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Overexpression of the disease resistance gene Pto in tomato induces gene expression changes similar to immune responses in human and fruitfly.

The Pto gene encodes a serine/threonine protein kinase that confers resistance in tomato (Lycopersicon esculentum) to Pseudomonas syringae pv tomato strains that express the type III effector protein AvrPto. Constitutive overexpression of Pto in tomato, in the absence of AvrPto, activates defense responses and confers resistance to several diverse bacterial and fungal plant pathogens. We have used a series of gene discovery and expression profiling methods to examine the effect of Pto overexpression in tomato leaves. Analysis of the tomato expressed sequence tag database and suppression subtractive hybridization identified 600 genes that were potentially differentially expressed in Pto-overexpressing tomato plants compared with a sibling line lacking Pto. By using cDNA microarrays, we verified changes in expression of many of these genes at various time points after inoculation with P. syringae pv tomato (avrPto) of the resistant Pto-overexpressing line and the susceptible sibling line. The combination of these three approaches led to the identification of 223 POR (Pto overexpression responsive) genes. Strikingly, 40% of the genes induced in the Pto-overexpressing plants previously have been shown to be differentially expressed during the human (Homo sapiens) and/or fruitfly (Drosophila melanogaster) immune responses.

Animals↗

The timing of senescence and response to pathogens is altered in the ascorbate-deficient Arabidopsis mutant vitamin c-1.

The ozone-sensitive Arabidopsis mutant vitamin c-1 (vtc1) is deficient in l-ascorbic acid (AsA) due to a mutation in GDP-Man pyrophosphorylase (Conklin et al., 1999), an enzyme involved in the AsA biosynthetic pathway (Smirnoff et al., 2001). In this study, the physiology of this AsA deficiency was initially investigated in response to biotic (virulent pathogens) stress and subsequently with regards to the onset of senescence. Infection with either virulent Pseudomonas syringae or Peronospora parasitica resulted in largely reduced bacterial and hyphal growth in the vtc1 mutant in comparison to the wild type. When vitamin c-2 (vtc2), another AsA-deficient mutant, was challenged with P. parasitica, growth of the fungus was also reduced, indicating that the two AsA-deficient mutants are more resistant to these pathogens. Induction of pathogenesis-related proteins PR-1 and PR-5 is significantly higher in vtc1 than in the wild type when challenged with virulent P. syringae. In addition, the vtc1 mutant exhibits elevated levels of some senescence-associated gene (SAG) transcripts as well as heightened salicylic acid levels. Presumably, therefore, low AsA is causing vtc1 to enter at least some stage(s) of senescence prematurely with an accompanying increase in salicylic acid levels that results in a faster induction of defense responses.

Arabidopsis↗

Fatty acid hydroperoxides and H2O2 in the execution of hypersensitive cell death in tobacco leaves.

We initially compared lipid peroxidation profiles in tobacco (Nicotiana tabacum) leaves during different cell death events. An upstream oxylipin assay was used to discriminate reactive oxygen species (ROS)-mediated lipid peroxidation from 9- and 13-lipoxygenase (LOX)-dependent lipid peroxidation. Free radical-mediated membrane peroxidation was measured during H(2)O(2)-dependent cell death in leaves of catalase-deficient plants. Taking advantage of these transgenic plants, we demonstrate that, under light conditions, H(2)O(2) plays an essential role in the execution of cell death triggered by an elicitor, cryptogein, which provokes a similar ROS-mediated lipid peroxidation. Under dark conditions, however, cell death induction by cryptogein was independent of H(2)O(2) and accompanied by products of the 9-LOX pathway. In the hypersensitive response induced by the avirulent pathogen Pseudomonas syringae pv syringae, both 9-LOX and oxidative processes operated concurrently, with ROS-mediated lipid peroxidation prevailing in the light. Our results demonstrate, therefore, the tight interplay between H(2)O(2) and lipid hydroperoxides and underscore the importance of light during the hypersensitive response.

Catalase↗

Gene-Expression Patterns and Levels of Jasmonic Acid in Rice Treated with the Resistance Inducer 2,6-Dichloroisonicotinic Acid.

