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.     

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.     

Ultraviolet light and ozone stimulate accumulation of salicylic acid,pathogenesis-related proteins and virus resistance in tobacco

In tobacco (Nicotiana tabacum L. cv. Xanthinc), salicylic acid (SA) levels increase in leaves inoculated by necrotizing pathogens and in healthy leaves located above the inoculated site. Systemic SA increase may trigger disease resistance and synthesis of pathogenesis-related proteins (PR proteins). Here we report that ultraviolet (UV)-C light or ozone induced biochemical responses similar to those induced by necrotizing pathogens. Exposure of leaves to UV-C light or ozone resulted in a transient ninefold increase in SA compared to controls. In addition, in UV-light-irradiated plants, SA increased nearly fourfold to 0.77 g·g^–1 fresh weight in leaves that were shielded from UV light. Increased SA levels were accompanied by accumulation of an SA conjugate and by an increase in the activity of benzoic acid 2-hydroxylase which catalyzes SA biosynthesis. In irradiated and in unirradiated leaves of plants treated with UV light, as well as in plants fumigated with ozone, PR proteins 1a and 1b accumulated. This was paralleled by the appearance of induced resistance to a subsequent challenge with tobacco mosaic virus. The results suggest that UV light, ozone fumigation and tobacco mosaic virus can activate a common signal-transduction pathway that leads to SA and PR-protein accumulation and increased disease resistance.Abbreviations PR protein pathogenesis-related protein - SA salicylic acid - TMV tobacco mosaic virus - UV ultraviolet This work was financed by grants from the U.S. Department of Agriculture (Competitive Research Grants Office), Division of Energy Biosciences of U.S. Department of Energy, the Rockefeller Foundation, the New Jersey Commission for Science and Technology, and the New Jersey Agricultural Experiment Station.     

Regulation by Cytokinins of Endogenous Levels of Jasmonic and Salicylic Acids in Mechanically Wounded Tobacco Plants

Plants respond differentially to wounding and pathogens usingdistinct signaling pathways, so that wound signals are transmittedto jasmonic acid (JA) which induces basic pathogenesis-related(PR) proteins, whereas pathogenic signals cause, in additionto JA, accumulation of salicylic acid (SA) which stimulatesproduction of acidic PR proteins. Transgenic tobacco plantsexpressing a gene for a small GTP binding protein respond abnormallyto mechanical wounding to produce SA and consequently acidicPR proteins, suggesting that wound signals cross with pathogensignaling pathways [Sano et al. (1994) Proc. Natl. Acad. Sci.USA 91: 10556]. This unusual signal crossing is associated witha highly sensitive wound-response of transgenic plants which,upon wounding, produce JA at least eighteen hours earlier thanwild-type plants. When wildtype plants are wounded in the presenceof the synthetic cytokinin, benzylaminopurine, production ofJA begins six hours earlier than in untreated samples, and alsoSA begins to accumulate. The cytokinin antagonist, 2-chloro-4-cyclohexylamino-6-ethylamino-s-triazine,erases these effects. Because transgenic plants constitutivelyproduce four-to six-fold higher amounts of endogenous cytokininsthan wild-type plants, it is concluded that cytokinins are indispensablefor control of endogenous levels of SA and JA. (Received April 23, 1996; Accepted June 10, 1996)     

An important role of a BAHD acyl transferase-like protein in plant innate immunity

Salicylic acid (SA) is an important regulator of plant resistance to biotrophic and hemi-biotrophic pathogens. The enhanced pseudomonas susceptibility 1 ( eps1 ) mutant in Arabidopsis thaliana is hypersusceptible to both virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae . Through positional cloning, the EPS1 gene was isolated and found to encode a novel member of the BAHD acyltransferase superfamily. Pathogen-induced accumulation of SA and expression of pathogenesis-related ( PR ) genes were compromised in the eps1 mutant. SA could induce PR1 gene expression and restore disease resistance in the eps1 mutant. These results suggest that EPS1 functions upstream of SA and may be involved directly in synthesis of a precursor or a regulatory molecule for SA biosynthesis. Mutations of EPS1 or other genes important for SA accumulation or signaling conferred enhanced resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola in the Nossen-0 background but had little effect in the Columbia-0 background. These results suggest that there is natural variation among Arabidopsis ecotypes with respect to the antagonistic cross-talk between defense signaling pathways against various types of microbial pathogens.     

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.     

Spermine Is a Salicylate-Independent Endogenous Inducer for Both Tobacco Acidic Pathogenesis-Related Proteins and Resistance against Tobacco Mosaic Virus Infection

Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.     

LSD1 regulates salicylic acid induction of copper zinc superoxide dismutase in Arabidopsis thaliana.

