Improved sulfur nutrition provides the basis for enhanced production of sulfur-containing defense compounds in Arabidopsis thaliana upon inoculation with Alternaria brassicicola

The antifungal activities of many sulfur-containing defense compounds suggest a connection between pathogen infection, primary sulfur metabolism and sulfate nutritional status of plants. This relationship was investigated using Arabidopsis thaliana plants that were cultivated under different sulfur regimes and challenged by Alternaria brassicicola. Plants grown with 500 μM sulfate were significantly less infected compared to plants grown on 50 μM sulfate. Upon infection, the formation of the sulfur-containing defense compound camalexin and the gene expression of the sulfur-rich defense peptide defensin were clearly enhanced in plants grown with an optimal compared to a sufficient sulfate supply in the growth medium. Elevated levels of sulfite and O-acetylserine and cysteine biosynthetic enzymes after infection indicated a stimulation of sulfur metabolism under the higher sulfate supply. The results suggest that, in addition to pathogen-triggered activation of sulfur metabolism and sulfur-containing defense compound formation, the sulfate nutritional status is sensed to contribute to plant defense.     

Endogenous Methyl Salicylate in Pathogen-Inoculated Tobacco Plants

The tobacco (Nicotiana tabacum) cultivar Xanthi-nc (genotype NN) produces high levels of salicylic acid (SA) after inoculation with the tobacco mosaic virus (TMV). Gaseous methyl salicylate (MeSA), a major volatile produced in TMV-inoculated tobacco plants, was recently shown to be an airborne defense signal. Using an assay developed to measure the MeSA present in tissue, we have shown that in TMV-inoculated tobacco plants the level of MeSA increases dramatically, paralleling increases in SA. MeSA accumulation was also observed in upper, noninoculated leaves. In TMV-inoculated tobacco shifted from 32 to 24°C, the MeSA concentration increased from nondetectable levels to 2318 ng/g fresh weight 12 h after the temperature shift, but subsequently decreased with the onset of the hypersensitive response. Similar results were observed in plants inoculated with Pseudomonas syringae pathovar phaseolicola, in which MeSA levels were highest just before the hypersensitive response-induced tissue desiccation. Transgenic NahG plants unable to accumulate SA also did not accumulate MeSA after TMV inoculation, and did not show increased resistance to TMV following MeSA treatment. Based on the spatial and temporal kinetics of its accumulation, we conclude that tissue MeSA may play a role similar to that of volatile MeSA in the pathogen-induced defense response.     

Local and systemic spread of tobacco mosaic virus in transgenic tobacco.

Expression of a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) in transgenic tobacco plants confers resistance to infection by TMV. We investigated the spread of TMV within the inoculated leaf and throughout the plant following inoculation. Plants that expressed the CP gene [CP(+)] and those that did not [CP(-)] accumulated equivalent amounts of virus in the inoculated leaves after inoculation with TMV-RNA, but the CP(+) plants showed a delay in the development of systemic symptoms and reduced virus accumulation in the upper leaves. Tissue printing experiments demonstrated that if TMV infection became systemic, spread of virus occurred in the CP(+) plants essentially as it occurred in the CP(-) plants although at a reduced rate. Through a series of grafting experiments, we showed that stem tissue with a leaf attached taken from CP(+) plants prevented the systemic spread of virus. Stem tissue without a leaf had no effect on TMV spread. All of these findings indicate that protection against systemic spread in CP(+) plants is caused by one or more mechanisms that, in correlation with the protection against initial infection upon inoculation, result in a phenotype of resistance to TMV.     

SOME EFFECTS OF VIRUS INFECTION ON LEAF WATER CONTENTS OF NICOTIANA SPECIES

Infection with tobacco mosaic virus decreases the water content which detached tobacco leaves attain when kept for 20 hr. in conditions of minimum water stress, and does so more when the plants are kept in light before inoculation than when they are kept in darkness. No such effects of infection during the first day after inoculation were obtained with tobacco leaves infected with either tobacco etch virus or potato virus X , or with Nicotiana glutinosa leaves infected with tobacco mosaic virus. These results, like those showing early effects of TMV on respiration and photosynthesis of tobacco leaves, suggest that inoculation with TMV affects deeper leaf tissues than the epidermis earlier in tobacco leaves than in other leaves, and earlier than other viruses in tobacco leaves.     

