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At least 505 records · Page 28Linked to original sources
PubMed2005

The biosynthetic gene cluster for the beta-lactam antibiotic tabtoxin in Pseudomonas syringae.

DNA sequence analysis revealed that the biosynthetic genes of the unusual beta-lactam antibiotic tabtoxin reside at the att site adjacent to the lysC tRNA gene in Pseudomonas syringae BR2. ORFs encoded within the region included ones with similarity to beta-lactam synthase and clavaminic acid synthase, as well as amino acid synthesis enzymes. Novel ORFs were present in a portion of the biosynthetic region associated with a toxin hypersensitivity phenotype. Tabtoxin resistance was associated with a fragment containing a major facilitator superfamily (MFS) transporter gene.

Anti-Bacterial Agents↗
PubMed2003

Virulence systems of Pseudomonas syringae pv. tomato promote bacterial speck disease in tomato by targeting the jasmonate signaling pathway.

Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) causes bacterial speck disease on tomato. The pathogenicity of Pst DC3000 depends on both the type III secretion system that delivers virulence effector proteins into host cells and the phytotoxin coronatine (COR), which is thought to mimic the action of the plant hormone jasmonic acid (JA). We found that a JA-insensitive mutant (jai1) of tomato was unresponsive to COR and highly resistant to Pst DC3000, whereas host genotypes that are defective in JA biosynthesis were as susceptible to Pst DC3000 as wild-type (WT) plants. Treatment of WT plants with exogenous methyl-JA (MeJA) complemented the virulence defect of a bacterial mutant deficient in COR production, but not a mutant defective in the type III secretion system. Analysis of host gene expression using cDNA microarrays revealed that COR works through Jai1 to induce the massive expression of JA and wound response genes that have been implicated in defense against herbivores. Concomitant with the induction of JA and wound response genes, the type III secretion system and COR repressed the expression of pathogenesis-related (PR) genes in Pst DC3000-infected WT plants. Resistance of jai1 plants to Pst DC3000 was correlated with a high level of PR gene expression and reduced expression of JA/wound response genes. These results indicate that COR promotes bacterial virulence by activating the host's JA signaling pathway, and further suggest that the type III secretion system might also modify host defense by targeting the JA signaling pathway in susceptible tomato plants.

Amino Acids↗
PubMed2004

Pseudomonas syringae pv. tomato cells encounter inhibitory levels of water stress during the hypersensitive response of Arabidopsis thaliana.

During plant defense against bacterial pathogens, the hypersensitive response (HR) functions to restrict pathogen growth and spread. The mechanisms driving this growth restriction are poorly understood. We used a water stress-responsive transcriptional fusion to quantify the water potential sensed by individual Pseudomonas syringae pv. tomato DC3000 cells during infection of Arabidopsis thaliana leaves. A nonpathogenic DC3000 hrcC mutant defective in type III secretion, as well as the saprophyte Pseudomonas fluorescens A506, sensed water potentials of -0.3 to -0.4 MPa at 48 h postinfiltration (hpi). During pathogenesis, DC3000 sensed lower water potentials (-0.4 to -0.9 MPa), demonstrating that it can modify the intercellular environment, and these water potentials were associated with optimal DC3000 growth in culture. During the HR, DC3000 cells sensed water potentials (-1.6 to -2.2 MPa) that were low enough to prevent cell division in the majority of cells in culture. This water potential decrease occurred within only 4 hpi and was influenced by avirulence gene expression, with avrRpm1 expression associated with lower water potentials than avrRpt2 or avrB expression at 48 hpi. The population sizes of the DC3000 variants tested were significantly correlated with the apoplastic water potential at 48 hpi, with a decrease of -0.9 MPa associated with a 10-fold decrease in cells per gram of leaf. These results suggest that the apoplastic water potential is a determinant of endophytic bacterial population size, and water stress, resulting from high osmolarity or tissue desiccation, is at least one factor restricting bacterial growth during the HR.

Arabidopsis↗
PubMed2004

Suppression of pathogen-inducible NO synthase (iNOS) activity in tomato increases susceptibility to Pseudomonas syringae.