We characterized the accumulation patterns of Arabidopsis thaliana proteins, two CuZnSODs, FeSOD, MnSOD, PR1, PR5, and GST1, in response to various pathogen-associated treatments. These treatments included inoculation with virulent and avirulent Pseudomonas syringae strains, spontaneous lesion formation in the lsd1 mutant, and treatment with the salicylic acid (SA) analogs INA (2,6-dichloroisonicotinic acid) and BTH (benzothiadiazole). The PR1, PR5, and GST1 proteins were inducible by all treatments tested, as expected from previous mRNA blot analysis. The two CuZnSOD proteins were induced by SA analogs and in conjunction with lsd1-mediated spreading cell death. Additionally, LSD1 is a part of a signaling pathway for the induction of the CuZnSOD proteins in response to SA but not in lsd1-mediated cell death. We suggest that the spreading lesion phenotype of lsd1 results from a lack of up-regulation of a CuZnSOD responsible for detoxification of accumulating superoxide before the reactive oxygen species can trigger a cell death cascade.     

The role of EDS1 (enhanced disease susceptibility) during singlet oxygen-mediated stress responses of Arabidopsis

Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen (1O2) that is restricted to the plastid compartment. Distinct sets of genes are activated that are different from those induced by hydrogen peroxide/superoxide. One of the genes that is rapidly upregulated is EDS1 (enhanced disease susceptibility). The EDS1 protein has been shown to be required for the resistance to biotrophic pathogens and the accumulation of salicylic acid (SA) that enhances the defenses of a plant by inducing the synthesis of pathogen-related (PR) proteins. Because of the similarity of its N-terminal portion to the catalytic site of lipases, EDS1 has also been implicated with the release of polyunsaturated fatty acids and the subsequent formation of various oxylipins. The release of singlet oxygen in the flu mutant triggers a drastic increase in the concentration of free SA and activates the expression of PR1 and PR5 genes. These changes depend on the activity of EDS1 and are suppressed in flu/eds1 double mutants. Soon after the beginning of singlet oxygen production, the synthesis of oxylipins such as jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) also start and plants stop growing and induce a cell-death response. The inactivation of EDS1 does not affect oxylipin synthesis, growth inhibition and the initiation of cell death, but it does allow plants to recover much faster from singlet oxygen-mediated growth inhibition and it also suppresses the spread of necrotic lesions in leaves. Hence, singlet oxygen activates a complex stress-response program with EDS1 playing a key role in initiating and modulating several steps of it. This program includes not only responses to oxidative stress, but also responses known to be activated during plant-pathogen interactions and wounding.     

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.     

Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4

Salicylic acid (SA) is an important regulator of plant defense responses, and a variety of Arabidopsis mutants impaired in resistance against bacterial and fungal pathogens show defects in SA accumulation, perception, or signal transduction. Nevertheless, the role of SA-dependent defense responses against necrotrophic fungi is currently unclear. We determined the susceptibility of a set of previously identified Arabidopsis mutants impaired in defense responses to the necrotrophic fungal pathogen Botrytis cinerea. The rate of development of B. cinerea disease symptoms on primary infected leaves was affected by responses mediated by the genes EIN2, JAR1, EDS4, PAD2, and PAD3, but was largely independent of EDS5, SID2/ICS1, and PAD4. Furthermore, plants expressing a nahG transgene or treated with a phenylalanine ammonia lyase (PAL) inhibitor showed enhanced symptoms, suggesting that SA synthesized via PAL, and not via isochorismate synthase (ICS), mediates lesion development. In addition, the degree of lesion development did not correlate with defensin or PR1 expression, although it was partially dependent upon camalexin accumulation. Although npr1 mutant leaves were normally susceptible to B. cinerea infection, a double ein2 npr1 mutant was significantly more susceptible than ein2 plants, and exogenous application of SA decreased B. cinerea lesion size through an NPR1-dependent mechanism that could be mimicked by the cpr1 mutation. These data indicate that local resistance to B. cinerea requires ethylene-, jasmonate-, and SA-mediated signaling, that the SA affecting this resistance does not require ICS1 and is likely synthesized via PAL, and that camalexin limits lesion development.     

Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.     

