling transduction may play a significant role in the SL-TMV-N. benthamiana interactions. This study reveals new functions of SLs in regulating plant immunity and provides a reference for controlling TMV diseases in production.

Transient Expression Vector Construction, Subcellular Localisation, and Evaluation of Antiviral Potential of Flagellin BP8-2.

This study used the DNA of Bacillus amyloliquefaciens Ba168 as a template to amplify the flagellin BP8-2 gene and ligate it into the fusion expression vector pCAMBIA1300-35S-EGFP after digestion for the construction of the expression vector pCAMBIA1300-EGFP-BP8-2. Next, using Nicotiana benthamiana as receptor material, transient expression was carried out under the mediation of Agrobacterium tumefaciens C58C1. Finally, the transient expression and subcellular localisation of flagellin BP8-2 protein were analysed using the imaging of co-transformed GFP under laser confocal microscopy. The results showed that flagellin BP8-2 was localised in the cell membrane and nucleus, and the RT-PCR results showed that the BP8-2 gene could be stably expressed in tobacco leaf cells. Furthermore, there was stronger antiviral activity against tobacco mosaic virus (TMV) infection in Nicotiana glutinosa than in BP8-2 and ningnanmycin, with an inhibitory effect of 75.91%, protective effect of 77.45%, and curative effect of 68.15%. TMV movement and coat protein expression were suppressed, and there was a high expression of PR-1a, PAL, and NPR1 in BP8-2-treated tobacco leaf. These results suggest that flagellin BP8-2 inhibits TMV by inducing resistance. Moreover, BP8-2 has low toxicity and is easily biodegradable and eco-friendly. These results further enrich our understanding of the antiviral mechanisms of proteins and provide alternatives for controlling viral diseases in agriculture.

Design, synthesis, and biological activities of arecoline derivatives containing 1,3,4-oxadiazole structure.

Pesticides play an important role in the development of agriculture, as they can prevent and control crop diseases and pests, improve crop yield and quality. However, the abuse and improper use of pesticides can lead to negative impacts such as environmental pollution and pest resistance issues. There is an urgent need to develop green, safe, and efficient pesticides. In this work, natural product arecoline was selected as parent structure, a series of arecoline derivatives were designed, synthesized, and systematically investigated antiviral activities against tobacco mosaic virus (TMV). These compounds were found to have good to excellent anti-TMV activities for the first time. The antiviral activities of 4a, 4 h, 4 l, 4p, 6a, 6c, and 6f are higher than that of ningnanmycin. Compounds 4 h (EC50 value 146 µg/mL) and 4p (EC50 value 161 µg/mL) with simple structures and excellent activities emerged as new antiviral candidates. We chose 4 h to further investigate the antiviral mechanism, which revealed that it can cause virus fragmentation by acting on the viral coat protein (CP). We further validated this result through molecular docking. These compounds also displayed broad-spectrum fungicidal activities against 8 plant pathogenic fungi. This work lays the theoretical foundation for the application of arecoline derivatives in the agricultural field.

Transcriptional responses and secondary metabolites variation of tomato plant in response to tobacco mosaic virus infestation.

The present study focused on the impact of infection with the tobacco mosaic virus (TMV). Specifically, changes in phytochemicals and gene activity related to pathogenesis-related and phenylpropanoid pathway genes in tomato plants (Solanum lycopersicum L.) during a period of 2-14 days post-inoculation (dpi). According to TEM investigation and coat protein sequence analysis, the purified TMV Egyptian AM isolate (PP133743) has a rod-shaped structure with a diameter of around 110 nm. The RT-qPCR analysis revealed that PR-1 showed an initial increase after TMV infection, as seen in the time-course analysis. In contrast, PR-2 was consistently elevated throughout the infection, suggesting a stronger reaction to the virus and suppressing PAL expression at 6 to 14 dpi. The expression levels of HQT and CHS transcripts exhibited alternating patterns of up-regulation and down-regulation at different time intervals. The HPLC and GC-MS analysis of control- and TMV-infected tomato extracts revealed that different phenolic, flavonoid, and fatty acid compounds were increased (such as naringenin, rutin, flavone, ferulic acid, and pyrogallol) or significantly decreased (such as salicylic acid and chlorogenic acid) after TMV infection. The ability of TMV to inhibit most polyphenolic compounds could potentially accelerate the viral life cycle. Consequently, focusing on enhancing the levels of such suppressed compounds may be critical for developing plant viral infection management strategies.