Acquired disease resistance can be induced in rice (Oryza sativa) by a number of synthetic or natural compounds, but the molecular mechanisms behind the phenomenon are poorly understood. One of the synthetic inducers of resistance, 2,6-dichloroisonicotinic acid (INA), efficiently protected rice leaves from infection by the rice blast fungus Magnaporthe grisea (Hebert) Barr. A comparison of gene-expression patterns in plants treated with INA versus plants inoculated with the compatible pathogen M. grisea or the incompatible pathogen Pseudomonas syringae pv syringae revealed only a marginal overlap: 6 gene products, including pathogenesis-related proteins (PR1-PR9), accumulated in both INA-treated and pathogen-attacked leaves, whereas 26 other gene products accumulated only in INA-treated or only in pathogen-attacked leaves. Lipoxygenase enzyme activity and levels of nonconjugated jasmonic acid (JA) were enhanced in leaves of plants treated with a high dose of INA (100 ppm). Exogenously applied JA enhanced the gene induction and plant protection caused by lower doses of INA (0.1 to 10 ppm) that by themselves did not give rise to enhanced levels of endogenous (-)-JA. These data suggest that INA, aside from activating a pathogen-induced signaling pathway, also induces events that are not related to pathogenesis. JA acts as an enhancer of both types of INA-induced reactions in rice.

Journal Article↗

Accumulation of salicylic acid and 4-hydroxybenzoic acid in phloem fluids of cucumber during systemic acquired resistance is preceded by a transient increase in phenylalanine ammonia-lyase activity in petioles and stems.

Cucumber (Cucumis sativa) leaves infiltrated with Pseudomonas syringae pv. syringae cells produced a mobile signal for systemic acquired resistance between 3 and 6 h after inoculation. The production of a mobile signal by inoculated leaves was followed by a transient increase in phenylalanine ammonia-lyase (PAL) activity in the petioles of inoculated leaves and in stems above inoculated leaves; with peaks in activity at 9 and 12 h, respectively, after inoculation. In contrast, PAL activity in inoculated leaves continued to rise slowly for at least 18 h. No increases in PAL activity were detected in healthy leaves of inoculated plants. Two benzoic acid derivatives, salicylic acid (SA) and 4-hydroxybenzoic acid (4HBA), began to accumulate in phloem fluids at about the time PAL activity began to increase, reaching maximum concentrations 15 h after inoculation. The accumulation of SA and 4HBA in phloem fluids was unaffected by the removal of all leaves 6 h after inoculation, and seedlings excised from roots prior to inoculation still accumulated high levels of SA and 4HBA. These results suggest that SA and 4HBA are synthesized de novo in stems and petioles in response to a mobile signal from the inoculated leaf.

Cucumis sativus↗

Isolation of ethylene-insensitive soybean mutants that are altered in pathogen susceptibility and gene-for-gene disease resistance

Plants commonly respond to pathogen infection by increasing ethylene production, but it is not clear if this ethylene does more to promote disease susceptibility or disease resistance. Ethylene production and/or responsiveness can be altered by genetic manipulation. The present study used mutagenesis to identify soybean (Glycine max L. Merr.) lines with reduced sensitivity to ethylene. Two new genetic loci were identified, Etr1 and Etr2. Mutants were compared with isogenic wild-type parents for their response to different soybean pathogens. Plant lines with reduced ethylene sensitivity developed similar or less-severe disease symptoms in response to virulent Pseudomonas syringae pv glycinea and Phytophthora sojae, but some of the mutants developed similar or more-severe symptoms in response to Septoria glycines and Rhizoctonia solani. Gene-for-gene resistance against P. syringae expressing avrRpt2 remained effective, but Rps1-k-mediated resistance against P. sojae races 4 and 7 was disrupted in the strong ethylene-insensitive etr1-1 mutant. Rps1-k-mediated resistance against P. sojae race 1 remained effective, suggesting that the Rps1-k locus may encode more than one gene for disease resistance. Overall, our results suggest that reduced ethylene sensitivity can be beneficial against some pathogens but deleterious to resistance against other pathogens.

Journal Article↗

Study of the role of antimicrobial glucosinolate-derived isothiocyanates in resistance of Arabidopsis to microbial pathogens.

