Down-regulation of Antioxidative Capacity in a Transgenic Tobacco which Fails to Develop Acquired Resistance to Necrotization Caused by TMV

Antioxidant status was assayed in leaves of two local lesion hosts of tobacco mosaic virus (TMV), namely in wild-type Xanthi-nc tobacco and in NahG transgenic tobacco, the latter of which is not able to accumulate salicylic acid (SA) and therefore is unable to develop systemic acquired resistance (SAR). Activities of several enzymes related to antioxidative defense, and the levels of glutathione, chlorogenic acid and rutin were studied. The majority of antioxidant enzymes were less active in uninfected NahG tobacco than in Xanthi-nc. Furthermore, important enzymatic and non-enzymatic antioxidants were down-regulated in TMV-infected NahG plants, as compared to Xanthi-nc. Correspondingly, SA pretreatment primed the leaves for stronger induction of antioxidants in infected Xanthi-nc, but not in NahG tobaccos. The antioxidant status of NahG tobacco even decreased after an attempted induction of SAR, while the antioxidative level increased in Xanthi-nc leaves in which the SAR was successfully induced. After infection, a greater accumulation of superoxide and H 2 O 2, and a more intensive necrotization was positively correlated with the reduced capability of NahG leaf tissue to detoxify reactive oxygen species.     

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.     

Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco.

Systemic induction of pathogenesis-related (PR) proteins in tobacco, which occurs during the hypersensitive response to tobacco mosaic virus (TMV), may be caused by a minimum 10-fold systemic increase in endogenous levels of salicylic acid (SA). This rise in SA parallels PR-1 protein induction and occurs in TMV-resistant Xanthi-nc tobacco carrying the N gene, but not in TMV-susceptible (nn) tobacco. By feeding SA to excised leaves of Xanthi-nc (NN) tobacco, we have shown that the observed increase in endogenous SA levels is sufficient for the systemic induction of PR-1 proteins. TMV infection became systemic and Xanthi-nc plants failed to accumulate PR-1 proteins at 32 degrees C. This loss of hypersensitive response at high temperature was associated with an inability to accumulate SA. However, spraying leaves with SA induced PR-1 proteins at both 24 and 32 degrees C. SA is most likely exported from the primary site of infection to the uninfected tissues. A computer model predicts that SA should move rapidly in phloem. When leaves of Xanthi-nc tobacco were excised 24 hr after TMV inoculation and exudates from the cut petioles were collected, the increase in endogenous SA in TMV-inoculated leaves paralleled SA levels in exudates. Exudation and leaf accumulation of SA were proportional to TMV concentration and were higher in light than in darkness. Different components of TMV were compared for their ability to induce SA accumulation and exudation: three different aggregation states of coat protein failed to induce SA, but unencapsidated viral RNA elicited SA accumulation in leaves and phloem. These results further support the hypothesis that SA acts as an endogenous signal that triggers local and systemic induction of PR-1 proteins and, possibly, some components of systemic acquired resistance in NN tobacco.     

Activity of nitric oxide is dependent on, but is partially required for function of, salicylic acid in the signaling pathway in tobacco systemic acquired resistance.

When tobacco plants were treated by injection with nitric oxide (NO)-releasing compounds, the sizes of lesions caused by Tobacco mosaic virus (TMV) on the treated leaves and on upper nontreated leaves were significantly reduced. The reduction in TMV lesion size was caused by NO released from the NO-releasing compounds; the byproduct formed after release of NO from the NO-releasing compound NOC-18, diethylenetriamine, did not itself alter lesion size. Treatment of tobacco plants with inhibitors of nitric oxide synthase or an NO scavenger attenuated but did not abolish the systemic acquired resistance (SAR) induced by salicylic acid (SA). In NahG transgenic tobacco plants, NO had no effect on lesion size following TMV infection. These results are consistent with the hypothesis that NO plays an important role in SAR induction in tobacco and that NO is required for the full function of SA as an SAR inducer. The activity of NO is fully dependent on the function of SA in the SAR signaling pathway in tobacco.     