Inducible NO synthase (iNOS) activity is induced upon pathogen inoculation in resistant, but not susceptible, tobacco and Arabidopsis plants. It was shown recently that a variant form of the Arabidopsis P protein (AtvarP) has iNOS activity. P protein is part of the glycine decarboxylase complex (GDC). It is unclear whether P protein also has iNOS activity and, if so, whether AtvarP, P, or both, play a role in plant defense. Here, we show that iNOS activity is induced in both resistant and susceptible tomato leaves upon inoculation with the Pseudomonas syringae pv. tomato strain DC3000. Virus-induced gene-silencing targeting LevarP, a putative tomato ortholog of AtvarP, led to complete suppression of DC3000-induced iNOS activation and an approximately 80% reduction in GDC activity; it also increased disease-symptom severity and DC3000 growth in both resistant and susceptible tomato. To determine whether enhanced susceptibility exhibited by LevarP-silenced, susceptible tomato was due to loss of (i) iNOS activity, (ii) GDC activity, or (iii) both, GDC activity was inhibited with or without concurrent suppression of iNOS. Treatment with methotrexate inhibited both iNOS and GDC activities and resulted in increased susceptibility, comparable with that observed in LevarP-silenced plants. When normal iNOS activity was maintained in the presence of methotrexate by the addition of tetrahydrobiopterin, there was no change in susceptibility, despite a dramatic reduction in GDC activity. Together, these results indicate that iNOS contributes to host defense response against DC3000.

Amino Acid Oxidoreductases↗
PubMed2005

Molecular characterization of proteolytic cleavage sites of the Pseudomonas syringae effector AvrRpt2.

During infection of Arabidopsis thaliana, the bacterium Pseudomonas syringae pv tomato delivers the effector protein AvrRpt2 into the plant cell cytosol. Within the plant cell, AvrRpt2 undergoes N-terminal processing and causes elimination of Arabidopsis RIN4. Previous work established that AvrRpt2 is a putative cysteine protease, and AvrRpt2 processing and RIN4 elimination require an intact predicted catalytic triad in that AvrRpt2. In this work, proteolytic events that depend on AvrRpt2 activity were characterized. The amino acid sequence surrounding the processing site of AvrRpt2 and two related sequences from RIN4 triggered Avr-Rpt2-dependent proteolytic cleavage of a synthetic substrate, demonstrating that these sequences are cleavage recognition sites for AvrRpt2 activity. Processing-deficient AvrRpt2 mutants were identified and shown to retain their ability to eliminate wild-type RIN4. Single amino acid substitutions were made in each of the two RIN4 cleavage sites, and mutation of both sites resulted in cleavage-resistant RIN4. Growth of Pseudomonas expressing AvrRpt2 was significantly higher than catalytically inactive mutants on Arabidopsis rin4/rps2 mutant plants, suggesting there are additional protein targets of AvrRpt2 that account for the virulence activity of this effector. Bioinformatics analysis identified putative Arabidopsis proteins containing sequences similar to the proteolytic cleavage sites conserved in AvrRpt2 and RIN4. Several of these proteins were eliminated in an AvrRpt2-dependent manner in a transient in planta expression system. These results identify amino acids important for AvrRpt2 substrate recognition and cleavage as well as demonstrate AvrRpt2 protease activity eliminates multiple Arabidopsis proteins in a transient expression system.

Amino Acid Sequence↗
PubMed2005

The Pseudomonas syringae effector AvrRpt2 cleaves its C-terminally acylated target, RIN4, from Arabidopsis membranes to block RPM1 activation.