Crude aqueous extracts from Arabidopsis leaves were subjected to chromatographic separations, after which the different fractions were monitored for antimicrobial activity using the fungus Neurospora crassa as a test organism. Two major fractions were obtained that appeared to have the same abundance in leaves from untreated plants versus leaves from plants challenge inoculated with the fungus Alternaria brassicicola. One of both major antimicrobial fractions was purified to homogeneity and identified by 1H nuclear magnetic resonance, gas chromatography/electron impact mass spectrometry, and gas chromatography/chemical ionization mass spectrometry as 4-methylsulphinylbutyl isothiocyanate (ITC). This compound has previously been described as a product of myrosinase-mediated breakdown of glucoraphanin, the predominant glucosinolate in Arabidopsis leaves. 4-Methylsulphinylbutyl ITC was found to be inhibitory to a wide range of fungi and bacteria, producing 50% growth inhibition in vitro at concentrations of 28 microM for the most sensitive organism tested (Pseudomonas syringae). A previously identified glucosinolate biosynthesis mutant, gsm1-1, was found to be largely deficient in either of the two major antimicrobial compounds, including 4-methylsulphinylbutyl ITC. The resistance of gsm1-1 was compared with that of wild-type plants after challenge with the fungi A. brassicicola, Plectosphaerella cucumerina, Botrytis cinerea, Fusarium oxysporum, or Peronospora parasitica, or the bacteria Erwinia carotovora or P. syringae. Of the tested pathogens, only F. oxysporum was found to be significantly more aggressive on gsm1-1 than on wild-type plants. Taken together, our data suggest that glucosinolate-derived antimicrobial ITCs can play a role in the protection of Arabidopsis against particular pathogens.

Arabidopsis↗

Evidence for the involvement of an oxidative stress in the initiation of infection of pear by Erwinia amylovora.

Involvement of an oxidative burst, usually related to incompatible plant/pathogen interactions leading to hypersensitive reactions, was investigated with Erwinia amylovora, the causal agent of fire blight of Maloideae subfamily of Rosaceae, in interaction with pear (Pyrus communis; compatible situation) and tobacco (Nicotiana tabacum; incompatible situation). As expected, this necrogenic bacterium induced in tobacco a sustained production of superoxide anion, lipid peroxidation, electrolyte leakage, and concomitant increases of several antioxidative enzymes (ascorbate peroxidases, glutathion reductases, glutathion-S-transferases, and peroxidases), in contrast to the compatible pathogen Pseudomonas syringae pv tabaci, which did not cause such reactions. In pear leaves, however, inoculations with both the disease- and the hypersensitive reaction-inducing bacteria (E. amylovora and P. syringae pv tabaci, respectively) resulted in superoxide accumulation, lipid peroxidation, electrolyte leakage, and enzyme induction at similar rates and according to equivalent time courses. The unexpected ability of E. amylovora to generate an oxidative stress even in compatible situation was linked to its functional hrp (for hypersensitive reaction and pathogenicity) cluster because an Hrp secretion mutant of the bacteria did not induce any plant response. It is suggested that E. amylovora uses the production of reactive oxygen species as a tool to provoke host cell death during pathogenesis to invade plant tissues. The bacterial exopolysaccharide could protect this pathogen against the toxic effects of oxygen species since a non-capsular mutant of E. amylovora induced locally the same responses than the wild type but was unable to further colonize the plant.

Ascorbate Peroxidases↗

Harpin induces activation of the Arabidopsis mitogen-activated protein kinases AtMPK4 and AtMPK6.

Mitogen-activated protein kinases (MAPKs) are key enzymes that mediate adaptive responses to various abiotic and biotic stresses, including pathogen challenge. The proteinaceous bacterial elicitor harpin (secreted by Pseudomonas syringae pv syringae) activates two MAPKs in suspension cultures of Arabidopsis var. Landsberg erecta. In this study, we show that harpin and exogenous hydrogen peroxide (H(2)O(2)) activate myelin basic protein kinases in Arabidopsis leaves. Using anti-AtMPK4 and anti-AtMPK6 antibodies, we identify the harpin-activated MAPKs in both leaves and suspension cultures as AtMPK4 and AtMPK6, and show that H(2)O(2), generated by Arabidopsis cells in response to challenge with harpin, activates only AtMPK6. However, treatments with catalase, which removes H(2)O(2), or diphenylene iodonium, which inhibits superoxide and H(2)O(2) production, do not inhibit harpin-induced activation of AtMPK4 or AtMPK6. In addition, activation of AtMPK4 but not AtMPK6 is inhibited by the MAPK kinase inhibitor PD98059. Neither harpin nor H(2)O(2) has any effect on AtMPK4 or AtMPK6 gene expression. In addition, the expression of AtMEKK1, AtMEK1, or AtMKK2, previously shown to be potential functional partners of AtMPK4, were not affected by either harpin or H(2)O(2) treatments. These data suggest that harpin activates several signaling pathways, one leading to stimulation of the oxidative burst and others leading to the activation of AtMPK4 or AtMPK6.

