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     

Construction of a Bacterial Artificial Chromosome Library and Characterization of hrp/hrc Gene Cluster of Pseudomonas Syringae Pathovar tagetis LMG5090

Pseudomonas syringae pv. tagetis causes apical chlorosis of several plant species in the Asteraceae, including marigold. As a means to facilitate the isolation of pathogenicity genes and to characterize the genome of this bacterium, we have constructed a bacterial artificial chromosome library of P. syringae pv. tagetis strain LMG5090. The library consists of 1,536 clones with insert size ranging from 30 to 160 kb and an average size of 86 kb. Based upon colony hybridization, the BAC clone 420E23 containing the hrp/hrc gene cluster encoding the type III secretion system was identified from this library and subsequently shotgun sequenced. The hrp/hrc gene cluster of P. syringae pv. tagetis has a 23 kb sequence which contains 27 open reading frames. Comparative analysis of the hrp/hrc gene cluster of P. syringae pv. tagetis LMG5090, P. syringae pv. tomato DC3000, P. syringae pv. syringae B728a, and P. syringae pv. phaseolicola 1448A revealed that the entire hrp/hrc gene cluster of P. syringae pv. tagetis is conserved and identically arranged in all four pathovars     

The genetic characterization of Pseudomonas syringae pv. tagetis based on the 16S–23S rDNA intergenic spacer regions

Pseudomonas syringae pv. tagetis, a plant pathogen being considered as a biological control agent of Canada thistle (Cirsium arvense), produces tagetitoxin, an inhibitor of RNA polymerase which results in chlorosis of developing shoot tissues. Although the bacterium is known to affect several plant species in the Asteraceae and has been reported in several countries, little is known of its genetic diversity. The genetic relatedness of 24 strains of P. syringae pv. tagetis with respect to each other and to other P. syringae and Pseudomonas savastanoi pathovars was examined using 16S–23S rDNA intergenic spacer (ITS) sequence analysis. The size of the 16S–23S rDNA ITS regions ranged from 508 to 548 bp in length for all 17 P. syringae and P. savastanoi pathovars examined. The size of the 16S–23S rDNA ITS regions for all the P. syringae pv. helianthi and all the P. syringae pv. tagetis strains examined were 526 bp in length. Furthermore, the 16S–23S rDNA ITS regions of both P. syringae pv. tagetis and P. syringae pv. helianthi had DNA signatures at specific nucleotides that distinguished them from the 15 other P. syringae and P. savastanoi pathovars examined. These results provide strong evidence that P. syringae pv. helianthi is a nontoxigenic form of P. syringae pv. tagetis. The results also demonstrated that there is little genetic diversity among the known strains of P. syringae pv. tagetis. The genetic differences that do exist were not correlated with differences in host plant, geographical origin, or the ability to produce toxin.     

Genes required for pathogenicity and hypersensitivity are conserved and interchangeable among pathovars of Pseudomonas syringae

Summary A group of pathogenicity genes was previously identified in Pseudomonas syringae pv. phaseolicola which controls the ability of the pathogen to cause disease on bean and to elicit the hypersensitive response on non-host plants. These genes, designated hrp, are located in a ca. 20 kb region which was referred to as the hrp cluster. Homologous sequences to DNA segments derived from this region were detected in several pathovars of P. syringae but not in symbiotic, saprophytic or other phytopathogenic bacteria. A Tn5-induced Hrp^- mutation was transferred from P. syringae pv. phaseolicola to P. syringae pv. tabaci and to three races of P. syringae pv. glycinea by marker exchange mutagenesis. The resulting progeny were phenotypically Hrp^-, i.e. no longer pathogenic on their respective hosts and unable to elicit the hypersensitive response on non-host plants. These mutants were restored to wild-type phenotype upon introduction of a recombinant plasmid carrying the corresponding wild-type locus from P. syringae pv. phaseolicola. The marker exchange mutants of P. syringae pv. glycinea psg0 and Psg5 which carry different avr genes for race specific avirulence did not elicit a hypersensitive response on incompatible soybean cultivars. It appears, therefore, that P. syringae pathovars possess common genes for pathogenicity which also control their interaction with non-host plants. Furthermore, the expression of race/cultivar specific incompatibility of P. syringae pv. glycinea requires a fully functional hrp region in addition to the avr genes which determine avirulence on single-gene differential cultivars of soybean.     

