Fire Blight Pathogen Erwinia Amylovora Research Articles

Expression profiles of differentially regulated genes during the early stages of apple flower infection with Erwinia amylovora.

To identify genes involved in the response to the fire blight pathogen Erwinia amylovora in apple (Malus×domestica), expression profiles were investigated using an apple oligo (70-mer) array representing 40, 000 genes. Blossoms of a fire blight-susceptible apple cultivar Gala were collected from trees growing in the orchard, placed on a tray in the laboratory, and spray-inoculated with a suspension of E. amylovora at a concentration of 108 cfu ml−1. Uninoculated detached flowers served as controls at each time point. Expression profiles were captured at three different time points post-inoculation at 2, 8, and 24 h, together with those at 0 h (uninoculated). A total of about 3500 genes were found to be significantly modulated in response to at least one of the three time points. Among those, a total of 770, 855, and 1002 genes were up-regulated, by 2-fold, at 2, 8, and 24 h following inoculation, respectively; while, 748, 1024, and 1455 genes were down-regulated, by 2-fold, at 2, 8, and 24 h following inoculation, respectively. Over the three time points post-inoculation, 365 genes were commonly up-regulated and 374 genes were commonly down-regulated. Both sets of genes were classified based on their functional categories. The majority of up-regulated genes were involved in metabolism, signal transduction, signalling, transport, and stress response. A number of transcripts encoding proteins/enzymes known to be up-regulated under particular biotic and abiotic stress were also up-regulated following E. amylovora treatment. Those up- or down-regulated genes encode transcription factors, signaling components, defense-related, transporter, and metabolism, all of which have been associated with disease responses in Arabidopsis and rice, suggesting similar response pathways are involved in apple blossoms.

Diversity, evolution, and functionality of clustered regularly interspaced short palindromic repeat (CRISPR) regions in the fire blight pathogen Erwinia amylovora.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system confers acquired heritable immunity against mobile nucleic acid elements in prokaryotes, limiting phage infection and horizontal gene transfer of plasmids. In CRISPR arrays, characteristic repeats are interspersed with similarly sized nonrepetitive spacers derived from transmissible genetic elements and acquired when the cell is challenged with foreign DNA. New spacers are added sequentially and the number and type of CRISPR units can differ among strains, providing a record of phage/plasmid exposure within a species and giving a valuable typing tool. The aim of this work was to investigate CRISPR diversity in the highly homogeneous species Erwinia amylovora, the causal agent of fire blight. A total of 18 CRISPR genotypes were defined within a collection of 37 cosmopolitan strains. Strains from Spiraeoideae plants clustered in three major groups: groups II and III were composed exclusively of bacteria originating from the United States, whereas group I generally contained strains of more recent dissemination obtained in Europe, New Zealand, and the Middle East. Strains from Rosoideae and Indian hawthorn (Rhaphiolepis indica) clustered separately and displayed a higher intrinsic diversity than that of isolates from Spiraeoideae plants. Reciprocal exclusion was generally observed between plasmid content and cognate spacer sequences, supporting the role of the CRISPR/Cas system in protecting against foreign DNA elements. However, in several group III strains, retention of plasmid pEU30 is inconsistent with a functional CRISPR/Cas system.

Genetic Analysis of Streptomycin-Resistant (SmR) Strains of Erwinia amylovora Suggests that Dissemination of Two Genotypes Is Responsible for the Current Distribution of SmR E. amylovora in Michigan

