Hydrogen peroxide does not function downstream of salicylic acid in the induction of PR protein expression

The roles of salicylic acid (SA) and H₂O₂ in the induction of PR proteins in tobacco have been examined. Studies were conducted on wild-type tobacco and plants engineered to express a bacterial salicylate hydroxylase capable of metabolizing SA to catechol (SH-L plants). Wild-type and PR-1a—GUS-transformed plants express PR-1a following challenge with Pseudomonas syringae pathovar syringae , SA or 2,6-dichloro-isonicotinic acid (INA). In contrast, SH-L plants failed to respond to SA but did express PR-1a following INA treatment. H₂O₂ and the irreversible catalase inhibitor 3-amino-1,2,4-triazole (3-AT) were found to be weak inducers of PR-1a expression (relative to SA) in wild-type tobacco but were unable to induce PR-1a in SH-L plants, suggesting that the action of these compounds depends upon the accumulation of SA. A model has been proposed suggesting that SA binds to and inhibits a catalase inducing an increase in H₂O₂ leading to PR protein expression. Catalase activity has been measured in tobacco and no significant changes in activity following infection with P. syringae pv. syringae were detected. Furthermore, inhibition of catalase activity in vitro in plant extracts requires pre-incubation and only occurs at SA concentrations above 250 µM. Leaf disks pre-incubated with 1 mM SA do accumulate SA to these levels and PR-1a is efficiently induced but there is no apparent inhibition of catalase activity. It is also shown that a SA-responsive gene, PR-1a, and a H₂O₂-sensitive gene, AoPR-1, are both relatively insensitive to 3-AT suggesting that induction of these genes is unlikely to be due entirely to inhibition of an endogenous catalase.     

Salicylic acid and the plant pathogen Erwinia carotovora induce defense genes via antagonistic pathways

Infection of tobacco plants with the plant pathogenic bacterium Erwinia carotovora subsp. carotovora or treatment of plants with Erwinia -derived elicitor preparations leads to the induction of a number of genes thought to play a role in plant defense response to pathogens. In order to determine the role of salicylic acid (SA) in the induction of the Erwinia responsive genes, the accumulation of mRNAs for these and other genes encoding pathogenesis-related proteins (PR genes) in response to both Erwinia elicitors and SA was determined. PR genes were identified which were preferentially induced by Erwinia elicitor preparations, one gene was induced by SA but not by Erwinia , and another gene was induced by both type of treatments. The differential expression of these genes and the timing of induction suggest that SA is not the signal molecule leading to the early response of plants to Erwinia . This was demonstrated by experiments using transgenic NahG plants that overproduce a salicylate hydroxylase inactivating SA. The elicitation of PR genes by Erwinia was similar in NahG and wild-type plants. Therefore, induction of plant defense genes by Erwinia and SA seems to be by two distinct pathways leading to expression of separate sets of genes. Furthermore, we could demonstrate that Erwinia elicitors antagonize the SA-mediated induction of PR genes. Similarly, SA appeared to inhibit the induction of PR genes elicited by Erwinia . The observed antagonism between the two signal transduction pathways indicates the presence of a common regulatory element in both pathways that acts downstream of SA in the SA-mediated response.     

Growth-Retarding Effect of 2-Aminoindan-2-phosphonic Acid on Spirodela punctata

2-Aminoindan-2-phosphonic acid (AIP) retarded the growth of duckweed, Spirodela punctata, and increased its dry mass. The accumulation of starch was observed at all concentrations of AIP at 8 days after treatment. The increase in starch was inversely proportional to the growth. The retarded growth of Spirodela by AIP was not limited only by excessive starch accumulation. Received December 8, 1997; accepted June 2, 1998     

Involvement of protein kinases and calcium in the * NO-signalling cascade for defence-gene induction in ozonated tobacco plants

