Fire Blight Pathogen Erwinia Amylovora Research Articles

A Novel IncX Plasmid Mediates High-Level Oxytetracycline and Streptomycin Resistance in Erwinia amylovora from Commercial Pear Orchards in California.

Isolates of the fire blight pathogen Erwinia amylovora with high-level resistance to oxytetracycline (minimal inhibitory concentration [MIC] > 100 μg/ml) and to streptomycin (MIC > 100 μg/ml) were recovered from four commercial pear orchards in California between 2018 and 2020. The two representative oxytetracycline- and streptomycin-resistant (OxyTcR-SmR) strains 32-10 and 33-1 were as virulent as the antibiotic susceptible strain 13-1 in causing blossom blight of pear and were recovered more than 50% of the time 7 days after co-inoculation to pear flowers with strain 13-1. In the field, inoculation of strain 32-10 to pear flowers that were pretreated with oxytetracycline at 200 μg/ml did not reduce disease compared with an untreated control. Four OxyTcR-SmR strains were subjected to draft genome sequencing to identify the genetic determinants of antibiotic resistance and their location. A 43.6-kb IncX plasmid, designated pX11-7, was detected in each of the four strains, and this plasmid encoded the tetracycline-resistance gene tetB and the streptomycin-resistance gene pair strAB within a large putatively mobile genetic element consisting of the transposon Tn10 that had inserted within the streptomycin-resistance transposon Tn6082. We also determined that pX11-7 was conjugative and was transferred at a rate that was 104 to 105 higher into an E. amylovora strain isolated in California compared with an E. amylovora strain that was isolated in Michigan. The occurrence of high levels of resistance to both oxytetracycline and streptomycin in E. amylovora strains from commercial pear orchards in California significantly limits the options for blossom blight management in these locations.

Fire blight rootstock infections causing apple tree death: A case study in high-density apple orchards with Erwinia amylovora strain characterization

A devastating outcome of fire blight in apple trees is the infection of rootstock, which leads to canker development. Fire blight cankers are infected zones of dead bark on perennial branches, trunk, or rootstock that develop after fire blight pathogen Erwinia amylovora invades wood. Cankers can girdle the trunk, branches and rootstock leading to tree death and production losses, especially significant in high-density orchards. An accurate diagnosis of trunk and rootstock blight is a top challenge for apple growers because fire blight cankers can be visually misdiagnosed with cankers caused by less frequent Oomycete and fungal pathogens (e.g., Phytophthora spp., Botryosphaeria dothidea, B. obtusa, Valsa mali). In addition, detecting E. amylovora in apple rootstocks is essential because this pathogen also causes asymptomatic infections. Accurate fire blight diagnosis is necessary to inform the complete removal of infected trees from the orchard and help replanting efforts while preventing further pathogen dissemination. To determine apple tree losses caused by fire blight rootstock infections, PCR was used to detect E. amylovora in symptomatic and asymptomatic rootstocks for two years. Rootstock canker incidence and tree death were rated in selected infection foci on seven commercial apple orchards in New York. Each infection focus consisted of central rootstock-blighted tree and the nearest surrounding edge trees showing no rootstock blight. E. amylovora strains collected from these seven orchards and other orchards in New York were characterized. In the first year, most of the orchards showed E. amylovora detection rates of 10.7 – 45.3% in asymptomatic rootstocks on the edge trees immediately surrounding visibly infected i.e. rootstock-blighted central trees. One year later, 20.8 – 56.3% cankered rootstocks were detected on the edge trees and from zero to 35.4% dead edge trees were recorded. However, the PCR from sampled edge rootstocks one year later showed no pathogen detections. E. amylovora rootstock strains showed slight variability in enzymatic activity, copper sensitivity, virulence, and exopolysaccharide production. These results elucidate the role and importance of rootstock infections for apple tree survival, the presence of latent fire blight infections, and the valuable utility of molecular detection methods to assist apple tree removal after epidemics.

A Polyketide Synthetase Gene Cluster Is Responsible for Antibacterial Activity of Burkholderia contaminans MS14.

