The HrpN(ea) harpin from Erwinia amylovora triggers differential responses on the nonhost Arabidopsis thaliana cells and on the host apple cells

Erwinia amylovora is a gram-negative necrogenic bacterium causing fire blight of the Maloideae subfamily of Rosaceae such as apple and pear. It provokes progressive necrosis in aerial parts of susceptible host plants (compatible interaction) and a hypersensitive reaction (HR) when infiltrated in nonhost plants (incompatible interaction). The HrpN(ea) harpin is a type three secretion system effector secreted by E. amylovora. This protein is involved in pathogenicity and HR-eliciting capacity of E. amylovora. In the present study, we showed that, in nonhost Arabidopsis thaliana cells, purified HrpN(ea) induces cell death and H2O2 production, two nonhost resistance responses, but failed to induce such responses in host MM106 apple cells. Moreover, HrpN(ea) induced an increase in anion current in host MM106 apple cells, at the opposite of the decrease of anion current previously shown to be necessary to induce cell death in nonhost A. thaliana cells. These results suggest that HrpN(ea) induced different signaling pathways, which could account for early induced compatible or incompatible interaction development.     

The role of luxS in the fire blight pathogen Erwinia amylovora is limited to metabolism and does not involve quorum sensing

Erwinia amylovora is a gram-negative phytopathogen that causes fire blight of pome fruit and related members of the family Rosaceae. We sequenced the putative autoinducer-2 (AI-2) synthase gene luxS from E. amylovora. Diversity analysis indicated that this gene is extremely conserved among E. amylovora strains. Quorum sensing mediated by LuxS has been implicated in coordinated gene expression, growth, and virulence in other enterobacteria; however, our evidence suggests this is not the function in E. amylovora. Mutational analysis pointed to a role in colonization of apple blossoms, the primary infection court for fire blight, although little if any role in virulence on apple shoots and pear fruit was observed. Expression of key virulence genes hrpL and dspA/E was reduced in mutants of two E. amylovora strains. Stronger effects on gene expression were observed for metabolic genes involved in the activated methyl cycle with mutants having greater levels of expression. No quorum-sensing effect was observed in coculture experiments with wild-type and mutant strains either in vitro or in apple blossoms. Known receptors essential for AI-2 quorum sensing, the LuxPQ sensor kinase or the Lsr ABC-transporter, are absent in E. amylovora, further suggesting a primarily metabolic role for luxS in this bacterium.     

NorM, an Erwinia amylovora multidrug efflux pump involved in in vitro competition with other epiphytic bacteria

Blossoms are important sites of infection for Erwinia amylovora, the causal agent of fire blight of rosaceous plants. Before entering the tissue, the pathogen colonizes the stigmatic surface and has to compete for space and nutrient resources within the epiphytic community. Several epiphytes are capable of synthesizing antibiotics with which they antagonize phytopathogenic bacteria. Here, we report that a multidrug efflux transporter, designated NorM, of E. amylovora confers tolerance to the toxin(s) produced by epiphytic bacteria cocolonizing plant blossoms. According to sequence comparisons, the single-component efflux pump NorM is a member of the multidrug and toxic compound extrusion protein family. The corresponding gene is widely distributed among E. amylovora strains and related plant-associated bacteria. NorM mediated resistance to the hydrophobic cationic compounds norfloxacin, ethidium bromide, and berberine. A norM mutant was constructed and exhibited full virulence on apple rootstock MM 106. However, it was susceptible to antibiotics produced by epiphytes isolated from apple and quince blossoms. The epiphytes were identified as Pantoea agglomerans by 16S rRNA analysis and were isolated from one-third of all trees examined. The promoter activity of norM was twofold greater at 18 degrees C than at 28 degrees C. The lower temperature seems to be beneficial for host infection because of the availability of moisture necessary for movement of the pathogen to the infection sites. Thus, E. amylovora might employ NorM for successful competition with other epiphytic microbes to reach high population densities, particularly at a lower temperature.     

