Pseudomonas Syringae Pv Tomato DC3000 Research Articles

Disruption of poly(ADP-ribosyl)ation mechanisms alters responses of Arabidopsis to biotic stress.

Poly(ADP-ribosyl)ation is a posttranslational protein modification in which ADP-ribose (ADP-Rib) units derived from NAD(+) are attached to proteins by poly(ADP-Rib) polymerase (PARP) enzymes. ADP-Rib groups are removed from these polymer chains by the enzyme poly(ADP-Rib) glycohydrolase (PARG). In animals, poly(ADP-ribosyl)ation is associated with DNA damage responses and programmed cell death. Previously, we hypothesized a role for poly(ADP-ribosyl)ation in plant defense responses when we detected defense-associated expression of the poly(ADP-ribosyl)ation-related genes PARG2 and NUDT7 and observed altered callose deposition in the presence of a chemical PARP inhibitor. The role of poly(ADP-ribosyl)ation in plant defenses was more extensively investigated in this study, using Arabidopsis (Arabidopsis thaliana). Pharmacological inhibition of PARP using 3-aminobenzamide perturbs certain innate immune responses to microbe-associated molecular patterns (flg22 and elf18), including callose deposition, lignin deposition, pigment accumulation, and phenylalanine ammonia lyase activity, but does not disrupt other responses, such as the initial oxidative burst and expression of some early defense-associated genes. Mutant parg1 seedlings exhibit exaggerated seedling growth inhibition and pigment accumulation in response to elf18 and are hypersensitive to the DNA-damaging agent mitomycin C. Both parg1 and parg2 knockout plants show accelerated onset of disease symptoms when infected with Botrytis cinerea. Cellular levels of ADP-Rib polymer increase after infection with avirulent Pseudomonas syringae pv tomato DC3000 avrRpt2(+), and pathogen-dependent changes in the poly(ADP-ribosyl)ation of discrete proteins were also observed. We conclude that poly(ADP-ribosyl)ation is a functional component in plant responses to biotic stress.

Gene Ontology annotation highlights shared and divergent pathogenic strategies of type III effector proteins deployed by the plant pathogen Pseudomonas syringae pv tomato DC3000 and animal pathogenic Escherichia coli strains

Genome-informed identification and characterization of Type III effector repertoires in various bacterial strains and species is revealing important insights into the critical roles that these proteins play in the pathogenic strategies of diverse bacteria. However, non-systematic discipline-specific approaches to their annotation impede analysis of the accumulating wealth of data and inhibit easy communication of findings among researchers working on different experimental systems. The development of Gene Ontology (GO) terms to capture biological processes occurring during the interaction between organisms creates a common language that facilitates cross-genome analyses. The application of these terms to annotate type III effector genes in different bacterial species – the plant pathogen Pseudomonas syringae pv tomato DC3000 and animal pathogenic strains of Escherichia coli – illustrates how GO can effectively describe fundamental similarities and differences among different gene products deployed as part of diverse pathogenic strategies. In depth descriptions of the GO annotations for P. syringae pv tomato DC3000 effector AvrPtoB and the E. coli effector Tir are described, with special emphasis given to GO capability for capturing information about interacting proteins and taxa. GO-highlighted similarities in biological process and molecular function for effectors from additional pathosystems are also discussed.

The Arabidopsis homologs of CCR4-associated factor 1 show mRNA deadenylation activity and play a role in plant defence responses

