Effect of yeast CTA1 gene expression on response of tobacco plants to tobacco mosaic virus infection

The response of tobacco (Nicotiana tabacum L. cv Xanthi-nc) plants with elevated catalase activity was studied after infection by tobacco mosaic virus (TMV). These plants contain the yeast (Saccharomyces cerevisiae) peroxisomal catalase gene CTA1 under the control of the cauliflower mosaic virus 35S promoter. The transgenic lines exhibited 2- to 4-fold higher total in vitro catalase activity than untransformed control plants under normal growth conditions. Cellular localization of the CTA1 protein was established using immunocytochemical analysis. Gold particles were detected mainly inside peroxisomes, whereas no significant labeling was detected in other cellular compartments or in the intercellular space. The physiological state of the transgenic plants was evaluated in respect to growth rate, general appearance, carbohydrate content, and dry weight. No significant differences were recorded in comparison with non-transgenic tobacco plants. The 3,3'-diaminobenzidine-stain method was applied to visualize hydrogen peroxide (H(2)O(2)) in the TMV infected tissue. Presence of H(2)O(2) could be detected around necrotic lesions caused by TMV infection in non-transgenic plants but to a much lesser extent in the CTA1 transgenic plants. In addition, the size of necrotic lesions was significantly bigger in the infected leaves of the transgenic plants. Changes in the distribution of H(2)O(2) and in lesion formation were not reflected by changes in salicylic acid production. In contrast to the local response, the systemic response in upper noninoculated leaves of both CTA1 transgenic and control plants was similar. This suggests that increased cellular catalase activity influences local but not systemic response to TMV infection.     

Phenylalanine ammonia-lyase levels in protoplasts isolated from hypersensitive tobacco pre-infected with tobacco mosaic virus

Leaves of tobacco varieties carrying the N gene for hypersensitiviy react to tobacco mosaic virus (TMV) infection by forming necrotic lesions and by localizing the virus in the vicinity of these lesions. These changes are accompanied in the host by an increased metabolic activity, in particular by an increased production of phenolic compounds derived from phenylalanine. Necrogenesis apparently destroys cells which have become heavily infected despite this strong defense reaction. However, it has been demonstrated previously (Otsuki et al., 1972) that protoplasts derived from leaves which normally respond in vivo to virus inoculation by forming necrotic local lesions, show no such response when inoculated in vitro. In the present study we have investigated the effect of pre-infecting hypersensitive leaves with TMV on the production or the non-production of the factor(s) of necrosis at the level of either protoplasts or mesophyll cells isolated from these preinfected leaves. Phenylalanine ammonia-lyase (PAL), whose rate of synthesis has been shown (Duchesne et al., 1977) to increase in stimulated cells of infected leaves, was used as a biochemical marker in the search for the stimulus preceding necrogenesis. We found that this stimulus concerning PAL activity was never elicited in either protoplasts or mesophyll cells which were prepared just before the appearance of necrotic local lesions. This result did not depend on the conditions of pre-infection or on the methods used to isolate the protoplasts or mesophyll cells. We also assayed samples derived from pre-infected leaves that were already carrying local lesions, i.e., in which the stimulus and necrogenesis were already operating: not only did the isolated protoplasts and mesophyll cells not sustain the stimulus concerning PAL activity, but the stimulated enzyme activity decreased abruptly and, in most of the experiments, had disappeared within the time necessary for maceration. Evidence is presented showing that the non-elicitation or the abrupt decrease of stimulated PAL activity could not result from a selection of unstimulated cells or from a preferential destruction of stimulated cells during maceration of the leaves.Our results support the view that hypertonic osmotic pressure is responsible for the non-occurence of the hypersensitive response by acting according to one or both of the following processes: it suppresses the contacts through plasmodesmata between neighboring cells and, hence, it also suppresses the cell-to-cell diffusion of the factor(s) eliciting the stimulus; and/or since hypertonic osmotic pressure causes striking differences between leaf cells and protoplasts in total RNA and protein synthesis, these differences might include the suppression of synthesis of the elicitor of hypersensitivity.Abbreviations OMT O-methyltransferase - PAL phenylalanine ammonia-lyase - TMV Tobacco mosaic virus     

Xanthine Oxidase Activity in the Susceptible and Hypersensitive Responses of Tobacco Leaves to Tobacco Mosaic Virus Infection

