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A bean cDNA expressed during a hypersensitive reaction encodes a putative calcium-binding protein.

The hypersensitive reaction (HR) is an inducible plant response that is associated with disease resistance. It is characterized by rapid, localized cell death at the site of infection and is believed to inhibit the spread of invading pathogens. We have isolated a cDNA clone, designated Hra32 (for hypersensitive reaction associated), corresponding to an RNA transcript that accumulates in bean during an HR. The predicted protein product of the Hra32 cDNA is an approximately 17 kDa protein of 161 amino acids, with four putative EF-hand calcium-binding domains. The temporal pattern of Hra32 transcript accumulation correlated closely with the onset of the HR in bean after inoculation with incompatible Pseudomonas syringae pv. tabaci and pv. tomato and with tobacco necrosis virus. Hra32 transcript also accumulated in bean in response to compatible P. syringae pv. phaseolicola and was correlated with necrotic cell death associated with disease lesion formation. A more transient pattern of Hra32 transcript accumulation occurred in bean in response to general stimuli that did not result in the HR or host cell death. These treatments included infiltration with a P. syringae pv. tabaci Hrp- mutant, P. syringae pv. tabaci cells treated with kanamycin, Escherichia coli, P. fluorescens, or glutathione, and in response to wounding. Thus, there was differential accumulation of the Hra32 transcript in response to specific stimuli resulting in the HR, compared with general stimuli that did not result in cell death. We hypothesize that the Hra32 product may be a component of the pathway that leads to hypersensitive cell death.

Amino Acid Sequence↗

Syringolin-mediated activation of the Pir7b esterase gene in rice cells is suppressed by phosphatase inhibitors.

Inoculation of rice plants (Oryza sativa) with the nonhost pathogen Pseudomonas syringae pv. syringae leads to the activation of defense-related genes and ultimately to induced resistance against the rice blast fungus Pyricularia oryzae. One of the molecular determinants of P. syringae pv. syringae that is recognized by the plant cells and evokes these defense responses is syringolin A, an elicitor that is secreted by the bacteria under appropriate conditions. In order to investigate signal transduction events elicited by syringolin A, the response of cultured rice cells to syringolin A application was analyzed. Cultured rice cells were able to sense syringolin A at concentrations in the nanomolar range as observed by the transient accumulation of Pir7b esterase transcripts. Syringolin A-mediated Pir7b transcript accumulation was inhibited by cycloheximide, indicating that de novo protein synthesis was required. Calyculin and okadaic acid, two protein phosphatase inhibitors, blocked Pir7b gene induction, whereas the serine/threonine protein kinase inhibitors staurosporine and K-252a had no effect on Pir7b transcript levels. Actin transcript levels were essentially not affected by inhibitor treatments over the experimental time span. These results imply that dephosphorylation of a phosphoprotein is an important step in the syringolin A-triggered signal transduction pathway.

Carbazoles↗

Characterization of a novel, defense-related Arabidopsis mutant, cir1, isolated by luciferase imaging.

In order to identify components of the defense signaling network engaged following attempted pathogen invasion, we generated a novel PR-1::luciferase (LUC) transgenic line that was deployed in an imaging-based screen to uncover defense-related mutants. The recessive mutant designated cir1 exhibited constitutive expression of salicylic acid (SA), jasmonic acid (JA)/ethylene, and reactive oxygen intermediate-dependent genes. Moreover, this mutation conferred resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and a virulent oomycete pathogen Peronospora parasitica Noco2. Epistasis analyses were undertaken between cir1 and mutants that disrupt the SA (nprl, nahG), JA (jar1), and ethylene (ET) (ein2) signaling pathways. While resistance against both P. syringae pv. tomato DC3000 and Peronospora parasitica Noco2 was partially reduced by npr1, resistance against both of these pathogens was lost in an nahG genetic background. Hence, cirl-mediated resistance is established via NPR1-dependent and -independent signaling pathways and SA accumulation is essential for the function of both pathways. While jar1 and ein2 reduced resistance against P. syringae pv. tomato DC3000, these mutations appeared not to impact cir1-mediated resistance against Peronospora parasitica Noco2. Thus, JA and ET sensitivity are required for cir1-mediated resistance against P. syringae pv. tomato DC3000 but not Peronospora parasitica Noco2. Therefore, the cir1 mutation may define a negative regulator of disease resistance that operates upstream of SA, JA, and ET accumulation.