Messenger RNA (mRNA) turnover in eukaryotic cells begins with shortening of the poly (A) tail at the 3' end, a process called deadenylation. In yeast, the deadenylation reaction is predominantly mediated by CCR4 and CCR4-associated factor 1 (CAF1), two components of the well-characterised protein complex named CCR4-NOT. We report here that AtCAF1a and AtCAF1b, putative Arabidopsis homologs of the yeast CAF1 gene, partially complement the growth defect of the yeast caf1 mutant in the presence of caffeine or at high temperatures. The expression of AtCAF1a and AtCAF1b is induced by multiple stress-related hormones and stimuli. Both AtCAF1a and AtCAF1b show deadenylation activity in vitro and point mutations in the predicted active sites disrupt this activity. T-DNA insertion mutants disrupting the expression of AtCAF1a and/or AtCAF1b are defective in deadenylation of stress-related mRNAs, indicating that the two AtCAF1 proteins are involved in regulated mRNA deadenylation in vivo. Interestingly, the single and double mutants of AtCAF1a and AtCAF1b show reduced expression of pathogenesis-related (PR) genes PR1 and PR2 and are more susceptible to Pseudomonas syringae pv tomato DC3000 (Pst DC3000) infection, whereas transgenic plants over-expressing AtCAF1a show elevated expression of PR1 and PR2 and increased resistance to the same pathogen. Our results suggest roles of the AtCAF1 proteins in regulated mRNA deadenylation and defence responses to pathogen infections.

Plant Pattern-Recognition Receptor FLS2 Is Directed for Degradation by the Bacterial Ubiquitin Ligase AvrPtoB

An important layer of active defense in plant immunity is the detection of pathogen-associated molecular patterns (PAMPs) mediated by cell-surface receptors. For the establishment of disease, pathogens depend on the ability to overcome PAMP perception and disable plant signaling pathways activated in response to PAMPs. Pattern recognition receptors (PRRs) are therefore prime targets for pathogen effectors. FLS2, its coreceptor BAK1, and EFR encode receptor-like kinases that play a role in immunity against bacterial pathogens. Here, we report that virulence of Pseudomonas syringae pv tomato DC3000 (PtoDC3000) in Arabidopsis is enhanced through the action of its effector AvrPtoB, which promotes degradation of FLS2. We show that AvrPtoB, through its N terminus, associates with FLS2 and BAK1, of which interaction with FLS2 is enhanced by flg22 activation. In vitro, AvrPtoB is active as an E3 ligase to catalyze polyubiquitination of the kinase domain of FLS2, a process confirmed in planta. Full enhancement of PtoDC3000 virulence appears to require the E3 ligase activity of AvrPtoB. AvrPtoB, initially identified through its activation of hypersensitive resistance in tomato cultivars expressing the Pto kinase, is composed of at least two functional domains: the N terminus is responsible for interaction with Pto, and the C terminus carries an E3 ligase activity. Based on our findings, we propose that both domains of AvrPtoB act together to support the virulence of PtoDC3000 in Arabidopsis through their ability to eliminate FLS2 from the cell periphery, and probably also other PAMP sensors that are constitutively expressed or induced after pathogen challenge.

BAH1/NLA, a RING-type ubiquitin E3 ligase, regulates the accumulation of salicylic acid and immune responses to Pseudomonas syringae DC3000.

Salicylic acid (SA) is a primary factor responsible for exerting diverse immune responses in plants and is synthesized in response to attack by a wide range of pathogens. The Arabidopsis (Arabidopsis thaliana) sid2 mutant is defective in a SA biosynthetic pathway involving ISOCHORISMATE SYNTHASE1 (ICS1) and consequently contains reduced levels of SA. However, the sid2 mutant as well as ICS-suppressed tobacco (Nicotiana benthamiana) still accumulate a small but significant level of SA. These observations along with previous studies suggest that SA might also be synthesized by another pathway involving benzoic acid (BA). Here we isolated a benzoic acid hypersensitive1-Dominant (bah1-D) mutant that excessively accumulated SA after application of BA from activation-tagged lines. This mutant also accumulated higher levels of SA after inoculation with Pseudomonas syringae pv tomato DC3000. Analysis of the bah1-D sid2 double mutant suggested that the bah1-D mutation caused both ICS1-dependent and -independent accumulation. In addition, the bah1-D mutant showed SA-dependent localized cell death in response to P. syringae pv tomato DC3000. The T-DNA insertional mutation that caused the bah1-D phenotypes resulted in the suppression of expression of the NLA gene, which encodes a RING-type ubiquitin E3 ligase. These results suggest that BAH1/NLA plays crucial roles in the ubiquitination-mediated regulation of immune responses, including BA- and pathogen-induced SA accumulation, and control of cell death.

