Identification of low-temperature-regulated genes in the fire blight pathogen Erwinia amylovora

Genes involved in pathogenicity of several plant pathogens were shown to be induced at relatively cold temperatures. Loci from the fire blight pathogen Erwinia amylovora (Burrill) induced at 18 degrees C were identified using the miniTn5 transposon that contains the promoterless reporter gene gusA coding for beta-glucuronidase (GUS). Certain mutants (2.7%) expressed GUS predominantly at 18 degrees C on minimal medium plates, indicating that the transposon had been inserted downstream of a putatively thermoregulated promoter. Those mutants were further screened with a quantitative GUS fluorometric assay. A total of 21 mutants were selected: 19 mutants had a transposon insertion in temperature-dependent genetic loci, with a 2.2- to 6.3-fold induction of gusA gene expression at 18 degrees C, and two mutants with impaired growth at 18 degrees C. Some of these genetic loci encoded (i) proteins implicated in flagella biosynthesis, biotin biosynthesis, multi-drug efflux, and type II secretion protein, and (ii) proteins of unknown function.     

Autoinducer-2 of the fire blight pathogen Erwinia amylovora and other plant-associated bacteria

Autoinducers are important for cellular communication of bacteria. The luxS gene has a central role in the synthesis of autoinducer-2 (AI-2). The gene was identified in a shotgun library of Erwinia amylovora and primers designed for PCR amplification from bacterial DNA. Supernatants of several Erwinia amylovora strains were assayed for AI-2 activity with a Vibrio harveyi mutant and were positive. Many other plant-associated bacteria also showed AI-2 activity such as Erwinia pyrifoliae and Erwinia tasmaniensis. The luxS genes of several bacteria were cloned, sequenced, and complemented Escherichia coli strain DH5alpha and a Salmonella typhimurium mutant, both defective in luxS, for synthesis of AI-2. Assays to detect AI-2 activity in culture supernatants of several Pseudomonas syringae pathovars failed, which may indicate the absence of AI-2 or synthesis of another type. Several reporter strains did not detect synthesis of an acyl homoserine lactone (AHL, AI-1) by Erwinia amylovora, but confirmed AHL-synthesis for Erwinia carotovora ssp. atroseptica and Pantoea stewartii.     

The fire blight pathogen Erwinia amylovora requires the rpoN gene for pathogenicity in apple

RpoN is a σ⁵⁴ factor regulating essential virulence gene expression in several plant pathogenic bacteria, including Pseudomonas syringae and Pectobacterium carotovorum. In this study, we found that mutation of rpoN in the fire blight pathogen Erwinia amylovora caused a nonpathogenic phenotype. The E. amylovora rpoN Tn5 transposon mutant rpoN1250::Tn5 did not cause fire blight disease symptoms on shoots of mature apple trees. In detached immature apple fruits, the rpoN1250::Tn5 mutant failed to cause fire blight disease symptoms and grew to population levels 12 orders of magnitude lower than the wild‐type. In addition, the rpoN1250::Tn5 mutant failed to elicit a hypersensitive response when infiltrated into nonhost tobacco plant leaves, and rpoN1250::Tn5 cells failed to express HrpN protein when grown in hrp (hypersensitive response and pathogenicity)‐inducing liquid medium. A plasmid‐borne copy of the wild‐type rpoN gene complemented all the rpoN1250::Tn5 mutant phenotypes tested. The rpoN1250::Tn5 mutant was prototrophic on minimal solid and liquid media, indicating that the rpoN1250::Tn5 nonpathogenic phenotype was not caused by a defect in basic metabolism or growth. This study provides clear genetic evidence that rpoN is an essential virulence gene of E. amylovora, suggesting that rpoN has the same function in E. amylovora as in P. syringae and Pe. carotovorum.     