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.     

First Report of Erwinia amylovora Fire Blight in Belarus

Erwinia amylovora is a phytopathogenic bacterium that causes fire blight, an economically important disease of Rosaceae . Several isolates from pears and apples with fire blight symptoms from Belarus were identified as E. amylovora . All tested isolates were yellow and mucoid on MM2Cu medium, positive in levan production and showed pathogenicity in immature pear fruits. These isolates have identical total protein patterns with E. amylovora 1/79. The PCR with specific primers for E. amylovora harpin gene also gave positive results.     

Role for tandem duplication and lon protease in AcrAB-TolC- dependent multiple antibiotic resistance (Mar) in an Escherichia coli mutant without mutations in marRAB or acrRAB

A spontaneous mutant (M113) of Escherichia coli AG100 with an unstable multiple antibiotic resistance (Mar) phenotype was isolated in the presence of tetracycline. Two mutations were found: an insertion in the promoter of lon (lon3::IS186) that occurred first and a subsequent large tandem duplication, dupIS186, bearing the genes acrAB and extending from the lon3::IS186 to another IS186 present 149 kb away from lon. The decreased amount of Lon protease increased the amount of MarA by stabilization of the basal quantities of MarA produced, which in turn increased the amount of multidrug effux pump AcrAB-TolC. However, in a mutant carrying only a lon mutation, the overproduced pump mediated little, if any, increased multidrug resistance, indicating that the Lon protease was required for the function of the pump. This requirement was only partial since resistance was mediated when amounts of AcrAB in a lon mutant were further increased by a second mutation. In M113, amplification of acrAB on the duplication led to increased amounts of AcrAB and multidrug resistance. Spontaneous gene duplication represents a new mechanism for mediating multidrug resistance in E. coli through AcrAB-TolC.     

DspA/E, a type III effector of Erwinia amylovora, is required for early rapid growth in Nicotiana benthamiana and causes NbSGT1-dependent cell death

DspA/E is a pathogenicity factor of Erwinia amylovora that is translocated into the plant cell cytoplasm through an Hrp type III secretion system. Transient expression of dspA/E in Nicotiana benthamiana or yeast induced cell death, as it does in N. tabacum and apple as described previously. DspA/E-induced cell death in N. benthamiana was not inhibited by coexpression of AvrPtoB of Pseudomonas syringae pv. tomato , which inhibits programmed cell death (PCD) induced by several other elicitors in plants. Silencing of NbSGT1 , the expression of which is required for PCD mediated by several resistance proteins of plants, prevented DspA/E-induced cell death in N. benthamiana. However, silencing of NbRAR1 , or two MAP kinase kinase genes, which are required for PCD associated with many resistance genes in plants, did not prevent cell death induced by DspA/E. Silencing of NbSGT1 also compromised non-host resistance against E. amylovora . E. amylovora grew rapidly within the first 24 h after infiltration in N. benthamiana , and DspA/E was required for this early rapid growth. However, bacterial cell numbers decreased after 24 h in TRV-vector-transformed plants, whereas a dspA/E mutant strain grew to high populations in NbSGT1 -silenced plants. Our results indicate that DspA/E enhances virulence of E. amylovora in N. benthamiana, but the bacteria are then recognized by the plant, resulting in PCD and death of bacterial cells or restriction of bacterial cell growth.     

