Anti-Tobacco Mosaic Virus Quassinoids from Ailanthus altissima (Mill.) Swingle.

Quassinoids are bitter constituents characteristic of the family Simaroubaceae. A total of 18 C20 quassinoids, including nine new quassinoid glycosides, named chuglycosides A-I (1-6 and 8-10), were identified from the samara of Ailanthus altissima (Mill.) Swingle. All of the quassinoids showed potent anti-tobacco mosaic virus (TMV) activity. A preliminary structure-anti-TMV activity relationship of quassinoids was discussed. The effects of three quassinoids, including chaparrinone (12), glaucarubinone (15), and ailanthone (16), on the accumulation of TMV coat protein (CP) were studied by western blot analysis. Ailanthone (16) was further investigated for its influence on TMV spread in the Nicotiana benthamiana plant.

RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases.

Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7.

Design, synthesis, anti-TMV, fungicidal, and insecticidal activity evaluation of 1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid derivatives based on virus inhibitors of plant sources.

By drawing the creation ideas of botanical pesticides, a series of tetrahydro-ß-carboline-3-carboxylic acid derivatives were designed and synthesized, and first evaluated for their anti-TMV, fungicidal and insecticidal activities. Most of these derivatives exhibited good antiviral activity against TMV both in vitro and in vivo. Especially, the activities of compounds 8 and 15 in vivo were higher than that of ribavirin. The compound 8 exhibited more than 70% fungicidal activities against Cercospora arachidicola Hori, Alternaria solani, Bipolaris maydis, and Rhizoctonia solani at 50mg/kg, compounds 16 and 20 exhibited more than 60% insecticidal activities against Mythimna separate and Ostrinia nubilalis.

A virus inhibitory protein isolated from Cyamopsis tetragonoloba (L.) Taub. upon induction of systemic antiviral resistance shares partial amino acid sequence homology with a lectin.

KEY MESSAGE: Two virus inhibitory proteins were purified from Cyamopsis tetragonoloba , induced to resist virus infections by CIP-29, a systemic resistance inducing protein from Clerodendrum inerme, and characterized. One of them shared homology with a lectin. CIP-29, a known 29 kDa systemic antiviral resistance inducing protein isolated from Clerodendrum inerme, has been used to induce systemic resistance in Cyamopsis tetragonoloba against Sunn-hemp rosette virus (SRV). Paper reports the detection of virus inhibitory activity in induced-resistant leaf sap of C. tetragonoloba, and the purification of two virus inhibitory agents (VIAs) thereof. VIA activity was recorded as a reduction in lesion number of SRV, Tobacco mosaic virus, and Papaya ringspot virus, when they were incubated separately with resistant sap and inoculated onto susceptible C. tetragonoloba, Nicotiana tabacum cv. Xanthi-nc, and Chenopodium quinoa, respectively. The two VIAs were isolated from resistant C. tetragonoloba plant leaves using combinations of column chromatography. Both were basic proteins, and since their M r was 32 and 62 kDa, these VIAs were called CT-VIA-32 and CT-VIA-62, respectively, on the basis of their molecular mass and the host. CT-VIA-62 displayed better activity, and was thus studied further. It tested positive for a glycoprotein, and was serologically detected only in leaf tissue post-induction. Tryptic peptides generated in-gel, post SDS-PAGE of CT-VIA-62, were sequenced through LC/MS/MS. All CT-VIA-62 peptides were found to share homologies with proteins from Medicago truncatula that possess a mannose-binding lectin domain.

A simple SPR-based method for the quantification of the effect of potential virus inhibitors.

Here, we describe a highly sensitive method that allows for the correct quantification of inhibition effect with a higher degree of accuracy directly at the molecular level. The protocol involves two stages, namely serological virus titration in comparison with the same procedure for virus-effector mixture. Owing to the robustness of the analysis this assay can be performed on crude cellular and plant extracts, and therefore it may be suitable for the routine analysis of clinical samples, or in the field. The efficiency of the approach to the quantification of the inhibition effect of polysaccharide glucuronoxylomannan (GXM) on the infection efficiency of the tobacco mosaic virus (TMV) was investigated using advanced serological approaches based on label-free surface plasmon resonance technique. It was shown that GXM drastically decreases the efficiency of TMV infection by blocking up to 70% of the virus shell. The obtained results are in conformity with the method of indicator plant infection.

