Npr1 Plants Research Articles

Salicylic acid-mediated alleviation in NO2 phytotoxicity correlated to increased expression levels of the genes related to photosynthesis and carbon metabolism in Arabidopsis

Nitrogen dioxide (NO2) is one of the major air pollutants affecting plant growth, development and yields. Salicylic acid (SA)-mediated alleviation in NO2-induced plant damage has been reported in Arabidopsis. However, the involved mechanisms are far from clear. In this study, Arabidopsis wild type (WT) and its SA-altering mutants, snc1 (suppressor of npr1-1, constitutive 1) with high SA, npr1-1 (nonexpressor of PR gene) with SA signaling blockage, and transgenic line nahG with low SA, were used to decipher the SA action mechanisms. NO2 fumigation at a concentration of 10 ppm for 8 h led to more severe injuries on nahG and npr1-1 plants, whereas a lesser on snc1 plants compared with WT plants. The expressions of SA biosynthesis-related genes were up-regulated leading to more SA accumulations in all the tested plants after NO2 treatment. The expression patterns of ethylene, oxylipin and nitric oxide biosynthesis-related genes were regularly altered among NO2-treated genotypes, among which the expression of dioxygenase-encoding gene was dramatically increased in snc1 plants. Total chlorophyll contents, net photosynthetic rate, maximum quantum and actual quantum efficiency of PSII were decreased, to a greater degree in nahG and npr1-1 plants, whereas a lesser in snc1 plants compared with WT plants after NO2 treatment. We analyzed the expression patterns of the selected genes related to chlorophyll biosynthesis, photosystem I and II structure and functions, electron transport, ATP synthesis, Calvin cycle, as well as photorespiration, pentose phosphate pathway and starch degradation. The expression levels of the most genes were up-regulated in snc1 and WT plants, whereas down-regulated in nahG and npr1-1 plants after NO2 treatment, among which the expression pattern of a photorespiration-related gene was particularly specific. The plant tolerant phenotypes were positively correlated with the gene expressions, suggesting that SA-mediated alleviation in NO2 phytotoxicity was associated with photosynthesis and carbon metabolisms, also with other signal molecules.

Multiple biological processes involved in the regulation of salicylic acid in Arabidopsis response to NO2 exposure

Salicylic acid (SA) has been extensively implicated in plant response to abiotic stresses. However, just a few studies addressed the regulatory role of SA in plants exposed to NO2. In this study, we used Arabidopsis wild type (WT) and its SA-related mutants, including snc1 (suppressor of npr1-1, constitutive 1) with high endogenous SA, transgenic line nahG with low SA level, and npr1-1 (nonexpressor of PR gene) with SA signaling blockage to explore the possible regulation mechanism of SA, focusing on NO2-induced gene expression patterns. Plants were fumigated with 10 ppm NO2 for 8 h. The results showed that NO2 fumigation led to oxidative stress damage to all the tested plants as indicated by productions of H2O2, superoxide anion (O2−) and malondialdehyde, and cell death shown by electrolyte leakage and Evans Blue staining. However, snc1 plants were injured to a much less degree, whereas nahG and npr1-1 plants to a larger degree as compared with WT plants. Correspondingly, the activities of superoxide dismutase, peroxidase, catalase and nitrate reductase were induced by NO2 fumigation to a higher extent in snc1 plants, and a less extent in nahG and npr1-1 plants relative to WT plants. The expressions of the genes related to antioxidative defense, disulfide bond reduction, SA-responsive and –biosynthesis pathway, flavonoid biosynthesis, nitrate metabolism and kinase were analyzed. The results showed that these genes were differentially expressed among the tested genotypes under NO2 fumigation, wherein the most were significantly up-regulated in snc1 plants relative to other tested genotypes. Based on the gene expression patterns, it was proposed that SA-mediated plant tolerance to NO2 stress was associated with multiple biological processes.