Burkholderia contaminans MS14, isolated from a soil sample in Mississippi, is known for producing the novel antifungal compound occidiofungin. In addition, MS14 exhibits a broad range of antibacterial activities against common plant pathogens. Random mutagenesis and gene complementation indicate that four genes are required for antibacterial activity of strain MS14 against the fire blight pathogen Erwinia amylovora. With the aim of finding the biosynthetic gene cluster for the unknown antibacterial compound, we used RNA-seq to analyze the transcriptome of MS14 wild type and mutants lacking antibacterial activity. The twofold lower expressed genes in all mutants were studied, and a polyketide synthase (PKS) gene cluster was predicted to be directly involved in MS14 antibacterial activities. The nptII-resistance cassette and CRISPR-Cas9 systems were used to mutate the PKS gene cluster. Plate bioassays showed that either insertion or frame-shifting one of the PKS genes resulted in a loss of antibacterial activity. Considering that the antibacterial-defective mutants maintain the same antifungal activities as the wild-type strain, the results suggest that this PKS gene cluster is highly likely to be involved in or directly responsible for the production of MS14 antibacterial activity. Purification efforts revealed that the antibacterial activity of the compound synthesized by the gene cluster is sensitive to UV radiation. Nevertheless, these findings have provided more insights to understand the antibacterial activity of strain MS14.

In-Orchard Population Dynamics of Erwinia amylovora on Apple Flower Stigmas.

Populations of the fire blight pathogen Erwinia amylovora Ea110 on apple flower stigmas were tracked over the course of apple bloom in field studies conducted between 2016 and 2019. In 18 of 23 experiments, flower stigmas inoculated on the first day of opening were found to harbor large (106 to 107 cells per flower) populations of E. amylovora when assessed 3 to 5 days postinoculation. However, populations inoculated on stigmas of flowers that were already open for 3 days did not reach 106 cells per flower, and populations inoculated on stigmas of flowers that were already open for 5 days never exceeded 104 cells per flower. During this study, ≥10-fold increases in E. amylovora stigma populations in a 24-h time period (termed population surges) were observed on 34.8, 20.0, and 4.0% of possible days on 1-, 3-, and 5-day-open flowers, respectively. Population surges occurred on days with average temperatures as high as 24.5 and as low as 6.1°C. Experiments incorporating more frequent sampling during days and overnight revealed that many population surges occurred between 10:00 p.m. and 2:00 a.m. A Pearson's correlation analysis of weather parameters occurring during surge events indicated that population surges were significantly associated with situations in which overnight temperatures increased or remained constant, in which wind speed decreased, and in which relative humidity increased. This study refines our knowledge of E. amylovora population dynamics and further indicates that E. amylovora is able to infect flowers during exposure to colder field temperatures than previously reported.

The power of unbiased phenotypic screens - cellulose as a first receptor for the Schitoviridae phage S6 of Erwinia amylovora.

SummaryBacteriophages, host‐dependent replicative non‐cellular entities which significantly shape the microbial genomes and consequently physiological and ecological properties of the microbial populations are exploited to restrict plant, animal and human pathogens. Unravelling of phage characteristics aids the understanding of the basic molecular mechanisms of phage infections which can subsequently lead to the development of rationalized strategies to combat microbial pathogens. In an unbiased screen to investigate the molecular basis of infectivity of the fire blight pathogen Erwinia amylovora by the lytic Schitoviridae phage S6, the biofilm extracellular matrix component cellulose has been identified as a cyclic di‐GMP dependent first receptor required for infection with the phage to possess beta‐1,4‐glucosidases to degrade the exopolysaccharide. This absolute receptor dependency allows maintenance of a phage‐microbe equilibrium with a low bacterial density.