Salicylic acid is not required for Cf-2- and Cf-9-dependent resistance of tomato to Cladosporium fulvum

Tomato leaves or cotyledons expressing the Cf-2 or Cf-9 Cladosporium fulvum resistance genes induce salicylic acid (SA) synthesis following infiltration with intercellular washing fluid (IF) containing the fungal peptide elicitors Avr2 and Avr9. We investigated whether SA was required for Cf gene-dependent resistance. Tomato plants expressing the bacterial gene nahG, encoding salicylate hydroxylase, did not accumulate SA in response to IF infiltration but remained fully resistant to C. fulvum. NahG Cf0 plants were as susceptible to C. fulvum as wild-type Cf0. Neither free nor conjugated salicylic acid accumulated in IF-infiltrated Cf2 and Cf9 NahG leaves and cotyledons but conjugated catechol did accumulate. The Cf-9-dependent necrotic response to IF was prevented in NahG plants and replaced by a chlorotic Cf-2-like response. SA also potentiated Cf-9-mediated necrosis in IF-infiltrated wild-type leaves. In contrast, the Cf-2-dependent IF response was retained in NahG leaves and chlorosis was more pronounced than in the wild-type. The distribution of cell death between different cell types was altered in both Cf2 and Cf9 NahG leaves after IF injection. IF-induced accumulation of three SA-inducible defence-related genes was delayed and reduced but not abolished in NahG Cf2 and Cf9 leaves and cotyledons. NahG Tm-22 tomato showed increased hypersensitive response (HR) lesion size upon TMV infection, as observed in TMV-inoculated N gene-containing NahG tobacco plants.     

Quantitative in situ assay of salicylic acid in tobacco leaves using a genetically modified biosensor strain of Acinetobacter sp. ADP1

Salicylic acid (SA) plays important roles in plants, most notably in the induction of systemic acquired resistance (SAR) against pathogens. A non-destructive in situ assay for SA would provide new insights into the functions of SA in SAR and other SA-regulated phenomena. We assessed a genetically engineered strain of Acinetobacter sp. ADP1, which proportionally produces bioluminescence in response to salicylates including SA and methylsalicylate, as a reporter for salicylate accumulation in the apoplast of plant leaves. SA was measured quantitatively in situ in NN genotype tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves inoculated with tobacco mosaic virus (TMV). The biosensor revealed accumulation of apoplastic SA before the visible appearance of hypersensitive response (HR) lesions. When the biosensor was infiltrated into TMV-inoculated leaves displaying HR lesions at 90 and 168 h post-inoculation, salicylate accumulation was detected predominantly in tissues surrounding the lesions and in veins adjacent to HR lesions. These images are consistent with previous data demonstrating that SA accumulation occurs prior to and following the onset of visible HR lesions. We also used the biosensor to observe apoplastic SA accumulation in tobacco leaves inoculated with virulent and HR-eliciting strains of the bacterial plant pathogen Pseudomonas syringae. The work demonstrates that the Acinetobacter sp. ADP1 biosensor is a useful new tool to non-destructively assay salicylates in situ and to map their spatial distribution in plant tissues.     

Is Salicylic Acid a Translocated Signal of Systemic Acquired Resistance in Tobacco?

Salicylic acid (SA) is a likely endogenous signal in the development of systemic acquired resistance (SAR) in some dicotyledonous plants. In tobacco mosaic virus (TMV)-resistant Xanthi-nc tobacco, SA levels increase systemically following the inoculation of a single leaf with TMV. To determine the extent to which systemic increases in SA result from SA export from the inoculated leaf, SA produced in TMV-inoculated or healthy leaves was noninvasively labeled with 18O2. Spatial and temporal distribution of 18O-SA indicated that most of the SA detected in the healthy tissues was synthesized in the inoculated leaf. No significant increase in the activity of benzoic acid 2-hydroxylase, the last enzyme involved in SA biosynthesis, was detected in upper uninoculated leaves, although the basal level of enzyme activity was relatively high. No increases in SA level, pathogenesis-related PR-1 gene expression, or TMV resistance in the upper uninoculated leaf were observed if the TMV-inoculated leaf was detached up to 60 hr after inoculation. Apart from the inoculated tissues, the highest increase in SA was observed in the leaf located directly above the inoculated leaf. The systemic SA increase observed during SAR may be explained by phloem transport of SA from the inoculation sites.     

Artificial elevation of glutathione contents in salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) reduces susceptibility to the powdery mildew pathogen Euoidium longipes

• The effects of elevated glutathione levels on defence responses to powdery mildew (Euoidium longipes) were investigated in a salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) and wild‐type cv. Xanthi plants, where salicylic acid (SA) contents are normal.
• Aqueous solutions of reduced glutathione (GSH) and its synthetic precursor R‐2‐oxothiazolidine‐4‐carboxylic acid (OTC) were injected into leaves of tobacco plants 3 h before powdery mildew inoculation.
• SA‐deficient NahG tobacco was hyper‐susceptible to E. longipes, as judged by significantly more severe powdery mildew symptoms and enhanced pathogen accumulation. Strikingly, elevation of GSH levels in SA‐deficient NahG tobacco restored susceptibility to E. longipes to the extent seen in wild‐type plants (i.e. enhanced basal resistance). However, expression of the SA‐mediated pathogenesis‐related gene (NtPR‐1a) did not increase significantly in GSH or OTC‐pretreated and powdery mildew‐inoculated NahG tobacco, suggesting that the induction of this PR gene may not be directly involved in the defence responses induced by GSH.
• Our results demonstrate that artificial elevation of glutathione content can significantly reduce susceptibility to powdery mildew in SA‐deficient tobacco.