Combined effect of ethylene- and salicylic acid-signaling insensitive mutation on Arabidopsis response to low temperature

The roles of ethylene (ET) or salicylic acid (SA) in plant response to low temperature (LT, 5 °C) have been implicated. However, the combined effect of ET- and SA-signaling on plant growth and metabolism under LT remains to be evaluated. In this study, we comparatively analyzed the response of Arabidopsis ethylene insensitive (ein) 2-1 (an ET insensitive mutant), nonexprressor of pathogenesis relative (npr)1-1 (an SA insensitive mutant) and double mutant ein2-1/npr1-1 plants to LT. The results show that a LT of 5 °C induced plant growth retardation to a less degree in ein2-1, an intermediate degree in npr1-1, but a much larger in ein2-1/npr1-1 compared to the wild-type (WT) plants. The LT susceptibility of the ein2-1/npr1-1 plants was correlated to a lower net photosynthetic rate and proline content, and a higher content of H2O2 and malondialdehyde and electrolyte leakage relative to the WT plants. Lower activities of superoxide dismutase, peroxidase, and catalase, as well as a lower glutathione content and a ratio of its reduced form to its oxidized form were also observed in the double mutant plants as compared with the WT plants. However, at normal conditions (23 °C), all the tested physiological and biochemical parameters were comparable between the ein2-1/npr1-1 and WT plants, and plant growth was even better in the double mutant than in the WT plants. On the contrary, most of the above-mentioned parameters were advantageous in the ein2-1 and npr1-1 plants over the WT plants under the LT conditions. These data suggest that a parallel function or physiological redundancy of nonexpressor of pathogenesis relative 1 and ethylene insensitive 2 existed in the Arabidopsis plant response to the LT. On the other hand, an interaction between ET- and SA-signaling occurred during this process.

Aluminum tolerance in Arabidopsis thaliana as affected by endogenous salicylic acid

Endogenous salicylic acid (SA) functions in plant response to an aluminum stress were assessed. We used different Arabidopsis thaliana genotypes including snc1 with a constitutively high content of SA, sid2 and nahG (transgenic lines) both with a low content of SA, SA insensitive mutant npr1-1, and snc1/nahG (i.e., the nahG expression in the snc1 background) with a similar SA content as in wild type (WT) plants. Results show that the snc1 plants displayed obvious growth retardation of roots and shoots under the Al3+ stress, whereas the sid2, nahG, and npr1-1 plants exhibited alleviated symptoms in comparison with the WT plants. The Al3+ content increased in all the tested genotypes with the increasing AlCl3 concentration applied, but no significant variations were detected among the tested genotypes. The snc1 had much higher superoxide dismutase and peroxidase activities, and a lower catalase activity and the ratio of reduced to oxidized glutathione accompanied by higher accumulations of H2O2 and malondialdehyde compared with the WT plants. These changes were largely reversed by the introduction of nahG; the sid2, nahG, and npr1-1 plants were less affected than WT plants in all the above-mentioned parameters. The Al3+ stress significantly enhanced malate exudation in all the tested genotypes, but no significant correlation was observed between the SA-involved response to the Al3+ stress and the malate exudation. Based on these data, it was concluded that the SA-related functions in Arabidopsis response to the Al3+ stress were associated with the control of oxidative stress, but not of malate exudation.

English

Symptomatological studies on Arabidopsis- Fusarium oxysporium f.sp. cubense (Foc) interaction has led to the identification of signaling pathways required for plant resistance to Foc, as well as key regulators of innate immunity against this type of vascular wilt pathogens. From the in planta symptom expressions of Foc on Arabidopsis, there is a clear indication of involvement of salicylic acid (SA) and jasmonic acid (JA) for disease resistance. Mutations occur in synthesis of salicylic acid and Jasmonic acid, which modulate disease resistance with the evident of severe veinal necrosis on leaves and petiole. The typical symptom of leaf rosetting was the clear indication of the active participation of SA biosynthesis for Foc resistance in nahG, npr-1 plants. This analysis revealed that salicylic acid, ethylene (ET) and jasmonic acid (JA) pathways influence the Foc disease outcome in Arabidopsis. All the three signaling pathways interact in a positive way in the activation of Arabidopsis resistance to Foc. Hence, there must be co-ordinate regulation of both SA and JA for Foc resistance in Arabidopsis. Constitutive expressions of some transcriptional regulators of these pathways are sufficient to confer enhanced resistance to Foc and it might be an oligogenic trait.             Key words: Fusarium oxysporium f.sp.cubense, Arabidopsis thaliana, disease resistance.