Cross protection in transgenic tobacco plants expressing a mild strain of tobacco mosaic virus

Summary Cross protection of plant viruses is a phenomenon in which plants infected with one strain of a virus are protected from the effects of superinfection by other related strains. Recently, we have succeeded in the introduction and expression of a cDNA copy of the tobacco mosaic virus (TMV) genomic RNA in transgenic tobacco plants. Using this system, we introduced a cDNA copy of a mild strain of TMV into tobacco plants. The transgenic plants did not develop any severe symptoms upon inoculation with a virulent TMV strain, indicating that these transgenic plants were cross protected against TMV infection. The system described here can be a useful model system to study the mechanism(s) of cross protection.     

Interaction of sulfur and nitrogen nutrition in tobacco (Nicotiana tabacum) plants: significance of nitrogen source and root nitrate reductase

Abstract: The significance of root nitrate reductase for sulfur assimilation was studied in tobacco (Nicotiana tabacum) plants. For this purpose, uptake, assimilation, and long-distance transport of sulfur were compared between wild-type tobacco and transformants lacking root nitrate reductase, cultivated either with nitrate or with ammonium nitrate. A recently developed empirical model of plant internal nitrogen cycling was adapted to sulfur and applied to characterise whole plant sulfur relations in wild-type tobacco and the transformant. Both transformation and nitrogen nutrition strongly affected sulfur pools and sulfur fluxes. Transformation decreased the rate of sulfate uptake in nitrate-grown plants and root sulfate and total sulfur contents in root biomass, irrespective of N nutrition. Nevertheless, glutathione levels were enhanced in the roots of transformed plants. This may be a consequence of enhanced APR activity in the leaves that also resulted in enhanced organic sulfur content in the leaves of the tranformants. The lack of nitrate reductase in the roots in the transformants caused regulatory changes in sulfur metabolism that resembled those observed under nitrogen deficiency. Nitrate nutrition reduced total sulfur content and all the major fractions analysed in the leaves, but not in the roots, compared to ammonium nitrate supply. The enhanced organic sulfur and glutathione levels in ammonium nitrate-fed plants corresponded well to elevated APR activity. But foliar sulfate contents also increased due to decreased re-allocation of sulfate into the phloem of ammonium nitrate-fed plants. Further studies will elucidate whether this decrease is achieved by downregulation of a specific sulfate transporter in vascular tissues.     

Effect of ubiquinone 50 and viral infection on phytohemagglutinin activity in development of induced resistance in tobacco plants

The effects of ubiquinone 50 (synthetic coenzyme Q₁₀) and viral infection on phytohemagglutinin activity were studied in the tobacco (Nicotiana tabacum L.) cultivar Samsun NN, which carries the necrosis-formation locus and exhibits hypersensitivity to tobacco mosaic virus (TMV) and ubiquinone (Q₁₀). The treatment with Q₁₀ and inoculation with TMV were accompanied by an increase in phytohemagglutinin activity, indicating that lectins may be involved in the development of defense responses in plants.     

Transgenic Plants Expressing Potato Virus X ORF2 Protein (p24) Are Resistant to Tobacco Mosaic Virus and Ob Tobamoviruses

The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.     

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.     