Dammarenediol-II production confers TMV tolerance in transgenic tobacco expressing Panax ginseng dammarenediol-II synthase.

Panax ginseng is one of the famous medicinal plants. Ginsenosides, a class of tetracyclic triterpene saponins, are mainly responsible for its pharmacological activity. Most ginsenosides are composed of dammarenediol-II aglycone with various sugar moieties. Dammarenediol-II synthase is the first enzyme in the biosynthesis of ginsenosides. Here, we report that transgenic tobacco expressing the P. ginseng dammarenediol-II synthase gene (PgDDS) produced dammarenediol-II, and conferred resistance to Tobacco mosaic virus (TMV). Upon infection with TMV, lesions developed more rapidly in transgenic tobacco plants, and their size was smaller than those of wild-type plants. Transgenic tobacco plants showed a low level of both the viral titer and mRNA accumulation of TMV coat protein (CP) compared with the wild type. The production of dammarenediol-II in transgenic tobacco stimulated the expression of tobacco pathogenesis-related genes (PR1 and PR2) under both virus-untreated and -treated conditions. When the leaves of wild-type plants were inoculated with a mixture of TMV and dammarenediol-II, the leaves exhibited a reduced viral concentration and TMV-CP expression than those receiving TMV treatment alone. When the leaves of P. ginseng were infected with TMV, transcription of PgDDS was significantly increased. Transgenic P. ginseng plants harboring a ß-glucuronidase (GUS) gene driven by the PgDDS promoter were constructed. The GUS expression was activated when the transgenic ginseng plants were treated with TMV. These results indicate that the medicinally important dammarenediol-II can be ectopically produced in tobacco, and the production of dammarenediol-II in tobacco plants allows them to adopt a viral defense system.

Silicon delays Tobacco ringspot virus systemic symptoms in Nicotiana tabacum.

Soluble silicon (Si) provides protection to plants against a variety of abiotic and biotic stress. However, the effects of Si on viral infections are largely unknown. To investigate the role of Si in viral infections, hydroponic studies were conducted in Nicotiana tabacum with two pathogens: Tobacco ringspot virus (TRSV) and Tobacco mosaic virus (TMV). Plants grown in elevated Si showed a delay in TRSV systemic symptom formation and a reduction in symptomatic leaf area, compared to the non-supplemented controls. TRSV-infected plants showed significantly higher levels of foliar Si compared to mock-inoculated plants. However, the Si effect appeared to be virus-specific, since the element did not alter TMV symptoms nor did infection by this virus alter foliar Si levels. Hence, increased foliar Si levels appear to correlate with Si-modulated protection against viral infection. This is all the more intriguing since N. tabacum is classified as a low Si accumulator.

Tobacco mosaic virus (TMV) inhibitors from Picrasma quassioides Benn.

To investigate natural inhibitors against tobacco mosaic virus (TMV) from plants, 10 known beta-carboline alkaloids and one quassinoid have been isolated from MeOH extract of the wood of Picrasma quassioides Benn. These compounds were screened for their inhibitory activities against tobacco mosaic virus (TMV). The activity of each compound against TMV infection and replication was tested using a half-leaf assay method, a leaf-disk method, and Western blotting analyses. All of the beta-carboline alkaloids showed moderate anti-TMV activities and exhibited synergistic effects when combined with the quassinoid nigakilactone B (11). To our knowledge, this is the first report on anti-TMV activity of beta-carbolines and their synergistic effects against TMV when combined with a quassinoid.

Isolation and characterization of ZH14 with antiviral activity against Tobacco mosaic virus.

