Cloning of genes required for hypersensitivity and pathogenicity inPseudomonas syringae pv.aptata

A genomic library ofPseudomonas syringae pv.aptata strain NCPPB 2664, which causes bacterial blight of sugar beet, lettuce and other plants, was constructed in the cosmid vector pCPP31. The 13.4 kbEcoRI fragment of the cosmid pHIR11, containing thehrp (hypersensitiveresponse andpathogenicity) gene cluster of the closely related bacteriumPseudomonas syringae pv.syringae strain 61, was used as a probe to identify a homologoushrp gene cluster inP. syringae pv.aptata. Thirty of 2500 cosmid clones, screened by colony hybridization, gave a strong hybridization signal with the probe, but none of these conferred to the non-pathogenic bacterium,Pseudomonas fluorescens, the ability to elicit the hypersensitive response (HR) in tobacco. Southern blot analysis ofEcoRI-digested genomic DNA ofP. syringae pv.aptata showed hybridizing bands of 12 kb and 4.4 kb. Only a 12 kb fragment hybridized in digests of the cosmids. Cosmid clone pCPP1069 was mutagenized with Tn10-minitet and marker-exchanged into the genome ofP. syringae pv.aptata. Three resulting prototrophic mutant strains failed to elicit the HR in tobacco and to cause disease in lettuce. The DNA flanking the Tn10-minitet insertions from mutated derivatives of pCPP1069 hybridized with the 10.6 kbBglII fragment of pHIR11. These results indicate thatP. syringae pv.aptata harbourshrp genes that are similar to, but arranged differently from, homologoushrp genes ofP. syringae pv.syringae.Abbreviations HR hypersensitive response - Hrp mutant unable to induce HR and pathogenicity - Psa Pseudomonas syringae pv.aptata - Pss Pseudomonas syringae pv.syringae - Ea Erwinia amylovora     

Multiple copies of a DNA sequence from Pseudomonas syringae pathovar phaseolicola abolish thermoregulation of phaseolotoxin production

Phaseolotoxin, a phytotoxin of Pseudomonas syringae pv. phaseolicola, is produced at 18°C but not at 28°C. Here we report that a fragment (24.4 kb) cloned from the wild-type strain, which does not harbour a gene(s) involved in phaseolotoxin biosynthesis, abolishes this thermoregulation in the wild type and suppresses a Tox^? mutant at both temperatures. A subclone harbouring a 465bp fragment contains motifs that are characteristic of DNA-binding sites. In mobility shift assays we have detected a protein(s) from the wild-type and the mutant strains, grown at appropriate temperatures, that specifically binds to the fragment containing the DNA-binding motifs. We propose that the binding protein is a repressor which is ‘titrated’ by this fragment when it is present in the cell on a multiple copy plasmid, thus allowing expression of phaseolotoxin genes.     

Development of a Method to Obtain Monospecific Antibodies Directed Against a 31 kD Protein of Pseudomonas syringae pv. syringae

We describe a simple method of purifying Pseudomonas syringae pv. syringae (P. s. pv. s.) specific polyclonal antibodies which are directed against a cell surface protein sized 31 kD. The actual purification step of the polyclonal antibodies occurs with denatured proteins after an SDS-PAGE gel electrophoresis and western blotting. Polyclonal antibodies were obtained which recognized a 31 kD protein. On a western blot no cross reaction of the purified polyclonal ‘monospecific 31 kD antibodies’ with other proteins from the same strain was observed. However, a positive “monospecific 31 kD antibody” reaction was only visible when either protein extracts from several selected pathogenic P. s. pv. s. strains from different hosts, or two isolates of Pseudomonas syringae pv. pisi (P. s. pv. p.) were used. An indication that this 31 kD protein is specific to P. s. pv. s. is that no cross reaction was found with protein extracts from the tested pathogenic and non-pathogenic pseudomonads. However, a differentiation of the tested P. s. pv. s. and P. s. pv. p. isolates was possible when cells were grown above 30 °C. Then protein extracts from P. s. pv. s. revealed the lack of the 31 kD protein but not P. s. pv. p. protein extracts. Polyclonal antibodies raised in rabbits against the 31 kD protein band from the western blot proved to be specific enough to agglutinate whole P. s. pv. s. strain R32 cells or for detecting a single 31 kD protein band on a western blot when whole cell protein extracts of P. s. pv. s. strain R32 were used.     