Plant pathogenic Pseudomonas syringae deliver type III effector proteins into the host cell, where they function to manipulate host defense and metabolism to benefit the extracellular bacterial colony. The activity of these virulence factors can be monitored by plant disease resistance proteins deployed to "guard" the targeted host proteins. The Arabidopsis RIN4 protein is targeted by three different type III effectors. Specific manipulation of RIN4 by each of them leads to activation of either the RPM1 or RPS2 disease resistance proteins. The type III effector AvrRpt2 is a cysteine protease that is autoprocessed inside the host cell where it activates RPS2 by causing RIN4 disappearance. RIN4 contains two sites related to the AvrRpt2 cleavage site (RCS1 and RCS2). We demonstrate that AvrRpt2-dependent cleavage of RIN4 at RCS2 is functionally critical in vivo. This event leads to proteasome-mediated elimination of all but a membrane-embedded approximately 6.4-kDa C-terminal fragment of RIN4. One or more of three consecutive cysteines in this C-terminal fragment are required for RIN4 localization; these are likely to be palmitoylation and/or prenylation sites. AvrRpt2-dependent cleavage at RCS2, and release of the remainder of RIN4 from the membrane, consequently prevents RPM1 activation by AvrRpm1 or AvrB. RCS2 is contained within the smallest tested fragment of RIN4 that binds AvrB in vitro. Thus, at least two bacterial virulence factors target the same domain of RIN4, a approximately 30-aa plant-specific signature sequence found in a small Arabidopsis protein family that may be additional targets for these bacterial virulence factors.

Amino Acid Sequence↗
PubMed2006

RAR1, a central player in plant immunity, is targeted by Pseudomonas syringae effector AvrB.

Pathogenic bacterial effectors suppress pathogen-associated molecular pattern (PAMP)-triggered host immunity, thereby promoting parasitism. In the presence of cognate resistance genes, it is proposed that plants detect the virulence activity of bacterial effectors and trigger a defense response, referred to here as effector-triggered immunity (ETI). However, the link between effector virulence and ETI at the molecular level is unknown. Here, we show that the Pseudomonas syringae effector AvrB suppresses PAMP-triggered immunity (PTI) through RAR1, a co-chaperone of HSP90 required for ETI. AvrB expressed in plants lacking the cognate resistance gene RPM1 suppresses cell wall defense induced by the flagellar peptide flg22, a well known PAMP, and promotes the growth of nonpathogenic bacteria in a RAR1-dependent manner. rar1 mutants display enhanced cell wall defense in response to flg22, indicating that RAR1 negatively regulates PTI. Furthermore, coimmunoprecipitation experiments indicated that RAR1 and AvrB interact in the plant. The results demonstrate that RAR1 molecularly links PTI, effector virulence, and ETI. The study supports that both pathogen virulence and plant disease resistance have evolved around PTI.

Arabidopsis↗
PubMed2004

The Pseudomonas syringae genome encodes a combined mannuronan C-5-epimerase and O-acetylhydrolase, which strongly enhances the predicted gel-forming properties of alginates.

Alginates are industrially important, linear copolymers of beta-d-mannuronic acid (M) and its C-5-epimer alpha-l-guluronic acid (G). The G residues originate from a postpolymerization reaction catalyzed by mannuronan C-5-epimerases (MEs), leading to extensive variability in M/G ratios and distribution patterns. Alginates containing long continuous stretches of G residues (G blocks) can form strong gels, a polymer type not found in alginate-producing bacteria belonging to the genus Pseudomonas. Here we show that the Pseudomonas syringae genome encodes a Ca(2+)-dependent ME (PsmE) that efficiently forms such G blocks in vitro. The deduced PsmE protein consists of 1610 amino acids and is a modular enzyme related to the previously characterized family of Azotobacter vinelandii ME (AlgE1-7). A- and R-like modules with sequence similarity to those in the AlgE enzymes are found in PsmE, and the A module of PsmE (PsmEA) was found to be sufficient for epimerization. Interestingly, an R module from AlgE4 stimulated Ps-mEA activity. PsmE contains two regions designated M and RTX, both presumably involved in the binding of Ca(2+). Bacterial alginates are partly acetylated, and such modified residues cannot be epimerized. Based on a detailed computer-assisted analysis and experimental studies another PsmE region, designated N, was found to encode an acetylhydrolase. By the combined action of N and A PsmE was capable of redesigning an extensively acetylated alginate low in G from a non gel-forming to a gel-forming state. Such a property has to our knowledge not been previously reported for an enzyme acting on a polysaccharide.