Induction of tomato stress protein mRNAs by ethephon, 2,6-dichloroisonicotinic acid and salicylate

To study the possible involvement of plant hormones in the synthesis of stress proteins in tomato upon inoculation with Cladosporium fulvum, we investigated the induction of mRNAs encoding PR proteins and ethylene biosynthesis enzymes by ethephon, 2,6-dichloroisonicotinic acid (INA) and salicylic acid (SA) by northern blot analysis. Ethephon slightly induced some but not all mRNAs encoding intra- and extracellular PR proteins. INA induced all PR protein mRNAs analysed, except for intracellular chitinase and extracellular PR-4. SA induced all PR protein mRNAs analyzed, except for intracellular chitinase and osmotin. None of the inducers affected the expression of ACC synthase mRNA, whereas all three induced ethylene-forming enzyme (EFE) mRNA.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - HR hypersensitive response - INA 2,6-dichloroisonicotinic acid - PR pathogenesis-related - SA salicylic acid - SAR systemic acquired resistance     

Accumulation of Pathogenesis-Related Proteins in Tobacco Leaves Irradiated with UV-B

Nicotiana tabacum L. (cv. Petit Havana SR1) were grown under ultraviolet-B (UV-B, 290–320 nm) irradiation, and soluble proteins were extracted from the leaves. Two-dimensional electrophoresis revealed that a minimum of 12 polypeptides were induced by UV-B. Polypeptides which were so abundant as to be detectable by Coomassie brilliant blue staining were then subjected to N-terminal amino acid sequence analyses. Two of the polypeptides were identified as a 23 kDa protein of PS II and 6 as a pathogenesis-related protein 5 (PR-5). Immunoblotting demonstrated that other PR proteins, PR-1 and PR-3 were also induced by UV-B. Salicylic acid (SA), which is an important component of signal transduction that leads to the expression of PR proteins and exhibition of acquired resistance to pathogens, increased in response to exposure to UV-B. In addition, the activity of phenylalanine ammonialyase, which catalyzes the synthesis from phenylalanine of trans-cinnamic acid, the endogenous precursor of SA, was transiently increased by UV-B irradiation. These results suggest that UV-B activates the signal transduction pathway, which is a common step in pathogen infection. Received 8 May 2000/ Accepted in revised form 29 August 2000     

Characterization of a new Arabidopsis mutant exhibiting enhanced disease resistance

In many plant-pathogen interactions, resistance is associated with the synthesis and accumulation of salicylic acid (SA) and pathogenesis-related (PR) proteins. At least two general classes of mutants with altered resistance to pathogen attack have been identified in Arabidopsis. One class exhibits increased susceptibility to pathogen infection; the other class exhibits enhanced resistance to pathogens. In an attempt to identify mutations in resistance-associated loci, we screened a population of T-DNA tagged Arabidopsis thaliana ecotype Wassilewskija (Ws) for mutants showing constitutive expression of the PR-1 gene (cep). A mutant was isolated and shown to constitutively express PR-1, PR-2, and PR-5 genes. This constitutive phenotype segregated as a single recessive trait in the Ws genetic background. The mutant also had elevated levels of SA, which are responsible for the cep phenotype. The cep mutant spontaneously formed hypersensitive response (HR)-like lesions on the leaves and cotyledons and also exhibited enhanced resistance to virulent bacterial and fungal pathogens. Genetic analyses of segregating progeny from outcrosses to other ecotypes unexpectedly revealed that alterations in more than one gene condition the constitutive expression of PR genes in the original mutant. One of the mutations, designated cpr20, maps to the lower arm of chromosome 4 and is required for the cep phenotype. Another mutation, which has been termed cpr21, maps to chromosome 1 and is often, but not always, associated with this phenotype. The recessive nature of the cep trait suggests that the CPR20 and CPR21 proteins may act as negative regulators in the disease resistance signal transduction pathway.     

Molecular and functional profiling of Arabidopsis pathogenesis-related genes: insights into their roles in salt response of seed germination

Pathogenesis-related (PR) proteins are a group of heterogeneous proteins encoded by genes that are rapidly induced by pathogenic infections and by salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). They are widely used as molecular markers for resistance response to pathogens and systemic acquired response (SAR). However, recent studies have shown that the PR genes are also regulated by environmental factors, including light and abiotic stresses, and by developmental cues, suggesting that they also play a role in certain stress responses and developmental processes. In this work, we systematically examined the expression patterns of Arabidopsis PR genes. We also investigated the effects of environmental stresses and growth hormones on the expression of PR genes. We found that individual PR genes are temporally and spatially regulated in distinct patterns. In addition, they are differentially regulated by plant growth hormones, including SA, ABA, JA, ET and brassinosteroid (BR), and by diverse abiotic stresses, supporting the contention that the PR proteins play a role in plant developmental processes other than disease resistance response. Interestingly, PR-3 was induced significantly by high salt in an ABA-dependent manner. Consistent with this, a T-DNA insertional knockout plant with disruption of the PR-3 gene showed a significantly reduced rate of seed germination in the presence of high salt. It is thus proposed that PR-3 mediates ABA-dependent salt stress signals that affect seed germination in Arabidopsis. PR-4 and PR-5 also contributed to salt regulation of seed germination, although their effects were not as evident as those of PR-3.