Induction of Systemic Resistance against Cucumber mosaic virus in Arabidopsis thaliana by Trichoderma asperellum SKT-1.

Trichoderma asperellum SKT-1 is a microbial pesticide that is very effective against various diseases. Our study was undertaken to evaluate T. asperellum SKT-1 for induction of resistance against yellow strain of Cucumber mosaic virus (CMV-Y) in Arabidopsis plants. Disease severity was rated at 2 weeks post inoculation (WPI). CMV titre in Arabidopsis leaves was determined by indirect enzyme-linked immunosorbent assay (ELISA) at 2 WPI. Our results demonstrated that among all Arabidopsis plants treated with barley grain inoculum (BGI) of SKT-1 NahG and npr1 plants showed no significant reduction in disease severity and CMV titre as compared with control plants. In contrast, disease severity and CMV titre were significantly reduced in all Arabidopsis plants treated with culture filtrate (CF) of SKT-1 as compared with control plants. RT-PCR results showed increased expression levels of SA-inducible genes, but not JA/ET-inducible genes, in leaves of BGI treated plants. Moreover, expression levels of SA- and JA/ET-inducible genes were increased in leaves of CF treated plants. In conclusion, BGI treatment induced systemic resistance against CMV through SA signaling cascade in Arabidopsis plants. While, treatment with CF of SKT-1 mediated the expression of a majority of the various pathogen related genes, which led to the increased defense mechanism against CMV infection.

Salicylic acid-altering Arabidopsis mutants response to salt stress

The role of salicylic acid (SA) in plant responses to salinity is still a matter of controversy. To address the effect of endogenous SA variation in level and signaling on plant responses to salinity, biochemical and physiological analyses were performed on SA-altering Arabidopsis mutants including snc1 with high level of SA, transgenic line nahG with low SA, npr1-1 with SA signaling blockage, snc1/nahG plants (expression of nahG in the snc1 background), as well as wild type plants. Plants were cultured in 1 × Hoagland solution under controlled conditions. For salt exposure, NaCl at final concentrations of 100 mM, 200 mM, and 300 mM, respectively, was added to the culture solution after 25 d of seed germination. Except where mentioned, plant leaves were harvested after 14 d of salt stress, and used for physiological and chemical analyses. Salt stress caused all plants growth retardation with a dose-effect relationship relative to control. However, compared to wild type plants, a greater growth inhibition occurred in snc1, while a less inhibition was observed in nahG and npr1-1 plants, and a comparable extent was detected in snc1/nahG plants in which the SA level was near to that in wild type plants. The snc1 plants had lower net photosynthetic rate, variable to maximum fluorescence ratio, quantum efficiency of photosystem 2, reduced glutathione/oxidized glutathione ratio, proline levels, and higher malondiadehyde levels and electrolyte leakage rates as compared to wild type plants under salt stress. These values were effectively reversed by the expression of nahG gene in snc1 plants. The nahG and npr1-1 plants always exhibited more tolerance to salinity in above-mentioned indices than wild type plants. However, higher activities of superoxide dismutase and peroxidase in snc1 plants did not contribute to salt tolerance. These data showed that SA deficit or signaling blockage in Arabidopsis plants was favorable to salt adaptation, while a high accumulation of SA potentiated salt-induced damage to Arabidopsis plants.