Structural Simplification of Podophyllotoxin: Discovery of γ-Butyrolactone Derivatives as Novel Antiviral Agents for Plant Protection.

Natural products are a valuable resource for the discovery of novel crop protection agents. A series of γ-butyrolactone derivatives, derived from the simplification of podophyllotoxin's structure, were synthesized and assessed for their efficacy against tobacco mosaic virus (TMV). Several derivatives exhibited notable antiviral properties, with compound 3g demonstrating the most potent in vivo anti-TMV activity. At 500 µg/mL, compound 3g achieved an inactivation effect of 87.8%, a protective effect of 71.7%, and a curative effect of 67.7%, surpassing the effectiveness of the commercial plant virucides ningnanmycin and ribavirin. Notably, the syn-diastereomer (syn-3g) exhibited superior antiviral activity compared to the anti-diastereomer (anti-3g). Mechanistic studies revealed that syn-3g could bind to the TMV coat protein and interfere with the self-assembly process of TMV particles. These findings indicate that compound 3g, with its simple chemical structure, could be a potential candidate for the development of novel antiviral agents for crop protection.

Engineered Resistance to Tobamoviruses.

Tobacco mosaic virus (TMV) was the first virus to be studied in detail and, for many years, TMV and other tobamoviruses, particularly tomato mosaic virus (ToMV) and tobamoviruses infecting pepper (Capsicum spp.), were serious crop pathogens. By the end of the twentieth and for the first decade of the twenty-first century, tobamoviruses were under some degree of control due to introgression of resistance genes into commercial tomato and pepper lines. However, tobamoviruses remained important models for molecular biology, biotechnology and bio-nanotechnology. Recently, tobamoviruses have again become serious crop pathogens due to the advent of tomato brown rugose fruit virus, which overcomes tomato resistance against TMV and ToMV, and the slow but apparently inexorable worldwide spread of cucumber green mottle mosaic virus, which threatens all cucurbit crops. This review discusses a range of mainly molecular biology-based approaches for protecting crops against tobamoviruses. These include cross-protection (using mild tobamovirus strains to 'immunize' plants against severe strains), expressing viral gene products in transgenic plants to inhibit the viral infection cycle, inducing RNA silencing against tobamoviruses by expressing virus-derived RNA sequences in planta or by direct application of double-stranded RNA molecules to non-engineered plants, gene editing of host susceptibility factors, and the transfer and optimization of natural resistance genes.

Magnetically Induced Thermal Effects on Tobacco Mosaic Virus-Based Nanocomposites for a Programmed Disassembly of Protein Cages.

Protein cages are promising tools for the controlled delivery of therapeutics and imaging agents when endowed with programmable disassembly strategies. Here, we produced hybrid nanocomposites made of tobacco mosaic virus (TMV) and magnetic iron oxide nanoparticles (IONPs), designed to disrupt the viral protein cages using magnetically induced release of heat. We studied the effects of this magnetic hyperthermia on the programmable viral protein capsid disassembly using (1) elongated nanocomposites of TMV coated heterogeneously with magnetic iron oxide nanoparticles (TMV@IONPs) and (2) spherical nanocomposites of polystyrene (PS) on which we deposited presynthesized IONPs and TMV via layer-by-layer self-assembly (PS@IONPs/TMV). Notably, we found that the extent of the disassembly of the protein cages is contingent upon the specific absorption rate (SAR) of the magnetic nanoparticles, that is, the heating efficiency, and the relative position of the protein cage within the nanocomposite concerning the heating sources. This implies that the spatial arrangement of components within the hybrid nanostructure has a significant impact on the disassembly process. Understanding and optimizing this relationship will contribute to the critical spatiotemporal control for targeted drug and gene delivery using protein cages.

Ferulic Acid Dimers as Potential Antiviral Agents by Inhibiting TMV Self-Assembly.