Identification and fine-mapping of a bacterial brown spot disease resistance gene in maize

Bacterial brown spot (BBS) in maize (Zea mays L.) is caused by Pseudomonas syringae pv. syringae Van Holl (Pss). In China, this disease is not prevalent in maize at present. Here, we report the identification and fine mapping of the gene, referred to as Psy1, which confers resistance to BBS. An F₂ population, derived from the cross P25/F349, was used for linkage analysis and mapping of the resistance gene Psy1. Analysis of a BC₈F₂ population, derived from the same parents, confirmed that Psy1 was located on chromosome 10L and inherited as a single dominant gene. For fine mapping of Psy1, two introgression lines, X41 and X44, homozygous at the resistant gene locus, were introduced to hybridize with the susceptible parent P25 respectively, and developed a mixed BC₁ population. We found the closest markers to Psy1 are EST1 and FG29-3, which located on two adjacent BACs respectively, based on the B73 BAC sequence. Sequence analysis of these two BAC sequences (~300 kb) revealed the presence of a homologous sequence of receptor-like kinase. Also a co-segregation marker was developed based on this homologous sequence. These results will be useful for cloning of Psy1 and for transferring or pyramiding Psy1 through MAS in maize breeding programs.     

Negative regulation of pathogenesis in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tabaci</Emphasis> 11528 by ATP-dependent lon protease

Pseudomonas syringae pv. tabaci causes wildfire disease in tobacco plants. The hrp pathogenicity island (hrp PAI) of P. syringae pv. tabaci encodes a type III secretion system (TTSS) and its regulatory system, which are required for pathogenesis in plants. Three important regulatory proteins-HrpR, HrpS, and HrpL-have been identified to activate hrp PAI gene expression. The bacterial Lon protease regulates the expression of various genes. To investigate the regulatory mechanism of the Lon protease in P. syringae pv. tabaci 11528, we cloned the lon gene, and then a Δlon mutant was generated by allelic exchange. lon mutants showed increased UV sensitivity, which is a typical feature of such mutants. The Δlon mutant produced higher levels of tabtoxin than the wild-type. The lacZ gene was fused with hrpA promoter and activity of β-galactosidase was measured in hrp-repressing and hrp-inducing media. The Lon protease functioned as a negative regulator of hrp PAI under hrp-repressing conditions. We found that strains with lon disruption elicited the host defense system more rapidly and strongly than the wild-type strain, suggesting that the Lon protease is essential for systemic pathogenesis.     

New disease resistance genes in soybean against Pseudomonas syringae pv glycinea: evidence that one of them interacts with a bacterial elicitor

Summary Soybean [Glycine max (L.) Merr.] cultivars Flambeau and Merit differed in their resistance to Pseudomonas syringae pv glycinea (Psg) race 4, carrying each of four different avirulence (avr) genes cloned from Psg or the related bacterium, Pseudomonas syringae pv tomato. Segregation data for F₂ and F₃ progeny of Flambeau x Merit crosses indicated that single dominant and nonallelic genes account for resistance to Psg race 4, carrying avirulence genes avrA, avrB, avrC, or avrD. Segregants were also recovered that carried all four or none of the disease resistance genes. One of the disease resistance genes (Rpg1, complementing bacterial avirulence gene B) had been described previously, but the other three genes — designated Rpg2, Rpg3, and Rpg4 — had not here to fore been defined. Rpg3 and Rpg4 are linked (40.5 ± 3.2 recombination units). Rpg4 complements avrD, cloned from Pseudomonas syringae pv tomato, but a functional copy of this avirulence gene has not thus far been observed in Pseudomonas syringae pv glycinea. Resistance gene Rpg4 therefore may account in part for the resistance of soybean to Pseudomonas syringae pv tomato and other pathogens harboring avrD.     