Role of endogenous salicylic acid in Arabidopsis response to elevated sulfur dioxide concentration

The response of Arabidopsis thaliana plants to elevated sulfur dioxide could be related to their endogenous salicylic acid (SA) content and signaling. The wild type (WT, ecotype Columbia) and its mutant snc1 with high SA content, npr1-1 with a blockage in SA signaling, transgenic line nahG with low SA content and double mutant snc1nahG plants were exposed to 0.5 mm3 dm-3 SO2 for 3 h d-1 for 14 d in a growth chamber. Under unstressed conditions, total SA contents in snc1 and npr1-1 were 7- and 2-fold higher than those in WT, respectively, but in nahG SA content was only 28 % of that in WT. The expression of nahG in snc1 plants decreased SA content to the WT level. Increased SA contents were observed in snc1, npr1-1 and WT after 12-h SO2 exposure, whereas no major changes were detected in nahG and snc1nahG plants. The snc1 plants exhibited higher tolerance to SO2 exposure than snc1nahG plants and especially nahG and npr1-1 plants according to plant biomass, total chlorophyll content and photosynthetic rate. The SO2 exposure decreased net photosynthetic rate, maximum photochemical efficiency (Fv/Fm) and actual quantum efficiency of photosystem 2 (ΦPS2). SO2-induced oxidative damage in the tested plants was confirmed by increased malondialdehyde (MDA) content and electrolyte leakage. Increases in superoxide dismutase (SOD) and peroxidase (POD) activity, reduced glutathione (GSH) content and a ratio of reduced/oxidized glutathione (GSSG) might be responsible for the decreased contents of H2O2 and alleviation of oxidative injury in snc1 plants compared with other lines exposed to SO2. These observations implied that endogenous SA content and signaling may play an essential role in plant responses to SO2 stress.

Autophagy Negatively Regulates Cell Death by Controlling NPR1-Dependent Salicylic Acid Signaling during Senescence and the Innate Immune Response inArabidopsis

Autophagy is an evolutionarily conserved intracellular process for vacuolar degradation of cytoplasmic components. In higher plants, autophagy defects result in early senescence and excessive immunity-related programmed cell death (PCD) irrespective of nutrient conditions; however, the mechanisms by which cells die in the absence of autophagy have been unclear. Here, we demonstrate a conserved requirement for salicylic acid (SA) signaling for these phenomena in autophagy-defective mutants (atg mutants). The atg mutant phenotypes of accelerated PCD in senescence and immunity are SA signaling dependent but do not require intact jasmonic acid or ethylene signaling pathways. Application of an SA agonist induces the senescence/cell death phenotype in SA-deficient atg mutants but not in atg npr1 plants, suggesting that the cell death phenotypes in the atg mutants are dependent on the SA signal transducer NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. We also show that autophagy is induced by the SA agonist. These findings imply that plant autophagy operates a novel negative feedback loop modulating SA signaling to negatively regulate senescence and immunity-related PCD.

Gene coding for SigA-binding protein from Arabidopsis appears to be transcriptionally up-regulated by salicylic acid and NPR1-dependent mechanisms

SigA-binding protein (SIB A) is a nuclear-encoded chloroplast-targeted protein that interacts with the plastid-encoded plastid RNA polymerase σ-factor SigA (Sig1). In this study, the SIB A gene responded rapidly to salicylic acid (SA) treatment, but responded slowly to ethylene (ET) and jasmonic acid (JA) treatments as determined with microarray and quantitative real-time RT-PCR analyses. Expression of the SIB A gene increased rapidly, with a peak at 2 h after SA treatment and then decreased gradually. In contrast, expression of the PR-1 gene, a marker gene on the SA defense signaling pathway, increased gradually between 0 and 24 h after SA treatment. In addition, transcription levels of SIB A and PR-1 decreased between 0 and 24 h in the benzothiadiazole (BTH)-treated npr1-1 plants. We suggest that the SIB A gene is downstream of NPR1 on the SA signaling pathway. From the results of our analysis of the 1.5-kbp promoter region of the SIB A gene in Arabidopsis, the isolated promoter region of the SIB A gene seems able to drive the transcription of the GUS gene to elevated levels more rapidly in transgenic Arabidopsis plants undergoing SA treatment compared with levels of PR-1. To analyze biological function of SIB A and investigate SIB A-regulated genes, we generated transgenic Arabidopsis plants in which SIB A was overexpressed. As a result, ROS-related genes, such as glutathione transferase, were up-regulated in plants overexpressing SIB A. However, SIB A-overexpressing plants were not resistant to Pseudomonas syringae pv. tomato DC3000 strain. The SIB A gene has promise as a new tool to analyze the SA-signaling pathway and the role of the chloroplast in plant defense responses.