Arabidopsis↗

Transient Activation of Plasmalemma K Efflux and H Influx in Tobacco by a Pectate Lyase Isozyme from Erwinia chrysanthemi.

A purified pectate lyase isozyme derived from Erwinia chrysanthemi induced rapid net K(+) efflux and H(+) influx in suspension-cultured tobacco cells. Comparable fluxes of other ions (Na(+), Cl(-)) were not observed. The K(+) efflux/H(+) influx response began within 15 minutes after addition of enzyme to cell suspensions and continued for approximately 1 hour after which cells resumed the net H(+) efflux exhibited prior to enzyme treatment. The response was not prolonged by a second enzyme dose 1 hour after the first. The K(+)/H(+) response was characterized by saturation at low enzymic activity (2 x 10(-3) units per milliliter), and inhibition by the protonophore, carbonyl cyanide m-chlorophenylhydrazone, and was not associated with membrane leakiness caused by structural cell wall damage. The total K(+) loss and H(+) uptake induced by enzyme was one-fourth to one-third that induced by Pseudomonas syringae pv. pisi and did not reduce cell viability. These results indicate that pectate lyase induces a K(+) efflux/H(+) influx response in tobacco similar to but of shorter duration than that induced by P. syringae pv. pisi during the hypersensitive response. Pectate lyase or other cell wall degrading enzymes may therefore influence the induction of hypersensitivity.

Journal Article↗

Involvement of plasma membrane calcium influx in bacterial induction of the k/h and hypersensitive responses in tobacco.

An early event in the hypersensitive response of tobacco to Pseudomonas syringae pv syringae is the initiation of a K(+)/H(+) response characterized by specific plasma membrane K(+) efflux, extracellular alkalinization, and intracellular acidification. We investigated the role of calcium in induction of these host responses. Suspension-cultured tobacco cells exhibited a baseline Ca(2+) influx of 0.02 to 0.06 micromole per gram per hour as determined from (45)Ca(2+) uptake. Following bacterial inoculation, uptake rates began to increase coincidently with onset of the K(+)/H(+) response. Rates increased steadily for 2 to 3 hours, reaching 0.5 to 1 micromole per gram per hour. This increased Ca(2+) influx was prevented by EGTA and calcium channel blockers such as La(3+), Co(2+), and Cd(2+) but not by verapamil and nifedipine. Lanthanum, cobalt, cadmium, and EGTA inhibited the K(+)/H(+) response in both suspension-cultured cells and leaf discs and prevented hypersensitive cell death in leaf discs. We conclude that increased plasmalemma Ca(2+) influx is required for the K(+)/H(+) and hypersensitive responses in tobacco.

Journal Article↗

The tomato transcription factor Pti4 regulates defense-related gene expression via GCC box and non-GCC box cis elements.