Arabidopsis↗

Expression of a bacterial ice nucleation gene in plants.

We have introduced an ice nucleation gene (inaZ) from Pseudomonas syringae pv. syringae into Nicotiana tabacum, a freezing-sensitive species, and Solanum commersonii, a freezing-tolerant species. Transformants of both species showed increased ice nucleation activity over untransformed controls. The concentration of ice nuclei detected at -10.5 degrees C in 15 different primary transformants of S. commersonii varied by over 1000-fold, and the most active transformant contained over 100 ice nuclei/mg of tissue. The temperature of the warmest freezing event in plant samples of small mass was increased from approximately -12 degrees C in the untransformed controls to -4 degrees C in inaZ-expressing transformants. The threshold nucleation temperature of samples from transformed plants did not increase appreciably with the mass of the sample. The most abundant protein detected in transgenic plants using immunological probes specific to the inaZ protein exhibited a higher mobility on sodium dodecyl sulfate polyacrylamide gels than the inaZ protein from bacterial sources. However, some protein with a similar mobility to the inaZ protein could be detected. Although the warmest ice nucleation temperature detected in transgenic plants is lower than that conferred by this gene in P. syringae (-2 degrees C), our results demonstrate that the ice nucleation gene of P. syringae can be expressed in plant cells to produce functional ice nuclei.

Journal Article↗

The Active Oxygen Response of Cell Suspensions to Incompatible Bacteria Is Not Sufficient to Cause Hypersensitive Cell Death.

The inoculation of tobacco (Nicotiana tabacum L.) suspension cells with bacterial pathogens that elicit the hypersensitive response (HR) in leaves has been shown to elicit production of active oxygen. This response occurs in two phases, the second of which occurs 1 to 3 h after bacterial addition and is unique to HR-causing interactions. The relationship between the phase II active oxygen response and the HR was characterized using Pseudomonas syringae pv syringae and P. fluorescens (pHIR11), which contains a cosmid clone of the hrp/hrm region from P. syringae pv syringae. TnphoA mutations in complementation groups II through XIII of the hrp cluster blocked the phase II active oxygen response, whereas mutations in the group I hrmA locus did not affect phase II. Despite the normal active oxygen response, bacteria with mutations in the hrmA region did not cause the HR in intact tobacco leaves nor did they induce hypersensitive cell death in cell suspensions. The data indicate that the bacteria do not require the hrmA region to elicit active oxygen production, but a full and intact hrp/hrm region is required to elicit hypersensitive cell death. Therefore, the phase II active oxygen response does not directly cause hypersensitive cell death nor is the response itself sufficient to trigger the HR.

Journal Article↗

Arabidopsis RIN4 negatively regulates disease resistance mediated by RPS2 and RPM1 downstream or independent of the NDR1 signal modulator and is not required for the virulence functions of bacterial type III effectors AvrRpt2 or AvrRpm1.

Bacterial pathogens deliver type III effector proteins into the plant cell during infection. On susceptible (r) hosts, type III effectors can contribute to virulence. Some trigger the action of specific disease resistance (R) gene products. The activation of R proteins can occur indirectly via modification of a host target. Thus, at least some type III effectors are recognized at site(s) where they may act as virulence factors. These data indicate that a type III effector's host target might be required for both initiation of R function in resistant plants and pathogen virulence in susceptible plants. In Arabidopsis thaliana, RPM1-interacting protein 4 (RIN4) associates with both the Resistance to Pseudomonas syringae pv maculicola 1 (RPM1) and Resistance to P. syringae 2 (RPS2) disease resistance proteins. RIN4 is posttranslationally modified after delivery of the P. syringae type III effectors AvrRpm1, AvrB, or AvrRpt2 to plant cells. Thus, RIN4 may be a target for virulence functions of these type III effectors. We demonstrate that RIN4 is not the only host target for AvrRpm1 and AvrRpt2 in susceptible plants because its elimination does not diminish their virulence functions. In fact, RIN4 negatively regulates AvrRpt2 virulence function. RIN4 also negatively regulates inappropriate activation of both RPM1 and RPS2. Inappropriate activation of RPS2 is nonspecific disease resistance 1 (NDR1) independent, in contrast with the established requirement for NDR1 during AvrRpt2-dependent RPS2 activation. Thus, RIN4 acts either cooperatively, downstream, or independently of NDR1 to negatively regulate RPS2 in the absence of pathogen. We propose that many P. syringae type III effectors have more than one target in the host cell. We suggest that a limited set of these targets, perhaps only one, are associated with R proteins. Thus, whereas any pathogen virulence factor may have multiple targets, the perturbation of only one is necessary and sufficient for R activation.