Gac two-component system in Pseudomonas syringae pv. tabaci is required for virulence but not for hypersensitive reaction

Pseudomonas syringae pv. tabaci 6605 causes wildfire disease on host tobacco plants. To investigate the regulatory mechanism of the expression of virulence, Gac two-component system-defective mutants, ΔgacA and ΔgacS, and a double mutant, ΔgacAΔgacS, were generated. These mutants produced smaller amounts of N-acyl homoserine lactones required for quorum sensing, had lost swarming motility, and had reduced expression of virulence-related hrp genes and the algT gene required for exopolysaccharide production. The ability of the mutants to cause disease symptoms in their host tobacco plant was remarkably reduced, while they retained the ability to induce hypersensitive reaction (HR) in the nonhost plants. These results indicated that the Gac two-component system of P. syringae pv. tabaci 6605 is indispensable for virulence on the host plant, but not for HR induction in the nonhost plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported in this paper have been submitted to the DDBJ/GenBank/EMBL databank with the accession numbers AB266103, AB266104, AB266105, AB266106, AB266107, AB266108.     

Phage biocontrol to combat Pseudomonas syringae pathogens causing disease in cherry

Bacterial canker is a major disease of Prunus species, such as cherry (Prunus avium). It is caused by Pseudomonas syringae pathovars, including P. syringae pv. syringae (Pss) and P. syringae pv. morsprunorum race 1 (Psm1) and race 2 (Psm2). Concerns over the environmental impact of, and the development of bacterial resistance to, traditional copper controls calls for new approaches to disease management. Bacteriophage-based biocontrol could provide a sustainable and natural alternative approach to combat bacterial pathogens. Therefore, seventy phages were isolated from soil, leaf and bark of cherry trees in six locations in the south east of England. Subsequently, their host range was assessed against strains of Pss, Psm1 and Psm2. While these phages lysed different Pss, Psm and some other P. syringae pathovar isolates, they did not infect beneficial bacteria such as Pseudomonas fluorescens. A subset of thirteen phages were further characterized by genome sequencing, revealing five distinct clades in which the phages could be clustered. No known toxins or lysogeny-associated genes could be identified. Using bioassays, selected phages could effectively reduce disease progression in vivo, both individually and in cocktails, reinforcing their potential as biocontrol agents in agriculture.     

Characterization and genetic diversity of Pseudomonas syringae isolates from stone fruits in north‐western Iran

From 33 Iranian fluorescent Pseudomonas isolates originating from symptomatic tissues of peach (Prunus persica), plum (Prunus domestica), sweet (Prunus avium) and sour cherry (Prunus cerasus), 27 were identified as Pseudomonas syringae using LOPAT tests. Further characterization of those isolates by GATTa and L‐lactate utilization tests and the detection of syringomycin and coronatine and yersiniabactin coding genes showed that five of them belonged to race 1 and four to race 2 of P. syringae pv. morsprunorum (Psm) and eighteen other isolates were identified as P. syringae pv. syringae (Pss). Based on the analysis of the fingerprint patterns generated by REP, ERIC and BOX‐PCR, the strains were differentiated into three main groups at the 67% similarity level. Strains of the groups 1, 2 and 3 belong to Psm race 1, Psm race 2 and Pss, respectively. Rep‐PCR analysis showed high intra‐pathovar variation within the Pss isolates, which grouped into four distinct clusters. Using the REP primers, the percentage of polymorphic loci was 74.61%, whereas with BOX and ERIC primers, it was 60.5 and 55.21%, respectively. Finally, this study is the first report of the isolation of P. syringae pv. morsprunorum race 1 and 2 strains from stone fruit trees in Iran.     