Long-distance transport of sulfur in Nicotiana tabacum

Sulfur reduction in tobacco plants is a light-enhanced process that predominantly takes place in the leaves rather than the roots. The amount of sulfate reduced in mature leaves can exceed their own requirement and enables an export of reduced sulfur, both basipetal toward the roots as well as acropetal toward the growing parts of the stem. Evidence is presented that translocation of reduced sulfur toward the roots occurs in the phloem. TLC and paper chromatography reveal that glutathione is the main transport form of reduced sulfur in tobacco plants; 67–70% of reduced ³⁵S was confined to glutathione, 27–30% to methionine, and 2–8% to cysteine.Abbreviation TLC thin-layer chromatography     

Inhibition of Programmed Cell Death in Tobacco Plants during a Pathogen-Induced Hypersensitive Response at Low Oxygen Pressure

The hypersensitive response (HR) of plants to invading pathogens is thought to involve a coordinated activation of plant defense mechanisms and programmed cell death (pcd). To date, little is known about the mechanism underlying death of plant cells during this response. In addition, it is not known whether suppression of pcd affects the induction of other defense mechanisms during the HR. Here, we report that death of tobacco cells (genotype NN) infected with tobacco mosaic virus (TMV) is inhibited at low oxygen pressure. In contrast, virus replication and activation of defense mechanisms, as measured by synthesis of the pathogenesis-related protein PR-1a, were not inhibited at low oxygen pressure. Bacterium-induced pcd was also inhibited at low oxygen pressure. However, pcd induced by TMV or bacteria was not inhibited in transgenic tobacco plants expressing the mammalian anti-pcd protein Bcl-XL. Our results suggest that ambient oxygen levels are required for efficient pcd induction during the HR of plants and that activation of defense responses can be uncoupled from cell death. Furthermore, pcd that occurs during the interaction of tobacco with TMV or bacteria may be distinct from some cases of pcd or apoptosis in animals that are insensitive to low oxygen or inhibited by the Bcl-XL protein.     

Downregulation of a pathogen-responsive tobacco UDP-Glc:phenylpropanoid glucosyltransferase reduces scopoletin glucoside accumulation,enhances oxidative stress,and weakens virus resistance

Plant UDP-Glc:phenylpropanoid glucosyltransferases (UGTs) catalyze the transfer of Glc from UDP-Glc to numerous substrates and regulate the activity of compounds that play important roles in plant defense against pathogens. We previously characterized two tobacco salicylic acid- and pathogen-inducible UGTs (TOGTs) that act very efficiently on the hydroxycoumarin scopoletin and on hydroxycinnamic acids. To identify the physiological roles of these UGTs in plant defense, we generated TOGT-depleted tobacco plants by antisense expression. After inoculation with Tobacco mosaic virus (TMV), TOGT-inhibited plants exhibited a significant decrease in the glucoside form of scopoletin (scopolin) and a decrease in scopoletin UGT activity. Unexpectedly, free scopoletin levels also were reduced in TOGT antisense lines. Scopolin and scopoletin reduction in TOGT-depleted lines resulted in a strong decrease of the blue fluorescence in cells surrounding TMV lesions and was associated with weakened resistance to infection with TMV. Consistent with the proposed role of scopoletin as a reactive oxygen intermediate (ROI) scavenger, TMV also triggered a more sustained ROI accumulation in TOGT-downregulated lines. Our results demonstrate the involvement of TOGT in scopoletin glucosylation in planta and provide evidence of the crucial role of a UGT in plant defense responses. We propose that TOGT-mediated glucosylation is required for scopoletin accumulation in cells surrounding TMV lesions, where this compound could both exert a direct antiviral effect and participate in ROI buffering.     

The mechanism(s) of coat protein-mediated resistance against tobacco mosaic virus

The resistance of transgenic plants express genes encoding viral coat proteins to infection by the viruses from which the genes are derived was termed coat protein-mediated resistance (CP-MR) and has been demonstrated for a variety of virus/host combinations. The mechanism of CP-MR is perhaps best understood in the tobacco/TMV system. CP-MR against TMV requires accumulation of CP and does not seem to involve the induction of plant defense mechanisms. The resistance appears to be mainly based on the inhibition of virion disassembly in transgenic cells although there is evidence that a later step of infection is also affected. CP-MR of tobacco to TMV shares some features with classical cross-protection and with CP-MR in some, but not all other host/virus combinations.     

