Wirkung von Virushemmstoffen auf die Infektion von Tabakprotoplasten mit dem Tabakmosaik-Virus

Effects of virus inhibitors on the infection of tobacco protoplasts with tobacco mosaic virus Yeast extract inhibits the infection of Nicotiana glutinosa plants with tobacco mosaic virus (TMV), whereas in N. sandérae yeast extract is not effective. This phenomena was compared with the effect of yeast extract on protoplasts, and on the infection of protoplasts of both tobacco species with TMV. Additionally, skim milk and ribonuclease were included in the experiments as further inhibitors of early stages of virus infection. It was examined whether these inhibitors damage non-inoculated protoplasts (a), and whether they affect virus infections in protoplasts as they do in cells of intact plants (b). To investigate protoplast damage by the inhibitors, conductivity measurements of protoplast suspensions containing inhibitors, and the ability of protoplasts for cell wall regeneration after treatment with the inhibitors, were used. Inhibitor concentrations which prevent virus infections in plants did not damage the protoplasts. The inhibitor effect on the course of infection was investigated by protoplast treatments before, during and after inoculation with TMV, and by addition of the substances to the culture medium. Measurements of virus content in protoplasts after cultivation revealed different results for the three inhibitors, however, there was no difference in the response of protoplasts from the two tobacco species to yeast extract. It is concluded that there are principal differences between the inhibition of plant and protoplast infections. Therefore, it is unlikely that protoplasts are a useful system for the mode of action studies on inhibitors of early stages of virus infection in plants.     

Expression of Pokeweed Antiviral Protein in Transgenic Plants Induces Virus Resistance in Grafted Wild-Type Plants Independently of Salicylic Acid Accumulation and Pathogenesis-Related Protein Synthesis

Pokeweed antiviral protein (PAP), a 29-kD protein isolated from Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. Transgenic tobacco (Nicotiana tabacum) plants expressing PAP or a variant (PAP-v) were shown to be resistant to a broad spectrum of plant viruses. Expression of PAP-v in transgenic plants induces synthesis of pathogenesis-related proteins and a very weak (<2-fold) increase in salicylic acid levels. Using reciprocal grafting experiments, we demonstrate here that transgenic tobacco rootstocks expressing PAP-v induce resistance to tobacco mosaic virus infection in both N. tabacum NN and nn scions. Increased resistance to potato virus X was also observed in N. tabacum nn scions grafted on transgenic rootstocks. PAP expression was not detected in the wild-type scions or rootstocks that showed virus resistance, nor was there any increase in salicylic acid levels or pathogenesis-related protein synthesis. Grafting experiments with transgenic plants expressing an inactive PAP mutant demonstrated that an intact active site of PAP is necessary for induction of virus resistance in wild-type scions. These results indicate that enzymatic activity of PAP is responsible for generating a signal that renders wild-type scions resistant to virus infection in the absence of increased salicylic acid levels and pathogenesis-related protein synthesis.     

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.     

EFFECTS OF DARKNESS ON THE CONSTITUTION OF TOBACCO LEAVES AND SUSCEPTIBILITY TO VIRUS INFECTION

An attempt was made to find the causes of increased susceptibility to virus infection when tobacco plants are kept in the dark before inoculation. The changes in certain nitrogen fractions, viz. insoluble-N, amino-N, amide-N, ammonia-N and nitrate-N, and in dry matter and water content were followed in tobacco plants subjected to a period of darkness before inoculation with tobacco aucuba mosaic virus. Only nitrate-N was strongly correlated with the susceptibility to infection, but the evidence suggests that the correlation is indirect and not causal.
Dry matter and water content, determined either as dry matter percentage of fresh weight or measured separately on a leaf-disk basis Ivere found to vary directly with variation in susceptibility.     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Changes in the activity of phosphogluconate dehydrogenase and its regulation in tobacco infected with PVY

The content and changes in the activity of phosphogluconate dehydrogenase were followed in leaf tissues of tobacco plants infected with the potato virus Y (PVY) in the acute phase of infection. The activity of the enzyme was higher in virus-infected tissues during the entire experimental period compared with the values found in healthy control plants in both crude homogenate and partially purified enzyme preparation. The courses of the activity curves of both the crude extract and partially purified enzyme preparations were consistent and correlated with the reproduction curve of the virus. These results suggest that increased activity of the enzyme was a result of its coarse regulation and indicate its immediate involvement in“de novo” biosynthesis of the virus via the oxidative pentose phosphate pathway.     