Apple proteins that interact with DspA/E, a pathogenicity effector of Erwinia amylovora, the fire blight pathogen

The disease-specific (dsp) gene dspA/E of Erwinia amylovora encodes an essential pathogenicity effector of 198 kDa, which is critical to the development of the devastating plant disease fire blight. A yeast two-hybrid assay and in vitro protein pull-down assay demonstrated that DspA/E interacts physically and specifically with four similar putative leucine-rich repeat (LRR) receptor-like serine/threonine kinases (RLK) from apple, an important host of E. amylovora. The genes encoding these four DspA/E-interacting proteins of Malus xdomestica (DIPM1 to 4) are conserved in all genera of hosts of E. amylovora tested. They also are conserved in all cultivars of apple tested that range in susceptibility to fire blight from highly susceptible to highly resistant. The four DIPMs have been characterized, and they are expressed constitutively in host plants. In silico analysis indicated that the DIPMs have similar sequence structure and resemble LRR RLKs from other organisms. Evidence is presented for direct physical interaction between DspA/E and the apple proteins encoded by the four identified clones, which may act as susceptibility factors and be essential to disease development. Knowledge of DIPMs and the interaction with DspA/E thus may facilitate understanding of fire blight development and lead to new approaches to control of disease.     

Partial characterization of an inhibitory strain of Erwinia herbicola with potential as a biocontrol agent for Erwinia amylovora, the fire blight pathogen

Erwinia herbicola Eh1087 isolated from apple blossom inhibits development of Erwinia amylovora in immature pear fruit and produces a broad spectrum antibiotic activity in vitro that is bactericidal for Erw. amylovora. The antibiotic activity is present in cell-free culture supernatant fluids of late log-early stationary phase cultures of Eh1087. This antibiotic activity is not inhibited by proteases, excess ferric ions or essential amino acids. It is stable to acidic and basic pH and is inactivated at high temperature. The antibiotic activity is inactivated by β-lactamase digestion.     

The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora

The enterobacterium Erwinia amylovora causes fire blight on members of the family Rosaceae, with economic importance on apple and pear. During pathogenesis, the bacterium is exposed to a variety of plant-borne antimicrobial compounds. In plants of Rosaceae, many constitutively synthesized isoflavonoids affecting microorganisms were identified. Bacterial multidrug efflux transporters which mediate resistance toward structurally unrelated compounds might confer tolerance to these phytoalexins. To prove this hypothesis, we cloned the acrAB locus from E. amylovora encoding a resistance nodulation division-type transport system. In Escherichia coli, AcrAB of E. amylovora conferred resistance to hydrophobic and amphiphilic toxins. An acrB-deficient E. amylovora mutant was impaired in virulence on apple rootstock MM 106. Furthermore, it was susceptible toward extracts of leaves of MM 106 as well as to the apple phytoalexins phloretin, naringenin, quercetin, and (+)-catechin. The expression of acrAB was determined using the promoterless reporter gene egfp. The acrAB operon was up-regulated in vitro by the addition of phloretin and naringenin. The promoter activity of acrR, encoding a regulatory protein involved in acrAB expression, was increased by naringenin. In planta, an induction of acrAB was proved by confocal laser scanning microscopy. Our results strongly suggest that the AcrAB transport system plays an important role as a protein complex required for virulence of E. amylovora in resistance toward apple phytoalexins and that it is required for successful colonization of a host plant.     

Autoinduction in Erwinia amylovora: evidence of an acyl-homoserine lactone signal in the fire blight pathogen

Erwinia amylovora causes fire blight disease of apple, pear, and other members of the Rosaceae. Here we present the first evidence for autoinduction in E. amylovora and a role for an N-acyl-homoserine lactone (AHL)-type signal. Two major plant virulence traits, production of extracellular polysaccharides (amylovoran and levan) and tolerance to free oxygen radicals, were controlled in a bacterial-cell-density-dependent manner. Two standard autoinducer biosensors, Agrobacterium tumefaciens NTL4 and Vibrio harveyi BB886, detected AHL in stationary-phase cultures of E. amylovora. A putative AHL synthase gene, eamI, was partially sequenced, which revealed homology with autoinducer genes from other bacterial pathogens (e.g., carI, esaI, expI, hsII, yenI, and luxI). E. amylovora was also found to carry eamR, a convergently transcribed gene with homology to luxR AHL activator genes in pathogens such as Erwinia carotovora. Heterologous expression of the Bacillus sp. strain A24 acyl-homoserine lactonase gene aiiA in E. amylovora abolished induction of AHL biosensors, impaired extracellular polysaccharide production and tolerance to hydrogen peroxide, and reduced virulence on apple leaves.     

