Antiviral activity of glucosylceramides isolated from Fusarium oxysporum against Tobacco mosaic virus infection.

Natural elicitors derived from pathogenic microorganisms represent an ecologic strategy to achieve resistance in plants against diseases. Glucosylceramides (GlcCer) are classified as neutral glycosphingolipids. GlcCer were isolated and purified from Fusarium oxysporum mycelium. F. oxysporum is a plant pathogenic fungus, abundant in soil and causing severe losses in economically important crops such as corn, tobacco, banana, cotton and passion fruit. In this study we evaluate the capacity of GlcCer in inducing resistance in N. tabacum cv Xanthi plants against Tobacco mosaic virus (TMV). Spraying tobacco plants with GlcCer before virus infection reduced the incidence of necrotic lesions caused by TMV. In addition, plants already infected with the virus showed a reduction in hypersensitive response (HR) lesions after GlcCer treatment, suggesting an antiviral effect of GlcCer. Our investigations showed that GlcCer stimulates the early accumulation of H2O2 and superoxide radicals. In addition, the expression of PR-1 (pathogenesis-related 1, with suggested antifungal action), PR-2 (ß-1,3-glucanase), PR-3 (Chitinase), PR-5 (Osmotin), PAL (Phenylalanine ammonia-lyase), LOX (Lipoxygenase) and POX (Peroxidase) genes was highly induced after treatment of tobacco plants with GlcCer and induction levels remained high throughout a period of 6 to 120 hours. Our experiments demonstrate that GlcCer induces resistance in tobacco plants against infection by TMV.

Influence of high hydrostatic pressure on epitope mapping of tobacco mosaic virus coat protein.

In this study, we investigated the effect of high hydrostatic pressure (HHP) on tobacco mosaic virus (TMV), a model virus in immunology and one of the most studied viruses to date. Exposure to HHP significantly altered the recognition epitopes when compared to sera from mice immunized with native virus. These alterations were studied further by combining HHP with urea or low temperature and then inoculating the altered virions into Balb-C mice. The antibody titers and cross-reactivity of the resulting sera were determined by ELISA. The antigenicity of the viral particles was maintained, as assessed by using polyclonal antibodies against native virus. The antigenicity of canonical epitopes was maintained, although binding intensities varied among the treatments. The patterns of recognition determined by epitope mapping were cross checked with the prediction algorithms for the TMVcp amino acid sequence to infer which alterations had occurred. These findings suggest that different cleavage sites were exposed after the treatments and this was confirmed by epitope mapping using sera from mice immunized with virus previously exposed to HHP.

Lichen depsides and depsidones reduce symptoms of diseases caused by tobacco mosaic virus (TMV) in tobacco leaves.

Two depsides and five depsidones, isolated from lichens, were tested to determine their in vivo protective effects on tobacco leaves challenged with tobacco mosaic virus (TMV). The results indicate that most of these compounds are able to reduce either the number and/or the size of necrotic lesions following virus infection. Pannarin, 1'-chloro-pannarin and stictic acid provided the more effective protective results, reducing by at least 45% the number and size of lesions. Real Time PCR assays were used to explore the target of action against TMV by examining the response behavior of genes involved in the plant defense mechanism. The application of the lichen substances did not lead to changes in the transcriptional levels of pathogen-related (PR1a), allene oxide synthase 2 (AOS2) or oxophytodienoate reductase (OPR3) genes. Thus, the protection observed in the tobacco leaves treated with the lichen compounds may be mediated by a mechanism which does not involved the SA- or JA-mediated defensive plant response. A possible structure-activity relationship is presented.

Different urea stoichiometries between the dissociation and denaturation of tobacco mosaic virus as probed by hydrostatic pressure.

Viruses are very efficient self-assembly structures, but little is understood about the thermodynamics governing their directed assembly. At higher levels of pressure or when pressure is combined with urea, denaturation occurs. For a better understanding of such processes, we investigated the apparent thermodynamic parameters of dissociation and denaturation by assuming a steady-state condition. These processes can be measured considering the decrease of light scattering of a viral solution due to the dissociation process, and the red shift of the fluorescence emission spectra, that occurs with the denaturation process. We determined the apparent urea stoichiometry considering the equilibrium reaction of TMV dissociation and subunit denaturation, which furnished, respectively, 1.53 and 11.1 mol of urea/mol of TMV subunit. The denaturation and dissociation conditions were arrived in a near reversible pathway, allowing the determination of thermodynamic parameters. Gel filtration HPLC, electron microscopy and circular dichroism confirmed the dissociation and denaturation processes. Based on spectroscopic results from earlier papers, the calculation of the apparent urea stoichiometry of dissociation and denaturation of several other viruses resulted in similar values, suggesting a similar virus-urea interaction among these systems.

Detection of antiviral and antitumoral fractions of Chenopodium amaranticolor leaf extract.

Based on the fact that Chenopodium amaranticolor extracts showed inhibitory activity against tobacco mosaic virus (TMV) and Ehrlich tumour (EA), tests were carried out to investigate whether the antiviral and antitumoral activity were caused by the same compounds. When the extract was purified by CM Sephadex C-25 column, after precipitation with 90% ammonium sulphate, twenty active fractions against TMV and two pools of fractions active against EA were obtained. Only one fraction with high absorbance values at 260 and 280 nm was able to inhibit both TMV and EA. When the extract was purified by Bio Gel P-60 column two active fractions against TMV and EA were obtained, suggesting that they were contained in the 0.01 M fraction of the CM Sephadex column. It is suggested that C. amaranticolor leaf extract contained at least two protein-like substances manifesting antiviral and antitumoral activity.

Antiviral activity of S-adenosylhomocysteine hydrolase inhibitors against plant viruses.

Three SAH hydrolase inhibitors, (RS)-3-adenin-9-yl-2-hydroxypropanoic acid (isobutyl ester) [(RS)-AHPA]; (RS)-9-(2,3-dihydroxypropyl)adenine [(RS)-DHPA] and the carbocyclic analog of 3-deazaadenosine (C-c3Ado) were evaluated for their inhibitory activity against tobacco mosaic virus (TMV) and potato virus X (PVX). Using the local lesion assay and ELISA, we demonstrated that all three compounds inhibit the replication of TMV and PVX. Whereas the three compounds proved about equally active against PVX, (RS)-AHPA was the most effective against TMV. (RS)-AHPA and C-c3Ado induced chlorosis in Nicotiana tabacum leaf discs. They also caused a substantial reduction in the growth of the main root of Phaseolus vulgaris. (RS)-DHPA was less phytotoxic than its two congeners.