A superoxide-producing xanthine oxidoreductase was isolated and quantified after polyacrylamide disc gel electrophoresis of tobacco leaf extracts. The results obtained indicate that, like uricase activity, a slight increase in tobacco xanthine oxidase activity takes place in the susceptible interaction with tobacco mosaic virus (TMV). In contrast, out of three hypersensitive tobacco cultivars tested, only two showed the same slight increase m activity during the late stage of hypersensitive response.
Allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] a specific and potent in vitro and in vivo inhibitor of xanthine oxidoreductase, applied to tobacco plants by root absorption, starting about 8 days before the inoculation, did not affect the hypersensitive response but weakened the hypersensitivity-linked virus localization and promoted the movement of a certain amount of TMV particles and/or virus related material from necrotic lesions which induced systemic necrotic symptoms in uninoculated leaves. However, due to the inefficacy of allopurinol in preventing necrotic lesion development, all results are consistent with the hypothesis that xanthine oxidoreductase, the first enzyme in purine oxidative degradation, plays only a secondary role during induction of primary hypersensitive cell death in TMV infected tobacco leaves.     

Effects of heat shock treatment on symptom development in tobacco leaves inoculated with potato mop-top virus

PMTV normally causes necrotic ringspot local lesions in Xanthi-nc tobacco leaves at 15 °C but not at 22°. Dipping the leaves once in hot water at different intervals after inoculation induced necrotic reactions even at 22°. Successive concentric rings, one for each day of the interval between inoculation and treatment, were sometimes induced at 22°; these even formed in continuous lighting, suggesting an intrinsic diurnal rhythm. No lesions were induced at 22° by dipping inoculated leaves in ice-cold water. Samsun tobacco inoculated with PMTV developed very few lesions at 15°. Many more were induced however, by dipping the leaves in hot water after inoculation. The results suggest some similarities between PMTV and TMV, and the two viruses have been reported as serologically related; but PMTV did not protect plants against TMV, for TMV challenge-inoculations induced lesions within existing PMTV lesions.     

Ornithine decarboxylase activity and the hypersensitive reaction to tobacco mosaic virus in Nicotiana tabacum

The activity of ornithine decarboxylase (ODC) is increased 20 fold in leaves of Nicotiana tabacum cv. Xanthi n.c. following infection with tobacco mosaic virus at 20°. The activity reaches its maximum when localized necrotic lesions appear. There is little or no increase in plants kept at 32° when infection is systemic. However, if the infected plants are transferred to 20°, a marked and rapid increase in ODC activity occurs in the upper leaves, which collapse seven to nine hours after the transfer. ODC activity therefore parallels the activity of phenylalanine ammonia lyase during the hypersensitive reaction. Tyrosine decarboxylase was found to be activated in the same conditions. By contrast no increase in arginine decarboxylase activity could be detected. Temperature has a much greater effect on the polyamine and tyramine content of Xanthi n.c. leaves than does infection with TMV.     

The 2b silencing suppressor of a mild strain of Cucumber mosaic virus alone is sufficient for synergistic interaction with Tobacco mosaic virus and induction of severe leaf malformation in 2b-transgenic tobacco plants

Tobacco plants infected simultaneously by Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) are known to produce a specific synergistic disease in which the emerging leaves are filiformic. Similar developmental malformations are also caused to a lesser extent by the severe strains (e.g., Fny) of CMV alone, but mild strains (e.g., Kin) cause them only in mixed infection with TMV. We show here that transgenic tobacco plants expressing 2b protein of CMV-Kin produce filiformic symptoms when infected with TMV, indicating that only 2b protein is needed from CMV-Kin for this synergistic relationship. On the other hand, transgenic plants that express either the wild-type TMV genome or a modified TMV genome with its coat protein deleted or movement protein (MP) inactivated also develop filiformic or at least distinctly narrow leaves, while plants expressing the MP alone do not develop any malformations when infected with CMV-Kin. These results show that either TMV helicase/replicase protein or active TMV replication are required for this synergistic effect. The effect appears to be related to an efficient depletion of silencing machinery, caused jointly by both viral silencing suppressors, i.e., CMV 2b protein and the TMV 126-kDa replicase subunit.     

Expression of the tobacco mosaic virus movement protein alters starch accumulation inNicotiana benthamiana

Nicotiana benthamiana plants were transformed with the movement protein (MP) gene of tobacco mosaic virus (TMV), usingAgrobacterium-mediated transformation. Plants regenerated from the transformed cells accumulated 30-kDa MP and complemented the activity of TMV MP when infected with chimeric TMVs containing defective MR These transgenic plants displayed stunting, pale-green leaves, and starch accumulations, indicating that TMV MP altered the carbon partitioning for leaves involved in TMV cell-to-cell movement.     