Identification of a pathogenicity locus in Xanthomonas campestris pv. vesicatoria

Summary Mutants of a tomato strain ofXanthomonas campestris pv.vesicatoria (XCV), causal agent of bacterial spot of tomato and pepper, were produced using the transposon Tn5 carried in the suicide plasmid pGS9. One prototrophic mutant, M461, was isolated which caused no visible reaction on tomato or pepper, but maintained the wild-type ability to induce a hypersensitive reaction (HR) on tobacco. This mutant showed similar growth characteristics to the wild-type in culture, but growth in planta was reduced. A genomic library of wild-type XCV was constructed in the broad host range cosmid vector pLAFR3. Clone p6AD4 restored pathogenicity to M461 on tomato and the ability to induce a HR on pepper. This clone contained ca. 22 kb of XCV DNA. The insertion in M461 was in a site corresponding to a 1.1 kbEcoRI fragment of p6AD4.     

Molecular cloning of a Pseudomonas syringae pv. syringae gene cluster that enables Pseudomonas fluorescens to elicit the hypersensitive response in tobacco plants.

A cosmid clone isolated from a genomic library of Pseudomonas syringae pv. syringae 61 restored to all Tn5 mutants of this strain studied the ability to elicit the hypersensitive response (HR) in tobacco. Cosmid pHIR11 also enabled Escherichia coli TB1 to elicit an HR-like reaction when high levels of inoculum (10(9) cells per ml) were infiltrated into tobacco leaves. The cosmid, which contains a 31-kilobase DNA insert, was mobilized by triparental matings into Pseudomonas fluorescens 55 (a nonpathogen that normally causes no plant reactions), P. syringae pv. syringae 226 (a tomato pathogen that causes the HR in tobacco), and P. syringae pv. tabaci (a tobacco pathogen that causes the HR in tomato). The plant reaction phenotypes of all of the transconjugants were altered. P. fluorescens(pHIR11) caused the HR in tobacco and tomato leaves and stimulated an apparent proton influx in suspension-cultured tobacco cells that was indistinguishable from the proton influx caused by incompatible pathogenic pseudomonads. P. syringae pv. tabaci(pHIR11) and P. syringae pv. syringae 226(pHIR11) elicited the HR rather than disease symptoms on their respective hosts and were no longer pathogenic. pHIR11 was mutagenized with TnphoA (Tn5 IS50L::phoA). One randomly chosen mutant, pHIR11-18, no longer conferred the HR phenotype to P. fluorescens. The mutation was marker-exchanged into the genomes of P. syringae pv. syringae strains 61 and 226. The TnphoA insertions in the two pseudomonads abolished their ability to elicit any plant reactions in all plants tested. The results indicate that a relatively small portion of the P. syringae genome is sufficient for the elicitation of plant reactions.     

The Pseudomonas syringae pv. tomato HrpW Protein Has Domains Similar to Harpins and Pectate Lyases and Can Elicit the Plant Hypersensitive Response and Bind to Pectate

The host-specific plant pathogen Pseudomonas syringae elicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the P. syringae genome. hrpW was found in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42.9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from Nectria haematococca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity in A₂₃₀ assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the P. syringae pv. syringae B728a hrpW was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the P. syringae pv. tomato hrpW were found in other P. syringae pathovars, Pseudomonas viridiflava, Ralstonia (Pseudomonas) solanacearum, and Xanthomonas campestris. ΔhrpZ::nptII or hrpW::ΩSp^r P. syringae pv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in a hrpZ hrpW double mutant. These features of hrpW and its product suggest that P. syringae produces multiple harpins and that the target of these proteins is in the plant cell wall.     

Structure and role in symbiosis of the exoB gene of Rhizobium leguminosarum bv trifolii

The Rhizobium leguminosarum bv trifolii exoB gene has been isolated by heterologous complementation of an exoB mutant of R. meliloti. We have cloned a chromosomal DNA fragment from the R. leguminosarum bv trifolii genome that contains an open reading frame of 981 bp showing 80% identity at the amino acid level to the UDP-glucose 4-epimerase of R. meliloti. This enzyme produces UDP-galactose, the donor of galactosyl residues for the lipid-linked oligosaccharide repeat units of various heteropolysaccharides of rhizobia. An R. leguminosarum bv trifoliiexoB disruption mutant differed from the wild type in the structure of both the acidic exopolysaccharide and the lipopolysaccharide. The acidic exopolysaccharide made by our wild-type strain is similar to the Type 2 exopolysaccharide made by other R. leguminosarum bv trifolii wild types. The exopolysaccharide made by the exoB mutant lacked the galactose residue and the substitutions attached to it. The exoB mutant induced the development of abnormal root nodules and was almost completely unable to invade plant cells. Our results stress the importance of exoB in the Rhizobium-plant interaction. Received: 31 May 1996 / Accepted: 18 December 1996     