An Erwinia amylovora inducible promoter for improvement of apple fire blight resistance

Key messagepPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes.Intragenesis is an important alternative to transgenesis to produce modified plants containing native DNA only. A key point to develop such a strategy is the availability of regulatory sequences controlling the expression of the gene of interest. With the aim of finding apple gene promoters either inducible by the fire blight pathogen Erwinia amylovora (Ea) or moderately constitutive, we focused on polyphenoloxidase genes (PPO). These genes encode oxidative enzymes involved in many physiological processes and have been previously shown to be upregulated during the Ea infection process. We found ten PPO and two PPO-like sequences in the apple genome and characterized the promoters of MdPPO16 (pPPO16) and MdKFDV02 PPO-like (pKFDV02) for their potential as Ea-inducible and low-constitutive regulatory sequences, respectively. Expression levels of reporter genes fused to these promoters and transiently or stably expressed in apple were quantified after various treatments. Unlike pKFDV02 which displayed a variable activity, pPPO16 allowed a fast and strong expression of transgenes in apple following Ea infection in a Type 3 Secretion System dependent manner. Altogether our results does not confirmed pKFDV02 as a constitutive and weak promoter whereas pPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes.

Multi-Omics Analysis Reveals the Dynamic Changes of RNA N 6 -Methyladenosine in Pear (Pyrus bretschneideri) Defense Responses to Erwinia amylovora Pathogen Infection.

N6-methylated adenine (m6A) is the most prevalent modification of mRNA methylation and can regulate many biological processes in plants, such as mRNA processing, development, and stress response. Some studies have increased our understanding of its various roles in model plants in recent years. Nevertheless, the distribution of m6A and the impact of m6A on the regulation of plant defense responses against pathogen inoculation are virtually unknown in pear. In this study, MeRIP-seq and RNA-seq data from healthy and inoculated plants were analyzed to assess the changes in the transcript levels and posttranscriptional modification of pear in response to the fire blight pathogen Erwinia amylovora. Following the analysis of 97,261 m6A peaks, we found that m6A preferred to modify duplicate genes rather than singleton genes and that m6A-methylated genes underwent stronger purifying selection. A total of 2,935 specific m6A sites were detected at the transcriptome level after inoculation, which may increase defense-related transcript abundance to enhance pear resistance. In addition, 1,850 transcripts were detected only in the mock-inoculated groups. The hypomethylated transcripts were mainly related to transcriptional regulation and various biological processes, such as chloroplast organization and sucrose biosynthetic processes. In addition, we found that the extent of m6A methylation was significantly positively correlated with the transcript level, suggesting a regulatory role for m6A in the plant response.

Effect of Kasugamycin, Oxytetracycline, and Streptomycin on In-orchard Population Dynamics of Erwinia amylovora on Apple Flower Stigmas.

We assessed the effect of three antibiotics (streptomycin, oxytetracycline, and kasugamycin) on populations of the fire blight pathogen Erwinia amylovora on apple flower stigmas during three field seasons. Application timing relative to E. amylovora presence on flower stigmas had little impact on population dynamics and subsequent disease incidence. Although E. amylovora populations on water-treated flowers increased to 106-7 cfu flower-1 after 4 to 5 days during each experiment, the antibiotics streptomycin and kasugamycin caused statistically significant reductions in stigma populations by as many as 4 to 5 logs over a 4- to 5-day period during two of the three experiments. In contrast, the effect of oxytetracycline on E. amylovora populations on stigmas was more variable, with reductions in E. amylovora populations only observed during one of the three experiments. In agreement with the population data, the disease incidence was significantly higher for oxytetracycline-treated flowers compared with the other antibiotic treatments during 2 of 3 years. Statistical analyses of the effects of weather parameters on antibiotic activity revealed that solar radiation and temperature negatively impacted the activity of both kasugamycin and oxytetracycline. We further assessed the potential for photodegradation of formulated kasugamycin (Kasumin 2L) and found that Kasumin 2L was susceptible to degradation in vitro after exposure to a 16-h photoperiod of daily light integrals (DLIs) varying from 6 to 35 mol⋅m-2⋅d-1. We further determined that exposure to three consecutive 16-h photoperiods of DLIs of 23 or 35 mol⋅m-2⋅d-1 reduced the available concentration of Kasumin 2L (assessed using a bioassay) from 100 μg⋅ml-1 to 10 to 20 μg⋅ml-1. Our results correlate the superior blossom blight control efficacy of kasugamycin and streptomycin with significant population reductions in E. amylovora on apple flower stigmas but indicate that, similar to oxytetracycline, kasugamycin is vulnerable to photodegradation, which would suggest that further considerations are necessary when applying this antibiotic.