Characterization of serracin P,a phage-tail-like bacteriocin,and its activity against Erwinia amylovora,the fire blight pathogen

Serratia plymithicum J7 culture supernatant displayed activity against many pathogenic strains of Erwinia amylovora, the causal agent of the most serious bacterial disease of apple and pear trees, fire blight, and against Klebsiella pneumoniae, Serratia liquefaciens, Serratia marcescens, and Pseudomonas fluorescens. This activity increased significantly upon induction with mitomycin C. A phage-tail-like bacteriocin, named serracin P, was purified from an induced culture supernatant of S. plymithicum J7. It was found to be the only compound involved in the antibacterial activity against sensitive strains. The N-terminal amino acid sequence analysis of the two major subunits (23 and 43 kDa) of serracin P revealed high homology with the Fels-2 prophage of Salmonella enterica, the coliphages P2 and 168, the phiCTX prophage of Pseudomonas aeruginosa, and a prophage of Yersinia pestis. This strongly suggests a common ancestry for serracin P and these bacteriophages.     

Genetics of galactose metabolism of Erwinia amylovora and its influence on polysaccharide synthesis and virulence of the fire blight pathogen.

Galactose metabolism mutants of Erwinia amylovora were created by transposon insertions and characterized for their growth properties and interaction with plant tissue. The nucleotide sequence of the galE gene was determined. The gene, which encodes UDP-galactose 4-epimerase, shows homology to the galE genes of Escherichia coli, Neisseria gonorrhoeae, Rhizobium meliloti, and other gram-negative bacteria. Cloned DNA with the galE and with the galT and galK genes did not share borders, as judged by the lack of common fragments in hybridization with chromosomal DNA. These genes are thus located separately on the bacterial chromosome. In contrast to the gal operon of E. coli, the galE gene of E. amylovora is constitutively expressed, independently of the presence of galactose in the medium. The function of the galE gene but not of the galT or galK gene is required for bacterial virulence on pear fruits and seedlings. In the absence of galactose, the galE mutant was deficient in amylovoran synthesis. Subsequently, the galE mutant cells elicited host defense reactions, and they were not stained by fluorescein isothiocyanate-labelled lectin, which efficiently binds to amylovoran capsules of E. amylovora. The mutation affected the side chains of bacterial lipopolysaccharide, but an intact O antigen was not required for virulence. This was shown with another mutant, which could be complemented for virulence but not for side chain synthesis of lipopolysaccharide.     

Assessment of fire blight tolerance in apple based on plant inoculations with Erwinia amylovora and DNA markers

Fire blight (Erwinia amylovora) causes serious damage to pome fruit orchards, and identification of germplasm with heritable disease resistance is therefore crucial. Two dominant SCAR (sequence characterised amplified region) marker alleles (AE10-375 and GE-8019), flanking a previously identified QTL (quantitative trait locus) for resistance to fire blight on ‘Fiesta’ linkage group 7 in apple cultivars related to ‘Cox’s Orange Pippin’, were screened on 205 apple cultivars. Both marker alleles were present in 22% of the cultivars, indicating presence of the QTL allele for tolerance, and both were lacking in 25%, indicating homozygosity for absence of the QTL tolerance allele. However, 33% had only the marker allele AE10-375, while 20% had only GE-8019, suggesting that some cultivars with the dominant alleles for both of the flanking markers can carry these on separate chromosomes and may lack the QTL allele for tolerance. In 2009 and 2010, terminal shoots of greenhouse-grown grafted trees of 21 cultivars (only 20 in 2010) were inoculated with Erwinia amylovora. ‘Idared’ (susceptible) and ‘Enterprise’ (tolerant) were included as controls. Disease severity for each cultivar was expressed as percentage of necrosis in relation to entire length of shoot, and the ranking of cultivars in 2009 and 2010 was compared with a Spearman rank correlation test, P < 0.01. A relationship between presence of both flanking marker alleles for tolerance and level of fire blight tolerance was confirmed with a Mann–Whitney U-test, P < 0.01 in 2009, and P < 0.05 in 2010. A PCO (principal coordinate) analysis based on band profiles obtained with 12 SSR (simple sequence repeat) loci produced three loose clusters, two of which contained known offspring of ‘Cox’s Orange Pippin’, and one with cultivars that were either unrelated or had an unknown origin. Cases where DNA markers did not predict level of fire blight damage as expected, were, however, as common among descendants of ‘Cox’s Orange Pippin’ as among apparently unrelated cultivars. Obviously the ‘Fiesta’ LG 7 QTL has some predictive value, both for known ‘Cox’ relatives and others, but more efficient markers would be desirable for marker-assisted selection.     

