Rapid and Transient Activation of a Myelin Basic Protein Kinase in Tobacco Leaves Treated with Harpin from Erwinia amylovora

Harpins are bacterial protein elicitors that induce hypersensitive response-like necrosis when infiltrated into nonhost plants such as tobacco (Nicotiana tabacum L.) (Z.-M. Wei, R.J. Laby, C.H. Zumoff, D.W. Bauer, S.Y. He, A. Collmer, S.V. Beer [1992] Science 257: 85-88). Activity of a 49-kD Mg2+-dependent and Ca2+-independent kinase in tobacco leaves increased 50-fold 15 min after infiltration of harpin from Erwinia amylovora (harpinEa). Much less pronounced and more transient activation was detected in water-infiltrated leaves. Biochemical characteristics of the harpinEa-activated protein kinase (HAPK) activity are consistent with those of the mitogen-activated protein kinase family. HAPK is cytosolic and phosphorylates myelin basic protein on serine/threonine residues. Treatment with a protein tyrosine phosphatase completely eliminated HAPK activity, suggesting that tyrosine phosphorylation is required for posttranslational activation. Sustained HAPK activation after cycloheximide treatment implies that HAPK may be negatively regulated by a translation-dependent mechanism. The extracellular Ca2+ chelator EGTA or the protein kinase inhibitor K252a, infiltrated in planta together with harpinEa, partially blocked HAPK activation. The Ca2+-channel blocker La3+ had no effect on HAPK activation, suggesting that phosphorylation events precede and/or do not depend on the entry of extracellular Ca2+ into the cell. These results suggest that early signal transduction events during harpinEa- induced hypersensitive response elicitation depend in part on the activation of HAPK.     

Histochemistry of Tobacco Mesophyll Cell Walls Pretreated with Bacterial Protein-lipopolysaccharides

Prevention of hypersensitive confluent-necrosis in tobacco mesophyll, caused by Pseudomonas syringae pv. aptata, and tolerance towards the incompatible bacterium are induced 48 h after intercellular injection of protein-lipopolysaccharide complexes. Histochemical analysis and ultrastructural observations were carried out to determine whether there was an association between this tolerance state and cell wall alterations due to callose, lignin and/or suberin deposition either before or after challenge with the incompatible bacterium. No evidence was obtained for such wall alterations.     

Comparison of Bacterial Community of Healthy and Erwinia amylovora Infected Apples

Fire blight disease, caused by Erwinia amylovora, could damage rosaceous plants such as apples, pears, and raspberries. In this study, we designed to understand how E. amylovora affected other bacterial communities on apple rhizosphere; twig and fruit endosphere; and leaf, and fruit episphere. Limited studies on the understanding of the microbial community of apples and changes the community structure by occurrence of the fire blight disease were conducted. As result of these experiments, the infected trees had low species richness and operational taxonomic unit diversity when compared to healthy trees. Rhizospheric bacterial communities were stable regardless of infection. But the communities in endosphere and episphere were significanlty affected by E. amylovora infection. We also found that several metabolic pathways differ significantly between infected and healthy trees. In particular, we observed differences in sugar metabolites. The finding provides that sucrose metabolites are important for colonization of E. amylovora in host tissue. Our results provide fundamental information on the microbial community structures between E. amylovora infected and uninfected trees, which will contribute to developing novel control strategies for the fire blight disease.     

Bacteriophages of Erwinia amylovora

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and PODOVIRIDAE:     

Bacteriophages of Erwinia amylovora

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and Podoviridae.     

Capsulation and virulence in Erwinia amylovora

Evidence is presented that capsulation may be one virulence determinant for Erwinia amylovora, the fireblight pathogen. When 15 virulent and seven avirulent strains were grown on a medium containing asparagine as the only source of carbon and nitrogen, or yeast peptone agar, or on a sugar medium containing an inorganic source of nitrogen, capsule production and virulence were not correlated. However, if a sugar or sugar alcohol was added to the asparagine medium or to yeast peptone agar all the virulent strains produced some or many capsulated cells whereas six of the avirulent ones did not. Capsules were also produced by all the virulent strains during infection. The existence of a seventh avirulent strain which was capsulated on all media except unsupplemented asparagine agar, suggested that capsule production was not the only virulence determinant.     

