Phenylpropanoid compounds and disease resistance in transgenic tobacco with altered expression of L-phenylalanine ammonia-lyase

Tobacco plants over-expressing L-phenylalanine ammonia-lyase (PAL(+)) produce high levels of chlorogenic acid (CGA) and exhibit markedly reduced susceptibility to infection with the fungal pathogen Cercospora nicotianae, although their resistance to tobacco mosaic virus (TMV) is unchanged. Levels of the signal molecule salicylic acid (SA) were similar in uninfected PAL(+) and control plants and also following TMV infection. In crosses of PAL(+) tobacco with tobacco harboring the bacterial NahG salicylate hydroxylase gene, progeny harboring both transgenes lost resistance to TMV, indicating that SA is critical for resistance to TMV and that increased production of phenylpropanoid compounds such as CGA cannot substitute for the reduction in SA levels. In contrast, PAL(+)/NahG plants showed strongly reduced susceptibility to Cercospora nicotianae compared to the NahG parent line. These results are consistent with a recent report questioning the role of PAL in SA biosynthesis in Arabidopsis, and highlight the importance of phenylpropanoid compounds such as CGA in plant disease resistance.     

Inverse relationship between systemic resistance of plants to microorganisms and to insect herbivory

Pre-inoculation of plants with a pathogen that induces necrosis leads to the development of systemic acquired resistance (SAR) to subsequent pathogen attack [1]. The phenylpropanoid-derived compound salicylic acid (SA) is necessary for the full expression of both local resistance and SAR [2] [3]. A separate signaling pathway involving jasmonic acid (JA) is involved in systemic responses to wounding and insect herbivory [4] [5]. There is evidence both supporting and opposing the idea of cross-protection against microbial pathogens and insect herbivores [6] [7]. This is a controversial area because pharmacological experiments point to negative cross-talk between responses to systemic pathogens and responses to wounding [8] [9] [10], although this has not been demonstrated functionally in vivo. Here, we report that reducing phenylpropanoid biosynthesis by silencing the expression of phenylalanine ammonialyase (PAL) reduces SAR to tobacco mosaic virus (TMV), whereas overexpression of PAL enhances SAR. Tobacco plants with reduced SAR exhibited more effective grazing-induced systemic resistance to larvae of Heliothis virescens, but larval resistance was reduced in plants with elevated phenylpropanoid levels. Furthermore, genetic modification of components involved in phenylpropanoid synthesis revealed an inverse relationship between SA and JA levels. These results demonstrate phenylpropanoid-mediated cross-talk in vivo between microbially induced and herbivore-induced pathways of systemic resistance.     

PeaT1-induced systemic acquired resistance in tobacco follows salicylic acid-dependent pathway

Systemic acquired resistance (SAR) is an inducible defense mechanism which plays a central role in protecting plants from pathogen attack. A new elicitor, PeaT1 from Alternaria tenuissima, was expressed in Escherichia coil and characterized with systemic acquired resistance to tobacco mosaic virus (TMV). PeaT1-treated plants exhibited enhanced systemic resistance with a significant reduction in number and size of TMV lesions on wild tobacco leaves as compared with control. The quantitative analysis of TMV CP gene expression with real-time quantitative PCR showed there was reduction in TMV virus concentration after PeaT1 treatment. Similarly, peroxidase (POD) activity and lignin increased significantly after PeaT1 treatment. The real-time quantitative PCR revealed that PeaT1 also induced the systemic accumulation of pathogenesis-related gene, PR-1a and PR-1b which are the markers of systemic acquired resistance (SAR), NPR1 gene for salicylic acid (SA) signal transduction pathway and PAL gene for SA synthesis. The accumulation of SA and the failure in development of similar level of resistance as in wild type tobacco plants in PeaT1 treated nahG transgenic tobacco plants indicated that PeaT1-induced resistance depended on SA accumulation. The present work suggested that the molecular mechanism of PeaT1 inducing disease resistance in tobacco was likely through the systemic acquired resistance pathway mediated by salicylic acid and the NPR1 gene.     

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 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.     