Streptomycin-resistant (Sm(R)) strains of the fire blight pathogen Erwinia amylovora were first isolated in southwest Michigan in 1991. Since that time, resistant strains have progressed northward to other apple-producing regions in the state. A total of 98.7% of Sm(R) strains isolated between 2003 and 2009 in Michigan harbored the strA-strB genes on transposon Tn5393. strA and strB encode phosphotransferase enzymes that modify streptomycin to a nonbactericidal form. Mutational resistance to streptomycin, caused by a point mutation-mediated target-site alteration of the ribosomal S12 protein, occurred in 1.3% of E. amylovora strains from Michigan. Tn5393 was originally introduced to E. amylovora on the plasmid pEa34; thus, the first Sm(R) strains isolated contained both pEa34 and the ubiquitous nonconjugative plasmid pEA29. More recently, we have observed Sm(R) strains in which Tn5393 is present on pEA29, suggesting that the transposon has moved via transposition from pEa34 to pEA29. Almost all of the strains containing Tn5393 on pEA29 had lost pEa34. Of 210 pEA29::Tn5393 plasmids examined, the transposon was inserted at either nucleotide position 1,515 or 17,527. Both of these positions were in noncoding regions of pEA29. Comparative sequencing of the housekeeping genes groEL and potentially variable sequences on pEA29 was done in an attempt to genetically distinguish Sm(R) strains from streptomycin-sensitive (Sm(S)) strains isolated in Michigan. Only 1 nucleotide difference within the total 2,660 bp sequenced from each strain was observed in 2 of 29 strains; multiple sequence differences were observed between the Michigan strains and E. amylovora control strains isolated in the western United States or from Rubus spp. Alterations in virulence observable using an immature pear fruit assay were detected in three of eight Sm(R) strains examined. Our current genetic data indicate that only two Sm(R) strain genotypes (strains containing pEA29::Tn5393 with Tn5393 inserted at either nucleotide position 1,515 or 17,527 on the plasmid) are responsible for the dissemination of Tn5393-encoded streptomycin resistance in Michigan, and that the Sm(R) and Sm(S) strains in Michigan compose a homogenous group.

Effect oftrans-resveratrol and ascorbigens on the fire blight pathogenErwinia amylovorain the BioArena system

On the basis of our earlier observations, preliminary experiments were conducted with the fire blight pathogen Erwinia amylovora with the BioArena system using the potential antibacterial substances 1′-methylascorbigen (MeAG) as formaldehyde (HCHO) precursor and trans-resveratrol (RES) as HCHO mobilizer, capturer, and scavenger. Results showed characteristic, strong, dose-dependent antibacterial effects of both MeAG and RES present as chromatographic spots on the adsorbent layer. It is supposed that the weak antibacterial effect of AG may have originated from MeAG formed in situ on the layer by partial enzymatic methylation of AG. Addition of HCHO capture compounds (l-arginine, glutathione, glucosamine) to the culture medium before inoculation partially or totally reduced the antibacterial effect of both molecules, that is, this simplest aliphatic aldehyde, the endogenous HCHO molecule, participates in the antibacterial activity of these compounds. It follows from these results that there is a possibility...

The outer membrane protein TolC is required for phytoalexin resistance and virulence of the fire blight pathogen Erwinia amylovora.

SummaryErwinia amylovora causes fire blight on several plant species such as apple and pear, which produce diverse phytoalexins as defence mechanisms. An evolutionary successful pathogen thus must develop resistance mechanisms towards these toxic compounds. The E. amylovora outer membrane protein, TolC, might mediate phytoalexin resistance through its interaction with the multidrug efflux pump, AcrAB. To prove this, a tolC mutant and an acrB/tolC double mutant were constructed. The minimal inhibitory concentrations of diverse antimicrobials and phytoalexins were determined for these mutants and compared with that of a previously generated acrB mutant. The tolC and arcB/tolC mutants were considerably more susceptible than the wild type but showed similar levels as the acrB mutant. The results clearly indicated that neither TolC nor AcrAB significantly interacted with other transport systems during the efflux of the tested toxic compounds. Survival and virulence assays on inoculated apple plants showed that pathogenicity and the ability of E. amylovora to colonize plant tissue were equally impaired by mutations of tolC and acrB/tolC. Our results allowed the conclusion that TolC plays an important role as a virulence and fitness factor of E. amylovora by mediating resistance towards phytoalexins through its exclusive interaction with AcrAB.