The Plant Growth-Promoting Fungus Penicillium simplicissimum GP17-2 Induces Resistance in Arabidopsis thaliana by Activation of Multiple Defense Signals

Arabidopsis thaliana grown in soil amended with barley grain inocula of Penicillium simplicissimum GP17-2 or receiving root treatment with its culture filtrate (CF) exhibited clear resistance to Pseudomonas syringae pv. tomato DC3000 (Pst). To assess the contribution of different defense pathways, Arabidopsis genotypes implicated in salicylic acid (SA) signaling expressing the NahG transgene or carrying disruption in NPR1 (npr1), jasmonic acid (JA) signaling (jar1) and ethylene (ET) signaling (ein2) were tested. All genotypes screened were protected by GP17-2 or its CF. However, the level of protection was significantly lower in NahG and npr1 plants than it was in similarly treated wild-type plants, indicating that the SA signaling pathway makes a minor contribution to the GP17-2-mediated resistance and is insufficient for a full response. Examination of local and systemic gene expression revealed that GP17-2 and its CF modulate the expression of genes involved in both the SA and JA/ET signaling pathways. Subsequent challenge of GP17-2-colonized plants with Pst was accompanied by direct activation of SA-inducible PR-2 and PR-5 genes as well as potentiated expression of the JA-inducible Vsp gene. In contrast, CF-treated plants infected with Pst exhibited elevated expression of most defense-related genes (PR-1, PR-2, PR-5, PDF1.2 and Hel) studied. Moreover, an initial elevation of SA responses was followed by late induction of JA responses during Pst infection of induced systemic resistance (ISR)-expressing plants. In conclusion, we hypothesize the involvement of multiple defense mechanisms leading to an ISR of Arabidopsis by GP17-2.

Silverleaf Whitefly Induces Salicylic Acid Defenses and Suppresses Effectual Jasmonic Acid Defenses

The basal defenses important in curtailing the development of the phloem-feeding silverleaf whitefly (Bemisia tabaci type B; SLWF) on Arabidopsis (Arabidopsis thaliana) were investigated. Sentinel defense gene RNAs were monitored in SLWF-infested and control plants. Salicylic acid (SA)-responsive gene transcripts accumulated locally (PR1, BGL2, PR5, SID2, EDS5, PAD4) and systemically (PR1, BGL2, PR5) during SLWF nymph feeding. In contrast, jasmonic acid (JA)- and ethylene-dependent RNAs (PDF1.2, VSP1, HEL, THI2.1, FAD3, ERS1, ERF1) were repressed or not modulated in SLWF-infested leaves. To test for a role of SA and JA pathways in basal defense, SLWF development on mutant and transgenic lines that constitutively activate or impair defense pathways was determined. By monitoring the percentage of SLWF nymphs in each instar, we show that mutants that activate SA defenses (cim10) or impair JA defenses (coi1) accelerated SLWF nymphal development. Reciprocally, mutants that activate JA defenses (cev1) or impair SA defenses (npr1, NahG) slowed SLWF nymphal development. Furthermore, when npr1 plants, which do not activate downstream SA defenses, were treated with methyl jasmonate, a dramatic delay in nymph development was observed. Collectively, these results showed that SLWF-repressed, JA-regulated defenses were associated with basal defense to the SLWF.

Plant growth-promoting rhizobacteria systemically protect Arabidopsis thaliana against Cucumber mosaic virus by a salicylic acid and NPR1-independent and jasmonic acid-dependent signaling pathway.