Sulfated fucan oligosaccharides elicit defense responses in tobacco and local and systemic resistance against tobacco mosaic virus

Sulfated fucans are common structural components of the cell walls of marine brown algae. Using a fucan-degrading hydrolase isolated from a marine bacterium, we prepared sulfated fucan oligosaccharides made of mono- and disulfated fucose units alternatively bound by alpha-1,4 and alpha-1,3 glycosidic linkages, respectively. Here, we report on the elicitor activity of such fucan oligosaccharide preparations in tobacco. In suspension cell cultures, oligofucans at the dose of 200 microg ml(-1) rapidly induced a marked alkalinization of the extracellular medium and the release of hydrogen peroxide. This was followed within a few hours by a strong stimulation of phenylalanine ammonia-lyase and lipoxygenase activities. Tobacco leaves treated with oligofucans locally accumulated salicylic acid (SA) and the phytoalexin scopoletin and expressed several pathogenesis-related (PR) proteins, but they displayed no symptoms of cell death. Fucan oligosaccharides also induced the systemic accumulation of SA and the acidic PR protein PR-1, two markers of systemic acquired resistance (SAR). Consistently, fucan oligosaccharides strongly stimulated both local and systemic resistance to tobacco mosaic virus (TMV). The use of transgenic plants unable to accumulate SA indicated that, as in the SAR primed by TMV, SA is required for the establishment of oligofucan-induced resistance.     

Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway

Pharmacological evidence implicates trans-cinnamic acid as a feedback modulator of the expression and enzymatic activity of the first enzyme in the phenylpropanoid pathway, L-phenylalanine ammonia-lyase (PAL). To test this hypothesis independently of methods that utilize potentially non-specific inhibitors, we generated transgenic tobacco lines with altered activity levels of the second enzyme of the pathway, cinnamic acid 4-hydroxylase (C4H), by sense or antisense expression of an alfalfa C4H cDNA. PAL activity and levels of phenylpropanoid compounds were reduced in leaves and stems of plants in which C4H activity had been genetically down-regulated. However, C4H activity was not reduced in plants in which PAL activity had been down-regulated by gene silencing. In crosses between a tobacco line over-expressing PAL from a bean PAL transgene and a C4H antisense line, progeny populations harboring both the bean PAL sense and C4H antisense transgenes had significantly lower extractable PAL activity than progeny populations harboring the PAL transgene alone. Our data provide genetic evidence for a feedback loop at the entry point into the phenylpropanoid pathway that had previously been inferred from potentially artifactual pharmacological experiments.     

Brassinosteroid functions in a broad range of disease resistance in tobacco and rice

Brassinolide (BL), considered to be the most important brassinosteroid (BR) and playing pivotal roles in the hormonal regulation of plant growth and development, was found to induce disease resistance in plants. To study the potentialities of BL activity on stress responding systems, we analyzed its ability to induce disease resistance in tobacco and rice plants. Wild-type tobacco treated with BL exhibited enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp. The measurement of salicylic acid (SA) in wild-type plants treated with BL and the pathogen infection assays using NahG transgenic plants indicate that BL-induced resistance does not require SA biosynthesis. BL treatment did not induce either acidic or basic pathogenesis-related (PR) gene expression, suggesting that BL-induced resistance is distinct from systemic acquired resistance (SAR) and wound-inducible disease resistance. Analysis using brassinazole 2001, a specific inhibitor for BR biosynthesis, and the measurement of BRs in TMV-infected tobacco leaves indicate that steroid hormone-mediated disease resistance (BDR) plays part in defense response in tobacco. Simultaneous activation of SAR and BDR by SAR inducers and BL, respectively, exhibited additive protective effects against TMV and Pst, indicating that there is no cross-talk between SAR- and BDR-signaling pathway downstream of BL. In addition to the enhanced resistance to a broad range of diseases in tobacco, BL induced resistance in rice to rice blast and bacterial blight diseases caused by Magnaporthe grisea and Xanthomonas oryzae pv. oryzae, respectively. Our data suggest that BDR functions in the innate immunity system of higher plants including dicotyledonous and monocotyledonous species.     

Biological and molecular comparison between localized and systemic acquired resistance induced in tobacco by a Phytophthora megasperma glycoprotein elicitin

We have compared localized (LAR) and systemic (SAR) acquired resistance induced in tobacco by a hypersensitive response (HR) inducing Phytophthora megasperma glycoprotein elicitin. Three different zones were taken into account: LAR, SAR_T and SAR_S. The LAR zone was 5–10 mm wide and surrounded the HR lesion. SAR_T was the tissue of the elicitor-treated leaf immediately beyond the LAR zone. The systemic leaf was called SAR_S. Glycoprotein-treated plants showed enhanced resistance to challenge infection by tobacco mosaic virus (TMV). Disease resistance was similar in SAR_T and SAR_S, and higher in LAR. The expression pattern, in glycoprotein-treated plants, of acidic and basic PR1, PR2, PR3 and PR5 proteins and of O-methyltransferases (OMT), enzymes of the phenylpropanoid pathway, was similar to that in TMV-infected plants. OMT was stimulated in LAR but not in SAR_T and SAR_S. The four classes of acidic and basic PR proteins accumulated strongly in LAR. Reduced amounts of acidic PR1, PR2, PR3 and only minute amounts of basic PR2 and PR3 accumulated in SAR_T and SAR_S. In glycoprotein-treated plants, expression of the acidic and basic PR proteins in LAR and SAR of transgenic NahG and ETR tobacco plants and in LAR of plants treated with inhibitors of salicylic acid accumulation and of ethylene biosynthesis indicated a salicylic acid-dependent signalling pathway for acidic isoform activation and an ethylene-dependent signalling pathway for basic isoform activation.     