A large number of bacteria were isolated from plant samples and screened for antiviral activity against the Tobacco mosaic virus (TMV). The bacterium ZH14, which was isolated from Chinese Anxi oolong tea, secreted the antiviral substances, having 94.2% virus inhibition when the bacterial culture filtrate and TMV extract were mixed at a ratio of 1:1. The ZH14 strain is a gram-positive, spore-forming rod and has the ability to degrade ribonucleic acid. Based on its effectiveness on virus inhibition, ZH14 was selected for characterization and was identified as a strain of the Bacillus cereus group based on phenotypic tests and comparative analysis of its 16S rDNA sequence. At the same time, we determined the antiviral product of ZH14 as an extracellular protein with high molecular mass, having an optimum temperature of 15-60 degrees C and an optimum pH of 6-10. Hence, the ZH14 strain and its culture filtrate have potential application in controlling plant diseases caused by TMV.

Functional study of hot pepper 26S proteasome subunit RPN7 induced by Tobacco mosaic virus from nuclear proteome analysis.

Two-dimensional gel electrophoresis (2-DE) was applied for the screening of Tobacco mosaic virus (TMV)-induced hot pepper (Capsicum annuum cv. Bugang) nuclear proteins. From differentially expressed protein spots, we acquired the matched peptide mass fingerprint (PMF) data, analyzed by MALDI-TOF MS, from the non-redundant hot pepper EST protein FASTA database using the VEMS 2.0 software. Among six identified nuclear proteins, the hot pepper 26S proteasome subunit RPN7 (CaRPN7) was subjected to further study. The level of CaRPN7 mRNA was specifically increased during incompatible TMV-P(0) interaction, but not during compatible TMV-P(1.2) interaction. When CaRPN7::GFP fusion protein was targeted in onion cells, the nuclei had been broken into pieces. In the hot pepper leaves, cell death was exacerbated and genomic DNA laddering was induced by Agrobacterium-mediated transient overexpression of CaPRN7. Thus, this report presents that the TMV-induced CaRPN7 may be involved in programmed cell death (PCD) in the hot pepper plant.

Thermal and chlorophyll-fluorescence imaging distinguish plant-pathogen interactions at an early stage.

Different biotic stresses yield specific symptoms, owing to their distinct influence on a plant's physiological status. To monitor early changes in a plant's physiological status upon pathogen attack, chlorophyll fluorescence imaging (Chl-FI) and thermography, which respectively visualize photosynthetic efficiency and transpiration, were carried out in parallel for two fundamentally different plant-pathogen interactions. These non-destructive imaging techniques were able to visualize infections at an early stage, before damage appeared. Under growth-room conditions, a robotized set-up captured time series of visual, thermal and chlorophyll fluorescence images from infected regions on attached leaves. As a first symptom of the plant-virus interaction between resistant tobacco and tobacco mosaic virus (TMV), thermal imaging detected a local rise in temperature while Chl-FI monitored a co-localized increase in fluorescence intensity. Chl-FI also revealed pre-symptomatic high-intensity spots for the plant-fungus system sugar beet-Cercospora beticola. Concomitantly, spots of lower temperature were monitored with thermography, in marked contrast with our observations on TMV-infection in tobacco. Knowledge of disease signatures for different plant-pathogen interactions could allow early identification of emerging biotic stresses in crops, facilitating the containment of disease outbreaks. Presymptomatic monitoring clearly opens perspectives for quantitative screening for disease resistance, either on excised leaf pieces or attached leaves.

Expression of the B subunit of the heat-labile enterotoxin of Escherichia coli in tobacco mosaic virus-infected Nicotiana benthamiana plants and its characterization as mucosal immunogen and adjuvant.