SOME EFFECTS OF TANNIC ACID AND OF LEAF EXTRACTS WHICH CONTAIN TANNINS ON THE INFECTIVITY OF TOBACCO MOSAIC AND TOBACCO NECROSIS VIRUSES

The inhibition of infection by tobacco necrosis and tobacco mosaic viruses by tannic acid, and by extracts of raspberry and strawberry leaves, was associated with the precipitation of the viruses. Precipitation and inhibition were reversible, and infective virus was obtained from the precipitate formed between the viruses and tannins. Infectivity was fully restored by diluting mixtures of virus and tannin adequately and partially restored by adding alumina or nicotine sulphate.
Viruses and tannins are thought to form non-infective complexes, in which the virus and tannin components are held together by co-ordinate linkages or hydrogen bonds.
Macerating tobacco leaves infected with tobacco mosaic virus together with raspberry leaves greatly decreased the infectivity of the extracts; adding nicotine sulphate to the mixture of leaves before it was ground increased the infectivity, even though nicotine sulphate alone decreases the infectivity of tobacco mosaic virus. Even in the presence of nicotine sulphate, much of the virus was precipitated by substances from the raspberry leaves.
Extracts of roots of Fragaria vesca plants, infected with a tobacco necrosis virus, were more infective when made by macerating the roots with four times their weight of buffer at pH 8 than when made without buffer. Various methods are suggested for facilitating the transmission of viruses from plants that contain tannin.     

THE INFLUENCE OF LIGHT INTENSITY ON THE SUSCEPTIBILITY OF PLANTS TO CERTAIN VIRUSES

Reducing the light intensity under which plants were grown in summer to one-third increased their susceptibility to infection with tobacco necrosis, tomato bushy stunt, tobacco mosaic and tomato aucuba mosaic viruses. With the first two viruses shading increased the average number of local lesions per leaf by more than ten times and by more than five times with the second two.
Reducing the light intensity increased the virus content of sap from leaves inoculated with Rothamsted tobacco necrosis virus by as much as twenty times. As it also reduced the total solid content of sap by about one-half, purification was greatly facilitated; crystalline preparations of the virus were readily made from shaded plants but not from unshaded controls.
Reducing the light intensity also increased the virus content of systemically infected leaves; the greatest effect was with tomato bushy stunt virus with which increases of up to ten times were obtained, but with tobacco mosaic and aucuba mosaic viruses there were also significant increases.
The importance of controlled illumination in raising plants for virus work and the possible mechanisms responsible for the variations in susceptibility are discussed.     

Chemical suppression of the symptoms of two virus diseases

Carbendazim applied at the rate of 2 g per plant to the roots of tobacco (Nicotiana tabacum cv. White Burley) plants before infection with tobacco mosaic virus (TMV) caused very considerable reduction in the severity of disease symptoms in systemically infected leaves but did not affect their virus content. Leaves of untreated, infected plants had a greatly reduced chlorophyll content 100 days after infection whereas the chlorophyll content of leaves of infected plants treated with carbendazim was similar to that of normal uninfected leaves. Carbendazim had no effect on the infectivity of TMV in vitro or on the local lesion reaction of N. glutinosa plants when inoculated with TMV. Carbendazim was applied to lettuce cv. Cobham Green at a total rate of o-i g per plant before and after they were infected with beet western yellows virus and the plants were then grown on in the field. At harvest time (50 days after infection) almost all the treated virus-infected plants were of a normal green appearance, whereas the untreated controls were almost all very severely yellowed and unmarketable.     

Role of polyisoprenoids in tobacco resistance against biotic stresses

Infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols, solanesol and a family of polyprenols [from polyprenol composed of 14 isoprene units (Pren-14) to -18, with Pren-16 dominating] in the leaves of resistant tobacco plants Nicotiana tabacum cv. Samsun NN. Upon TMV infection, solanesol content was increased seven- and eight-fold in the inoculated and upper leaves, respectively, while polyprenol content was increased 2.5- and 2-fold in the inoculated and upper leaves, respectively, on the seventh day post-infection. Accumulation of polyisoprenoid alcohols was also stimulated by exogenously applied hydrogen peroxide but not by exogenous salicylic acid (SA). On the contrary, neither inoculation of the leaves of susceptible tobacco plants nor wounding of tobacco leaves caused an increase in polyisoprenoid content. Taken together, these results indicate that polyisoprenoid alcohols might be involved in plant resistance against pathogens. A putative role of accumulated polyisoprenoids in plant response to pathogen attack is discussed. Similarly, the content of plastoquinone (PQ) was increased two-fold in TMV-inoculated and upper leaves of resistant plants. Accumulation of PQ was also stimulated by hydrogen peroxide, bacteria ( P.  syringae ) and SA. The role of PQ in antioxidant defense in cellular membranous compartments is discussed in the context of the enzymatic antioxidant machinery activated in tobacco leaves subjected to viral infection. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase was found in inoculated leaves of resistant tobacco plants but not in susceptible plants.     