The C-Terminal Half of the HrpN Virulence Protein of the Fire Blight PathogenErwinia amylovoraIs 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.

AtHIPM, an Ortholog of the Apple HrpN-Interacting Protein, Is a Negative Regulator of Plant Growth and Mediates the Growth-Enhancing Effect of HrpN in Arabidopsis

HrpN (harpin) protein is critical to the virulence of the fire blight pathogen Erwinia amylovora in host plants like apple (Malus x domestica). Moreover, exogenous treatment of Arabidopsis (Arabidopsis thaliana), a nonhost plant, with partially purified HrpN enhances growth. To address the bases of the effects of HrpN in disease, we sought a HrpN-interacting protein(s) in apple, using a yeast two-hybrid assay. A single positive clone, designated HIPM (HrpN-interacting protein from Malus), was found. HIPM, a 6.5-kD protein, interacted with HrpN in yeast and in vitro. Deletion analysis showed that the N-terminal 198 of 403 amino acids of HrpN are required for interaction with HIPM. HIPM orthologs were found in Arabidopsis (AtHIPM) and rice (Oryza sativa; OsHIPM). HrpN also interacted with AtHIPM in yeast and in vitro. In silico analyses revealed that the three plant proteins contain putative signal peptides and putative transmembrane domains. We showed that both HIPM and AtHIPM have functional signal peptides, and green fluorescent protein-tagged HIPM and AtHIPM associated, in clusters, with plasma membranes. Both HIPM and AtHIPM are expressed constitutively; however, they are expressed more strongly in apple and Arabidopsis flowers than in leaves and stems. The size of AtHIPM knockout mutant plants of Arabidopsis was slightly larger than the wild-type plants. Interestingly, the knockout mutant did not exhibit enhanced plant growth in response to treatment with HrpN. Overexpression of AtHIPM conversely resulted in smaller plants. These results indicate that AtHIPM functions as a negative regulator of plant growth and mediates enhanced growth that results from treatment with HrpN.

Identification of low-temperature-regulated genes in the fire blight pathogenErwinia 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.

Survival and Possible Spread of Erwinia amylovora and Related Plant-Pathogenic Bacteria Exposed to Environmental Stress Conditions

The fire blight pathogen Erwinia amylovora was assayed for survival under unfavourable conditions such as on nitrocellulose filters, in non-host plants as well as in inoculated mature apples and in infested apple stem sections. In a sterile dry environment, an E. amylovora EPS (exopolysaccharide) mutant, and to a lesser extent its parental wild-type strain decreased within 3 weeks to a low titre. However, under moist conditions the decrease of viable cells occurred only partially for both strains. Very low cell titres were recovered after application of E. amylovora onto the surface of tobacco leaves, whereas infiltration into the leaves produced lesions (hypersensitive response, HR), in which the bacteria survived in significant amounts. A similar effect was found for the necrotic zones of HR in tobacco leaves caused by E. pyrifoliae, by Pseudomonas syringae pathovars and HR-deficient E. amylovora mutants or mutants deficient in EPS synthesis and disease-specific genes. During 7 years of storage, the viability of E. amylovora in wood sections from fire blight-infested apple trees declined to a low titre. In tissue of mature apples, E. amylovora cells slowly dispersed and could still be recovered after several weeks of storage at room temperature. A minimal risk of accidental dissemination of E. amylovora apart from infested host plants can experimentally not be excluded, but other data confirm a very low incidence of any long distance distribution.

Note: Effectiveness of some environmentally friendly chemicals against the pear psyllaCacopsylla pyricola

The effectiveness of Vivere + Vivo phyto, AVA + vinegar, AVA + urea, Vivere + Vivo urticae, calcium oxide, and Surround WP against pear psyllaCacopsylla pyricola (Foerster) was evaluated by recording the number of eggs, the number of nymphs, and the amount of honeydew produced by the psylla. Very promising results were obtained with all chemicals used. The effectiveness against psylla of the AVA + urea combination was the best and similar to that achieved with conventional chemical treatments. Moreover, trees treated with the above chemicals showed a lower percentage of natural infection by the fire blight pathogenErwinia amylovora than the untreated control trees.