Cloning and Expression in Escherichia coli of the Polysaccharide Depolymerase Associated with Bacteriophage-Infected Erwinia amylovora

The bacteriophage-encoded polysaccharide depolymerase produced in Erwinia amylovora has been cloned and expressed in Escherichia coli. The bacteriophage ERA103 genome was observed to consist of five EcoRI fragments, labeled as follows: A, 7.5 kilobases (kb); B, 5.0 kb; C, 2.7 kb; D, 2.1 kb; and E, 1.8 kb. A restriction map for ERA103 was also prepared. Each of the fragments were cloned into the positive-selection vector pOP203(A₂⁺) and pBR322.     

Phenotypic diversity of Erwinia amylovora in Bulgaria

Fifty-one strains of Erwinia amylovora isolated from nine host plants in Bulgaria were characterized phenotypically and identified by the API 20E and BIOLOG system. The identification was confirmed by PCR amplification of a specific region of the plasmid pEA29 and the genome ams region. The phenotypic diversity of the strains was studied on the basis of their API 20E and BIOLOG metabolic profiles, as well as of their SDS-PAGE protein profile. Metabolic diversity among the strains was established, but no connection with the origin of the strains was revealed. The Bulgarian strains showed API 20E metabolic profiles not found in previous studies of E. amylovora. The strains formed a homogenous group on the basis of their protein profiles. All the strains were sensitive to the antibiotics streptomycin, tetracycline and oxytetracycline. This study was an initial step towards an investigation of the diversity and evolution in the Bulgarian population of E. amylovora, and it was the first characterization of E. amylovora strains isolated from different host plants in the period 1995-2005 in Bulgaria.     

Isolation and Characterization of Five Erwinia amylovora Bacteriophages and Assessment of Phage Resistance in Strains of Erwinia amylovora

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages Ea1 and Ea7 and 3 novel phages named Ea100, Ea125, and Ea116C, were identified based on differences in genome size and restriction fragment pattern. Ea1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages Ea100, Ea7, and Ea125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. Ea116C contained an approximately 75-kb genome. Ea1, Ea7, Ea100, Ea125, and Ea116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. Ea116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10⁵ CFU.     

Expression of bovine lactoferrin cDNA confers resistance to Erwinia amylovora in transgenic pear

The siderophore produced by Erwinia amylovora, the causal agent of fire blight of Maloideae, is one of the virulence factors of this bacterium. The production of siderophores enables E. amylovora to overcome the conditions of iron limitation met in plant tissue, and may also protect the bacteria against active oxygen species produced through the Fenton reaction. In this paper, we have examined the ability of an iron chelator protein, encoded by the bovine lactoferrin gene, to reduce fire blight susceptibility in pear (Pyrus communis L.). Transgenic pear clones expressing this gene controlled by the CaMV35S promoter were produced by Agrobacterium tumefaciens mediated transformation. Transformants were analysed by RT-PCR and western blot to determine lactoferrin expression levels. Most transgenic clones demonstrated significant reduction of susceptibility to fire blight in vitro and in the greenhouse when inoculated by E. amylovora. These transgenic clones also showed a significant reduction of symptoms when inoculated with two other pear bacterial pathogens : Pseudomonas syringae pv. syringae and Agrobacterium tumefaciens. Moreover, we have shown that this increase in bacterial resistance was correlated with an increase in root ferric reductase level activity and leaf iron content. Despite negative effects on the growth of a few clones, our results indicate the potential of lactoferrin gene transformation to protect pear from fire blight through increased iron chelation.     

Expression of lysozymes from Erwinia amylovora phages and Erwinia genomes and inhibition by a bacterial protein

Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ΦEa1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ΦEa104 and ΦEa116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ΦEa1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ΦEa1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned 'silent' lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme.     

Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages phiEa1 and phiEa7 and 3 novel phages named phiEa100, phiEa125, and phiEa116C, were identified based on differences in genome size and restriction fragment pattern. phiEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages phiEa100, phiEa7, and phiEa125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. phiEa116C contained an approximately 75-kb genome. phiEa1, phiEa7, phiEa100, phiEa125, and phiEa116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. phiEa116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10(5) CFU.     

Erwinia amylovora Infection of Hawthorn Blossom

Stamens of freshly opened flowers of hawthorn were inoculated with E;. amylovora and the development of blossom infection was monitored by viable bacterial counts and light and electron microscopy. Some bacterial multiplication occurred on the anther surface, over the dehiscence zone and over the junctions ot the anther-wall cells. bacteria invaded the anther loculc, via the ruptured dehiscence zone, and possibly also vid the stomata surrounding the filament insertion. bacteria within the locule multiplied rapidk with estimated doubling-times which were longer than those derived from in vitro data. Pollen grains Irom infected anthers were found to be heavily eontaminated with bacteria. The invasion of anther tissue, with the production of contaminated pollen, may be important epidemiologieally both as a phase of rapid bacterial multiplication and in the insect-mediated spread of this disease.     