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.     

Defense and resistance-inducing activities in tobacco of the sulfated beta-1,3 glucan PS3 and its synergistic activities with the unsulfated molecule

Laminarin, a beta-1,3 glucan with single beta-glucose branches at position 6, was chemically sulfated to produce PS3 with a degree of sulfation of 2.4. PS3 has previously been shown to activate the salicylic acid (SA) signaling pathway in infiltrated tobacco and Arabidopsis thaliana leaf tissues. Here, we investigated whether PS3 induces systemic defense and resistance responses in tobacco. Using a radiolabeled compound, it was first demonstrated that PS3 remains strictly localized to the infiltrated tissues. PS3 is also resistant to beta-glucanase degradation. In transgenic PR1-beta-glucuronidase (GUS) tobacco plants, PS3 causes a strong increase in GUS activity in treated tissues but none in untreated leaves. PS3-infiltrated tissues challenged with tobacco mosaic virus (TMV) 8 d after elicitor application show a decrease in both the lesion number and the lesion size, whereas treatment with laminarin, the unsulfated native glucan, affected only the lesion number. PS3 does not induce systemic acquired resistance to TMV. PS3 and laminarin show synergistic effects in promoting the oxidative burst in tobacco cell suspensions and in increasing the expression of genes encoding O-methyltransferases of the phenylpropanoid pathway in tobacco plants. No synergistic effect was observed on the expression of either the SA-dependent acidic PR1 gene or the ethylene-dependent basic PR5 gene in tobacco plants.     

Mutation of GT-1 binding sites in the Pr-1A promoter influences the level of inducible gene expression in vivo

Infection of Nicotiana tabacum Samsun NN with tobacco mosaic virus (TMV) results in a hypersensitive plant response and leads to systemic acquired resistance (SAR). The induction of SAR is mediated by the plant hormone salicylic acid (SA) and is accompanied by the induced expression of a number of genes including the pathogenesis-related (PR) gene 1a. Previously, it has been found that TMV infection and SA treatment resulted in a reduction of binding of nuclear protein GT-1 to far-upstream regions (–902 to –656) of the PR-1a gene. To test if GT-1 is a negative regulator of PR-1a gene expression, the effects of mutations in the seven putative GT-1 binding sites in this region were studied in vitro using dimethyl sulfate interference footprinting and band shift assays. This showed that at least one of the seven sites is indeed a GT-1 binding site. However, when tested in transgenic plants, the mutations did not result in constitutive expression of the chimeric PR-1a/GUS transgene, while inducible expression after SA treatment was decreased. The results suggest that binding of GT-1-like proteins to far-upstream PR-1a promoter regions indeed influences gene expression. A possible model for GT-1's mode of action in PR-1a gene expression is discussed.     

Changes in phenolic metabolism of tobacco plants during short-term boron deficiency

The effects of boron (B) deficiency on several phenolics and enzyme activities involved in the biosynthesis of these compounds were investigated in tobacco plants (Nicotiana tabacum L. cv. Gatersleben). The levels of phenylpropanoids (mainly the caffeic acid esters, chlorogenic acid and its isomers) as well as phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and polyphenoloxidase (PPO, EC 1.14.18.1) activities were determined in plants subjected to B starvation for 1–7 d. The results presented here show that a short-term B deficiency causes both quantitative and qualitative changes in the phenolic metabolism of tobacco plants, which are especially evident after 3 d of B starvation. Although the concentration of B decreased from the onset of B starvation, root B level was less affected than leaf B by a short-term B deficiency. The concentration of phenylpropanoids as well as PAL and PPO activities increased mainly in the leaves of tobacco plants during B starvation. Moreover, leaves starved of B for 7 d showed the accumulation of new compounds, one of which was identified as caffeoylputrescine. In addition, a positive correlation between PAL activity and phenylpropanoid concentration was observed in tobacco leaves, especially after 5–7 d of B starvation, suggesting that an increase in PAL activity during B starvation could be responsible for the enhancement in the levels of phenylpropanoids.     

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.     