The C-terminal half of the HrpN virulence protein of the fire blight pathogen Erwinia amylovora is essential for its secretion and for its virulence and avirulence activities

The HrpN (harpin) protein of the fire blight pathogen Erwinia amylovora is an essential virulence factor secreted via the bacterial type III secretion system. HrpN also has avirulence activity when delivered to tobacco by E. amylovora and has defense elicitor activity when applied to plants as a cell-free protein extract. Here, we characterize a series of random mutations in hrpN that altered the predicted amino acid sequence of the protein. Amino acid substitutions and deletions in the highly conserved, C-terminal portion of HrpN disrupted the virulence and avirulence activities of the protein. Several of these mutations produced a dominant-negative effect on E. amylovora avirulence on tobacco. None of the mutations clearly separated the virulence and avirulence activities of HrpN. Some C-terminal mutations abolished secretion of HrpN by E. amylovora. The results indicate that the C-terminal half of HrpN is essential for its secretion by E. amylovora, for its virulence activity on apple and pear, and for its avirulence activity on tobacco. In contrast, the C-terminal half of HrpN was not required for cell-free elicitor activity. This suggests that the N-terminal and C-terminal halves of HrpN mediate cell-free elicitor activity and avirulence activity, respectively.     

Two host-induced Ralstonia solanacearum genes, acrA and dinF, encode multidrug efflux pumps and contribute to bacterial wilt virulence

Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. We created mutations in the Ralstonia solanacearum acrA and dinF genes, which encode putative MDRs in the broad-host-range plant pathogen. Both mutations reduced the ability of R. solanacearum to grow in the presence of various toxic compounds, including antibiotics, phytoalexins, and detergents. Both acrAB and dinF mutants were significantly less virulent on the tomato plant than the wild-type strain. Complementation restored near-wild-type levels of virulence to both mutants. Addition of either dinF or acrAB to Escherichia coli MDR mutants KAM3 and KAM32 restored the resistance of these strains to several toxins, demonstrating that the R. solanacearum genes can function heterologously to complement known MDR mutations. Toxic and DNA-damaging compounds induced expression of acrA and dinF, as did growth in both susceptible and resistant tomato plants. Carbon limitation also increased expression of acrA and dinF, while the stress-related sigma factor RpoS was required at a high cell density (>10(7) CFU/ml) to obtain wild-type levels of acrA expression both in minimal medium and in planta. The type III secretion system regulator HrpB negatively regulated dinF expression in culture at high cell densities. Together, these results show that acrAB and dinF encode MDRs in R. solanacearum and that they contribute to the overall aggressiveness of this phytopathogen, probably by protecting the bacterium from the toxic effects of host antimicrobial compounds.     

Erwinia amylovora modifies phenolic profiles of susceptible and resistant apple through its type III secretion system

Fire blight is a disease affecting Maloideae caused by the necrogenic bacterium Erwinia amylovora , which requires the type III protein secretion system (TTSS) for pathogenicity. Profiles of methanol-extractable leaf phenolics of two apple ( Malus  ×  domestica ) genotypes with contrasting susceptibility to this disease were analyzed by HPLC after infection. Some qualitative differences were recorded between the constitutive compositions of the two genotypes but in both of them dihydrochalcones accounted for more than 90% of total phenolics. Principal component analysis separated leaves inoculated with a virulent wild-type strain from those inoculated with a non-pathogenic TTSS-defective mutant or with water. The changes in levels of the various groups of phenolics in response to the virulent bacterium were similar between the two genotypes, with a significant decrease of dihydrochalcones and a significant increase of hydroxycinnamate derivatives. Differences between genotypes were, however, recorded in amplitude and kinetic of variation in these groups. Occurrence of oxidation and polymerization reactions is proposed, based on the browning process of infected tissues, but whether some by-products act in defense as toxic compounds remain to be tested. Among direct antibacterial constitutive compounds present in apple leaves, the dihydrochalcone phloretin only was found at levels close to lethal concentrations in both genotypes. However, E.   amylovora exhibited the ability to stabilize this compound at sublethal levels even in the resistant apple, rejecting the hypothesis of its involvement in the resistance of this genotype.     