Instability of short-sequence DNA repeats of pear pathogenic Erwinia strains from Japan and Erwinia amylovora fruit tree and raspberry strains

An array of short-sequence DNA repeats (SSRs) occurs in the plasmid pEA29 of the fire blight pathogen Erwinia amylovora. A large number of "fruit tree" strains, mainly from Central and Western Europe, were screened for their SSR numbers, and the analyses were extended to five raspberry strains from North America and six pear pathogenic Erwinia strains from Japan. The repeat ATTACAGA present in all E. amylovorastrains was found to be reiterated 3 to 15 times. The Japanese strains contained the major repeat sequence GGATTCTG, which was reiterated 16 to 24 times. ATTACAGG, which resembles the SSR of E. amylovora, was reiterated two or three times. In a novel approach, sequencing gels were used to visualize the rare occurrence of shorter arrays (down to three repeats) in E. amylovoraand the Japanese Erwinia strains. Changes in the repeat numbers in E. amylovora were observed repeatedly when the bacteria had been exposed to stress conditions. The repeat structures of homo- and heteroduplices of PCR-amplified repeats were also analyzed by cleavage of annealed molecules with the single-strand-specific endonuclease from bacteriophage T4. Not only heteroduplexes, but also homoduplexes showed non-matching regions in the SSRs, which could arise from transient formation of loops due to strand slippage during the assays.

REGULATION AND BIOCHEMISTRY OF EXOPOLYSACCHARIDE SYNTHESIS OF THE FIRE BLIGHT PATHOGEN ERWINIA AMYLOVORA AND PEAR PATHOGENS FROM KOREA AND JAPAN

The fire blight pathogen Erwinia amylovora and the Asian pear pathogens synthesize similar capsular exopolysaccharides (EPS), which are strictly required for pathogenicity. The molecular weight of the EPS is at least 1 MDa. The repeating units of EPS synthesized by Erwinia pyrifoliae from Korea and the Erwinia strains from Japan do not bear a glucose residue as a second side chain found in 60% of the repeating units of amylovoran. Levan, a homopolymer of fructose synthesized by the secreted enzyme levansucrase of E. amylovora, is not produced by the Asian pear pathogens. Amylovoran and levan expression are controlled by regulatory genes and affected differently by the phosphate and chloride concentrations of the environment. Alignments of rcsB genes, and also of the nucleotide sequences upstream of the gene cluster for EPS synthesis, show a significant relationship between the pear pathogens and an evolutionary distance to E. amylovora. Three activator proteins for expression of levansucrase have been identified; RlsA, RlsB, and RlsC. The H-NS protein binds to a DNA fragment with the start of lsc. From a bacteriophage, a DNA fragment was sequenced with genes encoding a lysozyme and an EPS depolymerase. The enzyme was used to degrade amylovoran capsules and shown to interfere with colonization of pear slices by E. amylovora.

Characterization of an activator gene upstream of lsc, involved in levan synthesis of Erwinia amylovora