Sulfur-enhanced defence: effects of sulfur metabolism, nitrogen supply, and pathogen lifestyle

Evidence from field experiments indicates differential roles of sulfur and nitrogen supply for plant resistance against pathogens. Dissection of these observations in defined pathosystems and controlled nutritional conditions indicates an activation of plant sulfur metabolism in several incompatible and compatible interactions. Contents of cysteine and glutathione as markers of primary sulfate assimilation and stress response show increases in ARABIDOPSIS THALIANA upon infection, coinciding with the synthesis of sulfur-containing defence compounds. Similar increases of thiols were observed with necrotrophic, biotrophic, and hemibiotrophic pathogens. Sulfate supply was found to be neutral or beneficial for tolerance against fungal but neutral for bacterial pathogens under IN VITRO conditions. According to various reports and own observations the effects of nitrogen supply appeared to be neutral or harmful, depending on the pathogen. The activation of sulfur metabolism was a consequence of activation of gene expression as revealed by macroarray analysis of an A. THALIANA/ALTERNARIA BRASSICICOLA pathosystem. This activation appeared to be largely independent from sufficient or optimal sulfate supply and from the established sulfate deficiency response. The data suggest that plant-pathogen interactions and sulfur metabolism are linked by jasmonic acid as signal.     

Salicylic acid has cell-specific effects on tobacco mosaic virus replication and cell-to-cell movement

Tobacco mosaic virus (TMV) and Cucumber mosaic virus expressing green fluorescent protein (GFP) were used to probe the effects of salicylic acid (SA) on the cell biology of viral infection. Treatment of tobacco with SA restricted TMV.GFP to single-epidermal cell infection sites for at least 6 d post inoculation but did not affect infection sites of Cucumber mosaic virus expressing GFP. Microinjection experiments, using size-specific dextrans, showed that SA cannot inhibit TMV movement by decreasing the plasmodesmatal size exclusion limit. In SA-treated transgenic plants expressing TMV movement protein, TMV.GFP infection sites were larger, but they still consisted overwhelmingly of epidermal cells. TMV replication was strongly inhibited in mesophyll protoplasts isolated from SA-treated nontransgenic tobacco plants. Therefore, it appears that SA has distinct cell type-specific effects on virus replication and movement in the mesophyll and epidermal cell layers, respectively. Thus, SA can have fundamentally different effects on the same pathogen in different cell types.     

The Effect of Arachidonic Acid and Viral Infection on the Phytohemagglutinin Activity during the Development of Tobacco Acquired Resistance

The effects of arachidonic acid (AA) on the development of viral infection and the activity of phytohemagglutinins in Nicotiana tabacum L. plants were studied. Cv. Samsun NN was used, which displayed a genotypically determined hypersensitive response to tobacco mosaic virus (TMV) infection. When tobacco leaf disks were treated with 10^–9 to –10^–7 M AA, viral reproduction was suppressed by 90–100%. The AA concentration of 10^–8 M was optimal for the improvement of plant virus resistance. Tobacco leaves maintained virus resistance for at least two weeks. Both AA treatment and TMV inoculation were accompanied by an enhanced lectin activity, which may indicate the involvement of lectins in the development of plant defense responses. Lectin accumulation was observed in the intact plants developing systemic resistance and in the detached leaves characterized by local resistance.     

Constitutively Elevated Levels of Putrescine and Putrescine-Generating Enzymes Correlated with Oxidant Stress Resistance in Conyza bonariensis and Wheat