Identification of Erwinia amylovora genes induced during infection of immature pear tissue

The enterobacterium Erwinia amylovora is a devastating plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. In this study, we used a modified in vivo expression technology system to identify E. amylovora genes that are activated during infection of immature pear tissue, a process that requires the major pathogenicity factors of this organism. We identified 394 unique pear fruit-induced (pfi) genes on the basis of sequence similarity to known genes and separated them into nine putative function groups including host-microbe interactions (3.8%), stress response (5.3%), regulation (11.9%), cell surface (8.9%), transport (13.5%), mobile elements (1.0%), metabolism (20.3%), nutrient acquisition and synthesis (15.5%), and unknown or hypothetical proteins (19.8%). Known virulence genes, including hrp/hrc components of the type III secretion system, the major effector gene dspE, type II secretion, levansucrase (lsc), and regulators of levansucrase and amylovoran biosynthesis, were upregulated during pear tissue infection. Known virulence factors previously identified in E. (Pectobacterium) carotovora and Pseudomonas syringae were identified for the first time in E. amylovora and included HecA hemagglutinin family adhesion, Peh polygalacturonase, new effector HopPtoC(EA), and membrane-bound lytic murein transglycosylase MltE(EA). An insertional mutation within hopPtoC(EA) did not result in reduced virulence; however, an mltE(EA) knockout mutant was reduced in virulence and growth in immature pears. This study suggests that E. amylovora utilizes a variety of strategies during plant infection and to overcome the stressful and poor nutritional environment of its plant hosts.     

Characterization of hns genes from Erwinia amylovora

The small basic histone-like protein H-NS is known for bacteria to attenuate virulence of several animal pathogens. An hns homologue from E. amylovora was identified by complementing an E. coli hns-mutant strain with a cosmid library from E. amylovora. A 1.6 kb EcoRI-fragment complemented the mucoid phenotype and repressed the ss-glucosidase activity of E. coli PD32. The open reading frame encoding an H-NS-like protein of 134 amino acid was later shown to be located on plasmid pEA29 (McGhee and Jones 2000). A chromosomal hns gene was amplified with PCR consensus primers and localized near galU of E. amylovora. E. amylovora mutants were created by insertion of a resistance cassette, and the intact gene was inserted into a high copy number plasmid for constitutive expression. Purified chromosomal H-NS protein preferentially bound to a DNA fragment from the lsc region and bending was predicted for an adjacent fragment with the rlsB-promoter. Levan production was significantly increased by hns mutations. Synthesis of the capsular exopolysaccharide amylovoran and of levan were reduced, when hns from the E. amylovora plasmid was overexpressed. A mutation in chromosomal hns of E. amylovora increased amylovoran synthesis, and both mutations retarded symptom formation on immature pears.     

Penicillin-binding proteins from Erwinia amylovora: mutants lacking PBP2 are avirulent.

Radiolabelled penicillin G was used to examine penicillin-binding proteins (PBPs) from Erwinia amylovora (OT1). This procedure identified seven PBPs with molecular masses ranging from 22 to 83 kDa. E. amylovora PBPs were compared with those from Escherichia coli (JM101) and from two spherical, avirulent TnphoA mutants derived from OT1. Radiolabelled penicillin G bound to only six proteins from the spherical mutants which lacked a 69-kDa PBP. The spherical mutants could be complemented by the cloned E. coli pbpA-rodA operon, which restored both cell shape and virulence to apple seedlings. This suggested that the E. amylovora 69-kDa PBP is probably the functional equivalent of the E. coli PBP2 protein. Southern blot analysis using the E. coli rodA and pbpA genes as radiolabelled probes showed that TnphoA had inserted into the E. amylovora equivalent of the E. coli rodA-pbpA operon. Southern blots to chromosomal DNAs of the two spherical mutants, using the cloned hrp and dsp genes from E. amylovora as radiolabelled probes, confirmed that the TnphoA insertions were not located in the region of the E. amylovora chromosome postulated to encode known virulence factors. Both of the spherical TnphoA mutants synthesized amounts of extracellular polysaccharide equivalent to those synthesized by the wild-type strain (OT1), were resistant to lysis in distilled water and to lysozyme, and elicited the hypersensitive response on nonhost plants. These results indicate a possible role for cell shape in the virulence of this plant pathogen.     