Eukaryotic translation initiation factor 5A is involved in pathogen-induced cell death and development of disease symptoms in Arabidopsis.

Eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein found in all eukaryotic kingdoms. This study demonstrates that plant eIF5A is involved in the development of disease symptoms induced by a common necrotrophic bacterial phytopathogen. Specifically, AteIF5A-2, one of the three eIF5A genes in Arabidopsis (Arabidopsis thaliana), is shown to regulate programmed cell death caused by infection with virulent Pseudomonas syringae pv tomato DC3000 (Pst DC3000). Transgenic Arabidopsis plants with constitutively suppressed AteIF5A-2 exhibited marked resistance to programmed cell death induced by virulent Pst DC3000, and there was a corresponding reduction in pathogen growth and development of disease symptoms in the plant tissue. Constitutive overexpression of AteIF5A-2 circumvented the apparent posttranscriptional regulation of AteIF5A-2 protein expression characteristic of wild-type plants but did not increase susceptibility to virulent Pst DC3000 ingression. The transgenic plants with constitutive AteIF5A-2 overexpression did, however, display phenotypes consistent with precocious cell death. The results indicate that AteIF5A-2 is a key element of the signal transduction pathway resulting in plant programmed cell death.

A role for a menthone reductase in resistance against microbial pathogens in plants.

Plants elaborate a vast array of enzymes that synthesize defensive secondary metabolites in response to pathogen attack. Here, we isolated the pathogen-responsive CaMNR1 [menthone: (+)-(3S)-neomenthol reductase] gene, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, from pepper (Capsicum annuum) plants. Gas chromatography-mass spectrometry analysis revealed that purified CaMNR1 and its ortholog AtSDR1 from Arabidopsis (Arabidopsis thaliana) catalyze a menthone reduction with reduced nicotinamide adenine dinucleotide phosphate as a cofactor to produce neomenthol with antimicrobial activity. CaMNR1 and AtSDR1 also possess a significant catalytic activity for neomenthol oxidation. We examined the cellular function of the CaMNR1 gene by virus-induced gene silencing and ectopic overexpression in pepper and Arabidopsis plants, respectively. CaMNR1-silenced pepper plants were significantly more susceptible to Xanthomonas campestris pv vesicatoria and Colletotrichum coccodes infection and expressed lower levels of salicylic acid-responsive CaBPR1 and CaPR10 and jasmonic acid-responsive CaDEF1. CaMNR1-overexpressing Arabidopsis plants exhibited enhanced resistance to the hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000 and the biotrophic pathogen Hyaloperonospora parasitica isolate Noco2, accompanied by the induction of AtPR1 and AtPDF1.2. In contrast, mutation in the CaMNR1 ortholog AtSDR1 significantly enhanced susceptibility to both pathogens. Together, these results indicate that the novel menthone reductase gene CaMNR1 and its ortholog AtSDR1 positively regulate plant defenses against a broad spectrum of pathogens.

Plant Immunity: AvrPto Targets the Frontline

Bacterial pathogens must suppress host defences to cause disease. New research shows that the Pseudomonas effector protein AvrPto does so by directly targeting plant transmembrane receptor kinases involved in bacterial perception.

Natural variation in RPS2-mediated resistance among Arabidopsis accessions: correlation between gene expression profiles and phenotypic responses.