Arabidopsis thaliana ecotype Columbia plants (Col-0) treated with plant growth-promoting rhizobacteria (PGPR) Serattia marcescens strain 90-166 and Bacillus pumilus strain SE34 had significantly reduced symptom severity by Cucumber mosaic virus (CMV). In some cases, CMV accumulation was also significantly reduced in systemically infected leaves. The signal transduction pathway(s) associated with induced resistance against CMV by strain 90-166 was determined using mutant strains and transgenic and mutant Arabidopsis lines. NahG plants treated with strains 90-166 and SE34 had reduced symptom severity indicating that the resistance did not require salicylic acid (SA). Strain 90-166 naturally produces SA under iron-limited conditions. Col-0 and NahG plants treated with the SA-deficient mutant, 90-166-1441, had significantly reduced CMV symptom severity with reduced virus accumulation in Col-0 plants. Another PGPR mutant, 90-166-2882, caused reduced disease severity in Col-0 and NahG plants. In a time course study, strain 90-166 reduced virus accumulation at 7 but not at 14 and 21 days post-inoculation (dpi) on the non-inoculated leaves of Col-0 plants. NahG and npr1-1 plants treated with strain 90-166 had reduced amounts of virus at 7 and 14 dpi but not at 21 dpi. In contrast, no decrease in CMV accumulation occurred in strain 90-166-treated fad3-2 fad7-2 fad8 plants. These data indicate that the protection of Arabidopsis against CMV by strain 90-166 follows a signaling pathway for virus protection that is independent of SA and NPR1, but dependent on jasmonic acid.

Different signaling pathways of induced resistance by rhizobacteria in Arabidopsis thaliana against two pathovars of Pseudomonas syringae.

• The mechanisms by which plant growth-promoting rhizobacteria (PGPR) mediate induced systemic resistance are currently being intensively investigated from the viewpoint of signal transduction pathways within plants. • Here, we determined whether our well-characterized PGPR strains, which have demonstrated induced resistance on various plants, also elicit induced resistance in Arabidopsis thaliana. Nine different PGPR strains were evaluated for their capacity to cause induced resistance on Arabidopsis against two pathovars of Pseudomonas syringae. Six strains significantly reduced severity of P. syringae pv. tomato, whereas seven strains reduced severity of P. syringae pv. maculicola. • From the initial screenings, four strains (90-166, SE34, 89B61 and T4) were selected because of their consistent induced resistance capacity. Elicitation of induced resistance with these strains depended on how disease severity was measured. Three strains (90-166, 89B61 and T4) induced resistance in NahG plants (SA-deficient), indicating a salicylic acid-independent pathway, which agrees with the previously reported pathway for induced resistance by PGPR. However, differences from the reported pathway were noted with strain 89B61, which did not require jasmonic acid or ethylene signaling pathways for induced resistance, and with strain T4, which induced resistance in npr1 plants. • These results indicate that strains 89B61 and T4 induce resistance via a new pathway or possibly a variation of the previously reported pathway. This information will broaden our understanding of ways in which microorganisms can signal physiological changes in plants.

Arabidopsis sfd mutants affect plastidic lipid composition and suppress dwarfing, cell death, and the enhanced disease resistance phenotypes resulting from the deficiency of a fatty acid desaturase.

A loss-of-function mutation in the Arabidopsis SSI2/FAB2 gene, which encodes a plastidic stearoyl-acyl-carrier protein desaturase, has pleiotropic effects. The ssi2 mutant plant is dwarf, spontaneously develops lesions containing dead cells, accumulates increased salicylic acid (SA) levels, and constitutively expresses SA-mediated, NPR1-dependent and -independent defense responses. In parallel, jasmonic acid-regulated signaling is compromised in the ssi2 mutant. In an effort to discern the involvement of lipids in the ssi2-conferred developmental and defense phenotypes, we identified suppressors of fatty acid (stearoyl) desaturase deficiency (sfd) mutants. The sfd1, sfd2, and sfd4 mutant alleles suppress the ssi2-conferred dwarfing and lesion development, the NPR1-independent expression of the PATHOGENESIS-RELATED1 (PR1) gene, and resistance to Pseudomonas syringae pv maculicola. The sfd1 and sfd4 mutant alleles also depress ssi2-conferred PR1 expression in NPR1-containing sfd1 ssi2 and sfd4 ssi2 plants. By contrast, the sfd2 ssi2 plant retains the ssi2-conferred high-level expression of PR1. In parallel with the loss of ssi2-conferred constitutive SA signaling, the ability of jasmonic acid to activate PDF1.2 expression is reinstated in the sfd1 ssi2 npr1 plant. sfd4 is a mutation in the FAD6 gene that encodes a plastidic omega6-desaturase that is involved in the synthesis of polyunsaturated fatty acid-containing lipids. Because the levels of plastid complex lipid species containing hexadecatrienoic acid are depressed in all of the sfd ssi2 npr1 plants, we propose that these lipids are involved in the manifestation of the ssi2-conferred phenotypes.