This study analyses the signalling pathways triggered by nitric oxide (NO) in response to ozone (O(3)) fumigation of tobacco plants, with particular attention to protein kinase cascades and free cytosolic Ca(2+) in defence-gene activation. NO was visualized with the NO probe DAF-FM. Using a pharmacological approach, the effects of different inhibitors on the expression profiles of NO-dependent defence genes were monitored using RT-PCR. The assay of the kinase activity of the immunoprecipitates complexes shows that O(3) stimulates a 48 kDa salicylic acid (SA)-induced protein kinase (SIPK) in an NO-dependent manner. The O(3)-induced alternative oxidase 1a (AOX1a) and phenylalanine ammonia lyase a (PALa) genes are modulated by phosphorylation by protein kinases, and SIPK might have a role in this up-regulation. By contrast, protein dephosphorylation mediates pathogenesis-related protein 1a (PR1a) expression in O(3)-treated tobacco plants. Ca(2+) is essential, but not sufficient, to promote NO accumulation in ozonated tobacco plants. Intracellular Ca(2+) transients are also essential for PALa up-regulation and cGMP-induced PR1a expression. Partial dependence on intracellular Ca(2+) suggests two different pathways of SA accumulation and PR1a induction. A model summarizing the signalling networks involving NO, SA, and the cellular messengers in this O(3)-induced defence gene activation is proposed.     

Constitutive expression of Arabidopsis NPR1 confers enhanced resistance to the early instars of Spodoptera litura in transgenic tobacco

In Arabidopsis , NPR1 ( AtNPR1 ) regulates salicylic acid (SA)-mediated activation of PR genes at the onset of systemic acquired resistance. AtNPR1 also modulates SA-induced suppression of jasmonic acid-responsive gene expression, and npr1 mutants manifest enhanced herbivore resistance. We have raised stable transgenic tobacco lines, expressing AtNPR1 constitutively, which showed elevated expression of PR1 and PR2 genes upon SA treatment. Herbivore bioassays with a generalist polyphagous pest, Spodoptera litura , revealed that the transgenic lines exhibited enhanced resistance compared to the wild-type plants, particularly with respect to younger larval populations. Insect-mediated injury induced several protease inhibitors (PIs), more significantly a 40-kDa serine PI in all the tobacco lines, but the induction was higher in the transgenic plants. We show in this communication that heterologous expression of AtNPR1 provides enhanced resistance to early larval populations of the herbivore, Spodoptera in transgenic tobacco plants.     

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.     

Production of 6-methylsalicylic acid by expression of a fungal polyketide synthase activates disease resistance in tobacco

Salicylic acid (SA) has been shown to act as a signal molecule that is produced by many plants subsequent to the recognition of potentially pathogenic microbes. Increases in levels of SA often trigger the activation of plant defenses and can result in increased resistance to subsequent challenge by pathogens. We observed that the polyketide 6-methylsalicylic acid (6-MeSA), a compound that apparently is not endogenous to tobacco, can mimic SA. Tobacco leaves treated with 6-MeSA show enhanced accumulation of the pathogenesis-related (PR) proteins PR1, beta-1,3-glucanase, and chitinase and also develop increased resistance to tobacco mosaic virus. We transformed tobacco with 6msas, the 6-methylsalicylic acid synthase (6MSAS) gene from Penicillium patulum, to generate plants that constitutively accumulate 6-MeSA. Analysis of primary transformants and the first generation progeny of 6MSAS tobacco revealed that plants can be engineered to accumulate significant amounts of 6-MeSA as a conjugate. Levels of total 6-MeSA increased with plant age. Increased 6-MeSA accumulation correlated with increased levels of PR1 and chitinase proteins and resulted in enhanced resistance of NN genotype 6MSAS tobacco to tobacco mosaic virus. Our results demonstrate that a multistep biosynthetic pathway can be engineered into plants using a single fungal polyketide synthase gene. The functional expression of 6msas can be used to activate disease resistance pathways that normally are induced by SA.     