In addition to activation by rlsA, a second gene for positive regulation of levansucrase expression of the fire blight pathogen Erwinia amylovora has been detected. The gene rlsB was cloned from a genomic library by suppression of a levan-deficient strain and is located adjacent to the start of lsc in the opposite orientation. The gene rlsB presumably encodes a 6.9kDa basic protein and is preceded by a potential promoter and a ribosome binding site. RlsB belongs to the phage P2 Ogr protein superfamily, which includes other transcriptional activators containing the zinc-finger CCCC motif. An rlsB mutant was created from the wild-type strain Ea1/79 by gene disruption, which was reduced in levan synthesis and was complemented by the intact rlsB gene. Expression of rlsB also restored extracellular levan synthesis of several natural levan-deficient E. amylovora strains, but did not affect amylovoran synthesis. The lsc promoter inserted in an intermediate-copy number plasmid diminished levan synthesis, which could be restored by high expression of rlsB. PCR primers, deduced from rlsB, produced a specific DNA fragment only with E. amylovora strains. The genes rlsB and lsc of E. amylovora were not detected in the chromosome of Escherichia coli, but the genes at both borders localized in the complete genomic map of E. coli. It can be assumed that the lsc -gene cluster was introduced into the fire blight pathogen at a late stage of evolution.

Molecular Detection and Differentiation of Erwinia pyrifoliae and Host Range Analysis of the Asian Pear Pathogen.

The recently described pathogen Erwinia pyrifoliae, isolated from Nashi pear fruit trees in Korea, resembles the fire blight pathogen Erwinia amylovora in some of its properties. The two pathogens were classified into different species by DNA hybridization kinetics and microbiological criteria. From the nucleotide sequences of the 16S rRNA and the internal transcribed spacer (ITS) region as well as extracellular polysaccharide (EPS)-encoding genes, polymerase chain reaction (PCR) primers were designed that specifically detect E. pyrifoliae but not the fire blight pathogen Erwinia amylovora, and these primers were also applied to identify E. pyrifoliae in necrotic plant material. The genomes of several strains were digested with the restriction enzyme SpeI, and the DNA fragments were analyzed by pulsed-field gel electrophoresis (PFGE). Three groups of patterns could be distinguished for the isolated E. pyrifoliae strains, all different from various E. amylovora strains, which produce a relatively homogeneous PFGE pattern after SpeI digests. Typical fire blight host plants were assayed in a growth chamber or an experimental field for their susceptibility to E. pyrifoliae. A strong preference was found for pear varieties, whereas apple, cotoneaster, hawthorn, or raspberry rarely produced necrotic symptoms. E. pyrifoliae was readily detected in samples from pear orchards in South Korea during 1995 to 1998; however, the Asian pear pathogen was not recovered in necrotic plant tissue from 1999 and 2000.

Occurrence and Spread of Fire Blight Pathogen (Erwinia amylovora) on Spontaneous and Ornamental Plants in Yugoslavia

During a ten-year-investigation on the occurrence and spread of fire blight (Erwinia amylovora) pathogen on spontaneous and ornamental plants in Yugoslavia, the pathogen was found only on hawthorn, wild pear and firethorn. No bacterium was isolated from others species among these two groups of hosts.

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.

Molecular Analysis of Sucrose Metabolism of Erwinia amylovora and Influence on Bacterial Virulence

Sucrose is an important storage and transport sugar of plants and an energy source for many phytopathogenic bacteria. To analyze regulation and biochemistry of sucrose metabolism of the fire blight pathogen Erwinia amylovora, a chromosomal fragment which enabled Escherichia coli to utilize sucrose as sole carbon source was cloned. By transposon mutagenesis, the scr regulon of E. amylovora was tagged, and its nucleotide sequence was determined. Five open reading frames, with the genes scrK, scrY, scrA, scrB, and scrR, had high homology to genes of the scr regulons from Klebsiella pneumoniae and plasmid pUR400. scrB and scrR of E. amylovora were fused to a histidine tag and to the maltose-binding protein (MalE) of E. coli, respectively. ScrB (53 kDa) catalyzed the hydrolysis of sucrose with a K(m) of 125 mM. Binding of a MalE-ScrR fusion protein to an scrYAB promoter fragment was shown by gel mobility shifts. This complex dissociated in the presence of fructose but not after addition of sucrose. Expression of the scr regulon was studied with an scrYAB promoter-green fluorescent protein gene fusion and measured by flow cytometry and spectrofluorometry. The operon was affected by catabolite repression and induced by sucrose or fructose. The level of gene induction correlated to the sucrose concentration in plant tissue, as shown by flow cytometry. Sucrose mutants created by site-directed mutagenesis did not produce significant fire blight symptoms on apple seedlings, indicating the importance of sucrose metabolism for colonization of host plants by E. amylovora.