Natural variation in gene expression (expression traits or e-traits) is increasingly used for the discovery of genes controlling traits. An important question is whether a particular e-trait is correlated with a phenotypic trait. Here, we examined the correlations between phenotypic traits and e-traits among 10 Arabidopsis thaliana accessions. We studied defense against Pseudomonas syringae pv tomato DC3000 (Pst), with a focus on resistance gene-mediated resistance triggered by the type III effector protein AvrRpt2. As phenotypic traits, we measured growth of the bacteria and extent of the hypersensitive response (HR) as measured by electrolyte leakage. Genetic variation among accessions affected growth of Pst both with (Pst avrRpt2) and without (Pst) the AvrRpt2 effector. Variation in HR was not correlated with variation in bacterial growth. We also collected gene expression profiles 6 h after mock and Pst avrRpt2 inoculation using a custom microarray. Clusters of genes whose expression levels are correlated with bacterial growth or electrolyte leakage were identified. Thus, we demonstrated that variation in gene expression profiles of Arabidopsis accessions collected at one time point under one experimental condition has the power to explain variation in phenotypic responses to pathogen attack.

ARGONAUTE4 Is Required for Resistance toPseudomonas syringaeinArabidopsis

Here, we report the characterization of the Arabidopsis thaliana ocp11 (for overexpressor of cationic peroxidase11) mutant, in which a beta-glucuronidase reporter gene under the control of the H(2)O(2)-responsive Ep5C promoter is constitutively expressed. ocp11 plants show enhanced disease susceptibility to the virulent bacterium Pseudomonas syringae pv tomato DC3000 (P.s.t. DC3000) and also to the avirulent P.s.t. DC3000 carrying the effector avrRpm1 gene. In addition, ocp11 plants are also compromised in resistance to the nonhost pathogen P. syringae pv tabaci. Genetic and molecular analyses reveal that ocp11 plants are not affected in salicylic acid perception. We cloned OCP11 and show that it encodes ARGONAUTE4 (AGO4), a component of the pathway that mediates the transcriptional gene silencing associated with small interfering RNAs that direct DNA methylation at specific loci, a phenomenon known as RNA-directed DNA methylation (RdDM). Thus, we renamed our ocp11 mutant ago4-2, as it represents a different allele to the previously characterized recessive ago4-1. Both mutants decrease the extent of DNA cytosine methylation at CpNpG and CpHpH (asymmetric) positions present at different DNA loci and show commonalities in all of the molecular and phenotypic aspects that we have considered. Interestingly, we show that AGO4 works independently of other components of the RdDM pathway in mediating resistance to P.s.t. DC3000, and loss of function in other components of the pathway operating upstream of AGO4, such as RDR2 and DCL3, or operating downstream, such as DRD1, CMT3, DRM1, and DRM2, does not compromise resistance to this pathogen.

Microarray analysis of the Arabidopsis thaliana cir1 ( constitutively induced resistance 1) mutant reveals candidate defence response genes against Pseudomonas syringae pv tomato DC3000

Microarray expression profiling on the model plant Arabidopsis thaliana has contributed to the elucidation of plant defence responses and resistance against disease. An Arabidopsis mutant, cir1 ( constitutively induced resistance 1), previously showed enhanced resistance to the pathogenic biotrophic bacterium Pseudomonas syringae pv tomato ( Pst) DC3000. It was hypothesised that induced or repressed genes in cir1 may play a role in conferring resistance against this pathogen. We investigated differential gene expression in wild type and cir1 plants without pathogen challenge using a custom 500-probe microarray, biased towards defence-response and signalling genes, to identify transcripts, which may be required for resistance in cir1. Several genes were found to be induced in cir1 at a significance threshold of − log 10( p) equal to 3 ( p < 0.001) using a mixed model ANOVA approach. The induction of the genes encoding AtACP1 (sodium inducible calcium binding protein), AtP2C-HA (protein phosphatase 2C), AtGSTF7 (glutathione- S-transferase), tryptophan synthase beta-like and AtPAL1 (phenylalanine ammonia lyase 1) and the repression of AtEREBP-4 (ethylene response element binding protein 4) and HFR1 (long hypocotyl in far-red 1) in cir1 correlates with publicly available microarray data which shows the same genes differentially expressed in a similar manner in Arabidopsis plants infected with Pst. This observation supports our hypothesis that these genes contribute to disease resistance in cir1.