The expression of the t-SNARE AtSNAP33 is induced by pathogens and mechanical stimulation.

The fusion of vesicles in the secretory pathway involves the interaction of t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) on the target membrane and v-SNAREs on the vesicle membrane. AtSNAP33 is an Arabidopsis homolog of the neuronal t-SNARE SNAP-25 involved in exocytosis and is localized at the cell plate and at the plasma membrane. In this paper, the expression of AtSNAP33 was analyzed after different biotic and abiotic stresses. The expression of AtSNAP33 increased after inoculation with the pathogens Plectosporium tabacinum and virulent and avirulent forms of Peronospora parasitica and Pseudomonas syringae pv tomato. The expression of PR1 transcripts encoding the secreted pathogenesis-related protein 1 also increased after inoculation with these pathogens and the expression of AtSNAP33 preceded or occurred at the same time as the expression of PR1. AtSNAP33 was also expressed in npr1 plants that do not express PR1 after pathogen inoculation as well as in cpr1 plants that overexpress PR1 in the absence of a pathogen. The level of AtSNAP33 decreased slightly in leaves inoculated with P. parasitica in the NahG plants, and eds5 and sid2 mutants that are unable to accumulate salicylic acid (SA) after pathogen inoculation, indicating a partial dependence on SA. AtSNAP33 was also expressed in systemic noninoculated leaves of plants inoculated with P. syringae. In contrast to the situation in infected leaves, the expression of AtSNAP33 in systemic leaves was fully SA dependent. Thus, the expression of AtSNAP33 after pathogen attack is regulated by SA-dependent and SA-independent pathways. Mechanical stimulation also led to an increase of AtSNAP33 transcripts.

Ozone-induced ethylene production is dependent on salicylic acid, and both salicylic acid and ethylene act in concert to regulate ozone-induced cell death.

Ethylene is known to influence plant defense responses including cell death in response to both biotic and abiotic stress factors. However, whether ethylene acts alone or in conjunction with other signaling pathways is not clearly understood. Ethylene overproducer mutants, eto1 and eto3, produced high levels of ethylene and developed necrotic lesions in response to an acute O3 exposure that does not induce lesions in O3-tolerant wild-type Col-0 plants. Treatment of plants with ethylene inhibitors completely blocked O3-induced ethylene production and partially attenuated O3-induced cell death. Analyses of the responses of molecular markers of specific signaling pathways indicated a relationship between salicylic acid (SA)- and ethylene-signaling pathways and O3 sensitivity. Both eto1 and eto3 plants constitutively accumulated threefold higher levels of total SA and exhibited a rapid increase in free SA and ethylene levels prior to lesion formation in response to O3 exposure. SA pre-treatments increased O3 sensitivity of Col-0, suggesting that constitutive high SA levels prime leaf tissue to exhibit increased magnitude of O3-induced cell death. NahG and npr1 plants compromised in SA signaling failed to produce ethylene in response to O3 and other stress factors suggesting that SA is required for stress-induced ethylene production. Furthermore, NahG expression in the dominant eto3 mutant attenuated ethylene-dependent PR4 expression and rescued the O3-induced HR (hypersensitive response) cell death phenotype exhibited by eto3 plants. Our results suggest that both SA and ethylene act in concert to influence cell death in O3-sensitive genotypes, and that O3-induced ethylene production is dependent on SA.