Alginates↗
PubMed2005

Use of dominant-negative HrpA mutants to dissect Hrp pilus assembly and type III secretion in Pseudomonas syringae pv. tomato.

The Hrp pilus plays an essential role in the long-distance type III translocation of effector proteins from bacteria into plant cells. HrpA is the structural subunit of the Hrp pilus in Pseudomonas syringae pv. tomato (Pst) DC3000. Little is known about the molecular features in the HrpA protein for pilus assembly or for transporting effector proteins. From previous collections of nonfunctional HrpA derivatives that carry random pentapeptide insertions or single amino acid mutations, we identified several dominant-negative mutants that blocked the ability of wild-type Pst DC3000 to elicit host responses. The dominant-negative phenotype was correlated with the disappearance of the Hrp pilus in culture and inhibition of wild-type HrpA protein self-assembly in vitro. Dominant-negative HrpA mutants can be grouped into two functional classes: one class exerted a strong dominant-negative effect on the secretion of effector proteins AvrPto and HopPtoM in culture, and the other did not. The two classes of mutant HrpA proteins carry pentapeptide insertions in discrete regions, which are interrupted by insertions without a dominant-negative effect. These results enable prediction of possible subunit-subunit interaction sites in the assembly of the Hrp pilus and suggest the usefulness of dominant-negative mutants in dissection of the role of the wild-type HrpA protein in various stages of type III translocation: protein exit across the bacterial cell wall, the assembly and/or stabilization of the Hrp pilus in the extracellular space, and Hrp pilus-mediated long-distance transport beyond the bacterial cell wall.

Amino Acid Sequence↗
PubMed2006

Visualization of dynamics of plant-pathogen interaction by novel combination of chlorophyll fluorescence imaging and statistical analysis: differential effects of virulent and avirulent strains of P. syringae and of oxylipins on A. thaliana.

Pathogen infection leads to defence induction as well as to changes in carbohydrate metabolism of plants. Salicylic acid and oxylipins are involved in the induction of defence, but it is not known if these signalling molecules also mediate changes in carbohydrate metabolism. In this study, the effect of application of salicylic acid and the oxylipins 12-oxo-phytodienoic acid (OPDA) and jasmonic acid on photosynthesis was investigated by kinetic chlorophyll fluorescence imaging and compared with the effects of infection by virulent and avirulent strains of Pseudomonas syringae. Both pathogen strains and OPDA caused a similar change in fluorescence parameters of leaves of Arabidopsis thaliana. The response to OPDA appeared faster compared with that to the pathogens and persisted only for a short time. Infiltration with jasmonic acid or salicylic acid did not lead to a localized and distinct fluorescence response of the plant. To capture the faint early symptoms of the plant response, a novel algorithm was applied identifying the unique fluorescence signature-the set of images that, when combined, yield the highest contrast between control and infected leaf segments. Unlike conventional fluorescence parameters, this non-biased approach indeed detected the infection as early as 6 h after inoculation with bacteria. It was posssible to identify distinct fluorescence signatures characterizing the early and late phases of the infection. Fluorescence signatures of both infection phases were found in leaves infiltrated with OPDA.

Arabidopsis↗
PubMed2005

An avrPto/avrPtoB mutant of Pseudomonas syringae pv. tomato DC3000 does not elicit Pto-mediated resistance and is less virulent on tomato.