AtERF14, a member of the ERF family of transcription factors, plays a nonredundant role in plant defense.

We had previously shown that several transcription factors of the ethylene (ET) response factor (ERF) family were induced with different but overlapping kinetics following challenge of Arabidopsis (Arabidopsis thaliana) with Pseudomonas syringae pv tomato DC3000 (avrRpt2). One of these genes, a transcriptional activator, AtERF14, was induced at the same time as ERF-target genes (ChiB, basic chitinase). To unravel the potential function of AtERF14 in regulating the plant defense response, we have analyzed gain- and loss-of-function mutants. We show here that AtERF14 has a prominent role in the plant defense response, since overexpression of AtERF14 had dramatic effects on both plant phenotype and defense gene expression and AtERF14 loss-of-function mutants showed impaired induction of defense genes following exogenous ET treatment and increased susceptibility to Fusarium oxysporum. Moreover, the expression of other ERF genes involved in defense and ET/jasmonic acid responses, such as ERF1 and AtERF2, depends on AtERF14 expression. A number of ERFs have been shown to function in the defense response through overexpression. However, the effect of loss of AtERF14 function on defense gene expression, pathogen resistance, and regulation of the expression of other ERF genes is unique thus far. These results suggest a unique role for AtERF14 in regulating the plant defense response.

Modifications to the Arabidopsis defense proteome occur prior to significant transcriptional change in response to inoculation with Pseudomonas syringae.

Alterations in the proteome of Arabidopsis (Arabidopsis thaliana) leaves during responses to challenge by Pseudomonas syringae pv tomato DC3000 were analyzed using two-dimensional gel electrophoresis. Protein changes characteristic of the establishment of disease, basal resistance, and resistance-gene-mediated resistance were examined by comparing responses to DC3000, a hrp mutant, and DC3000 expressing avrRpm1, respectively. The abundance of each protein identified was compared with that of selected transcripts obtained from comparable GeneChip experiments. We report changes in three subcellular fractions: total soluble protein, chloroplast enriched, and mitochondria enriched over four time points (1.5-6 h after inoculation). In total, 73 differential spots representing 52 unique proteins were successfully identified. Many of the changes in protein spot density occurred before significant transcriptional reprogramming was evident between treatments. The high proportion of proteins represented by more than one spot indicated that many of the changes to the proteome can be attributed to posttranscriptional modifications. Proteins found to show significant change after bacterial challenge are representative of two main functional groups: defense-related antioxidants and metabolic enzymes. Significant changes to photosystem II and to components of the mitochondrial permeability transition were also identified. Rapid communication between organelles and regulation of primary metabolism through redox-mediated signaling are supported by our data.

The Arabidopsis Gene CAD1 Controls Programmed Cell Death in the Plant Immune System and Encodes a Protein Containing a MACPF Domain

To clarify the processes involved in plant immunity, we have isolated and characterized a single recessive Arabidopsis mutant, cad1 (constitutively activated cell death 1), which shows a phenotype that mimics the lesions seen in the hypersensitive response (HR). This mutant shows spontaneously activated expression of pathogenesis-related (PR) genes, and leading to a 32-fold increase in salicylic acid (SA). Inoculation of cad1 mutant plants with Pseudomonas syringae pv tomato DC3000 shows that the cad1 mutation results in the restriction of bacterial growth. Cloning of CAD1 reveals that this gene encodes a protein containing a domain with significant homology to the MACPF (membrane attack complex and perforin) domain of complement components and perforin proteins that are involved in innate immunity in animals. Furthermore, cell death is suppressed in transgenic cad1 plants expressing nahG, which encodes an SA-degrading enzyme. We therefore conclude that the CAD1 protein negatively controls the SA-mediated pathway of programmed cell death in plant immunity.