The symptom difference induced by Tobacco mosaic virus and Tomato mosaic virus in tobacco plants containing the N gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaicvirus (ToMV) are members of the genus Tobamoviruswith a world-wide distribution, and cause severe dis-eases on many economically important crops. TMVand ToMV have very close relationship and both havessRNA genome with a length of about 6400 nucleo-tides, encoding at least three nonstructural proteinsand a 17.6 kD coat protein (CP). Both 126 kD and 183kD proteins function as components of replicase, andthe 30 kD protein is involved in viral ce…     

Inhibitory effect of fucoidan from brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus in tobacco leaves of two cultivars

The effect of fucoidan from the brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus (TMV) was investigated in the leaves of tobacco (Nicotiana tabacum L.) of two cultivars (Ksanti-nk and Samsun). In the leaves of cv. Ksanti-nk inoculated with a mixture of TMV preparation (2 μg/ml) and fucoidan (1 mg/ml), the number of local necrotic lesions induced by the virus decreased by more than 90% as compared with the leaves inoculated with the virus alone. In tobacco leaves of cv. Samsun, virulence and the concentration of the virus 3 days after inoculation with the same mixture of TMV and fucoidan were by 62 and 66%, respectively, lower than in the leaves inoculated with TMV alone. As the infection spread, the inhibitory effect of fucoidan decreased. When the leaves were treated with fucoidan before and after the inoculation with TMV, its antiviral activity was less pronounced than when a mixture of the virus and the polysaccharide was used as inoculum. Electron microscopic investigation of TMV mixed with fucoidan often showed agglutinated virions. The highest virulence of the mixture (TMV preparation, 12 μg/ml, plus fucoidan, 1 mg/ml) was observed upon its twofold dilution, and after that it decreased. It was concluded that, when the leaves were inoculated with the mixture of TMV and fucoidan, the latter affected not only the plant but the virus as well. Treatment of tobacco leaves, cv. Ksanti-nk, with actinomycin D (10 μg/ml) 24 h before the inoculation with TMV almost completely suppressed the effect of fucoidan, indicating that fucoidan acted at a gene level.     

Induction of Systemic Resistance to Tobacco Powdery Mildew by Tobacco Mosaic Virus, Tobacco Necrosis Virus or Ethephon

Local infections of either TMV or TNV in tobacco plants cv. Havana 425 (hypersensitive to TMV) proved effective in inducing systemic resistance to subsequent inoculation with the powdery mildew fungus Erysiphe cichoracearum DC. The proportion of leaf surface invaded by this pathogen and the amount of conidia it produced were both significantly lower in virus inoculated plants than in non-inoculated controls. However, the decrease in sporulation rate was less regularly observed than the reduction in leaf area infected. TMV was more effective than TNV in protecting tobacco plants from powdery mildew. E. cichoracearum is thus added to the list of challenge pathogens to which TMV or TNV are known to induce resistance in the host plants. Necrotic lesions caused to the leaves by local treatment with Ethephon (an ethylene-releasing compound) also conferred to tobacco some degree of systemic resistance to the same fungal pathogen, more frequently visible as a reduction of leaf area invaded. The protection due to the Ethephon lesions was in present experiments less marked than that of TMV. No effects against subsequent powdery mildew infection were obtained when point freeze necrotic lesions were provoked on the plants.     

NTH201, a novel class II KNOTTED1-like protein, facilitates the cell-to-cell movement of Tobacco mosaic virus in tobacco

NTH201, a novel class II KNOTTED1-like protein gene, was cloned from tobacco (Nicotiana tabacum cv. Xanthi) and its role in Tobacco mosaic virus (TMV) infection was analyzed. Virus-induced gene silencing of NTH201 caused a delay in viral RNA accumulation as well as virus spread in infected tobacco plants. Overexpression of the gene in a transgenic tobacco plant (N. tabacum cv. Xanthi nc) infected by TMV showed larger local lesions than those of the nontransgenic plant. NTH201 exhibited no intercellular trafficking ability but did exhibit colocalization with movement protein (MP) at the plasmodesmata. When NTH201-overexpressing tobacco BY-2 cultured cells were infected with TMV, the accumulation of MP but not of viral genomic and subgenomic RNA clearly was accelerated compared with those in nontransgenic cells at an early infection period. The formation of virus replication complexes (VRC) also was accelerated in these transgenic cells. Conversely, NTH201-silenced cells showed less MP accumulations and fewer VRC formations than did nontransgenic cells. These results suggested that NTH201 might indirectly facilitate MP accumulation and VRC formation in TMV-infected cells, leading to rapid viral cell-to-cell movement in plants at an early infection stage.     