AvrPto and AvrPtoB are type III effector proteins expressed by Pseudomonas syringae pv. tomato strain DC3000, a pathogen of both tomato and Arabidopsis spp. Each effector physically interacts with the tomato Pto kinase and elicits a hypersensitive response when expressed in tomato leaves containing Pto. An avrPto deletion mutant of DC3000 previously was shown to retain avirulence activity on Pto-expressing tomato plants. We developed an avrPtoB deletion mutant of DC3000 and found that it also retains Pto-specific avirulence on tomato. These observations suggested that avrPto and avrPtoB both contribute to avirulence. To test this hypothesis, we developed an deltaavrPtodeltaavrPtoB double mutant in DC3000. This double mutant was able to cause disease on a Pto-expressing tomato line. Thus, avrPto and avrPtoB are the only avirulence genes in DC3000 that elicit Pto-mediated defense responses in tomato. When inoculated onto susceptible tomato leaves and compared with wild-type DC3000, the mutants DC3000deltaavrPto and DC3000deltaavrPtoB each caused slightly less severe disease symptoms, although their growth rate was unaffected. However, DC3000deltaavr PtodeltaavrPtoB caused even less severe disease symptoms than the single mutants and grew more slowly than them on susceptible leaves. Our results indicate that AvrPto and AvrPtoB have phenotypically redundant avirulence activity on Pto-expressing tomato and additive virulence activities on susceptible tomato plants.

Bacterial Proteins↗
PubMed2005

Expression profiling soybean response to Pseudomonas syringae reveals new defense-related genes and rapid HR-specific downregulation of photosynthesis.

Transcript profiling during susceptible (S) and hypersensitive response-associated resistance (R) interactions was determined in soybean (Glycine max). Pseudomonas syringae pv. glycinea carrying or lacking the avirulence gene avrB, was infiltrated into cultivar Williams 82. Leaf RNA was sampled at 2, 8, and 24 h postinoculation (hpi). Significant changes in transcript abundance were observed for 3,897 genes during the experiment at P < or = 0.000005. Many of the genes showed a similar direction of increase or decrease in abundance in both the S and R responses, but the R response generally showed a significantly greater degree of differential expression. More than 25% of these responsive genes had not been previously reported as being associated with pathogen interactions, as 704 had no functional annotation and 378 had no homolog in National Center for Biotechnology Information databases. The highest number of transcriptional changes was noted at 8 hpi, including the downregulation of 94 chloroplast-associated genes specific to the R response. Photosynthetic measurements were consistent with an R-specific reduction in photosystem II operating efficiency (phiPSII) that was apparent at 8 hpi for the R response with little effect in the S or control treatments. Imaging analyses suggest that the decreased phiPSII was a result of physical damage to PSII reaction centers.

Analysis of Variance↗
PubMed2005

Membrane release and destabilization of Arabidopsis RIN4 following cleavage by Pseudomonas syringae AvrRpt2.

The Arabidopsis RIN4 protein mediates interaction between the Pseudomonas syringae type III effector proteins AvrB, AvrRpm1, and AvrRpt2 and the Arabidopsis disease-resistance proteins RPM1 and RPS2. Confocal laser-scanning fluorescence microscopy following particle bombardment of tobacco leaf epidermal cells was used to examine the subcellular localization of fusions between GFP and RIN4 or several of its homologs and to examine the effects of cobombardment with AvrRpt2 or AvrRpml. This study showed that RIN4 was attached to the plasma membrane at its carboxyl terminus and that a carboxyl-terminal CCCFxFxxx prenylation or acylation (typically palmitoylation) motif, or both, was essential for this attachment. RIN4 was cleaved by AvrRpt2 at two PxFGxW motifs, one releasing a large portion of RIN4 from the plasma membrane and both exposing amino-terminal residues that destabilized the carboxyl-terminal cleavage products by targeting them for N-end ubiquitylation and proteasomal degradation. Plasma-membrane localization of RIN4 was not affected by AvrRpml. RIN4 was found to be part of a protein family comprising two full-length homologs and at least 11 short carboxyl-terminal homologs. Representatives of this family, comprising a full-length RIN4 homolog and two short carboxyl-terminal RIN4 homologs, were also attached to the plasma membrane and cleaved near their amino termini by AvrRpt2, but in contrast to RIN4, the major portions of these proteins remained on the plasma membrane. N-end degradation may play a minor role in RIN4 degradation but probably plays a major role in the degradation of RIN4 homologs and is, therefore, a major pathogenic consequence of AvrRpt2 cleavage.