Genes for phaseolotoxin synthesis are located on the chromosome ofPseudomonas syringae pv.phaseolicola

Transposon mutagenesis was used to locate the genes for phaseolotoxin biosynthesis inPseudomonas syringae pathovar.phaseolicola. Mutants unable to produce toxin were obtained that carried Tn5 on different chromosomal restriction fragments. None of the Tn5-induced nontoxigenic mutants carried the transposon in plasmid DNA. The insertion of Tn5 intotox DNA was confirmed by site-directed mutagenesis. The results reported here suggest the involvement of at least five chromosomal genes in phaseolotoxin biosynthesis. All of the toxinminus mutants retained both full pathogenicity on beans and resistance to the toxin.     

A cAMP‐Dependent Protein Kinase Gene,aapk1, Is Required for Mycelia Growth,Toxicity and Pathogenicity of Alternaria alternata on Tobacco

The fungus Alternaria alternata is a common spot‐producing plant pathogen. During the past decade, tobacco brown spot disease caused by this fungus has became prevalent in China and lead to significant losses. To better understand the molecular pathogenesis of this fungus, the aapk1 gene encoding a cAMP‐dependent protein kinase catalytic subunit was cloned, sequenced and characterized. The aapk1 deletion mutants were identified from hygromycin‐resistant transformants by PCR strategy and confirmed by Southern blot analysis and RT‐PCR. The aapk1 deletion mutant exhibited reduced vegetative growth and was less toxic than the wild‐type strain sd1. Deletion of aapk1 also delayed disease development on detached tobacco leaves. Thus, we propose that the cAMP signalling pathway is involved in mycelia growth and pathogenic phenotype of Alternaria alternata.     

Transgenic tobacco cultivars resistant to Pseudomonas syringae pv. tabaci

 Six oriental cultivars of tobacco (Nicotiana tabacum L.) were evaluated for transformation and foreign gene expression. Leaf-disc explant tissue was transformed with Agrobacterium tumefaciens strain LBA4404 carrying the plasmid pARK21, which contains NPTII gene and ttr (tabtoxin resistance) gene conferring the resistance to Pseudomonas syringae pv. tabaci. The disease resistance of regenerated plants and segregation of this trait up to R₇ progeny were investigated in a greenhouse and under field conditions. Our results indicated that the resistance to Pseudomonas syringae pv. tabaci introduced by transformation is heritable. Received: 10 June 1997 / Accepted: 31 March 1998     

Two native plasmids of Pseudomonas syringae pathovar tomato strain PT23 share a large amount of repeated DNA, including replication sequences

Strain PT23 of Pseudomonas syringae pv, tomato contains four native plasmids, designated A, B, C, and D. By DNA hybridization of genomic and plasmid DNA digests from the wild type and a plasmid-cured strain, we determined that c. 61 kb (c. 74%) of pPT23B is repeated in pPT23A and only c. 17 kb (c. 21%) is in single copy in strain PT23. pPT23B also contains DNA repeated in the chromosome that occurs in three DNA fragments of 0.6, 4.6, and 9.6 kb that might be transposable elements. Additionally, the 9.6 kb fragment also shares sequences with the three other plasmids of strain PT23. By DNA hybridization with the origin of replication from a native plasmid of P. syringae pv. syringae and in vivo replication tests, we identified the origins of replication of plasmids A, B, and D and showed that they cross-hybridize. The putative par region from pPT23 A has also been identified and is not conserved in the other three native plasmids from strain PT23. By using the defined minimal origin of replication from pPT23 A as a probe, we showed that it is highly conserved in 14 strains belonging to nine different pathovars of P. syringae and that as many as five different native plasmids with closely related origins of replication coexist in the same cell. The duplication and reorganization of plasmids might therefore occur at high frequency and could be responsible for the existence of large numbers of native plasmids in P. syringae strains.     