The Arabidopsis NPR1/NIM1 Protein Enhances the DNA Binding Activity of a Subgroup of the TGA Family of bZIP Transcription Factors

The Arabidopsis NPR1 gene is essential in activating systemic, inducible plant defense responses. To gain a better understanding of NPR1 function, we conducted a yeast two-hybrid screening procedure and identified a differential interaction between NPR1 and all known members of the Arabidopsis TGA family of basic leucine zipper transcription factors. In the electrophoretic mobility shift assay, NPR1 substantially increased the binding of TGA2 to its cognate promoter element (as-1) as well as to a positive salicylic acid-inducible element (LS7) and a negative element (LS5) in the promoter of the pathogenesis-related PR-1 gene. Proteins encoded by npr1 mutants interacted poorly with TGA2 and did not substantially increase TGA2 binding to the as-1, LS5, or LS7 elements, thus establishing a link between the loss of disease resistance and the loss of TGA2 interaction and NPR1-enhanced DNA binding. Coupled with observations that the DNA binding activity of TGA factors is deregulated in npr1 plants, the results suggest that NPR1-mediated DNA binding of TGA2 is critical for activation of defense genes.

A novel signaling pathway controlling induced systemic resistance in Arabidopsis.

Plants have the ability to acquire an enhanced level of resistance to pathogen attack after being exposed to specific biotic stimuli. In Arabidopsis, nonpathogenic, root-colonizing Pseudomonas fluorescens bacteria trigger an induced systemic resistance (ISR) response against infection by the bacterial leaf pathogen P. syringae pv tomato. In contrast to classic, pathogen-induced systemic acquired resistance (SAR), this rhizobacteria-mediated ISR response is independent of salicylic acid accumulation and pathogenesis-related gene activation. Using the jasmonate response mutant jar1, the ethylene response mutant etr1, and the SAR regulatory mutant npr1, we demonstrate that signal transduction leading to P. fluorescens WCS417r-mediated ISR requires responsiveness to jasmonate and ethylene and is dependent on NPR1. Similar to P. fluorescens WCS417r, methyl jasmonate and the ethylene precursor 1-aminocyclopropane-1-carboxylate were effective in inducing resistance against P. s. tomato in salicylic acid-nonaccumulating NahG plants. Moreover, methyl jasmonate-induced protection was blocked in jar1, etr1, and npr1 plants, whereas 1-aminocyclopropane-1-carboxylate-induced protection was affected in etr1 and npr1 plants but not in jar1 plants. Hence, we postulate that rhizobacteria-mediated ISR follows a novel signaling pathway in which components from the jasmonate and ethylene response are engaged successively to trigger a defense reaction that, like SAR, is regulated by NPR1. We provide evidence that the processes downstream of NPR1 in the ISR pathway are divergent from those in the SAR pathway, indicating that NPR1 differentially regulates defense responses, depending on the signals that are elicited during induction of resistance.

The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance.

The cpr5 mutant was identified from a screen for constitutive expression of systemic acquired resistance (SAR). This single recessive mutation also leads to spontaneous expression of chlorotic lesions and reduced trichome development. The cpr5 plants were found to be constitutively resistant to two virulent pathogens, Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2; to have endogenous expression of the pathogenesis-related gene 1 (PR-1); and to have an elevated level of salicylic acid (SA). Lines homozygous for cpr5 and either the SA-degrading bacterial gene nahG or the SA-insensitive mutation npr1 do not express PR-1 or exhibit resistance to P. s. maculicola ES4326. Therefore, we conclude that cpr5 acts upstream of SA in inducing SAR. However, the cpr5 npr1 plants retained heightened resistance to P. parasitica Noco2 and elevated expression of the defensin gene PDF1.2, implying that NPR1-independent resistance signaling also occurs. We conclude that the cpr5 mutation leads to constitutive expression of both an NPR1-dependent and an NPR1-independent SAR pathway. Identification of this mutation indicates that these pathways are connected in early signal transduction steps and that they have overlapping functions in providing resistance.