Protection of apple against fire blight induced by an hrpL mutant of Erwinia amylovora

A regulatory hrpL non-virulent mutant of Erwinia amylovora is effective in controlling fire blight disease when inoculated on apple seedlings simultaneously with the pathogenic parental strain. Mechanisms involved in this protective effect were investigated. The use of two marker genes, uidA and lacZ, expressed in the hrpL mutant and the pathogenic strain, respectively, allowed to localize simultaneously the two inoculated strains in plant tissue. An anti-β-glucuronidase antibody was also used to detect the hrpL mutant. Both techniques indicated that the two strains localized mainly in separate areas of the leaf tissue. In addition, leaves infiltrated with the hrpL mutant exhibited a significant increase in peroxidase activity in contrast to a hrp secretion mutant known to be less effective in the protection. It is suggested that protection obtained with the hrpL mutant relies on the physical separation between the mutant and the parental strain after co-inoculation and the rapid and sustained activation of plant defense mechanisms in reactive tissue, i.e. not invaded by the virulent strain.     

Characterization of the rcsB gene from Erwinia amylovora and its influence on exoploysaccharide synthesis and virulence of the fire blight pathogen.

RcsB belongs to a family of positive regulators of exopolysaccharide synthesis in various enterobacteria. The rcsB gene of the fire blight pathogen Erwinia amylovora was cloned by PCR amplification with consensus primers, and its role in exopolysaccharide (EPS) synthesis was investigated. Its overexpression from high-copy-number plasmids stimulated the synthesis of the acidic EPS amylovoran and suppressed expression of the levan-forming enzyme levansucrase. Inactivation of rcsB by site-directed mutagenesis created mutants that were deficient in amylovoran synthesis and avirulent on host plants. In addition, a cosmid which complemented rcsB mutants was selected from a genomic library. The spontaneous E. amylovora mutant E8 has a similar phenotype and was complemented by the cloned rcsB gene. The rcsB region of strain E8 was also amplified by PCR, and the mutation was characterized as a nine-nucleotide deletion at the start of the rcsB gene. Nucleotide sequence analysis of the E. amylovora rcsB region and the predicted amino acid sequence of RcsB revealed extensive homology to rcsB and the encoded protein of other bacteria such as Escherichia coli and Erwinia stewartii. In all three organisms, rcsB is localized adjacent to the rcsC gene, which is transcribed in the opposite direction of rcsB. The E. amylovora rcsB gene has now been shown to strongly affect the formation of disease symptoms of a plant pathogen.     

Phenotypic and genetic diversity of Erwinia amylovora: the causal agent of fire blight

Erwinia amylovora is a polyphagous bacterium causing fire blight on apple, pear and over 130 other plant species belonging mainly to the Rosaceae family. Although E. amylovora is regarded as a very homogenous species, the particular strains can differ in pathogenic ability as far as their host range is concerned (e.g. those originating from Rubus or Maloidae plants) as well as by the extent of the disease they cause. It was found that strains originating from North America are generally more genetically heterogeneous than those from Europe. Diversity of E. amylovora is also related to streptomycin resistance as a result of its application to control of fire blight. The level of genetic heterogeneity of E. amylovora is so low (comparative genome analysis revealed a similarity of over 99% for the two genomes tested) that standard DNA-based techniques fail in detection of intra-species variability. Amplified fragment length polymorphism was found to be most useful for differentiation of strains of fire blight causal agent as well as techniques ensuing release of pan-genome sequences of two E. amylovora strains: multi-locus variable number of tandem repeats analysis and clustered regularly interspaced short palindrome repeats.