PopW of Ralstonia solanacearum,a new two‐domain harpin targeting the plant cell wall

Harpins are extracellular glycine‐rich proteins eliciting a hypersensitive response (HR). In this study, we identified a new harpin, PopW, from Ralstonia solanacearum strain ZJ3721. This 380‐amino‐acid protein is acidic, rich in glycine and serine, and lacks cysteine. When infiltrated into the leaves of tobacco (non‐host), PopW induced a rapid tissue collapse via a heat‐stable but protease‐sensitive HR‐eliciting activity. PopW has an N‐terminal harpin domain (residues 1–159) and a C‐terminal pectate lyase (PL) domain (residues 160–366); its HR‐eliciting activity depends on its N‐terminal domain. Analyses of subcellular localization and plasmolysis demonstrated that PopW targeted the onion cell wall. This was further confirmed by its ability to specifically bind to calcium pectate, a major component of the plant cell wall. However, PopW had no detectable PL activity. Western blotting revealed that PopW was secreted by the type III secretion system in an hrpB‐dependent manner. Gene sequencing indicated that popW is conserved among 20 diverse strains of R. solanacearum. A popW‐deficient mutant retained the ability of wild‐type strain ZJ3721 to elicit HR in tobacco and to cause wilt disease in tomato (a host). We conclude that PopW is a new cell wall‐associated, hrpB‐dependent, two‐domain harpin that is conserved across the R. solanacearum species complex.     

In vitro Antibiosis by Pseudomonas syringae Pss22d,Acting Against the Bacterial Blight Pathogen of Soybean Plants,Does Not Influence In planta Biocontrol

The epiphyte Pseudomonas syringae pv. syringae 22d / 93 (Pss22d), isolated from soybean leaves, had been characterized as a promising and species‐specific biocontrol strain in vitro and in planta against the plant pathogen P. syringae pv. glycinea (Psg), which causes bacterial blight of soybean. Three toxins are known to be produced by Pss22d: syringomycin, syringopeptin and 3‐methylarginine (MeArg). In contrast to syringopeptin and syringomycin, MeArg inhibited the growth of Psg in vitro. To examine if the toxins produced by Pss22d are responsible for antagonistic effects in planta, the pathogen Psg was co‐inoculated with either Pss22d wild‐type, a syringopeptin/syringomycin‐negative double mutant (Pss22d.ΔsypA/syrE), or a MeArg‐negative mutant (Pss22d.1) into wounds of pin‐pricked leaves of greenhouse‐grown soybean plants, respectively. In all three cases, the wild‐type Pss22d and its toxin‐deficient mutants prevented development of disease symptoms normally caused by Psg. These results indicated that neither syringopeptin, nor syringomycin, nor MeArg was required for Pss22d’s antagonistic activity in planta. Consequently, factors other than the three toxins may contribute to the intra‐species antagonism in planta.     

Progesterone moderates damage in Arabidopsis thaliana caused by infection with Pseudomonas syringae or P. fluorescens

Brassinosteroids are known to protect plants against various abiotic and biotic stresses, however, very limited information is available about the role of progesterone. Therefore the effects of Pseudomonas syringae pv. syringae (P.s.) wild type strain 61, its hrcC mutant, and the saprophytic P. fluorescens (P.f.) strain 55 were investigated in wild type Arabidopsis thaliana cv. Columbia and its rbohF knock-out mutant, with and without progesterone pre-treatment. The reactions of wild type and rbohF mutant Arabidopsis to bacterial inoculations were similar, although 2 h after injection of P.s. a larger increase of electrolyte leakage was measured in wild type than in rbohF knockout mutant leaves. The hrcC mutant caused weak necrotic symptoms and increased leakage in both types of Arabidopsis, although to a much lesser extent than P.s. The P.f. did not induce any visible symptom, but slightly increased the electrolyte leakage in both types of Arabidopsis. Inoculation by all Pseudomonas bacteria led to significant alterations in photosystem 2 efficiency as compared to control plants. Pre-treatment of leaves with progesterone diminished the necrotic symptoms, the electrolyte leakage and improve the efficiency of photosystem 2 caused by Pseudomonas bacteria.     

Characterization of eight excision plasmids of Pseudomonas syringae pv. phaseolicola

Summary Pseudomonas syringae pv. phaseolicola strain LR719 contains a 150 kilobase pair (kb) plasmid pMC7105, stably integrated into its chromosome. Occasionally, single colony isolates of this strain contain an excision plasmid. Eight unique excision plasmids were selected and characterized by BamHI restriction endonuclease and blot hybridization analyses. These plasmids ranged in size from 35 to 270 kb; the largest contained approximately 130 kb of chromosomal DNA sequences. Restriction maps of pMC7105 were developed to deduce the site of integration and to identify the fragments in which recombination occurred to produce each excision plasmid. The eight excision plasmids were arranged into five classes based on the sites where excision occurs. A 20 kb region of pMC7105, which includes BamHI fragment 9 and portions of adjacent fragments, is present in all excision plasmids and thought to contain the origin of replication. The site of integration on pMC7105 maps within BamHI fragment 8. This fragment shows homology with seven other BamHI fragments of pMC7105 and with five chromosomal fragments identified among the excision plasmids. The data strongly suggest that the integration of pMC7105 may have occurred at a repetitive sequence present on the chromosome and on the plasmid.     