Several New Putative Bacterial ADP-Ribosyltransferase Toxins Are Revealed from In Silico Data Mining, Including the Novel Toxin Vorin, Encoded by the Fire Blight Pathogen Erwinia amylovora.

Mono-ADP-ribosyltransferase (mART) toxins are secreted by several pathogenic bacteria that disrupt vital host cell processes in deadly diseases like cholera and whooping cough. In the last two decades, the discovery of mART toxins has helped uncover the mechanisms of disease employed by pathogens impacting agriculture, aquaculture, and human health. Due to the current abundance of mARTs in bacterial genomes, and an unprecedented availability of genomic sequence data, mART toxins are amenable to discovery using an in silico strategy involving a series of sequence pattern filters and structural predictions. In this work, a bioinformatics approach was used to discover six bacterial mART sequences, one of which was a functional mART toxin encoded by the plant pathogen, Erwinia amylovora, called Vorin. Using a yeast growth-deficiency assay, we show that wild-type Vorin inhibited yeast cell growth, while catalytic variants reversed the growth-defective phenotype. Quantitative mass spectrometry analysis revealed that Vorin may cause eukaryotic host cell death by suppressing the initiation of autophagic processes. The genomic neighbourhood of Vorin indicated that it is a Type-VI-secreted effector, and co-expression experiments showed that Vorin is neutralized by binding of a cognate immunity protein, VorinI. We demonstrate that Vorin may also act as an antibacterial effector, since bacterial expression of Vorin was not achieved in the absence of VorinI. Vorin is the newest member of the mART family; further characterization of the Vorin/VorinI complex may help refine inhibitor design for mART toxins from other deadly pathogens.

Genome-wide identification of the Sec-dependent secretory protease genes in Erwinia amylovora and analysis of their expression during infection of immature pear fruit.

The general secretory (Sec) pathway represents a common mechanism by which bacteria secrete proteins, including virulence factors, into the extracytoplasmic milieu. However, there is little information about this system, as well as its associated secretory proteins, in relation to the fire blight pathogen Erwinia amylovora. In this study, data mining revealed that E. amylovora harbors all of the essential components of the Sec system. Based on this information, we identified putative Sec-dependent secretory proteases in E. amylovora on a genome-wide scale. Using the programs SignalP, LipoP, and Phobius, a total of 15 putative proteases were predicted to contain the N-terminal signal peptides (SPs) that might link them to the Sec-dependent pathway. The activities of the predicted SPs were further validated using an Escherichia coli-based alkaline phosphatase (PhoA) gene fusion system that confirmed their extracytoplasmic property. Transcriptional analyses showed that the expression of 11 of the 15 extracytoplasmic protease genes increased significantly when E. amylovora was used to inoculate immature pears, suggesting their potential roles in plant infection. The results of this study support the suggestion that E. amylovora might employ the Sec system to secrete a suite of proteases to enable successful infection of plants, and shed new light on the interaction of E. amylovora with host plants.

Virulence Genetics of an Erwinia amylovora Putative Polysaccharide Transporter Family Member.