A series of ferulic acid dimers were designed, synthesized, and evaluated for anti-TMV activity. Biological assays demonstrated that compounds A6, E3, and E5 displayed excellent inactivating against tobacco mosaic virus (TMV) with EC50 values of 62.8, 94.4, and 85.2 µg mL-1, respectively, which were superior to that of ningnanmycin (108.1 µg mL-1). Microscale thermophoresis indicated that compounds A6, E3, and E5 showed strong binding capacity to TMV coat protein with binding affinity values of 1.862, 3.439, and 2.926 µM, respectively. Molecular docking and molecular dynamics simulation revealed that compound A6 could firmly bind to the TMV coat protein through hydrogen and hydrophobic bonds. Transmission electron microscopy and self-assembly experiments indicated that compound A6 obviously destroyed the integrity of the TMV particles and blocked the virus from infecting the host. This study revealed that A6 can be used as a promising leading structure for the development of antiviral agents by inhibiting TMV self-assembly.

LW-1 induced resistance to TMV in tobacco was mediated by nitric oxide and salicylic acid pathway.

The objective of this study was to investigate the mechanism underlying LW-1-induced resistance to TMV in wild-type and salicylic acid (SA)-deficient NahG transgenic tobacco plants. Our findings revealed that LW-1 failed to induce antivirus infection activity and increase SA content in NahG tobacco, indicating the crucial role of SA in these processes. Meanwhile, LW-1 triggered defense-related early-signaling nitric oxide (NO) generation, as evidenced by the emergence of NO fluorescence in both types of tobacco upon treatment with LW-1, however, NO fluorescence was stronger in NahG compared to wild-type tobacco. Notably, both of them were eliminated by the NO scavenger cPTIO, which also reversed LW-1-induced antivirus activity and the increase of SA content, suggesting that NO participates in LW-1-induced resistance to TMV, and may act upstream of the SA pathway. Defense-related enzymes and genes were detected in tobacco with or without TMV inoculation, and the results showed that LW-1 regulated both enzyme activity (ß-1,3-glucanase [GLU], catalase [CAT] and phenylalanine ammonia-lyase [PAL]) and gene expression (PR1, PAL, WYKY4) through NO signaling in both SA-dependent and SA-independent pathways.

A highly sensitive fluorescence sensor for tobacco mosaic virus RNA based on DSN cycle and ARGET ATRP double signal amplification.

Early and sensitive detection of tobacco mosaic virus (TMV) is of great significance for improving crop yield and protecting germplasm resources. Herein, we constructed a novel fluorescence sensor to detect TMV RNA (tRNA) through double strand specific nuclease (DSN) cycle and activator regenerative electron transfer atom transfer radical polymerization (ARGET ATRP) dual signal amplification strategy. The hairpin DNA complementarily paired with tRNA was used as a recognition unit to specifically capture tRNA. By the double-stranded DNA hydrolyzed with DSN, tRNA is released to open more hairpin DNA, and more complementary DNA (cDNA) is bound to the surface of the magnetic beads (MBs) to achieve the first amplification. After binding with the initiator, the cDNA employed ARGET ATRP to attach more fluorescent signal molecules to the surface of MBs, thus achieving the second signal amplification. Under the optimal experimental conditions, the logarithm of fluorescence intensity versus tRNA concentration showed a good linear relationship in the range of 0.01-100 pM, with a detection limit of 1.03 fM. The limit of detection (LOD) was calculated according to LOD = 3 N/S. Besides, the sensor showed good reproducibility and stability, which present provided new method for early and highly sensitive detection for plant viruses.

CDC48 regulates immunity pathway in tobacco plants.

The CDC48 protein, highly conserved in the living kingdom, is a player of the ubiquitin proteasome system and contributes to various cellular processes. In plants, CDC48 is involved in cell division, plant growth and, as recently highlighted in several reports, in plant immunity. In the present study, to further extend our knowledge about CDC48 functions in plants, we analysed the incidence of its overexpression on tobacco development and immune responses. CDC48 overexpression disrupted plant development and morphology, induced changes in plastoglobule appearance and exacerbated ROS production. In addition, levels of salicylic acid (SA) and glycosylated SA were higher in transgenic plants, both in the basal state and in response to cryptogein, a protein produced by the oomycete Phytophthora cryptogea triggering defence responses. The expression of defence genes, notably those coding for some pathogenesis-related (PR) proteins, was also exacerbated in the basal state in transgenic plant lines. Finally, tobacco plants overexpressing CDC48 did not develop necrosis in response to tobacco mosaic virus (TMV) infection, suggesting a role for CDC48 in virus resistance.