Identification and expression of the Pseudomonas syringae pv. aptata hrpZPsa gene which encodes an harpin elicitor

A sequence homologous to an internal fragment 0.75 kb BstXI of the Pseudomonas syringae pv. syringae hrpZ gene was identified in Pseudomonas syringae pv. aptata NCPPB 2664, the causal agent of bacterial blight in sugar beet, lettuce and other plants, and in E. coli DH10B (pCCP1069) containing the P. syringae pv. aptata hrp gene cluster. PCR with oligonucleotides, based on the hrpZ_Pss gene and used as primers with the total genomic DNA of P. syringae pv. aptata, amplified a 1 kb fragment that hybridized with the probe in highly stringent conditions. The amplicon was cloned into the pGEM-T® plasmid vector, amplified in E. coli DH5 and sequenced. The sequence showed 95%, 83% and 61% identity with those of hrpZ_Pss, hrpZ_Psg and hrpZ_Pst genes encoding the harpins of the P. syringae pv. syringae, glycinea and tomato, respectively. The amplicon was cloned into the pMAL® expression system. The expressed protein, fused with maltose-binding protein, was cleaved with a specific protease factor Xa, and purified using affinity chromatography. On the basis of the amino acid sequence and its ability to induce HR in tobacco leaves, it was identified as a P. syringae pv. aptata harpin.     

Plant Innate Immunity Induced by Flagellin Suppresses the Hypersensitive Response in Non-Host Plants Elicited by Pseudomonas syringae pv. averrhoi

A new pathogen, Pseudomonas syringae pv. averrhoi (Pav), which causes bacterial spot disease on carambola was identified in Taiwan in 1997. Many strains of this pathovar have been isolated from different locations and several varieties of hosts. Some of these strains, such as HL1, are nonmotile and elicit a strong hypersensitive response (HR) in nonhost tobacco leaves, while other strains, such as PA5, are motile and elicit a weak HR. Based on the image from a transmission electron microscope, the results showed that HL1 is flagellum-deficient and PA5 has normal flagella. Here we cloned and analyzed the fliC gene and glycosylation island from Pav HL1 and PA5. The amino acid sequences of FliC from HL1 and PA5 are identical to P. s. pvs. tabaci (Pta), glycinea and phaseolicola and share very high similarity with other pathovars of P. syringae. In contrast to the flagellin mutant PtaΔfliC, PA5ΔfliC grows as well as wild type in the host plant, but it elicits stronger HR than wild type does in non-host plants. Furthermore, the purified Pav flagellin, but not the divergent flagellin from Agrobacterium tumefaciens, is able to impair the HR induced by PA5ΔfliC. PA5Δfgt1 possessing nonglycosylated flagella behaved as its wild type in both bacterial growth in host and HR elicitation. Flagellin was infiltrated into tobacco leaves either simultaneously with flagellum-deficient HL1 or prior to the inoculation of wild type HL1, and both treatments impaired the HR induced by HL1. Moreover, the HR elicited by PA5 and PA5ΔfliC was enhanced by the addition of cycloheximide, suggesting that the flagellin is one of the PAMPs (pathogen-associated molecular patterns) contributed to induce the PAMP-triggered immunity (PTI). Taken together, the results shown in this study reveal that flagellin in Pav is capable of suppressing HR via PTI induction during an incompatible interaction.     