The Gram-negative enterobacterium Erwinia amylovora causes fire blight disease in apple and pear trees. Lipopolysaccharides and the exopolysaccharide amylovoran are essential E. amylovora virulence factors. We found that mutations in rfbX disrupted amylovoran production and virulence in apple fruits and tree shoots and that the deletion of yibD suppressed the rfbX mutant phenotype. The level of expression of yibD was about 10-fold higher in the ΔrfbX mutant than the wild type. A forward genetic suppressor screen in the ΔrfbX mutant uncovered multiple mutations in yibD and supported the conclusion that the virulence defect of rfbX mutants is due to reduced amylovoran production. The yibD and rfbX genes are expressed as a two-gene operon, yibD rfbX The rfbX gene encodes a previously uncharacterized putative polysaccharide subunit transporter, while yibD encodes a predicted glycosyltransferase. Mutation of rfbX did not have a detectable effect on lipopolysaccharide patterns; however, the overexpression of yibD in both the wild-type and ΔyibD ΔrfbX genetic backgrounds disrupted both amylovoran and lipopolysaccharide production. Additionally, the overexpression of yibD in the ΔyibD ΔrfbX mutant inhibited bacterial growth in amylovoran-inducing medium. This growth inhibition phenotype was used in a forward genetic suppressor screen and reverse-genetics tests to identify several genes involved in lipopolysaccharide production, which, when mutated, restored the ability of the ΔyibD ΔrfbX mutant overexpressing yibD to grow in amylovoran-inducing medium. Remarkably, all the lipopolysaccharide gene mutants tested were defective in lipopolysaccharide and amylovoran production. These results reveal a genetic connection between amylovoran and lipopolysaccharide production in E. amylovoraIMPORTANCE This study discovered previously unknown genetic connections between exopolysaccharide and lipopolysaccharide production in the fire blight pathogen Erwinia amylovora This represents a step forward in our understanding of the biology underlying the production of these two macromolecules. Fire blight is an economically important disease that impacts the production of apples and pears worldwide. Few fire blight control measures are available, and growers rely heavily on antibiotic applications at bloom time. Both exopolysaccharide and lipopolysaccharide are E. amylovora virulence factors. Our results indicate that the overexpression of the yibD gene in E. amylovora disrupts both lipopolysaccharide production and exopolysaccharide production. This effect could potentially be used as the basis for the development of an antivirulence treatment for the prevention of fire blight disease.

Effectors, chaperones, and harpins of the Type III secretion system in the fire blight pathogen Erwinia amylovora: a review

Erwinia amylovora is a plant pathogenic bacterium that causes the disease fire blight in a wide range of Rosaceous plants including apples and pears. This pathogen utilizes the hypersensitive response and pathogenicity (hrp)-type III secretion system (T3SS), an essential pathogenicity factor of E. amylovora, to deliver proteins from bacteria to plant apoplasts or the cytoplasm to regulate host immune responses and physiology. Proteins secreted by the E. amylovora T3SS include five effectors: DspA/E, Eop1, Eop3, Eop4 (AvrRpt2Ea), and HopPtoCEa, two harpins: HrpN and HrpW, and other proteins such as Eop2, HrpJ, and HrpK. Homologs of these T3SS-secreted proteins have been characterized in plant pathogens like Pseudomonas syringae, Xanthomonas spp., Ralstonia spp., plant symbionts like Rhizobium spp. and animal pathogens such as Yersinia spp., implicating a central role of these effector proteins in manipulating bacterial interactions with diverse host species. Translocation of effectors is crucial for T3SS-mediated disease development in hosts. It is thought to be mediated by cognate chaperones, but the translocation of E. amylovora DspA/E was recently shown to be regulated by several different chaperones as well as a previously reported harpin, HrpN, in addition to its cognate chaperone DspB/F. This suggests that the dynamics of effector translocation are more complicated than previously expected in E. amylovora. In this review, we summarize the current knowledge of E. amylovora T3SS effectors, chaperones, and harpins together with their homologs from other plant-associated bacterial species. We also discuss the roles of T3SS-secreted proteins in pathogenicity and fitness via exploring their localization, activity, and direct or indirect targets in host plants. Finally, we provide future perspectives on studying effector biology and the T3SS in E. amylovora and how this fundamental knowledge provides potential applications in preventing fire blight.