Comparative analysis of induction pattern of programmed cell death and defense-related responses during hypersensitive cell death and development of bacterial necrotic leaf spots in eggplant

Pseudomonas cichorii causes necrotic leaf spots (NLS), while Pseudomonas syringae pv. tabaci induces a hypersensitive response (HR) in eggplant. P. cichorii induced cell death at 9 h after inoculation (HAI), reaching a maximum of around 24–30 HAI. On the other hand, cell death was induced 6 HAI with P. syringae pv. tabaci, reaching a maximum of around 12–18 HAI. Superoxide generation was observed in eggplant inoculated with both bacteria. DNA fragmentation, cytochrome c release into the cytosol and expression of defense-related genes such as PR-1 and hsr203J was also induced by inoculation with both bacteria, but these plant reactions were more rapidly induced in eggplant inoculated with P. syringae pv. tabaci rather than those with P. cichorii. Lipid peroxidation and induction of lipoxygenase (LOX) was drastically induced in eggplant inoculated with P. syringae pv. tabaci compared to P. cichorii-inoculated eggplant. Pharmacological studies showed that induction of the cell death, and the NLS or the HR in response to both bacteria was commonly associated with de novo protein synthesis, reactive oxygen species and caspase III-like protease. Interestingly, involvement of lipid peroxidation, LOX, serine protease, and DNase differed between induction of NLS and HR. These results suggest that programmed cell death might be closely associated not only with the HR but also NLS. However, there may be differences not only in the induction kinetics and level of plant responses but also in the infection-related responses between HR and NLS.     

Comparative Analysis of argK-tox Clusters and Their Flanking Regions in Phaseolotoxin-Producing Pseudomonas syringae Pathovars

DNA fragments containing argK-tox clusters and their flanking regions were cloned from the chromosomes of Pseudomonas syringae pathovar (pv.) actinidiae strain KW-11 (ACT) and P. syringae pv. phaseolicola strain MAFF 302282 (PHA), and then their sequences were determined. Comparative analysis of these sequences and the sequences of P. syringae pv. tomato DC3000 (TOM) (Buell et al., Proc Natl Acad Sci USA 100:10181–10186, 2003) and pv. syringae B728a (SYR) (Feil et al., Proc Natl Acad Sci USA 102:11064–11069, 2005) revealed that the chromosomal backbone regions of ACT and TOM shared a high similarity to each other but presented a low similarity to those of PHA and SYR. Nevertheless, almost-identical DNA regions of about 38 kb were confirmed to be present on the chromosomes of both ACT and PHA, which we named “tox islands.” The facts that the GC content of such tox islands was 6% lower than that of the chromosomal backbone regions of P. syringae, and that argK-tox clusters, which are considered to be of exogenous origin based on our previous studies (Sawada et al., J Mol Evol 54:437–457, 2002), were confirmed to be contained within the tox islands, suggested that the tox islands were an exogenous, mobile genetic element inserted into the chromosomes of P. syringae strains. It was also predicted that the tox islands integrated site-specifically into the homologous sites of the chromosomes of ACT and PHA in the same direction, respectively, wherein 34 common gene coding sequences (CDSs) existed. Furthermore, at the left end of the tox islands were three CDSs, which encoded polypeptides and had similarities to the members of the tyrosine recombinase family, suggesting that these putative site-specific recombinases were involved in the recent horizontal transfer of tox islands. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Oligomerization,Conformational Stability and Thermal Unfolding of Harpin,HrpZPss and Its Hypersensitive Response-Inducing C-Terminal Fragment,C-214-HrpZPss