The tomato transcription factor Pti4, an ethylene-responsive factor (ERF), interacts physically with the disease resistance protein Pto and binds the GCC box cis element that is present in the promoters of many pathogenesis-related (PR) genes. We reported previously that Arabidopsis plants expressing Pti4 constitutively express several GCC box-containing PR genes and show reduced disease symptoms compared with wild-type plants after inoculation with Pseudomonas syringae pv tomato or Erysiphe orontii. To gain insight into how genome-wide gene expression is affected by Pti4, we used serial analysis of gene expression (SAGE) to compare transcripts in wild-type and Pti4-expressing Arabidopsis plants. SAGE provided quantitative measurements of >20,000 transcripts and identified the 50 most highly expressed genes in Arabidopsis vegetative tissues. Comparison of the profiles from wild-type and Pti4-expressing Arabidopsis plants revealed 78 differentially abundant transcripts encoding defense-related proteins, protein kinases, ribosomal proteins, transporters, and two transcription factors (TFs). Many of the genes identified were expressed differentially in wild-type Arabidopsis during infection by Pseudomonas syringae pv tomato, supporting a role for them in defense-related processes. Unexpectedly, the promoters of most Pti4-regulated genes did not have a GCC box. Chromatin immunoprecipitation experiments confirmed that Pti4 binds in vivo to promoters lacking this cis element. Potential binding sites for ERF, MYB, and GBF TFs were present in statistically significantly increased numbers in promoters regulated by Pti4. Thus, Pti4 appears to regulate gene expression directly by binding the GCC box and possibly a non-GCC box element and indirectly by either activating the expression of TF genes or interacting physically with other TFs.

Arabidopsis↗

The gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses, and cell growth.

We isolated a dominant gain-of-function Arabidopsis mutant, accelerated cell death 6 (acd6), with elevated defenses, patches of dead and enlarged cells, reduced stature, and increased resistance to Pseudomonas syringae. The acd6-conferred phenotypes are suppressed by removing a key signaling molecule, salicylic acid (SA), by using the nahG transgene, which encodes SA hydroxylase. This suppression includes phenotypes that are not induced by application of SA to wild-type plants, indicating that SA acts with a second signal to cause many acd6-conferred phenotypes. acd6-nahG plants show hyperactivation of all acd6-conferred phenotypes after treatment with a synthetic inducer of the SA pathway, benzo(1,2, 3)thiadiazole-7-carbothioic acid (BTH), suggesting that SA acts with and also modulates the levels and/or activity of the second defense signal. acd6 acts partially through a NONEXPRESSOR OF PR 1 (NPR1) gene-independent pathway that activates defenses and confers resistance to P. syringae. Surprisingly, BTH-treated acd6-nahG plants develop many tumor-like abnormal growths, indicating a possible role for SA in modulating cell growth.

Arabidopsis↗

A disease resistance gene in Arabidopsis with specificity for two different pathogen avirulence genes.

The RPS3 and RPM1 disease resistance loci of Arabidopsis confer resistance to Pseudomonas syringae strains that carry the avirulence genes avrB and avrRpm1, respectively. We have previously shown that RPS3 and RPM1 are closely linked genetically. Here, we show that RPS3 and RPM1 are in fact the same gene. We screened a mutagenized Arabidopsis population with a P. syringae strain carrying avrB and found 12 susceptible mutants. All 12 mutants were also susceptible to an isogenic strain carrying avrRpm1, indicating a loss of both RPS3 and RPM1 functions. No mutants were recovered that lost only RPS3 function. Genetic analysis of four independent mutants revealed that the lesions were in RPS3. Thus, a single gene in Arabidopsis confers resistance that is specific to two distinct pathogen avirulence genes--a gene-for-genes interaction. This observation suggests that the RPS3/RPM1 gene product can bind multiple pathogen ligands, or alternatively, that it does not function as a receptor.

Arabidopsis↗

Interference between Two Specific Pathogen Recognition Events Mediated by Distinct Plant Disease Resistance Genes.

We demonstrate that the interaction of the avirulence gene avrRpt2 and the cognate resistance gene RPS2 interferes with the interaction of avrRpm1-RPM1 in Arabidopsis. Interference is mediated outside of the bacterial pathogen Pseudomonas syringae, presumably at the level of recognition of avr-dependent signals, yet does not require the wild-type RPS2 product. A numerical excess of P. syringae expressing avrRpm1 can overcome this interference in mixed inoculations. The interference of avrRpt2-RPS2 engagement with RPM1-dependent functions is mirrored by transcriptional activation of genes preferentially expressed during RPM1- or RPS2-mediated disease resistance reactions. This demonstration of interference between two plant disease resistance genes suggests that their products compete for a common element(s) in a signal transduction pathway leading to disease resistance.

Journal Article↗

Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression.