The stringent response regulator (p) ppGpp mediates virulence gene expression and survival in Erwinia amylovora

BackgroundThe nucleotide second messengers, i.e., guanosine tetraphosphate and pentaphosphate [collectively referred to as (p) ppGpp], trigger the stringent response under nutrient starvation conditions and play an essential role in virulence in the fire blight pathogen Erwinia amylovora. Here, we present transcriptomic analyses to uncover the overall effect of (p) ppGpp-mediated stringent response in E. amylovora in the hrp-inducing minimal medium (HMM).ResultsIn this study, we investigated the transcriptomic changes of the (p) ppGpp0 mutant under the type III secretion system (T3SS)-inducing condition using RNA-seq. A total of 1314 differentially expressed genes (DEGs) was uncovered, representing more than one third (36.8%) of all genes in the E. amylovora genome. Compared to the wild-type, the (p) ppGpp0 mutant showed down-regulation of genes involved in peptide ATP-binding cassette (ABC) transporters and virulence-related processes, including type III secretion system (T3SS), biofilm, and motility. Interestingly, in contrast to previous reports, the (p) ppGpp0 mutant showed up-regulation of amino acid biosynthesis genes, suggesting that it might be due to that these amino acid biosynthesis genes are indirectly regulated by (p) ppGpp in E. amylovora or represent specific culturing condition used. Furthermore, the (p) ppGpp0 mutant exhibited up-regulation of genes involved in translation, SOS response, DNA replication, chromosome segregation, as well as biosynthesis of nucleotide, fatty acid and lipid.ConclusionThese findings suggested that in HMM environment, E. amylovora might use (p) ppGpp as a signal to activate virulence gene expression, and simultaneously mediate the balance between virulence and survival by negatively regulating DNA replication, translation, cell division, as well as biosynthesis of nucleotide, amino acid, fatty acid, and lipid. Therefore, (p) ppGpp could be a promising target for developing novel control measures to fight against this devastating disease of apples and pears.

Resistance to Two Vinylglycine Antibiotic Analogs Is Conferred by Inactivation of Two Separate Amino Acid Transporters in Erwinia amylovora.

Erwinia amylovora is the causal agent of fire blight of apple and pear trees. Several bacteria have been shown to produce antibiotics that antagonize E. amylovora, including pantocins, herbicolins, dapdiamides, and the vinylglycines, 4-formylaminooxyvinylglycine (FVG) and 4-aminoethoxyvinylglycine (AVG). Pantoea ananatis BRT175 was previously shown to exhibit antibiotic activity against E. amylovora via the production of Pantoea natural product 1 (PNP-1), later shown to be FVG; however, exposure of E. amylovora to FVG results in spontaneously resistant mutants. To identify the mechanism of resistance, we used genome variant analysis on spontaneous FVG-resistant mutants of E. amylovora and identified null mutations in the l-asparagine permease gene ansP Heterologous expression of ansP in normally resistant Escherichia coli was sufficient to impart FVG susceptibility, suggesting that FVG is imported through this permease. Because FVG and AVG are structurally similar, we hypothesized that resistance to AVG would also be conferred through inactivation of ansP; however, ansP mutants were not resistant to AVG. We found that spontaneously resistant Ea321 mutants also arise in the presence of AVG, with whole-genome variant analysis revealing that resistance was due to inactivation of the arginine ABC transporter permease subunit gene artQ Heterologous expression of the predicted lysE-like transporter encoded within the Pantoea ananatis BRT175 FVG biosynthetic cluster, which is likely responsible for antibiotic export, was sufficient to confer resistance to both FVG and AVG. This work highlights the important roles of amino acid transporters in antibiotic import into bacteria and the potential utility of antimicrobial amino acid analogs as antibiotics.IMPORTANCE The related antibiotics formylaminooxyvinylglycine (FVG) and aminoethoxyvinylglycine (AVG) have been shown to have activity against the fire blight pathogen Erwinia amylovora; however, E. amylovora can develop spontaneous resistance to these antibiotics. By comparing the genomes of mutants to those of the wild type, we found that inactivation of the l-asparagine transporter conferred resistance to FVG, while inactivation of the l-arginine transporter conferred resistance to AVG. We also show that the transporter encoded by the FVG biosynthetic cluster can confer resistance to both FVG and AVG. Our work indicates the important role that amino acid transporters play in the import of antibiotics and highlights the possible utility in designer antibiotics that enter the bacterial cell through amino acid transporters.