Evaluation of Pathogenicity and of Cultural and Biochemical Tests for Identification of Pseudomonas syringae Pathovars syringae, morsprunorum and persicae from Fruit Trees

Comparative studies were based on 216 isolates of the Pseudomonas syringae pathovars syringae, morsprunorum and persicae, mainly originating from fruit trees in several European countries, but also from USA, USSR, South Africa, New Zealand and Turkey. All the identified morsprunorum isolates were obtained from stonefruit trees, which were also a source of some syringae isolates. Fluorescent strains HR negative on tobacco leaves were regarded as saprophytic pseudomonads. Nearly all the HR positive but none of the HR negative isolates caused lesions on sweet cherry shoots and fruitlets. The largest lesions on sweet cherry shoots were caused by the morsprunorum isolates. Syringae strains always caused lesions on pear fruitlets, whereas the morsprunorum strains never did so. Some degree of specialization within the pathovar syringae seemed to exist. Cultural and biochemical tests well suited to differentiate the three pathovars were as follows: colour after growth in sucrose nutrient broth, liquefaction of gelatin, activity of β-glucosidase (arbutin) and tyrosinase, use of L-leucine, L(+)tartrate and DL-lactate as sole carbon source, fluorescent pigment production, crystaline inclusions in the nutrient agar medium, and growth rate in 0.2% yeast extract nutrient broth. Tests for longevity on nutrient agar with 5% sucrose turned out to be fairly unreliable. A tube assay gave the most consistent results when studying utilization of organic substrates. The capability for gelatin liquefaction correlated with virulence, and mucoid isolates showed a tendency for higher aggressiveness than rough colony variants. Page of protein extracts obtained from freeze-pressed bacterial cells revealed that the pathovars syringae and morsprunorum could be differentiated due to specific isoenzyme patterns for esterases and acid phosphatases. It was concluded that several pathogenicity, cultural and biochemical tests allowed an unequivocal differentiation of the Pseudomonas syringae pvs. syringae and morsprunorum. Some of the discriminating biochemical characters may, play a role during pathogenesis.     

Early Detection of Bacterium-induced Basal Resistance in Tobacco Leaves with Diaminobenzidine and Dichlorofluorescein Diacetate

The present study demonstrate that in tobacco leaves the diaminobenzidine (DAB) and 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) staining is a useful indicator of the basal (also known as general or innate) defence‐associated reactions, especially of the early developing form of basal resistance (EBR). DAB and DCFH‐DA, in the presence of H₂O₂ and peroxidase converts to a brown polymer and fluorescent DCF respectively. In the present study, the hypersensitive response (HR)‐inducing avirulent Pseudomonas syringae pv. syringae 61, its HR‐negative hrp/hrc mutants and even non‐pathogenic bacteria such as P. fluorescens and Escherichia coli caused DAB and DCFH‐DA staining, if the dyes were injected 3–4 h after bacterial inoculation into tobacco leaves. The conditions that enable the staining of plant leaves infiltrated with HR‐negative bacteria were persisted for 1 to several days depending on the physiological state of the plant, and plant activity was required to the development of the staining. The live virulent P. syringae pv. tabaci was able to suppress the development of the staining reaction. Bacteria that induced more intensive staining reaction triggered stronger local resistance response, which was verified by its ability to inhibit the HR by challenging avirulent bacteria and by expression analysis of genes that are activated during the basal defence response. The peroxidase enzyme activity increased in bacterially treated tobacco tissue, and inhibition of peroxidase activity blocked the development of the staining. The results showed that in tobacco leaves the staining reactions were associated with the general recognition and basal defence reaction of tobacco plant and can be used as markers in tobacco leaves for testing the occurrence of this type of defence.     

NudC Nudix hydrolase from Pseudomonas syringae,but not its counterpart from Pseudomonas aeruginosa,is a novel regulator of intracellular redox balance required for growth,motility and biofilm formation

Nudix pyrophosphatases, ubiquitous in all organisms, have not been well studied. Recent implications that some of them may be involved in response to stress and in pathogenesis indicate that they play important biological functions. We have investigated NudC Nudix proteins from the plant pathogen Pseudomonas syringae pv. tomato str. DC3000 and from the human pathogen Pseudomonas aeruginosa PAO1161. We found that these homologous enzymes are homodimeric and in vitro preferentially hydrolyse NADH. The P. syringae mutant strain deficient in NudC accumulated NADH and displayed significant defects in growth, motility and biofilm formation. The wild type copy of the nudC gene with its cognate promoter delivered in trans into the nudC mutant restored its fitness. However, introduction of the P. syringae nudC gene under the control of the strong tacp promoter into either P. syringae or P. aeruginosa cells had a toxic effect on both strains. Opposite to P. syringae NudC, the P. aeruginosa NudC deficiency as well as its overproduction had no visible impact on cells. Moreover, P. aeruginosa NudC does not compensate the lack of its counterpart in the P. syringae mutant. These results indicate that NudC from P. syringae, but not from P. aeruginosa is vital for bacteria.