Systemic acquired resistance is a pathogen-inducible defense mechanism in plants. The resistant state is dependent on endogenous accumulation of salicylic acid (SA) and is characterized by the activation of genes encoding pathogenesis-related (PR) proteins. Recently, selected nonpathogenic, root-colonizing biocontrol bacteria have been shown to trigger a systemic resistance response as well. To study the molecular basis underlying this type of systemic resistance, we developed an Arabidopsis-based model system using Fusarium oxysporum f sp raphani and Pseudomonas syringae pv tomato as challenging pathogens. Colonization of the rhizosphere by the biological control strain WCS417r of P. fluorescens resulted in a plant-mediated resistance response that significantly reduced symptoms elicited by both challenging pathogens. Moreover, growth of P. syringae in infected leaves was strongly inhibited in P. fluorescens WCS417r-treated plants. Transgenic Arabidopsis NahG plants, unable to accumulate SA, and wild-type plants were equally responsive to P. fluorescens WCS417r-mediated induction of resistance. Furthermore, P. fluorescens WCS417r-mediated systemic resistance did not coincide with the accumulation of PR mRNAs before challenge inoculation. These results indicate that P. fluorescens WCS417r induces a pathway different from the one that controls classic systemic acquired resistance and that this pathway leads to a form of systemic resistance independent of SA accumulation and PR gene expression.

Arabidopsis↗

Cloning of a large gene cluster involved in Erwinia amylovora CFBP1430 virulence.

Phage MudIIPR13 insertional mutagenesis of Erwinia amylovora CFBP1430 allowed us to isolate 6900 independent CmR mutants. The frequencies of different auxotrophs in this population indicated that MudIIPR13 had inserted randomly in E. amylovora. Screening of 3500 CmR mutants on (i) apple calli and (ii) pear and apple seedlings led to the isolation of 19 non-pathogenic prototrophic single mutants, four of which expressed a LacZ+ hybrid protein. Expression of the fusion proteins was temperature sensitive. The 19 mutants could be separated into two classes according to their behaviour on tobacco: 13 were unable to elicit the hypersensitive response on tobacco (Hrp-) while six still could (Dsp-). The 19 MudIIPR13 insertions all mapped in the same virulence region. The MudIIPR13 insertions of Hrp- mutants were all clustered on the left part of this region, while the MudIIPR13 insertions of Dsp- mutants were located on the right part. All of the mutants except one, which proved to have a large deletion of the entire virulence region, could be complemented functionally by cosmids from an E. amylovora CFBP1430 genomic library. No hybridization was observed between the cosmid pPV130, which complemented 12 hrp::MudIIPR13 mutations, and the hrp genes from Pseudomonas syringae pv. phaseolicola (Lindgren et al., 1986), P. syringae pv. tomato (N.J. Panopoulos, unpublished data) or P. solanacearum (Boucher et al., 1987). Further analysis of the large virulence region will allow mapping of the border of the virulence region and facilitate the study of the function and regulation of the hrp and dsp genes.

Cloning, Molecular↗

Eukaryotic cyclophilin as a molecular switch for effector activation.

Gram-negative phytopathogenic bacteria, such as Pseudomonas syringae, deliver multiple effector proteins into plant cells during infection. It is hypothesized that certain plant and mammalian effector proteins need to traverse the type III secretion system unfolded and are delivered into host cells as inactive enzymes. We have previously identified cyclophilin as the Arabidopsis eukaryotic activator of AvrRpt2, a P. syringae effector that is a cysteine protease. Cyclophilins are general folding catalysts and possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. In this paper, we demonstrate the mechanism of AvrRpt2 activation by the Arabidopsis cyclophilin ROC1. ROC1 mutants lacking PPIase enzymatic activity were unable to activate AvrRpt2. Furthermore, nuclear magnetic resonance spectroscopy revealed a structural change in AvrRpt2 from an unfolded to a folded state in the presence of ROC1. Using in vitro binding assays, ROC1's consensus binding sequence was identified as GPxL, a motif present at four sites within AvrRpt2. The GPxL motifs are located in close proximity to AvrRpt2's catalytic triad and are required for protease activity both in vitro and in planta. These data suggest that after delivery into the plant cell during infection, cyclophilin binds AvrRpt2 at four sites and properly folds the effector protein by peptidyl-prolyl cis/trans isomerization.

Amino Acid Sequence↗