Changes in ascorbate and glutathione levels and in activities of ascorbate peroxidase, catalase, dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST), and superoxide dismutase (SOD) were investigated in tobacco mosaic virus (TMV)-inoculated lower leaves and in non-inoculated upper leaves of Nicotiana tabacum L. cv Xanthi-nc. In separate experiments the effects of exogenous salicylic acid (SA) were also studied. Symptom appearance after TMV inoculation was preceded by a slight, transient decline of ascorbate peroxidase, GR, GST, and SOD activities in the inoculated lower leaves, but after the onset of necrosis these activities and the glutathione level substantially increased. Ascorbic acid level and DHAR activity declined and dehydroascorbate accumulated in the inoculated leaves. In upper leaves, the glutathione level and the activities of GR, GST, and SOD increased 10 to 14 d after TMV inoculation of the lower leaves, concomitantly with the development of systemic acquired resistance. From the six distinct SOD isoenzymes found in tobacco leaves, only the activities of Cu,Zn-SOD isoenzymes were affected by TMV. SA injection induced DHAR, GR, GST, and SOD activities. Catalase activities were not modified by TMV infection or SA treatment. It is supposed that stimulated antioxidative processes contribute to the suppression of necrotic symptom development in leaves with systemic acquired resistance.     

Mechanism Determining the Host Specificity of Tobacco Mosaic Virus

In order to know the mechanism controling the host specificity of tobacco mosaic virus (TMV), three species of plants showing various degrees of resistance to TMV or TMV-RNA infection were selected and the fate of infecting viral genome was studied. Extract was obtained from leaves 0.5–6 hr after inoculation of ³²P-TMV or ³²P-TMV-RNA and analyzed by sucrose density gradient centrifugation. It was found that polysomes containing infecting ³²P-RNA were formed in plants resistant to TMV to the same extent as in susceptible tobacco plants, suggesting that the host specificity of TMV is determined at a stage of viral multiplication later than the step of translation of infecting viral genome.     

Altered response to viral infection by tobacco plants perturbed in ubiquitin system.

Tobacco plants can be perturbed in their ubiquitin system by expression of a ubiquitin variant with a lys to arg change at position 48. Plants expressing this ubiquitin variant have a tendency to form necrotic lesions resembling those occurring as defense reactions against some pathogens. Using the interaction between tobacco and tobacco mosaic virus (TMV), it was investigated whether the ubiquitin variant influences plant responses to pathogen infection. Expression of the ubiquitin variant leads to changes in the response of both resistant and susceptible tobacco varieties. Resistant plants form fewer, but darker lesions. In sensitive plants, TMV replication is inhibited, but not completely abolished. Moreover, susceptible plants expressing the ubiquitin variant induce PR-1 mRNA in response to virus, whereas controls do not express PR-1 under these conditions.     

Observations and experiments on the use of an avirulent mutant strain of tobacco mosaic virus as a means of controlling tomato mosaic

In 1973 tobacco mosaic virus (TMV) strain M II-16 was successfully used by growers in the United Kingdom to protect commercial tomato crops against the severe effects of naturally occurring strains of TMV. However, plants in many crops had mosaic leaf symptoms which were occasionally severe, so possible reasons for symptom appearance were examined. The concentration of the mutant strain in commercially produced inocula (assessed by infectivity and spectrophotometry) ranged from 28 to 1220 μg virus/ml; nevertheless all samples contained sufficient virus to infect a high percentage of inoculated tomato seedlings. Increasing the distance between the plants and the spray gun used for inoculation from 5 to 15 cm resulted in a significant decrease in the number of tomato seedlings infected. When M II-16 infected tomato plants were subsequently inoculated with each of fifty-three different isolates of TMV, none showed severe symptoms of the challenging isolates within 4 wk, although some isolates of strain o induced atypically mild leaf symptoms. In a further experiment, M II-16 infected plants showed conspicuous leaf symptoms only 7 wk after inoculation with a virulent TMV isolate. M II-16 multiplied more slowly in tomato plants and had a lower specific infectivity than a naturally occurring strain of TMV. More than 50% of plants in crops inoculated with strain M II-16 which subsequently showed conspicuous leaf mosaic contained TMV strain 1 or a form intermediate between strains o and 1. It is suggested that the production of TMV symptoms in commercial crops previously inoculated with strain M II-16 may result from an initially low level of infection, due to inefficient inoculation, which allows subsequent infection of unprotected plants by virulent strains. Incomplete protection by strain M II-16 against all naturally occurring strains may also be an important factor.