A dysfunctional movement protein of tobacco mosaic virus that partially modifies the plasmodesmata and limits virus spread in transgenic plants

A chimeric gene encoding a dysfunctional tobacco mosaic virus (TMV) movement protein (MP) mutant lacking amino acids 3, 4 and 5 (MPΔ3–5), was expressed in transgenic Nicotiana tabacum Xanthi and Xanthi NN plants. Immunogold labeling studies of tissues from transgenic plants indicated that while wild-type MP accumulated in the plasmodesmata, MPΔ3–5 did not. Tissue fractionation studies confirmed that only a low level of the mutant MP accumulated in the cell wall-enriched fraction compared with the accumulation of the wild-type MP. Dye coupling studies showed that MPΔ3–5 enabled the movement between leaf mesophyll cells of a fluorescently labeled dextran of 3 kDa, while 9.4 kDa molecules failed to move. In contrast, in transgenic plants expressing the wild-type MP gene the 9.4 kDa probe did move from cell to cell. Seedlings from self-fertilized transgenic plants were inoculated with TMV and observed for disease symptoms. Transgenic Xanthi NN plants that expressed the MPΔ3–5 gene developed fewer and smaller necrotic local lesions compared with control plants following inoculation with TMV. Transgenic Xanthi nn plants were delayed in the development of systemic symptoms. Inoculating the transgenic plants with TMV-RNA, and the tobamo-viruses TMGMV and SHMV, essentially produced the same results, i.e. inhibition of disease development. These results demonstrate that transgenic plants expressing an inactive MP can inhibit virus disease spread presumably by interfering with its cell-to-cell movement.     

Characterization of tobacco plants expressing a bacterial salicylate hydroxylase gene

Transgenic tobacco plants that express the bacterial nahG gene encoding salicylate hydroxylase have been shown to accumulate very little salicylic acid and to be defective in their ability to induce systemic acquired resistance (SAR). In recent experiments using transgenic NahG tobacco and Arabidopsis plants, we have also demonstrated that salicylic acid plays a central role in both disease susceptibility and genetic resistance. In this paper, we further characterize tobacco plants that express the salicylate hydroxylase enzyme. We show that tobacco mosaic virus (TMV) inoculation of NahG tobacco leaves induces the accumulation of the nahG mRNA in the pathogen infected leaves, presumably due to enhanced stabilization of the bacterial mRNA. SAR-associated genes are expressed in the TMV-infected leaves, but this is localized to the area surrounding necrotic lesions. Localized acquired resistance (LAR) is not induced in the TMV-inoculated NahG plants suggesting that LAR, like SAR, is dependent on SA accumulation. When SA is applied to nahG-expressing leave's SAR gene expression does not result. We have confirmed earlier reports that the salicylate hydroxylase enzyme has a narrow substrate specificity and we find that catechol, the breakdown product of salicylic acid, neither induces acquired resistance nor prevents the SA-dependent induction of the SAR genes.     

The symptom difference induced by <Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> in tobacco plants containing the <Emphasis Type="Italic">N</Emphasis> gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was replaced by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in tobacco plants containing the N gene results from the functional difference of their MP genes.     

Manifestations of non-specific antiviral factor originating in tobacco leaves during virus infection

A non-specific effect of antiviral factor (AVF) was proved by interference of five different viruses (CMV, PVX, PVY, TMVe, TMV vulgare) with CBRV. The non-specific AVF originates in non-infected tissues around TMV lesions. Transmitted mechanically to other plants this AVF exhibits the same suppressive effect on multiplication of TMV and CBRV. The AVF is formed in the non-infected apical part of the leaf which was infected in its basal region. The susceptibility of leaves is decreased even when AVF is added to virus inoculum and applied mechanically on the leaf. The apical part of the leaf the base of which is met with necrotic reaction to TMV, is considerably less susceptible and TMV multiplication in this region is decreased. The necrosis on the leaf base induced by mechanical injury or by cysteine hydrochloride may decrease the susceptibility of the leaf to some extent, but has no effect on multiplication of the virus. The activity of AVF in healthy apical region of the leaf increases till the fifth to the eighth day after its basal part was infected. The apical region then exhibits lower susceptibility (decrease in number of lesions) and the growth of lesions is decreased. This indicates the virus multiplication being suppressed.