A new catalytic activity from tobacco converting 2-coumaric acid to salicylic aldehyde

Salicylic acid (SA) mediates plant response to pathogen invasion, resulting in hypersensitive response and in the formation of systemic acquired resistance. It is well known that Nicotiana tabacum and other plants respond to Tobacco Mosaic Virus (TMV) infection by increasing the content of SA but the details of SA biosynthesis are still not fully understood. Generally, SA may originate directly from isochorismate ( Arabidopsis thaliana ), or its C₆–C₁ skeleton could be synthesized via the phenylpropanoid pathway by β-oxidation of trans -cinnamic acid ( N. tabacum ), 2-coumaric acid (OCA) ( Gaulteria procumbens , Lycopersicum esculentum ) or by retro-aldol reaction of trans -cinnamoyl-CoA ( Hypericum androsaemum ). We report here a novel putative enzyme activity from tobacco, salicylic aldehyde synthase (SAS), catalysing non-oxidative formation of salicylic aldehyde (SALD) directly from OCA. This chain-shortening activity is similar to that of 4-hydroxybenzaldehyde synthase from Vanilla planifolia , Lithospermum erythrorhizon , Daucus carota , Solanum tuberosum and Polyporus hispidus but the enzyme differs in the kinetics of the reaction, substrate specificity and requirements for reducing cofactors. SAS activity is constitutively expressed in healthy tobacco leaves and doubles as a result of infection with TMV. Moreover, the product of SAS activity—SALD, applied exogenously on tobacco leaves, stimulates peroxidase activity and enhances resistance to consecutive infection with TMV. These observations could suggest a contribution of SAS and SALD to the response of tobacco to TMV infection.     

Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance

After a hypersensitive response to invading pathogens, plants show elevated accumulation of salicylic acid (SA), induced expression of plant defense genes, and systemic acquired resistance (SAR) to further infection by a broad range of pathogens. There is compelling evidence that SA plays a crucial role in triggering SAR. We have transformed tobacco with two bacterial genes coding for enzymes that convert chorismate into SA by a two-step process. When the two enzymes were targeted to the chloroplasts, the transgenic (CSA, constitutive SA biosynthesis) plants showed a 500- to 1,000-fold increased accumulation of SA and SA glucoside compared to control plants. Defense genes, particularly those encoding acidic pathogenesis-related (PR) proteins, were constitutively expressed in CSA plants. This expression did not affect the plant phenotype, but the CSA plants showed a resistance to viral and fungal infection resembling SAR in nontransgenic plants.     

A strobilurin fungicide enhances the resistance of tobacco against tobacco mosaic virus and Pseudomonas syringae pv tabaci

The strobilurin class of fungicides comprises a variety of synthetic plant-protecting compounds with broad-spectrum antifungal activity. In the present study, we demonstrate that a strobilurin fungicide, F 500 (Pyraclostrobin), enhances the resistance of tobacco (Nicotiana tabacum cv Xanthi nc) against infection by either tobacco mosaic virus (TMV) or the wildfire pathogen Pseudomonas syringae pv tabaci. F 500 was also active at enhancing TMV resistance in NahG transgenic tobacco plants unable to accumulate significant amounts of the endogenous inducer of enhanced disease resistance, salicylic acid (SA). This finding suggests that F 500 enhances TMV resistance in tobacco either by acting downstream of SA in the SA signaling mechanism or by functioning independently of SA. The latter assumption is the more likely because in infiltrated leaves, F 500 did not cause the accumulation of SA-inducible pathogenesis-related (PR)-1 proteins that often are used as conventional molecular markers for SA-induced disease resistance. However, accumulation of PR-1 proteins and the associated activation of the PR-1 genes were elicited upon TMV infection of tobacco leaves and both these responses were induced more rapidly in F 500-pretreated plants than in the water-pretreated controls. Taken together, our results suggest that F 500, in addition to exerting direct antifungal activity, may also protect plants by priming them for potentiated activation of subsequently pathogen-induced cellular defense responses.     

Signaling of systemic acquired resistance in tobacco depends on ethylene perception

The hypersensitive interaction between Tobacco mosaic virus (TMV) and tobacco results in accumulation of salicylic acid (SA), defense gene expression, and development of systemic acquired resistance (SAR) in uninfected leaves. The plant hormones SA and ethylene have been implicated in SAR. From a study with ethylene-insensitive (Tetr) tobacco, we concluded that ethylene perception is required to generate the systemic signal molecules in TMV-infected leaves that trigger SA accumulation, defense gene expression, and SAR development in uninfected leaves. Ethylene perception was not required for the responses of the plant to the systemic signal that leads to SAR development.