In Vivo Delivery of Spherical and Cylindrical In Vitro Reconstituted Virus-like Particles Containing the Same Self-Amplifying mRNA.

The dramatic effectiveness of recent mRNA (mRNA)-based COVID vaccines delivered in lipid nanoparticles has highlighted the promise of mRNA therapeutics in general. In this report, we extend our earlier work on self-amplifying mRNAs delivered in spherical in vitro reconstituted virus-like particles (VLPs), and on drug delivery using cylindrical virus particles. In particular, we carry out separate in vitro assemblies of a self-amplifying mRNA gene in two different virus-like particles: one spherical, formed with the capsid protein of cowpea chlorotic mottle virus (CCMV), and the other cylindrical, formed from the capsid protein of tobacco mosaic virus (TMV). The mRNA gene is rendered self-amplifying by genetically fusing it to the RNA-dependent RNA polymerase (RdRp) of Nodamura virus, and the relative efficacies of cell uptake and downstream protein expression resulting from their CCMV- and TMV-packaged forms are compared directly. This comparison is carried out by their transfections into cells in culture: expressions of two self-amplifying genes, enhanced yellow fluorescent protein (EYFP) and Renilla luciferase (Luc), packaged alternately in CCMV and TMV VLPs, are quantified by fluorescence and chemiluminescence levels, respectively, and relative numbers of the delivered mRNAs are measured by quantitative real-time PCR. The cellular uptake of both forms of these VLPs is further confirmed by confocal microscopy of transfected cells. Finally, VLP-mediated delivery of the self-amplifying-mRNA in mice following footpad injection is shown by in vivo fluorescence imaging to result in robust expression of EYFP in the draining lymph nodes, suggesting the potential of these plant virus-like particles as a promising mRNA gene and vaccine delivery modality. These results establish that both CCMV and TMV VLPs can deliver their in vitro packaged mRNA genes to immune cells and that their self-amplifying forms significantly enhance in situ expression. Choice of one VLP (CCMV or TMV) over the other will depend on which geometry of nucleocapsid is self-assembled more efficiently for a given length and sequence of RNA, and suggests that these plant VLP gene delivery systems will prove useful in a wide variety of medical applications, both preventive and therapeutic.

Repairing Host Damage Caused by Tobacco Mosaic Virus Stress: Design, Synthesis, and Mechanism Study of Novel Oxadiazole and Arylhydrazone Derivatives.

Tobacco mosaic virus (TMV), as one of the most traditional and extensive biological stresses, poses a serious threat to plant growth and development. In this work, a series of 1-phenyl/tertbutyl-5-amino-4-pyrazole oxadiazole and arylhydrazone derivatives was synthesized. Bioassay evaluation demonstrated that the title compounds (P1-P18) without a "thioether bond" lost their anti-TMV activity, while some of the ring-opening arylhydrazone compounds exhibited superior in vivo activity against TMV in tobacco. The EC50 value of title compound T8 for curative activity was 139 µg/mL, similar to that of ningnanmycin (NNM) (EC50 = 152 µg/mL). Safety analysis revealed that compound T8 had no adverse effects on plant growth or seed germination at a concentration of 250 µg/mL. Morphological observation revealed that compound T8 could restore the leaf tissue of a TMV-stressed host and the leaf stomatal aperture to normal. A mechanism study further revealed that compound T8 not only restored the photosynthetic and growth ability of the damaged host to normal levels but also enhanced catalase (CAT) activity and reduced the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in the damaged host, thereby reducing the oxidation damage to the host. TMV-green fluorescent protein (GFP) experiments further demonstrated that compound T8 not only slowed the transmission speed of TMV in the host but also inhibited its reproduction. All of the experimental results demonstrated that compound T8 could reduce the oxidative damage caused by TMV stress and regulate the photosynthetic ability of the host, achieving the ability to repair damage, to make the plant grow normally.