Molecular characterization of a protease secreted by Erwinia amylovora

A protease with a molecular mass of 48 kDa is secreted by the fire blight pathogen Erwinia amylovora in minimal medium. We characterized this activity as a metalloprotease, since the enzyme was inhibited by EDTA and o-phenanthroline. A gene cluster was determined to encode four genes connected to protease expression, including a structural gene (prtA) and three genes (prtD, prtE, prtF) for secretion of the protease, which are transcribed in the same direction. The organization of the protease gene cluster in E. amylovora is different from that in other Gram-negative bacteria, such as Erwinia chrysanthemi, Pseudomonas aeruginosa and Serratia marcescens. On the basis of the conservative motif of metalloproteases, PrtA was identified to be a member of the metzincin subfamily of zinc-binding metalloproteases, and was confirmed to be the 48 kDa protease on gels by sequencing of tryptic peptide fragments derived from the protein. The protease is apparently secreted into the external medium through the type I secretion pathway via PrtD, PrtE and PrtF which share more than 90% identity with the secretion apparatus for lipase of S. marcescens. A protease mutant was created by Tn5-insertions, and the mutation localized in the prtD gene. The lack of protease reduced colonization of an E. amylovora secretion mutant labelled with the gene for the green fluorescent protein (gfp) in the parenchyma of apple leaves.

Molecular analysis of the rlsA gene regulating levan production by the fireblight pathogen Erwinia amylovora

Natural Erwinia amylovora can display a reduced level of levan synthesis. A gene (rlsA) responsible for the suppression of low levansucrase synthesis by E. amylovora was cloned by complementation of natural levan-deficient strains lacking levansucrase expression. The rlsA region was sequenced, and an open reading frame of 266 amino acids was found. Sequence analysis revealed that RlsA resembles a transcriptional activator and may be a member of the LysR family. Overexpression of rlsA from a high copy number plasmid led to a two-fold increase of levan production, but had no influence on amylovoran synthesis. A mutant created by site-directed mutagenesis of wild type strain Eal/79 had the same phenotype as natural levan-deficient strains. The rlsA gene of natural levan deficient strains (with the exception of strain PD494) could be amplified by PCR, yielding the same DNA fragments as wild type strains. The gene was absent in a strain with a large deletion affecting the hrp region of E. amylovora . A match of the nucleotide sequence of rlsA with a sequence database revealed its identity with an incomplete open reading frame located downstream of two recently characterised dsp genes.

Reduction of lesion growth rate of late blight plant disease in transgenic potato expressing harpin protein

Using harpin protein gene from apple fire blight pathogen Erwinia amylovora and potato prp1-1 promoter as main DNA elements, the feasibility of using pathogen infection-induced hypersensitive response was explored as a new strategy of engineering fungal disease resistance. Three plant transformation vectors were constructed and 68 transgenic potato plants were produced through Agrobacterium mediated transformation method. Southern, Northern and Western blot analysis demonstrated the insertion, transcription and protein expression of harpin protein gene in transgenic plants. Disease resistance test using a complex race of Phytophthora infestans as challenging pathogen showed that both constitutive and pathogen infection-induced expression of harpin protein gene in transgenic potato reduced the lesion growth rate of fungus. Among plants where harpin protein gene expression was induced only by fungus infection, two plants were found to be highly resistant to P.infestans infection. Fungal hyphae were not produced on total of 30 inoculated leaves from the two resistant plants and necrotic lesion was limited to inoculation area. The results highlighted that engineered hypersensitive response in plants was a very promising approach to produce fungal disease resistant genotype.