Local and Systemic Biosynthesis of Salicylic Acid in Infected Cucumber Plants

Radiolabeling studies showed that salicylic acid (SA), an essential component in the signal transduction pathway leading to systemic acquired resistance, is synthesized from phenylalanine (Phe) and benzoic acid in cucumber (Cucumis sativus L.) plants inoculated with pathogens. Leaf discs from plants inoculated with either tobacco necrosis virus or Pseudomonas lachrymans incorporated more [14C]Phe into [14C]SA than mock-inoculated controls. The identity of SA was confirmed by gas chromatography-mass spectrometry. No reduction in specific activity of [14C]SA was observed for either free or bound SA between control and infected plants after feeding [14C]Phe. A specific inhibitor of Phe ammonia-lyase, 2-aminoindan-2-phosphonic acid, completely inhibited the incorporation of [14C]Phe into [14C]SA, although plants treated with 2-aminoindan-2-phosphonic acid could still produce [14C]SA from [14C]benzoic acid. Biosynthesis of SA in tissue inoculated with tobacco necrosis virus followed a transient pattern with the highest induction occurring 72 h postinoculation. Uninfected tissues from an infected plant synthesized de novo more SA than did controls. This suggests the involvement of a systemic signal triggering SA synthesis in tissue distant from the site of infection that display systemic acquired resistance.     

Extracellular pyridine nucleotides induce PR gene expression and disease resistance in Arabidopsis

Although it is well known that the pyridine nucleotides NAD and NADP function inside the cell to regulate intracellular signaling processes, recent evidence from animal studies suggests that NAD(P) also functions in the extracellular compartment (ECC). Extracellular NAD(P) [eNAD(P)] can either directly bind to plasma membrane receptors or be metabolized by ecto-enzymes to produce cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate, and/or may ADP-ribosylate cell-surface receptors, resulting in activation of transmembrane signaling. In this study, we report that, in plants, exogenous NAD(P) induces the expression of pathogenesis-related ( PR ) genes and resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. Chelation of Ca²⁺ by EGTA significantly inhibits the induction of PR genes by exogenous NAD(P), suggesting that exogenous NAD(P) may induce PR genes through a pathway that involves Ca²⁺ signaling. We show that exogenous application of NAD(P) causes accumulation of the defense signal molecule salicylic acid (SA), and induces both SA/NPR1-dependent and -independent PR gene expression and disease resistance. Furthermore, we demonstrate that NAD(P) leaks into the plant ECC after mechanical wounding and pathogen infection, and that the amount of NAD(P) leaking into the ECC after P. syringae pv. tobacco DC3000/ avrRpt2 infection is sufficient for induction of both PR gene expression and disease resistance. We propose that NAD(P) leakage from cells losing membrane integrity upon environmental stress may function as an elicitor to activate plant defense responses. Our data provide evidence that eNAD(P) functions in plant signaling, and illustrate the potential importance of eNAD(P) in plant innate immunity.     

Cholera toxin elevates pathogen resistance and induces pathogenesis-related gene expression in tobacco.

In animals, plants and fungi, cholera toxin (CTX) can activate signalling pathways dependent on heterotrimeric GTP binding proteins (G-proteins). We transformed tobacco plants with a chimeric gene encoding the A1 subunit of CTX regulated by a light-inducible wheat Cab-1 promoter. Tissues of transgenic plants expressing CTX showed greatly reduced susceptibility to the bacterial pathogen Pseudomonas tabaci, accumulated high levels of salicylic acid (SA) and constitutively expressed pathogenesis-related (PR) protein genes encoding PR-1 and the class II isoforms of PR-2 and PR-3. In contrast, the class I isoforms of PR-2 and PR-3 known to be induced in tobacco by stress, by ethylene treatment and as part of the hypersensitive response to infection, were not induced and displayed normal regulation. In good agreement with these results, microinjection experiments demonstrated that CTX or GTP-gamma-S induced the expression of a PR1-GUS reporter gene but not that of a GLB-GUS reporter gene containing the promoter region of a gene encoding the class I isoform of PR-2. Microinjection and grafting experiments strongly suggest that CTX-sensitive G-proteins are important in inducing the expression of a subset of PR genes and that these G-proteins act locally rather than systemically upstream of SA induction.     