Arabidopsis↗

Long-term storage of plant-pathogenic bacteria in sterile distilled water.

This study was made to determine the effectiveness of the preservation of plant-pathogenic bacteria in sterile distilled water. After 20 or 24 years of storage in distilled water, a very high percentage (90 to 92%) of the isolates of Agrobacterium tumefaciens and Pseudomonas spp. were still alive. Moreover, 12 of 13 viable (after 24 years) isolates of P. syringae subsp. syringae maintained their ability to produce syringomycin and were pathogenic to bean seedlings. The water-stored cells of two isolates of P. syringae subsp. syringae, when observed by electron microscopy, were smaller than cells of 24-h-old subcultures of bacterial cells grown in nutrient broth; the water-stored cells appeared plasmolysed with an electron-dense cytoplasm and thickened cell wall.

Journal Article↗

Accumulation of copper and other metals by copper-resistant plant-pathogenic and saprophytic pseudomonads.

Copper-resistant strains of Pseudomonas syringae carrying the cop operon produce periplasmic copper-binding proteins, and this sequestration outside the cytoplasm has been proposed as a resistance mechanism. In this study, strain PS61 of P. syringae carrying the cloned cop operon accumulated more total cellular copper than without the operon. Several other copper-resistant pseudomonads with homology to cop were isolated from plants, and these bacteria also accumulated copper. Two highly resistant species accumulated up to 115 to 120 mg of copper per g (dry weight) of cells. P. putida 08891 was more resistant to several metals than P. syringae pv. tomato PT23, but this increased resistance was not correlated with an increased accumulation of metals other than copper. Several metals were accumulated by both PT23 and P. putida, but when copper was added to induce the cop operon, there was generally no increase of accumulation of the other metals, suggesting that the cop operon does not contribute to accumulation of these other metals. The exceptions were aluminum for PT23 and iron for P. putida, which accumulated to higher levels when copper was added to the cultures. The results of this study support the role of copper sequestration in the copper resistance mechanism of P. syringae and suggest that this mechanism is common to several copper-resistant Pseudomonas species found on plants to which antimicrobial copper compounds are applied for plant disease control.

Journal Article↗

Regulation of resistance to copper in Xanthomonas axonopodis pv. vesicatoria.

Copper-resistant strains of Xanthomonas axonopodis pv. vesicatoria were previously shown to carry plasmid-borne copper resistance genes related to the cop and pco operons of Pseudomonas syringae and Escherichia coli, respectively. However, instead of the two-component (copRS and pcoRS) systems determining copper-inducible expression of the operons in P. syringae and E. coli, a novel open reading frame, copL, was found to be required for copper-inducible expression of the downstream multicopper oxidase copA in X. axonopodis. copL encodes a predicted protein product of 122 amino acids that is rich in histidine and cysteine residues, suggesting a possible direct interaction with copper. Deletions or frameshift mutations within copL, as well as an amino acid substitution generated at the putative start codon of copL, caused a loss of copper-inducible transcriptional activation of copA. A nonpolar insertion of a kanamycin resistance gene in copL resulted in copper sensitivity in the wild-type strain. However, repeated attempts to complement copL mutations in trans failed. Analysis of the genomic sequence databases shows that there are copL homologs upstream of copAB genes in X. axonopodis pv. citri, X. campestris pv. campestris, and Xylella fastidiosa. The cloned promoter area upstream of copA in X. axonopodis pv. vesicatoria did not function in Pseudomonas syringae or in E. coli, nor did the P. syringae cop promoter function in Xanthomonas. However, a transcriptional fusion of the Xanthomonas cop promoter with the Pseudomonas copABCDRS was able to confer resistance to copper in Xanthomonas, showing divergence in the mechanisms of regulation of the resistance to copper in phytopathogenic bacteria.