Amino Acid Sequence↗
PubMed2006

Genome-wide gene expression analysis of Pseudomonas syringae pv. tomato DC3000 reveals overlapping and distinct pathways regulated by hrpL and hrpRS.

Pseudomonas syringae pv. tomato DC3000 is a model pathogen infecting tomato and Arabidopsis plants. Genes encoding the type III secretion system and substrate proteins (collectively called TTSS genes) of this bacterium are induced in plants and in minimal medium (MM). The induction of TTSS genes is mediated by HrpL, an alternative sigma factor recognizing the hrp box in the promoter of TTSS genes. The transcription of hrpL is activated by HrpR and HrpS, two homologous DNA-binding proteins encoded by the hrpRS operon. Microarray analysis was conducted to evaluate the DC3000 genes regulated by hrpL and hrpRS in MM. The analysis identified a number of novel hrpL-activated genes with a putative TTSS-independent function. Genes regulated by hrpL were mostly regulated by hrpRS in the same manner, but a large number of genes regulated by hrpRS were hrpL-independent, indicating that hrpL represents one branch of the regulatory pathways downstream of hrpRS. The induction of the TTSS genes was associated with downregulation of the housekeeping genes, indicating that the activation of the TTSS has a cost on the basic cellular activities. The novel genes and pathways identified by the microarray provide new insight into the bacterial functions coordinating with the TTSS.

Bacterial Proteins↗
PubMed2003

GacA, the response regulator of a two-component system, acts as a master regulator in Pseudomonas syringae pv. tomato DC3000 by controlling regulatory RNA, transcriptional activators, and alternate sigma factors.

Concerted investigations of factors affecting host-pathogen interactions are now possible with the model plant Arabidopsis thaliana and its model pathogen Pseudomonas syringae pv. tomato DC3000, as their whole genome sequences have become available. As a prelude to analysis of the regulatory genes and their targets, we have focused on GacA, the response regulator of a two-component system. The DC3000 gene was cloned by testing for the reversal of phenotypes of an Erwinia GacA- mutant. A GacA- mutant of DC3000 constructed by marker exchange produces much-reduced levels of transcripts of three alternate sigma factors: HrpL, required for the production of effector proteins and their translocation via the type III secretion system; RpoS, required for stress responses and secondary metabolite production; and RpoN, required for an assortment of metabolic processes and expression of hrpL. GacA deficiency also reduces the expression of hrpR and hrpS, which specify enhancer-binding proteins of the NtrC family required for hrpL transcription; ahlI and ahlR, the genes for quorum sensing signal; salA, a regulatory gene known to control virulence; CorS, a sensor kinase; CorR, the cognate response regulator that controls coronatine biosynthetic genes; and rsmB and rsmZ, which specify untranslatable regulatory RNA species. gacA expression itself is regulated by environmental conditions in DC3000, since transcript levels are affected by growth phase and media composition. The observations that high levels of gacA RNA occur in the hrp-inducing medium and GacA deficiency reduces the levels of rpoS expression implicate an important role of GacA in stress responses of DC3000. Consistent with the effects on hrpL expression, the GacA- mutant produces lower levels of transcripts of avr, hrp, and hop genes controlled by HrpL. In addition, GacA deficiency results in reduced levels of transcripts of several HrpL-independent genes. As would be expected, these effects on gene expression cause drastic changes in bacterial behavior: virulence towards A. thaliana and tomato; multiplication in planta; efficiency of the induction of the hypersensitive reaction (HR); production of pigment and N-acyl-homoserine lactone (AHL), the presumed quorum-sensing signal; and swarming motility. Our findings establish that GacA, located at the top in a regulatory cascade in DC3000, functions as a central regulator by controlling an assortment of transcriptional and posttranscriptional factors.

Bacterial Proteins↗
PubMed2004

Mutations in the Pseudomonas syringae avrRpt2 gene that dissociate its virulence and avirulence activities lead to decreased efficiency in AvrRpt2-induced disappearance of RIN4.