Antibiotic Production by Erwinia herbicola Eh1087: Its Role in Inhibition of Erwinia amylovora and Partial Characterization of Antibiotic Biosynthesis Genes

Mutants of Erwinia herbicola Eh1087 (Ant⁻), which did not produce antibiotic activity against Erwinia amylovora, the fire blight pathogen, were selected after TnphoA mutagenesis. In immature pear fruit Ant⁻ mutants grew at the same rate as wild-type strain Eh1087 but did not suppress development of the disease caused by E. amylovora. These results indicated that antibiosis plays an important role in the suppression of disease by strain Eh1087. All of the Ant⁻ mutations obtained were located in a 2.2-kb region on a 200-kb indigenous plasmid. Sequence analysis of the mutated DNA region resulted in identification of six open reading frames, designated ORF1 through ORF6, four of which were essential to antibiotic expression. One gene was identified as a gene which encodes a translocase protein which is probably involved in antibiotic secretion. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasmid proteins produced in Escherichia coli minicells confirmed the presence of proteins whose sizes corresponded to the sizes of the predicted open reading frame products.     

Discrimination and Detection of Erwinia amylovora and Erwinia pyrifoliae with a Single Primer Set

Erwinia amylovora and Erwinia pyrifoliae cause fire blight and black-shoot blight, respectively, in apples and pears. E. pyrifoliae is less pathogenic and has a narrower host range than that of E. amylovora. Fire blight and black-shoot blight exhibit similar symptoms, making it difficult to distinguish one bacterial disease from the other. Molecular tools that differentiate fire blight from black-shoot blight could guide in the implementation of appropriate management strategies to control both diseases. In this study, a primer set was developed to detect and distinguish E. amylovora from E. pyrifoliae by conventional polymerase chain reaction (PCR). The primers produced amplicons of different sizes that were specific to each bacterial species. PCR products from E. amylovora and E. pyrifoliae cells at concentrations of 10⁴ cfu/ml and 10⁷ cfu/ml, respectively, were amplified, which demonstrated sufficient primer detection sensitivity. This primer set provides a simple molecular tool to distinguish between two types of bacterial diseases with similar symptoms.     

New host plants of Erwinia amylovora in Bulgaria

Nine strains of Erwinia amylovora were isolated from new host plants in Bulgaria--chokeberry and strawberry. The strains were characterized morphologically and biochemically using the API 20E and BIOLOG system. It was established that they showed three different API 20E metabolic profiles, not found by previous studies of E. amylovora. All strains were identified as E. amylovora due to their metabolic fingerprint patterns obtained by the BIOLOG system. The identification was confirmed by PCR amplification of a specific region of plasmid pEA29 and genome ams-region. This study is the first characterization and identification of E. amylovora strains isolated from chokeberry and strawberry by the API 20E and BIOLOG system and by polymerase chain reaction.     

Altered Expression of Immune-Related Genes in Children with Down Syndrome

Individuals with Down syndrome (DS) have a high incidence of immunological alterations with increased susceptibility to bacterial and viral infections and high frequency of different types of hematologic malignancies and autoimmune disorders. In the current study, we profiled the expression pattern of 92 immune-related genes in peripheral blood mononuclear cells (PBMCs) of two different groups, children with DS and control children, to identify differentially expressed genes that might be of pathogenetic importance for the development and phenotype of the immunological alterations observed in individuals with DS. PBMCs samples were obtained from six DS individuals with karyotypically confirmed full trisomy 21 and six healthy control individuals (ages 2–6 years). Gene expression was profiled in duplicate according to the manufacturer''s instructions provided by commercially available TaqMan Human Immune Array representing 92 immune function genes and four reference genes on a 96-plex gene card. A set of 17 differentially expressed genes, not located on chromosome 21 (HSA21), involved in immune and inflammatory pathways was identified including 13 genes (BCL2, CCL3, CCR7, CD19, CD28, CD40, CD40LG, CD80, EDN1, IKBKB, IL6, NOS2 and SKI) significantly down-regulated and four genes (BCL2L1, CCR2, CCR5 and IL10) significantly up-regulated in children with DS. These findings highlight a list of candidate genes for further investigation into the molecular mechanism underlying DS pathology and reinforce the secondary effects of the presence of a third copy of HSA21.