Bacterial Proteins↗

Cloning and expression of bacterial ice nucleation genes in Escherichia coli.

Epiphytic populations of Pseudomonas syringae and Erwinia herbicola are important sources of ice nuclei that incite frost damage in agricultural crop plants. We have cloned and characterized DNA segments carrying the genes (ice) responsible for the ice-nucleating ability of these bacteria. The ice region spanned 3.5 to 4.0 kilobases and was continuous over this region in P. syringae Cit7R1. The cloned fragments imparted ice-nucleating activity in Escherichia coli. Substantial increases in the nucleating activity of both E. coli and P. syringae were obtained by subcloning the DNA fragments on multicopy plasmid vectors. Southern blot analysis showed substantial homology between the ice regions of P. syringae and E. herbicola, although individual restriction sites within the ice regions differed between the two species.

Chromosome Mapping↗

hrpL activates Erwinia amylovora hrp gene transcription and is a member of the ECF subfamily of sigma factors.

hrpL of Erwinia amylovora Ea321 encodes a 21.7-kDa regulatory protein, similar to members of the ECF (extra cytoplasmic functions) subfamily of eubacterial RNA polymerase sigma factors. hrpL is a single-gene operon in complementation group VI of the E. amylovora hrp gene cluster. Its product is required by Ea321 to elicit the hypersensitive response (HR) and to cause disease. HrpL controls the expression of five independent hrp loci, including hrpN, which encodes harpin, a proteinaceous elicitor of the HR. hrpL is environmentally regulated, and its expression is affected by hrpS, another regulatory gene of the hrp gene cluster of E. amylovora. pCPP1078, a multicopy plasmid carrying hrpL, is able to restore HR-eliciting ability to hrpS mutants. A conserved motif was identified upstream of the hrpI and hrpN operons, which are transcriptionally regulated by hrpL. This conserved motif shares a high degree of similarity with other biochemically defined or putative ECF-dependent promoter sequences, including sequences upstream of Streptomyces coelicolor dagA P2, Pseudomonas aeruginosa algD, Pseudomonas syringae pv. syringae 61 hrpZ, and P. syringae pv. tomato avrD. In spite of the similarity between the hrpL genes of E. amylovora and P. syringae 61, no functional cross-complementation was observed.

Amino Acid Sequence↗

The hrpA and hrpC operons of Erwinia amylovora encode components of a type III pathway that secretes harpin.

A 6.2-kb region of DNA corresponding to complementation groups II and III of the Erwinia amylovora hrp gene cluster was analyzed. Transposon mutagenesis indicated that the two complementation groups are required for secretion of harpin, an elicitor of the hypersensitive reaction. The sequence of the region revealed 10 open reading frames in two putative transcription units: hrpA, hrpB, hrcJ, hrpD, and hrpE in the hrpA operon (group III) and hrpF, hrpG, hrcC, hrpT, and hrpV in the hrpC operon (group II). From promoter regions of the hrpA, hrpC, and hrpN operons, sequences similar to those of the HrpL-dependent promoters of Pseudomonas syringae pathovars were identified with a consensus sequence of 5'-GGAAC-N17-18-CACTNAA-3'. The protein products of seven genes, hrpA, hrcJ, hrpE, hrpF, hrpG, hrcC, and hrpV, were visualized with a T7 polymerase/promoter expression system. HrcC, HrcJ, and HrpT sequences contained potential signal peptides, and HrcC appeared to be envelope associated based on a TnphoA translational fusion. Comparison of deduced amino acid sequences indicated that many of the proteins are homologous to proteins that function in the type III protein secretion pathway. HrcC is a member of the YscC-containing subgroup in the PulD/pIV superfamily of outer membrane proteins. HrcJ is a member of a lipoprotein family that includes YscJ of Yersinia spp., MxiJ of Shigella flexneri, and NolT of Rhizobim fredii. Additional similarities were detected between HrpB and YscI and between HrpE and YscL. HrcJ and HrpE were similar to flagellar biogenesis proteins FliF and FliH, respectively. In addition, HrpA, HrpB, HrcJ, HrpD, HrpE, HrpF, and HrcC showed various degrees of similarity to corresponding proteins of P. syringae. Comparison of hrp clusters with respect to gene organization and similarity of individual proteins confirms that the hrp systems of E. amylovora and P. syringae are closely related to each other and distinct from those of Ralstonia (Pseudomonas) solanacearum and Xanthomonas campestris. Possible implications of extensive similarities between the E. amylovora and P. syringae hrp systems in pathogenesis mechanisms are discussed.