A complete structural characterization of the desferrioxamine E biosynthetic pathway from the fire blight pathogen Erwinia amylovora.

The Gram-negative bacterium Erwinia amylovora is the etiological agent of fire blight, a devastating disease which affects Rosaceae such as apple, pear and quince. The siderophore desferrioxamine E plays an important role in bacterial pathogenesis by scavenging iron from the host. DfoJ, DfoA and DfoC are the enzymes responsible for desferrioxamine production starting from lysine. We have determined the crystal structures of each enzyme in the desferrioxamine E pathway and demonstrate that the biosynthesis involves the concerted action of DfoJ, followed by DfoA and lastly DfoC. These data provide the first crystal structures of a Group II pyridoxal-dependent lysine decarboxylase, a cadaverine monooxygenase and a desferrioxamine synthetase. DfoJ is a homodimer made up of three domains. Each monomer contributes to the completion of the active site, which is positioned at the dimer interface. DfoA is the first structure of a cadaverine monooxygenase. It forms homotetramers whose subunits are built by two domains: one for FAD and one for NADP+ binding, the latter of which is formed by two subdomains. We propose a model for substrate binding and the role of residues 43-47 as gate keepers for FAD binding and the role of Arg97 in cofactors turnover. DfoC is the first structure of a desferrioxamine synthetase and the first of a multi-enzyme siderophore synthetase coupling an acyltransferase domain with a Non-Ribosomal Peptide Synthetase (NRPS)-Independent Siderophore domain (NIS).

The virulence-associated protein HsvA from the fire blight pathogen Erwinia amylovora is a polyamine amidinotransferase

Studies of virulence determinants in the bacterial phytopathogen Erwinia amylovora, the cause of devastating fire blight disease in apple and pear, have shown that HsvA, a putative amidinotransferase enzyme located in the Hrp pathogenicity island, is required for systemic infection in apple. However, the mechanism by which HsvA contributes to virulence is unclear. To investigate the role of HsvA in virulence, we carried out a series of biochemical and structural studies to characterize the amidinotransferase activity of HsvA. We found that HsvA displays a preference for linear aliphatic polyamines as the amidino acceptor substrate, especially for spermidine and putrescine (Km values of 33 μm and 3.9 mm, respectively). The three-dimensional structure, determined at 2.30 Å resolution using X-ray crystallography, revealed that the overall architecture of HsvA is similar to that of the human arginine-glycine amidinotransferase in the creatine biosynthesis pathway. The active site is located in the core of the protein at the base of a long, narrow substrate access channel. Specific amino acids near the entrance of the channel may serve as major determinants of the substrate specificity, including a glutamate residue at the rim of the channel entrance that appears to be positioned to interact with the distal primary amine in the putrescine substrate as well as the internal and distal amines in the spermidine substrate. These results suggest potential in vivo functions for HsvA as a virulence factor in fire blight and may also provide a basis for strategies to control fire blight by inhibiting HsvA activity.

Exploration of Using Antisense Peptide Nucleic Acid (PNA)-cell Penetrating Peptide (CPP) as a Novel Bactericide against Fire Blight Pathogen Erwinia amylovora