Inducible expression of the gene of Zinnia elegans coding for extracellular ribonuclease in Nicotiana tabacum plants

The gene of Zinnia elegans L. coding for S-like extracellular ribonuclease (ZRNase II) was used to produce transgenic tobacco plants with an increased ribonuclease activity. The protein-coding part of ZRNase II included the signal peptide sequence so the transgenic protein was located extracellularly. The cDNA of ZRNase II was cloned under the control of 2′-promoter of the mannopine synthase (MAS 2′) gene from Ti-plasmid of Agrobacterium tumefaciens. It was shown that the resultant transgenic plants had an increased ribonuclease activity of the crude extracts and the induction of MAS 2′ promoter by wounding additionally increased the activity. The plants of two transforming lines characterized by different ribonuclease activities were used to analyze the transgene influence on plant resistance to tobacco mosaic virus. The plants demonstrated either absence of disease symptoms or a significant delay in their appearance, depending on the virus content in the inoculum and ribonuclease activity.     

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.     

Virus Resistance Induced with Mannan Sulphates in Hypersensitive Host Plants

Mannan sulphates (MS), obtained by sulphatation of extracellular linear mannan (LM) from Rhodotorula rubra, induced resistance to tobacco mosaic virus (TMV) in hypersensitive plants, retained in isolated protoplasts. MS-induced resistance in tobacco is accompanied by a marked increase of lytic processes in cells and a decrease of total (acidic and alkaline) protein content. In addition to this, new protein components, including PR-proteins and antiviral substances, of the inhibitor of virus replication (IVR) type, appeared in treated plant tissues. MS-induced resistance resembles localized resistance induced by TMV in hypersensitive tobacco plants and could serve as an experimental model for study of the latter.     

Effects of Salicylate on Systemic Invasion of Tobacco Plants by Various Viruses

Salicylate watered onto soil in which White Burley tobacco plants were grown represents a reversible stress characterized by stomatal closure, slight slackening of plant growth and low chlorophyll loss. Salicylate affected viral pathogenesis in opposite ways. It had no effect against local and systemic infections by potato virus X (PVX), potato virus Y⁰ (PVY⁰) or tobacco mosaic virus (TMV), whereas it completely prevented systemic infection by alfalfa mosaic virus (AIMV) or tobacco, rattle virus (TRV) in a high proportion of treated plants. When infection moved from leaves inoculated with AIMV or TRV, the tendency to limit systemic spread was shown by the restriction of systemic infection to very limited areas erratically distributed in some uninoculated leaves. The salicylate-induced restriction of AIMV or TRV infectivity to inoculated leaves did not appear due to inhibition of virus multiplication because the inoculation of potentially resistant leaves of salicylate-reated plants resulted in virus antigen accumulation comparable to that of untreated controls. Salicylate may therefore inhibit some long distance virus transport function. Salicylate appears able to evoke true hypersensitivity only against systemic viruses able to induce local necrotic lesions, probably by activating some genetic information for resistance that is normally not expressed.     

Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses

Transgenic tobacco plants expressing the coat protein (CP) gene of tobacco mosaic virus were tested for resistance against infection by five other tobamoviruses sharing 45-82% homology in CP amino acid sequence with the CP of tobacco mosaic virus. The transgenic plants (CP+) showed significant delays in systemic disease development after inoculation with tomato mosaic virus or tobacco mild green mosaic virus compared to the control (CP-) plants, but showed no resistance against infection by ribgrass mosaic virus. On a transgenic local lesion host, the CP+ plants showed greatly reduced numbers of necrotic lesions compared to the CP- plants after inoculation with tomato mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, and Odontoglossum ringspot virus but not ribgrass mosaic virus. The implications of these results are discussed in relation to the possible mechanism(s) of CP-mediated protection.     

Application of exogenous substances reduces tobacco-specific nitrosamines content by regulating biosynthesis of nicotine and nitrite in burley tobacco

Tobacco-specific nitrosamines (TSNAs) are carcinogenic chemicals found in tobacco plants. The increasing health consciousness of individuals had led to an increased interest in research on reducing TSNAs content. The aim of this study was to use a pot experiment in which exogenous substances were applied to burley tobacco to dissect the mechanism of TSNAs production. The results indicated that spraying the exogenous substances IAA, NAA, SA and combination thereof on burley tobacco after topping decreased TSNAs content by 2.69–29.4 % in upper leaves and 0.23–39.3 % in middle leaves without affecting total sugar, total nitrogen, potassium and chlorine contents. The application of exogenous substances could down-regulate expression of the NR gene and the activity of the NR enzyme, resulting in less accumulation of the TSNAs precursor nitrite. The exogenous substances significantly reduced nicotine accumulation, which was consistent with low enzyme activities and the down-regulated expressions of genes involved in nicotine biosynthesis, especially significant in the case of quinolinate phosphoribosyltransferase. These results suggested that the application of exogenous substances on burley tobacco after topping could reduce TSNAs content which may be attributed to the regulation of exogenous substances on nitrite and nicotine. This also implies one potential improvement to agronomic practices aimed at controlling the accumulation of TSNAs in burley tobacco.     