HrpZ—a harpin from Pseudomonas syringae—is a highly thermostable protein that exhibits multifunctional abilities e.g., it elicits hypersensitive response (HR), enhances plant growth, acts as a virulence factor, and forms pores in plant plasma membranes as well as artificial membranes. However, the molecular mechanism of its biological activity and high thermal stability remained poorly understood. HR inducing abilities of non-overlapping short deletion mutants of harpins put further constraints on the ability to establish structure-activity relationships. We characterized HrpZ_Pss from Pseudomonas syringae pv. syringae and its HR inducing C-terminal fragment with 214 amino acids (C-214-HrpZ_Pss) using calorimetric, spectroscopic and microscopic approaches. Both C-214-HrpZ_Pss and HrpZ_Pss were found to form oligomers. We propose that leucine-zipper-like motifs may take part in the formation of oligomeric aggregates, and oligomerization could be related to HR elicitation. CD, DSC and fluorescence studies showed that the thermal unfolding of these proteins is complex and involves multiple steps. The comparable conformational stability at 25°C (∼10.0 kcal/mol) of HrpZ_Pss and C-214-HrpZ_Pss further suggest that their structures are flexible, and the flexibility allows them to adopt proper conformation for multifunctional abilities.     

Early detection of bean infection by Pseudomonas syringae in asymptomatic leaf areas using chlorophyll fluorescence imaging

Chlorophyll fluorescence imaging has been used to analyse the response elicited in Phaseolus vulgaris after inoculation with Pseudomonas syringae pv. phaseolicola 1448A (compatible interaction) and P. syringae pv. tomato DC3000 (incompatible interaction). With the aim of modulating timing of symptom development, different cell densities were used to inoculate bean plants and the population dynamics of both bacterial strains was followed within the leaf tissue. Fluorescence quenching analysis was carried out and images of the different chlorophyll fluorescence parameters were obtained for infected as well as control plants at different timepoints post-infection. Among the different parameters analysed, we observed that non-photochemical quenching maximised the differences between the compatible and the incompatible interaction before the appearance of visual symptom. A decrease in non-photochemical quenching, evident in both infiltrated and non-infiltrated leaf areas, was observed in P. syringae pv. phaseolicola-infected plants as compared with corresponding values from controls and P. syringae pv. tomato-infected plants. No photoinhibitory damage was detected, as the maximum photosystem II quantum yield remained stable during the infection period analysed.     

Homology between the HrpO protein of Pseudomonas solanacearum and bacterial proteins implicated in a signal peptide-independent secretion mechanism

A region of approximately 22 kb of DNA defines the large hrp gene cluster of strain GMI1000 of Pseudomonas solanacearum. The majority of mutants that map to this region have lost the ability to induce disease symptoms on tomato plants and are no longer able to elicit a hypersensitive reaction (HR) on tobacco, a nonhost plant. In this study we present the complementation analysis and nucleotide sequence of a 4772 by region of this hrp gene cluster. Three complete open reading frames (ORFs) are predicted within this region. The corresponding putative proteins, HrpN, HrpO and HpaP, have predicted sizes of 357, 690 and 197 amino acids, respectively, and predicted molecular weights of 38607, 73 990 and 21959 dalton, respectively. HrpN and HrpO are both predicted to be hydrophobic proteins with potential membrane-spanning domains and HpaP is rich in proline residues. A mutation in hpaP (for hrp associated) does not affect the HR on tobacco or the disease on tomato plants. None of the proteins is predicted to have an N-terminal signal sequence, which would have indicated that the proteins are exported. Considerable sequence similarities were found between HrpO and eight known or predicted prokaryotic proteins: LcrD of Yersinia pestis and Y. enterocolitica, FlbF of Caulobacter crescentus, F1hA of Bacillus subtilis, MxiA and VirH of Shigella flexneri, InvA of Salmonella typhimurium and HrpC2 of Xanthomonas campestris pv. vesicatoria. These homologies suggest that certain hrp genes of phytopathogenic bacteria code for components of a secretory system, which is related to the systems for secretion of flagellar proteins, Ipa proteins of Shigella flexneri and the Yersinia Yop proteins. Furthermore, these homologous proteins have the common feature of being implicated in a distinct secretory mechanism, which does not require the cleavage of a signal peptide. The sequence similarity between HrpO and HrpC2 is particularly high (66% identity and 81 % similarity) and the amino acid sequence comparison between these two proteins presented here reveals the first such sequence similarity to be shown between Hrp proteins of P. solanacearum and X. campestris. An efflux of plant electrolytes was found to be associated with the interactions between P. solanacearum and both tomato and tobacco leaves. This phenomenon may be part of the mechanism by which hrp gene products control and determine plant-bacterial interactions, since hrpO mutants induced levels of leakage which were significantly lower than those induced by the wild type on each plant.     