Amino Acid Sequence↗

The role of NDR1 in avirulence gene-directed signaling and control of programmed cell death in Arabidopsis.

Arabidopsis plants containing the ndr1-1 mutation are incapable of mounting a hypersensitive response to bacteria carrying avrRpt2, but show an exaggerated cell death response to bacteria carrying avrB (Century et al., 1995). We show here that ndr1-1 plants are severely impaired in induction of systemic acquired resistance and PR1-driven transcription of a reporter gene in response to Pseudomonas syringae strains carrying avrRpt2 but not in response to P. syringae carrying avrB. The ndr1-1 mutation also impaired salicylic acid (SA) accumulation in response to treatments that produced reactive oxygen species (ROS) and impaired induction of systemic acquired resistance in response to in situ production of ROS. Hydrogen peroxide accumulated in wild-type Arabidopsis leaves beginning 4 to 7 h postinoculation with P. syringae carrying either avrRpt2 or avrB. In ndr1-1 plants, P. syringae carrying avrRpt2 elicited no detectable hydrogen peroxide production. Hydrogen peroxide production in response to bacteria carrying avrB was similar to that of Columbia in kinetics but of lesser intensity at early time points. These data are interpreted to indicate that NDR1 links ROS generation to SA production and that the phenotypic consequences of the ndr1-1 mutation are caused by a reduced ability to accumulate SA upon pathogen infection.

Apoptosis↗

Dead flower buds of pear: effect of tree growth control, and Alternaria alternata as causal agent.

Dead (dormant) flower buds of pear is an important phenomenon in pear production in the Netherlands. Vigourous or unbalanced tree growth and Pseudomonas syringae pv. syringae are mentioned as likely causes of dead flower buds. Several tree growth control treatments including ethephon. Regalis (Prohexadione-Ca) and root pruning were evaluated. Regalis increased disease incidence. The plant stimulant (foliar fertilizer) Resistim (potassium phosphonate) reduced disease incidence. Pseudomonas syringae pv. syringae was occasionally isolated from diseased flower buds. However, Alternaria alternata was nearly always isolated from diseased buds. Indicating a strong relation between this fungus and dead flower buds of pear.

Alternaria↗

[Fatty acids composition of cellular lipids of the collected and newly isolated Pseudomonas lupini strains].

Fatty acid composition of cellular lipids of 23 Pseudomonas lupini strains (Beltjukova et Koroljova 1968) has been investigated. Cellular fatty acids which contained from C10 to C19 carbon atoms have been identified. Basic fatty acid of those Pseudomonas cells are hexadecanoic, hexadecenoic and octadecanoic acids. The 3-hydroxydecanoic (C10:0 3OH), 3-hydroxydodecanoic (C12:0 3OH), 2-hydroxydodecanoic (C12:0 2OH) and cyclopropane fatty acids which contain 17 and 19 carbon atoms have been detected in cellular lipids. The cellular fatty acids spectra of 22 P. lupini strains are similar to cellular fatty acids spectrum of the type strain Pseudomonas syringae pv. syringae 8511. Pathogenic isolate 2, which fatty acid content of cell lipids significantly differ from lipids of cell fatty acids from P. lupini strains and cell lipids of fatty acids of typical strains Pseudomonas syringae pv. syringae 8511 and Pseudomonas savastanoi pv. phaseolicola 9066 is the exception.

Carbohydrate Conformation↗

Antisense downregulation of polyphenol oxidase results in enhanced disease susceptibility.