We have produced biologically active recombinant (r) LTB, the nontoxic B subunit of heat-labile toxin (LT) of Escherichia coli in tobacco mosaic virus (TMV)-infected Nicotiana benthamiana plants. We amplified the LTB encoding sequence with its leader and introduced a hexahistidyl tag and an endoplasmic reticulum retention signal. The resulting product was ligated into a TMV-based plant viral expression vector that was used for the generation of recombinant viral RNA. Eighty-nine percent of N. benthamiana plants inoculated with the recombinant viral RNA were systemically infected as determined by anti-TMV enzyme-linked immunosorbent assay (ELISA) experiments. The rLTB monomer was identified by LT-specific as well as by histidyl-tag-specific immunoblots. rLTB from plant extracts of TMV-infected N. benthamiana leaves was purified to give 75 microg rLTB pentamers per gram fresh plant material and was capable of binding G(M)1 ganglioside. The immunogenicity of the plant-produced rLTB was tested in mice and showed that intranasal application of rLTB (15 microg per mouse) induced LTB-specific IgG1 antibodies. To prove its adjuvanticity, rLTB was intranasally co-administered with the Hevea latex allergen Hev b 3, leading to allergen-specific IgG1 and IgG2a antibody production. The fact that intranasal application of rLTB and Hev b 3 prior to systemic challenge with the allergen enhanced the Th2 responses at the humoral and cellular level indicated that rLTB promoted immune responses that were naturally induced by the antigen/allergen. In conclusion, these results indicate that the plant viral expression system is suitable for the rapid large-scale production of biologically active LTB with strong mucosal adjuvant capacity.

Superoxide production by plant homologues of the gp91(phox) NADPH oxidase. Modulation of activity by calcium and by tobacco mosaic virus infection.

Genes encoding homologs of the gp91(phox) subunit of the plasma membrane NADPH oxidase complex have been identified in plants and are hypothesized to be a source of reactive oxygen species during defense responses. However, the direct involvement of the gene products in superoxide (O(2)(-)) production has yet to be shown. A novel activity gel assay based on protein fractionation in native or sodium dodecyl sulfate (SDS)-denaturing polyacrylamide gels was developed. In native polyacrylamide gel electrophoresis, one or two major O(2)(-)-producing formazan bands were detected in tomato (Lycopersicum esculentum Mill. cv Moneymaker) and tobacco (Nicotiana tabacum var. Samsun, NN) plasma membranes, respectively. Denaturing fractionation of tomato and tobacco plasma membrane in SDS-polyacrylamide gel electrophoresis, followed by regeneration of the in-gel activity, revealed NADPH-dependent O(2)(-)-producing formazan bands of 106-, 103-, and 80- to 75-kD molecular masses. The SDS and native activity bands were dependent on NADPH and completely inhibited by diphenylene iodonium or CuZn- O(2)(-) dismutase, indicating that the formazan precipitates were due to reduction by O(2)(-) radicals catalyzed by an NADPH-dependent flavin containing enzyme. The source of the plasma membrane activity bands was confirmed by their cross-reaction with antibody prepared from the C terminus of the tomato gp91(phox) homolog. Membrane extracts as well as the in-gel NADPH oxidase activities were stimulated in the presence of Ca(2+). In addition, the relative activity of the gp91(phox) homolog was enhanced in the plasma membrane of tobacco mosaic virus-infected leaves. Thus, in contrast to the mammalian gp91(phox), the plant homolog can produce O(2)(-) in the absence of additional cytosolic components and is stimulated directly by Ca(2+).

Virus-induced permeability transition in mitochondria.

Isolated rat liver mitochondria undergo permeability transition after supplementation with a suspension of tobacco mosaic virus. Four mitochondrial parameters proved the opening of the permeability transition pore in the inner mitochondrial membrane: increased oxygen consumption, collapse of the membrane potential, release of calcium ions from mitochondria, and high amplitude mitochondrial swelling. All virus-induced changes in mitochondria were prevented by cyclosporin A. These effects were not observed if the virus was treated with EGTA or disrupted by heating. Protein component of the virus particle in the form of 20S aggregate A-protein, or helical polymer, as well as supernatant of the heat-disrupted virus sample, had no effect on mitochondrial functioning. Electron microscopy revealed the direct interaction of the virus particles with isolated mitochondria. The possible role of the mitochondrial permeability transition pore in virus-induced apoptosis is discussed.