The Serological Heterogeneity of Pseudomonas syringae pv. atrofaciens Strains and Their Ecological Niches

The paper deals with a comparative analysis of the serological and ecological properties of Pseudomonas syringae pv. atrofaciens strains from the collections of microbial cultures at the Malkov Institute for Plant Genetic Resources and Zabolotny Institute of Microbiology and Virology. All of the strains from the Bulgarian collection, except for one, fall into five serogroups (II through VI) of the classification system of Pastushenko and Simonovich. The P. syringae pv. atrofaciens strains isolated from Bulgarian and Ukrainian wheats belong mainly to serogroups II and IV, respectively. The strains that were isolated from rye plants belong to serogroup I. The strains isolated from sorghum and Sudan grass belong to serogroups II, IV, and VI. Serogroup III includes the P. syringae pv. atrofaciens strains that were isolated from cereals in the United Kingdom but not in Ukraine.     

Cloning,characterization and phenotypic expression in Escherichia coli of catF,which encodes the catalytic subunit of catalase isozyme CatF of Pseudomonas syringae

The phytopathogenic, gram-negative bacterium Pseudomonas syringae pv. syringae 61 contains three isozymes of catalase (EC 1.11.1.6), which have been proposed to play a role in the bacterium's responses to various environmental stresses. To study the role of individual isozymes, the gene coding for the catalytic subunit of one catalase isozyme was cloned from a cosmid library hosted in Escherichia coli DH5 by using a designed catalase-specific DNA probe for the screening. One out of four clones with a catalase-positive genotype was subcloned and a pUC19-based 2.7 × 10³-base (2.7-kb) insert subclone, pMK3E5, was used to transform catalase-deficient E. coli strain UM255 (HPI^–, HPII^–). The transformants contained a single isozyme of catalase that had electrophoretic and enzymic properties similar to catalase isozyme CatF from P. syringae pv. syringae 61. Analysis of the sequenced 2.7-kb insert DNA revealed six putative open-reading frames (ORF). The 1542-base-pair DNA sequence of ORF2, called catF, encodes a peptide of 513 amino acid residues with a calculated molecular mass of 66.6 kDa. The amino acid sequence deduced from catF had homology to the primary structure of true catalases from mammals, plants, yeasts and bacteria. The activity of the recombinant catalase was inhibited by 3-amino-1,2,4-triazole and azide and stimulated by chloramphenicol. The N terminus contained a signal sequence of 26 amino acids necessary for secretion into the periplasm, a so-far unique property of Pseudomonas catalases.Paper no. 4552 of the Utah Agricultural Experiment station     

Location of the O-methyl groups in the O polysaccharide of Pseudomonas syringae pv. phaseolicola

The O-methylation pattern of the O polysaccharide (OPS) of the lipopolysaccharide of Pseudomonas syringae pv. phaseolicola GSPB 1552 was revealed by methylation (CD₃I) analysis, Smith degradation, and NMR spectroscopy. Together with the major O repeats consisting of -rhamnopyranose ( -Rhap) and -fucofuranose ( -Fucf), there are minor repeats (30%) containing 3-O-methyl- -rhamnose ( -acofriose), which is 2-substituted in the interior repeats and occupies the terminal non-reducing end of the OPS. It was suggested that the methylated O repeats are linked to each other nearby the non-reducing end of the OPS and that the ‘biological’ O repeat of the OPS has the following structure:

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