Erwinia amylovora is a Gram-negative bacterial plant pathogen in the family Enterobacteriaceae and is the causal agent of fire blight, a devastating disease of apple and pear. Fire blight is traditionally managed by the application of the antibiotic streptomycin during bloom, but this strategy has been challenged by the development and spread of streptomycin resistance. Thus, there is an urgent need for effective, specific, and sustainable control alternatives for fire blight. Antisense antimicrobials are oligomers of nucleic acid homologs with antisense sequence of essential genes in bacteria. The binding of these molecules to the mRNA of essential genes can result in translational repression and antimicrobial effect. Here, we explored the possibility of developing antisense antimicrobials against E. amylovora and using these compounds in fire blight control. We determined that a 10-nucleotide oligomer of peptide nucleic acid (PNA) targeting the start codon region of an essential gene acpP is able to cause complete growth inhibition of E. amylovora. We found that conjugation of cell penetrating peptide (CPP) to PNA is essential for the antimicrobial effect, with CPP1 [(KFF)3K] being the most effective against E. amylovora. The minimal inhibitory concentration (MIC) of anti-acpP-CPP1 (2.5 μM) is comparable to the MIC of streptomycin (2 μM). Examination of the antimicrobial mechanisms demonstrated that anti-acpP-CPP1 caused dose-dependent reduction of acpP mRNA in E. amylovora upon treatment and resulted in cell death (bactericidal effect). Anti-acpP-CPP1 (100 μM) is able to effectively limit the pathogen growth on stigmas of apple flowers, although less effective than streptomycin. Finally, unlike streptomycin that does not display any specificity in inhibiting pathogen growth, anti-acpP-CPP1 has more specific antimicrobial effect against E. amylovora. In summary, we demonstrated that PNA–CPP can cause an effective, specific antimicrobial effect against E. amylovora and may provide the basis for a novel approach for fire blight control.

Development of an enzyme immunoassay diagnostic test for detection of fire blight pathogen Erwinia amylovora

Development of an enzyme immunoassay diagnostic test for detection of fire blight pathogen Erwinia amylovora

HrcU and HrpP are pathogenicity factors in the fire blight pathogen Erwinia amylovora required for the type III secretion of DspA/E.

BackgroundMany Gram-negative bacterial pathogens mediate host-microbe interactions via utilization of the type III secretion (T3S) system. The T3S system is a complex molecular machine consisting of more than 20 proteins. Collectively, these proteins translocate effectors across extracellular space and into the host cytoplasm. Successful translocation requires timely synthesis and allocation of both structural and secreted T3S proteins. Based on amino acid conservation in animal pathogenic bacteria, HrcU and HrpP were examined for their roles in regulation of T3S hierarchy.ResultsBoth HrcU and HrpP were shown to be required for disease development in an immature pear infection model and respective mutants were unable to induce a hypersensitive response in tobacco. Using in vitro western blot analyses, both proteins were also shown to be required for the secretion of DspA/E, a type 3 effector and an important pathogenicity factor. Via yeast-two hybridization (Y2H), HrpP and HrcU were revealed to exhibit protein-protein binding. Finally, all HrcU and HrpP phenotypes identified were shown to be dependent on a conserved amino acid motif in the cytoplasmic tail of HrcU.ConclusionsCollectively, these data demonstrate roles for HrcU and HrpP in regulating T3S and represent the first attempt in understanding T3S heirarchy in E. amylovora.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0702-y) contains supplementary material, which is available to authorized users.

Fire blight disease reactome: RNA-seq transcriptional profile of apple host plant defense responses to Erwinia amylovora pathogen infection.

The molecular basis of resistance and susceptibility of host plants to fire blight, a major disease threat to pome fruit production globally, is largely unknown. RNA-sequencing data from challenged and mock-inoculated flowers were analyzed to assess the susceptible response of apple to the fire blight pathogen Erwinia amylovora. In presence of the pathogen 1,080 transcripts were differentially expressed at 48 h post inoculation. These included putative disease resistance, stress, pathogen related, general metabolic, and phytohormone related genes. Reads, mapped to regions on the apple genome where no genes were assigned, were used to identify potential novel genes and open reading frames. To identify transcripts specifically expressed in response to E. amylovora, RT-PCRs were conducted and compared to the expression patterns of the fire blight biocontrol agent Pantoea vagans strain C9-1, another apple pathogen Pseudomonas syringae pv. papulans, and mock inoculated apple flowers. This led to the identification of a peroxidase superfamily gene that was lower expressed in response to E. amylovora suggesting a potential role in the susceptibility response. Overall, this study provides the first transcriptional profile by RNA-seq of the host plant during fire blight disease and insights into the response of susceptible apple plants to E. amylovora.