     

Pyrazolecarboxylic acid derivative induces systemic acquired resistance in tobacco

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through asalicylic acid (SA)-mediated pathway. Here, we characterized 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA) as an effective SAR inducer in tobacco. Soil drench application of CMPA induced PR gene expression and a broad range of disease resistance without antibacterial activity in tobacco. Both analysis of CMPA's effects on NahG transgenic tobacco plants and SA measurement in wild-type plants indicated that CMPA-induced resistance enhancement does not require SA. Therefore, it is suggested that CMPA induces SAR by triggering the signaling at the same level as or downstream of SA accumulation as do both benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester and N-cyanomethyl-2-chloroisonicotinamide.     

Chloroisonicotinamide derivative induces a broad range of disease resistance in rice and tobacco

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of pathogens. SAR in dicotyledonous plants such as tobacco and Arabidopsis has been partially elucidated and is mediated by salicylic acid (SA). However, the SAR mechanism of monocotyledonous rice plants remains to be clarified, although some similarities between SAR mechanisms in both types have been reported. Here we have characterized N-cyanomethyl-2-chloroisonicotinamide (NCI) as an effective SAR inducer in both plant species. Soil drench application of NCI induces a broad range of disease resistance in tobacco and rice and, more specifically, PR gene expression in tobacco. Both SA measurements in wild-type NCI-treated tobacco and pathogenic infection studies using NahG transgenic tobacco plants indicate that NCI-induced resistance enhancement does not require SA. Therefore, it is suggested that NCI induces SAR by triggering signaling at the same level as or downstream of SA accumulation as do both benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester and 2,6-dichloroisonicotinic acid. The fact that all of these chemicals are effective in rice and tobacco suggests that several common components function in disease resistance in both plant species.     

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.     

2种植物生长调节剂诱导烟草抗链格孢菌研究

以链格孢菌Alternaria alternata和烟草Nicotiana tabacum品种云烟87为试材,研究不同浓度茉莉酸甲酯(MeJA)和水杨酸(SA)处理下,链格孢菌菌丝生长情况以及成熟期烟叶防御酶活性与多酚含量变化,探讨植物生长调节剂对烟草抗链格孢菌的影响。结果表明,1 mmol·L^–1 MeJA对链格孢菌的抑制效果最好,抑菌率高达59%以上,其次是3.5 mmol·L^–1SA;MeJA和SA对链格孢菌的抑制效果随药剂浓度升高而增强;0.1 mmol·L^–1 MeJA和2.5 mmol·L^–1 SA能诱导烟草叶片SOD、POD、CAT等防御酶活性,并能降低H₂O₂活性氧含量,尤其以MeJA诱导效果较好。2种植物生长调节剂处理并接种链格孢菌能诱导提高烟叶多酚代谢相关酶PPO及PAL活性,但对烟草多酚类物质影响较小;成熟烟叶中含量较高的前3种多酚物质是绿原酸、芸香苷、隐绿原酸。     

Interconversion of the salicylic acid signal and its glucoside in tobacco

Salicylic acid (SA) has been proposed to play a role in the induction of pathogenesis-related (PR) proteins and systemic acquired resistance (SAR) in tobacco. Since SA is rapidly converted to salicylic acid β-glucoside (SAG) in tobacco, we have attempted to assess the role of SAG in pathogenesis by application of chemically synthesized SAG to tobacco leaves. SAG was as active as SA in induction of PR-1 gene expression. This induction was preceded by a transient release of SA, which occurred in the extracellular spaces. The existence of a mechanism that releases SA from SAG suggests a possible role for SAG in SAR.     