[Determination of tobacco mosaic viruses and tar carcinogens using electromagnetic resonance in smokers]. / Sledování viru tabákové mozaiky a dehtových kancerogenu u kuráku elektromagnetickou rezonancí.

Distance monitoring of viral diseases in the zone of MHz frequencies makes rapid non-contact investigation of the presence of tobacco mosaic viruses (VTM) in cigarettes and secretions of smokers possible by using electromagnetic resonance. The incidence of VTM in common cigarettes was assessed as well as in several smokers, in dermatitis, arthralgias and in tumours (of the large intestiner, in mastopathy). Also in neuritis of the optic nerve, in suspected sclerosis multiplex of smokers. After sanation of VTM by resonance therapy with Sanator (Bradna AO 272,361) VTM disappeared as well as symptoms of this viral mosaic disease. Electromagnetic resonance with tar made it possible to make this investigation in cigarettes filters after smoking as well as in the secretions of smokers. The authors proved the hastening effect of cigarette smoke with tar on the growth of VTM on cultivation as well as an increase of the bioelectric activity of tumours. It proved possible to abolish the electromagnetic distance action of tar by interaction with pyridsoxine similarly as in foods containing tar (smoked meat, fish, frankfurters, black coffee, cocoa). Filters with pyridoxine proved useful. VTM monitoring was quick, in tumours it was possible to follow the accompanying viral agent as well as the action of cancerogens on their bioelectromagnetic activity. It also made it possible to follow up disintegrating action of MHz resonance frequencies of the Sanator.

Molecular cloning of a metallothionein-like gene from Nicotiana glutinosa L. and its induction by wounding and tobacco mosaic virus infection.

The cloning and characterization of genes expressed in plant disease resistance could be an initial step toward understanding the molecular mechanisms of disease resistance. A metallothionein-like gene that is inducible by tobacco mosaic virus and by wounding was cloned in the process of subtractive cloning of disease resistance-response genes in Nicotiana glutinosa. One 530-bp cDNA clone (KC9-10) containing an open reading frame of 81 amino acids was characterized. Genomic Southern blot hybridization with the cDNA probe revealed that tobacco metallothionein-like genes are present in few or in one copy per diploid genome. Northern blot hybridization detected strong induction of a 0.5-kb mRNA by wounding and tobacco mosaic virus infection, but only mild induction was detected when copper was tested as an inducer. Methyl jasmonate, salicylic acid, and ethylene were also tested as possible inducers of this gene, but they had no effect on its expression. The possible role of this gene in wounded and pathogen-stressed plants is discussed.

The coat protein of potato virus X is a strain-specific elicitor of Rx1-mediated virus resistance in potato.

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a nonvirion form of the coat protein.

On the inhibition of plant virus multiplication by ribavirin.

The inhibition of the replication of potato virus X (PVX), belladonna mottle virus, tobacco mosaic virus, potato virus Y (PVY), and tobacco necrosis virus by ribavirin and pyrazofurin is described with emphasis on the inhibition of PVX by ribavirin. Ribavirin inhibits an early step of PVX replication. The inhibition is reversed to different degrees by all ribo- and deoxyribonucleosides, most strongly by thymidine. In tobacco leaves, nucleosides compete with ribavirin for phosphorylation to monophosphate by a nucleoside phosphotransferase. However, the final and main phosphorylation product of ribavirin is triphosphate. It is suggested that ribavirin triphosphate is the antiviral form and that it acts by inhibiting the capping of viral RNAs. This mode of action cannot be applied to the inhibition of PVY, the RNA of which is probably covalently linked to a protein at the 5'-terminus.

The effects of legume seed extracts on plant virus infection.

Extracts from the seeds of 15 species of legume inhibited the infection of plants by viruses. Extracts could be divided into those with marked inhibitory activity reducible on heating and those with less marked inhibitory activity which increased on heating. Evidence is given is given to suggest that seed extracts contain both virus inhibitors and augmenters and that the inhibitors are high molecular weight proteins possibly related to lectins.