The avrRpt2 gene from Pseudomonas syringae pv. tomato exhibits avirulence activity on Arabidopsis expressing the resistance gene RPS2 but promotes bacterial virulence on susceptible rps2 Arabidopsis. To understand the functional relationship between the avirulence and virulence activities of avrRpt2, we analyzed a series of six avrRpt2 mutants deficient in eliciting the RPS2-dependent hypersensitive response. We show that the mutants are also severely impaired in triggering RSP2-dependent resistance. Four of these mutants are severely impaired in their virulence activity, whereas two alleles, encoding C-terminal deletions of AvrRpt2, retain significant but slightly reduced virulence activity. Thus, the avirulence and virulence activities of avrRpt2 can be genetically uncoupled. We tested the ability of the two C-terminal deletion mutants to trigger AvrRpt2-induced elimination of the Arabidopsis RIN4 protein and show that they retain this activity but are less efficient than wild-type AvrRpt2. Thus, reduced AvrRpt2 virulence activity is correlated with reduced efficiency in the induction of RIN4 disappearance. This suggests that an alteration in kinetics of RIN4 disappearance triggered by the C-terminal deletion mutants may provide the mechanistic basis for the uncoupling of the avirulence and virulence activities of avrRpt2.

Apoptosis↗
PubMed2004

Topological and deletion analysis of CorS, a Pseudomonas syringae sensor kinase.

A modified two-component regulatory system consisting of two response regulators, CorR and CorP, and the histidine protein kinase CorS, regulates the thermoresponsive production of the phytotoxin coronatine (COR) in Pseudomonas syringae PG4180. COR is produced at the virulence-promoting temperature of 18 degrees C, but not at 28 degrees C, the optimal growth temperature of PG4180. Assuming that the highly hydrophobic N-terminus of CorS might be involved in temperature-signal perception, the membrane topology of CorS was determined using translational phoA and lacZ fusions, leading to a topological model for CorS with six transmembrane domains (TMDs). Interestingly, three PhoA fusions located downstream of the sixth TMD showed a thermoresponsive phenotype. Enzymic activity, immunoblot, and protease-sensitivity assays were performed to localize the CorS derivatives, to analyse the expression level of hybrid proteins and to examine the model. In-frame deletions of the last four, or all six TMDs gave rise to non-functional CorS. The results indicated that the transmembrane region is important for CorS to function as a temperature sensor, and that the membrane topology of CorS might be involved in signal perception.

Alkaline Phosphatase↗
PubMed2005

Laser photoacoustic detection allows in planta detection of nitric oxide in tobacco following challenge with avirulent and virulent Pseudomonas syringae Pathovars.

We demonstrate the use of laser photoacoustic detection (LPAD) as a highly sensitive method to detect in planta nitric oxide ((*)NO) production from tobacco (Nicotiana tabacum). LPAD calibration against (*)NO gas demonstrated a linear relationship over 2 orders of magnitude with a detection threshold of <20 pmol h(-1) (1 part per billion volume [ppbv]). The specificity of the photoacoustic signal for (*)NO when adding gas or the (*)NO donor, sodium nitroprusside, on injection into plant leaves, was demonstrated by its abolition with O(3) ((*)NO + O(3) --> NO(2) + O(2)). The utility of the LPAD method was shown by examination of a nonhost hypersensitive response and a disease induced by Pseudomonas syringae (P. s.) pv phaseolicola and P. s. pv tabaci in tobacco. (*)NO was detected within 40 min of challenge with P. s. pv phaseolicola, some 5 h before the initiation of visible tissue collapse. The wildfire tobacco pathogen P. s. pv tabaci initiated (*)NO generation at 2 h postinfection. The use of (*)NO donors, the scavenger CPTIO ([4-carboxyphenyl]-4,5-dihydro-4,4,5,5-tetramethyl-3-oxide), and the mammalian nitric oxide synthase inhibitor l-NMMA (N(G)-monomethyl-l-arginine) indicated that (*)NO influenced the kinetics of cell death and resistance to both avirulent and virulent bacteria in tobacco. These observations suggest that (*)NO is integral to the elicitation of cell death associated with these two bacterial pathogens in tobacco.

Acoustics↗