Pathway of Salicylic Acid Biosynthesis in Healthy and Virus-Inoculated Tobacco

Salicylic acid (SA) is a likely endogenous regulator of localized and systemic disease resistance in plants. During the hypersensitive response of Nicotiana tabacum L. cv Xanthi-nc to tobacco mosaic virus (TMV), SA levels rise dramatically. We studied SA biosynthesis in healthy and TMV-inoculated tobacco by monitoring the levels of SA and its likely precursors in extracts of leaves and cell suspensions. In TMV-inoculated leaves, stimulation of SA accumulation is accompanied by a corresponding increase in the levels of benzoic acid. 14C-Tracer studies with cell suspensions and mock-or TMV-inoculated leaves indicate that the label moves from trans-cinnamic acid to SA via benzoic acid. In healthy and TMV-inoculated tobacco leaves, benzoic acid induced SA accumulation. o-Coumaric acid, which was previously reported as a possible precursor of SA in other species, did not increase SA levels in tobacco. In healthy tobacco tissue, the specific activity of newly formed SA was equal to that of the supplied [14C]benzoic acid, whereas in TMV-inoculated leaves some isotope dilution was observed, presumably because of the increase in the pool of endogenous benzoic acid. We observed accumulation of pathogen-esis-related-1 proteins and increased resistance to TMV in benzoic acid- but not in o-coumaric acid-treated tobacco leaves. This is consistent with benzoic acid being the immediate precursor of SA. We conclude that in healthy and virus-inoculated tobacco, SA is formed from cinnamic acid via benzoic acid.     

Induction of Benzoic Acid 2-Hydroxylase in Virus-Inoculated Tobacco

Salicylic acid (SA) plays an important role in the induction of plant resistance to pathogens. An accompanying article (N. Yalpani, J. Leon, M.A. Lawton, I. Raskin [1993] Plant Physiol 103: 315-321) shows that SA is synthesized via the decarboxylation of cinnamic acid to benzoic acid (BA), which is, in turn, hydroxylated to SA. Leaf extracts of tobacco (Nicotiana tabacum L. cv Xanthi-nc) catalyze the 2-hydroxylation of BA to SA. The monooxygenase catalyzing this reaction, benzoic acid 2-hydroxylase (BA2H), required NAD(P)H or reduced methyl viologen as an electron donor. BA2H activity was detected in healthy tobacco leaf extracts (1-2 nmol h-1 g-1 fresh weight) and was significantly increased upon inoculation with tobacco mosaic virus (TMV). This increase paralleled the levels of free SA in the leaves. Induction of BA2H activity was restricted to tissue expressing a hypersensitive response at 24[deg]C. TMV induction of BA2H activity and SA accumulation were inhibited when inoculated tobacco plants were incubated at 32[deg]C. However, when inoculated plants were incubated for 4 d at 32[deg]C and then transferred to 24[deg]C, they showed a 15-fold increase in BA2H activity and a 65-fold increase in free SA content compared with healthy plants incubated at 24[deg]C. Treatment of leaf tissue with the protein synthesis inhibitor cycloheximide blocked the induction of BA2H activity by TMV. The effect of TMV inoculation on BA2H could be duplicated by infiltrating leaf discs of healthy plants with BA. This response was observed even when applied levels of BA were much lower than the levels observed in vivo after virus inoculation. Feeding tobacco leaves with phenylalanine, cinnamic acid, or o-coumaric acid (putative precursors of SA) failed to trigger the induction of BA2H activity. BA2H appears to be a pathogen-inducible protein with an important regulatory role in SA accumulation during the development of induced resistance to TMV in tobacco.     

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.     

Activation of a diverse set of genes during the tobacco resistance response to TMV is independent of salicylic acid; induction of a subset is also ethylene independent

Through differential screening of a cDNA library, we cloned six groups of genes that are expressed relatively early in the inoculated leaves of tobacco resisting infection by tobacco mosaic virus (TMV). Induction of all these genes was subsequently detected in the uninoculated leaves; thus, their expression is associated with the development of both local and systemic acquired resistance. Exogenously applied salicylic acid (SA) was observed to induce these genes transiently. However, analyses with transgenic NahG plants, which are unable to accumulate SA, demonstrated that expression of these genes in TMV-inoculated leaves is mediated via an SA-independent pathway. Because the expression kinetics of these genes differ from those associated with the well-characterized pathogenesis-related protein (PR-1) and phenylalanine ammonia-lyase (PAL) genes, we propose that they belong to a group which we designate SIS, for SA-independent, systemically induced genes. Interestingly, the expression of several SIS genes in the uninoculated leaves of TMV-infected NahG plants was delayed and/or reduced, raising the possibility that SA is involved in activating some of these genes in systemic tissue. Most of the SIS genes were induced by exogenous ethylene. However, analyses of infected NahG plants treated with ethylene action and/or synthesis inhibitors indicated that the TMV-induced expression of several SIS genes is independent of ethylene as well as SA.