The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants

As the processing mechanism of all known potyviruses involves the activity of cysteine proteinases, we asked whether constitutive expression of a rice cysteine proteinase inhibitor gene could induce resistance against two important potyviruses, tobacco etch virus (TEV) and potato virus Y (PVY), in transgenic tobacco plants. Tobacco lines expressing the foreign gene at varying levels were examined for resistance against TEV and PVY infection. There was a clear, direct correlation between the level of oryzacystatin message, inhibition of papain (a cysteine proteinase), and resistance to TEV and PVY in all lines tested. The inhibitor was ineffective against tobacco mosaic virus (TMV) infection because processing of this virus does not involve cysteine proteinases. These results show that plant cystatins can be used against different potyviruses and potentially also against other viruses, whose replication involves cysteine proteinase activity.     

Protection of transgenic tobacco plants expressing bovine pancreatic ribonuclease against tobacco mosaic virus

Transgenic tobacco plants (Nicotiana tabacum cv. SR1) expressing extracellular pancreatic ribonuclease from Bos taurus and characterized by an increased level of ribonuclease activity in leaf extracts were challenged with tobacco mosaic virus. The transgenic plants exhibited a significantly higher level of protection against the virus infection than the control non-transformed plants. The protection was evidenced by the absence (or significant delay) of the appearance of typical mosaic symptoms and the retarded accumulation of infectious virus and viral antigen. These results demonstrate that modulation of extracellular nuclease expression can be efficiently used in promoting protection against viral diseases.     

PVY侵染后烟草营养成分的变化及其对介体烟蚜生长发育的影响

【目的】明确马铃薯Y病毒(potato virus Y,PVY)侵染后诱导的烟草营养成分的变化及其对烟蚜Myzus persicae生命特性的影响,旨在进一步解析PVY-烟草-烟蚜三者间的互作机制。【方法】通过蒽酮比色法和氨基酸自动分析仪测定了PVY不同侵染时期烟株体内的可溶性糖和游离氨基酸含量的变化;测定和比较了感病与健康烟草植株上烟蚜种群生长发育、成虫寿命、繁殖力和有翅蚜产生量的差异性。【结果】PVY侵染前、中、后期(分别为侵染后5,12和20 d)的烟草叶片中游离氨基酸的总量均显著高于健康烟草叶片。相较于健康烟草叶片,在PVY侵染前期的烟草叶片中,谷氨酸、脯氨酸、天冬氨酸、色氨酸、缬氨酸、赖氨酸和组氨酸的含量显著增加;PVY侵染中期,感病叶片中丝氨酸含量显著下降,谷氨酸、天冬氨酸、色氨酸、缬氨酸、亮氨酸、苯丙氨酸、精氨酸和组氨酸含量显著提高;PVY侵染后期,感病叶片中甘氨酸含量显著下降,谷氨酸、脯氨酸、天冬氨酸、苏氨酸、缬氨酸、亮氨酸、丙氨酸、苯丙氨酸、组氨酸、酪氨酸和精氨酸含量显著提高。在PVY侵染的前期和中期,感病叶片中的可溶性糖含量显著高于健康烟叶,而在侵染后期感病叶片中可溶性糖含量显著低于健康烟草叶片的。PVY侵染前期和中期的烟草叶片中总糖和总游离氨基酸的含量比值显著高于健康烟草叶片中的。在PVY侵染的烟草植株和健康烟草植株上取食的烟蚜其发育历期、若蚜历期、成蚜繁殖期、繁殖后期、寿命、烟蚜种群的内禀增长率、周限增长率和平均世代周期均无显著差异,但在感病烟草植株上取食的烟蚜成蚜繁殖前期显著缩短,其繁殖力和净生殖率显著提高。相较于健康烟草植株,在PVY侵染烟草植株上定殖的烟蚜种群有翅蚜发生的高峰期提前。【结论】PVY侵染前期和中期提高了寄主烟草的营养品质,从而提高了烟蚜的繁殖力。侵染后期烟草营养品质的下降,促使烟蚜种群有翅蚜的产生和扩散,从而有利于PVY自身的传播。