The AcrAB RND efflux system from the live vaccine strain of Francisella tularensis is a multiple drug efflux system that is required for virulence in mice

The ability of bacterial pathogens to infect and cause disease is dependent upon their ability to resist antimicrobial components produced by their host, such as bile acids, fatty acids and other detergent-like molecules, and products of the innate immune system (e.g. cationic antimicrobial peptides). Bacterial resistance to the antimicrobial effects of such compounds is often mediated by active efflux systems belonging to the resistance-nodulation-division (RND) family of transporters. RND efflux systems have been implicated in antibiotic resistance and virulence extending their clinical relevance. In this report the hypothesis that the Francisella tularensis AcrAB RND efflux system contributes to antimicrobial resistance and pathogenesis has been tested. A null mutation was generated in the gene encoding the AcrB RND efflux pump protein of the live vaccine strain of F. tularensis. The resulting mutant exhibited increased sensitivity to multiple antibiotics and antimicrobial compounds. Murine challenge experiments revealed that the acrB mutant was attenuated. Collectively these results suggest that the F. tularensis AcrAB RND efflux system encodes a multiple drug efflux system that is important for virulence.     

Development and Properties of Streptomycin Resistant Cultures of Erwinia amylovora Derived from English Isolates

Resistance to 1000 p/m, streptomycin was developed in 3 out of 16 virulent strains of Erwinia amylovora by continuous subculturing on increasing concentrations of the drug. Resistance to various antibiotics, including streptomycin, was more readily developed in strains of Erwinia herbicola . Streptomycin resistance carried on an R factor was transferred by conjugation from Escherichia coli to E. amylovora and to E. herbicola . Resistance to streptomycin was associated with attenuation or, in one case, complete loss of virulence. Doubling times of resistant cultures were greater than those of the parent culture both in shaken broth culture and (with the two attenuated cultures) in apple seedlings. The avirulent culture appeared to persist longer in vivo in the presence of the virulent culture than alone.     

Visualization of harpin secretion in planta during infection of apple seedlings by Erwinia amylovora

Erwinia amylovora is a Gram-negative pathogenic bacterium that infects pear and apple trees as well as other plants from the Rosaceae family. E. amylovora pathogenicity is dependent on a functional Hrp type III secretion system. Harpin, a protein playing a major role in virulence, has been shown to be exported in vitro via the type III secretion apparatus. The data presented here focus on harpin detection in planta after infection of apple seedlings with the wild-type strain CFPB 1430. Using a specific harpin antiserum, harpin was not detected inside the host plant cells, but was found associated with the bacteria and secreted. The extracellular localization of harpin is in agreement with the physiological effects induced by purified harpin when applied as an exogenous elicitor. Harpin was not found associated with the host plant cell wall, a result that weakens its postulated role in cell wall loosening. A differential labelling was observed at the bacterial level: for some bacteria, harpin was exclusively cytoplasmic, whereas in others, it appeared as small patches over the bacterial outer membrane or associated with extracellular linear structures. All the bacteria present within the same area were similarly labelled, suggesting co-ordination in the secretion process. All observations suggest that harpin is synthesized in the bacterial cytoplasm and that secretion occurs from this cytoplasmic pool upon sensing of a plant or bacterial signal.     

Identification and Characterization of the Erwinia amylovora rpoS Gene: RpoS Is Not Involved in Induction of Fireblight Disease Symptoms

The Erwinia amylovora rpoS gene, encoding the alternative sigma factor RpoS, has been cloned and characterized. Though highly sensitive to a number of environmental stresses, an E. amylovora rpoS mutant was not compromised in its ability to grow or cause disease symptoms within apple seedlings or in an overwintering model.