Extracellular ATP is a regulator of pathogen defence in plants

In healthy plants extracellular ATP (eATP) regulates the balance between cell viability and death. Here we show an unexpected critical regulatory role of eATP in disease resistance and defensive signalling. In tobacco, enzymatic depletion of eATP or competition with non-hydrolysable ATP analogues induced pathogenesis-related ( PR ) gene expression and enhanced resistance to tobacco mosaic virus and Pseudomonas syringae pv. tabaci . Artificially increasing eATP concentrations triggered a drop in levels of the important defensive signal chemical salicylic acid (SA) and compromised basal resistance to viral and bacterial infection. Inoculating tobacco leaf tissues with bacterial pathogens capable of activating PR gene expression triggered a rapid decline in eATP. Conversely, inoculations with mutant bacteria unable to induce defence gene expression failed to deplete eATP. Furthermore, a collapse in eATP concentration immediately preceded PR gene induction by SA. Our study reveals a previously unsuspected role for eATP as a negative regulator of defensive signal transduction and demonstrates its importance as a key signal integrating defence and cell viability in plants.     

Temperature-Dependent Induction of Salicylic Acid and Its Conjugates during the Resistance Response to Tobacco Mosaic Virus Infection

Increases in endogenous salicylic acid (SA) levels and induction of several families of pathogenesis-related genes (PR-1 through PR-5) occur during the resistance response of tobacco to tobacco mosaic virus infection. We found that at temperatures that prevent the induction of PR genes and resistance, the increases in SA levels were eliminated. The addition of exogenous SA to infected plants at these temperatures was sufficient to induce the PR genes but not the hypersensitive response. However, when the resistance response was restored by shifting infected plants to permissive temperatures, SA levels increased dramatically and preceded PR-1 gene expression and necrotic lesion formation associated with resistance. SA was also found in a conjugated form whose levels increased in parallel with the free SA levels. The majority of the conjugates appeared to be SA glucosides. The same glucoside was formed when plants were supplied with exogenous SA. These results provide further evidence that endogenous SA signals the induction of certain defense responses and suggests additional complexity in the modulation of this signal.     

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.     

Probenazole-induced accumulation of salicylic acid confers resistance to Magnaporthe grisea in adult rice plants

Probenazole (PBZ) is the active ingredient of Oryzemate, an agrochemical which is used for the protection of rice plants from Magnaporthe grisea (blast fungus). While PBZ was reported to function upstream of salicylic acid (SA) in Arabidopsis, little is known about the mechanism of PBZ-induced resistance in rice. The role of SA in blast fungus resistance is also unclear. The recommended application period for Oryzemate is just before the Japanese rainy season, at which time rice plants in the field have reached the 8-leaf stage with adult traits. Thus, the involvement of SA in PBZ-induced resistance was studied in compatible and incompatible blast fungus-rice interactions at two developmentally different leaf morphology stages. Pre-treatment of inoculated fourth leaves of young wild-type rice plants at the 4-leaf stage with PBZ did not influence the development of whitish expanding lesions (ELs) in the susceptible interaction without the accumulation of SA and pathogenesis-related (PR) proteins. However, PBZ pre-treatment increased accumulation of SA and PR proteins in the eighth leaves of adult plants at the 8-leaf stage, resulting in the formation of hypersensitive reaction (HR) lesions (HRLs). Exogenous SA induced resistance in adult but not young plants. SA concentrations in blast fungus-inoculated young leaves were essentially the same in compatible and incompatible interactions, suggesting that PBZ-induced resistance in rice is age-dependently regulated via SA accumulation.     

N-cyanomethyl-2-chloroisonicotinamide induces systemic acquired resistance in arabidopsis without salicylic acid accumulation

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through the salicylic acid-mediated pathway. N-cyanomethyl-2-chloroisonicotinamide (NCI) is able to induce a broad range of disease resistance in tobacco and rice and induces SAR marker gene expression without SA accumulation in tobacco. To clarify the detailed mode of action of NCI, we analyzed its ability to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with NCI exhibited increased expression of several pathogenesis-related genes and enhanced resistance to the bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. NCI induced disease resistance and PR gene expression in NahG transgenic plants, but not in the npr1 mutant. NCI could induce PR gene expression in the etr1-1, ein2-1 and jar1-1 mutants. Thus, NCI activates SAR, independently from ethylene and jasmonic acid, by stimulating the site between SA and NPR1.