An Arabidopsis Homeodomain Transcription Factor, OVEREXPRESSOR OF CATIONIC PEROXIDASE 3, Mediates Resistance to Infection by Necrotrophic Pathogens

The mechanisms controlling plant resistance to necrotrophic fungal pathogens are poorly understood. We previously reported on Ep5C, a gene shown to be induced by the H(2)O(2) generated during a plant-pathogen interaction. To identify novel plant components operating in pathogen-induced signaling cascades, we initiated a large-scale screen using Arabidopsis thaliana plants carrying the beta-glucuronidase reporter gene under control of the H(2)O(2)-responsive Ep5C promoter. Here, we report the identification and characterization of a mutant, ocp3 (for overexpressor of cationic peroxidase 3), in which the reporter construct is constitutively expressed. Healthy ocp3 plants show increased accumulation of H(2)O(2) and express constitutively the Glutathione S-transferase1 and Plant Defensine 1.2 marker genes, but not the salicylic acid (SA)-dependent pathogenesis-related PR-1 gene. Strikingly, the ocp3 mutant shows enhanced resistance to the necrotrophic pathogens Botrytis cinerea and Plectosphaerella cucumerina. Conversely, resistance to virulent forms of the biotrophic oomycete Hyaloperonospora parasitica and the bacterial pathogen Pseudomonas syringae pv tomato DC3000 remains unaffected in ocp3 plants when compared with wild-type plants. Consistently with this, ocp3 plants are not affected in SA perception and express normal levels of PR genes after pathogen attack. To analyze signal transduction pathways where ocp3 operates, epistasis analyses between ocp3 and pad4, nahG, npr1, ein2, jin1, or coi1 were performed. These studies revealed that the resistance signaling to necrotrophic infection in ocp3 is fully dependent on appropriate perception of jasmonic acid through COI1 and does not require SA or ethylene perception through NPR1 or EIN2, respectively. The OCP3 gene encodes a homeodomain transcription factor that is constitutively expressed in healthy plants but repressed in response to infection by necrotrophic fungi. Together, these results suggest that OCP3 is an important factor for the COI1-dependent resistance of plants to infection by necrotrophic pathogens.

Lineage‐specific regions in Pseudomonas syringae pv tomato DC3000

SUMMARY Comparative analyses of the chromosome of Pseudomonas syringae pv tomato DC3000 with the finished, complete genomes of Pseudomonas aeruginosa PAO1, an animal pathogen, and the non-pathogenic soil inhabitant Pseudomonas putida KT2440 revealed a high degree of sequence conservation in genes involved in 'housekeeping functions'. However, divergence is present among these three fluorescent pseudomonads, yielding 'suites' of species-specific genes that may provide the genetic basis for adaptation to an ecological niche and lifestyle. For DC3000, 1053 genes located on the chromosome were specific to DC3000 and not present in PAO1 or KT2440. The majority of these DC3000-specific genes either lack a known function or are mobile genetic elements. However, these genes do share features among themselves such as association with regions of atypical trinucleotides, unusual G+C content and localization within large tracts of DC3000-specific sequence, suggestive of lateral gene transfer events. Indeed, a comparison of syntenic blocks among these three complete Pseudomonas genomes revealed that a substantial portion (533) of the DC3000-specific chromosomal genes (1053) were located in lineage-specific regions (defined as being larger than 2 kb and enriched in mobile genetic elements and/or genes specific to DC3000 in this three-way comparison). A large proportion of mobile genetic elements (199 of 318 genes; 63%), which are highly enriched in DC3000, were present within such regions. Similarly, most of the genes encoding type III secretion system virulence effectors were located in lineage-specific regions. Consistent with the plasticity of the DC3000 genome, a putative chromosomal inversion mediated by identical copies of ISPsy6 involving 2838 kb (44%) of the DC3000 genome was detected. These data suggest that a substantial portion of the differentiation of DC3000, a plant pathogen, from an animal pathogen and a soil inhabitant has involved transfer of a large number of novel genes coupled with amplification of mobile genetic elements.

Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production.

Relatively little is known about the exact mechanisms used by Bacillus subtilis in its behavior as a biocontrol agent on plants. Here, we report the development of a sensitive plant infection model demonstrating that the bacterial pathogen Pseudomonas syringae pv tomato DC3000 is capable of infecting Arabidopsis roots both in vitro and in soil. Using this infection model, we demonstrated the biocontrol ability of a wild-type B. subtilis strain 6051 against P. syringae. Arabidopsis root surfaces treated with B. subtilis were analyzed with confocal scanning laser microscopy to reveal a three-dimensional B. subtilis biofilm. It is known that formation of biofilms by B. subtilis is a complex process that includes secretion of surfactin, a lipopeptide antimicrobial agent. To determine the role of surfactin in biocontrol by B. subtilis, we tested a mutant strain, M1, with a deletion in a surfactin synthase gene and, thus, deficient in surfactin production. B. subtilis M1 was ineffective as a biocontrol agent against P. syringae infectivity in Arabidopsis and also failed to form robust biofilms on either roots or inert surfaces. The antibacterial activity of surfactin against P. syringae was determined in both broth and agar cultures and also by live-dead staining methods. Although the minimum inhibitory concentrations determined were relatively high (25 microg mL(-1)), the levels of the lipopeptide in roots colonized by B. subtilis are likely to be sufficient to kill P. syringae. Our results collectively indicate that upon root colonization, B. subtilis 6051 forms a stable, extensive biofilm and secretes surfactin, which act together to protect plants against attack by pathogenic bacteria.

NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol.

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signal transduction pathways in which salicylic acid (SA) and jasmonic acid (JA) play key roles. In this study, we investigated the molecular mechanism of the antagonistic effect of SA on JA signaling. Arabidopsis plants unable to accumulate SA produced 25-fold higher levels of JA and showed enhanced expression of the JA-responsive genes LOX2, PDF1.2, and VSP in response to infection by Pseudomonas syringae pv tomato DC3000, indicating that in wild-type plants, pathogen-induced SA accumulation is associated with the suppression of JA signaling. Analysis of the Arabidopsis mutant npr1, which is impaired in SA signal transduction, revealed that the antagonistic effect of SA on JA signaling requires the regulatory protein NPR1. Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression, is not required for the suppression of JA signaling, indicating that cross-talk between SA and JA is modulated through a novel function of NPR1 in the cytosol.

Enhanced expression and activation of the alternative oxidase during infection of Arabidopsis with Pseudomonas syringae pv tomato.

Cyanide-resistant ("alternative") respiration was studied in Arabidopsis during incompatible and compatible infection with Pseudomonas syringae pv tomato DC3000. Total leaf respiration increased as the leaves became necrotic, as did the cyanide-resistant component that was sensitive to salicylhydroxamic acid. Infiltration of leaves with an avirulent strain rapidly induced alternative oxidase (AOX) mRNA, whereas the increase was delayed in the compatible combination. The increase in mRNA correlated with the increase in AOX protein. Increased expression was confined to the infected leaves, in contrast to the pathogenesis-related protein-1, which was induced systemically. Virtually all of the AOX protein was in the reduced (high-activity) form. Using transgenic NahG and mutant npr1-1 and etr1-1 plants, we established that the rapid induction of the AOX was associated with necrosis and that ethylene, but not salicylic acid, was required for its induction. Increased pyruvate levels in the infected leaves suggested that increased substrate levels were respired through the alternative pathway; however, in the control leaves and the infected leaves, respiration was not inhibited by salicylhydroxamic acid alone. Increased respiration appeared to be associated primarily with symptom expression rather than resistance reactions.