Polyphenol oxidases (PPOs; EC 1.14.18.1 or EC 1.10.3.2) catalyze the oxidation of phenolics to quinones, highly reactive intermediates whose secondary reactions are responsible for much of the oxidative browning that accompanies plant senescence, wounding, and responses to pathogens. To assess the impact of PPO expression on resistance to Pseudomonas syringae pv. tomato we introduced a chimeric antisense potato PPO cDNA into tomato (Lycopersicon esculentum L.). Oxidation of caffeic acid, the dominant o-diphenolic aglycone of tomato foliage, was decreased ca. 40-fold by antisense expression of PPO. All members of the PPO gene family were downregulated: neither immunoreactive PPO nor PPO-specific mRNA were detectable in the transgenic plants. In addition, the antisense PPO construct suppressed inducible increases in PPO activity. Downregulation of PPO in antisense plants did not affect growth, development, or reproduction of greenhouse-grown plants. However, antisense PPO expression dramatically increased susceptibility to P. syringae expressing the avirulence gene avrPto in both Pto and pto backgrounds. In a compatible (pto) interaction, plants constitutively expressing an antisense PPO construct exhibited a 55-fold increase in bacterial growth, three times larger lesion area, and ten times more lesions cm(-2) than nontransformed plants. In an incompatible (Pto) interaction, antisense PPO plants exhibited 100-fold increases in bacterial growth and ten times more lesions cm(-2) than nontransformed plants. Although it is not clear whether hypersusceptibility of antisense plants is due to low constitutive PPO levels or failure to induce PPO upon infection, these findings suggest a critical role for PPO-catalyzed phenolic oxidation in limiting disease development. As a preliminary effort to understand the role of induced PPO in limiting disease development, we also examined the response of PPO promoter::beta-glucuronidase constructs when plants are challenged with P. syringae in both Pto and pto backgrounds. While PPO B inducibility was the same in both compatible and incompatible interactions, PPO D, E and F were induced to higher levels and with different expression patterns in incompatible interactions.

Catechol Oxidase↗

Involvement of acetosyringone in plant-pathogen recognition.

In this study, acetosyringone was identified as one of the major extracellular phenolics in tobacco suspension cells and was shown to have bioactive properties that influence early events in plant-bacterial pathogenesis. In our model system, tobacco cell suspensions treated with bacterial isolate Pseudomonas syringae WT (HR+) undergo a resistant interaction characterized by a burst in oxygen uptake several hours after inoculation. When the extracellular concentration of acetosyringone in tobacco cell suspensions was supplemented with exogenous acetosyringone, the burst in oxygen uptake occurred as much as 1.5h earlier. The exogenous acetosyringone had no effect on tobacco suspensions undergoing susceptible interactions with Pseudomonas tabaci or a non-resistant interaction with a near-isogenic mutant derivative of isolate P. syringae WT (HR+). Resistant interactions with isolate P. syringae WT (HR+) also produce an oxidative burst which oxidizes the extracellular acetosyringone. This study demonstrates that acetosyringone, and likely other extracellular phenolics, may have bioactive characteristics that can influence plant-bacterial pathogenesis.

Acetophenones↗

VPEgamma exhibits a caspase-like activity that contributes to defense against pathogens.

BACKGROUND: Caspases are a family of aspartate-specific cysteine proteases that play an essential role in initiating and executing programmed cell death (PCD) in metazoans. Caspase-like activities have been shown to be required for the initiation of PCD in plants, but the genes encoding those activities have not been identified. VPEgamma, a cysteine protease, is induced during senescence, a form of PCD in plants, and is localized in precursor protease vesicles and vacuoles, compartments associated with PCD processes in plants. RESULTS: We show that VPEgamma binds in vivo to a general caspase inhibitor and to caspase-1-specific inhibitors, which block the activity of VPEgamma. A cysteine protease inhibitor, cystatin, accumulates to 20-fold higher levels in vpegamma mutants. Homologs of cystatin are known to suppress hypersensitive cell death in plant and animal systems. We also report that infection with an avirulent strain of Pseudomonas syringae results in an increase of caspase-1 activity, and this increase is partially suppressed in vpegamma mutants. Plants overexpressing VPEgamma exhibit a greater amount of ion leakage during infection with P. syringae, suggesting that VPEgamma may regulate cell death progression during plant-pathogen interaction. VPEgamma expression is induced after infection with P. syringae, Botrytis cinerea, and turnip mosaic virus, and knockout of VPEgamma results in increased susceptibility to these pathogens. CONCLUSIONS: We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions.

Apoptosis↗