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

KEY MESSAGE: pPPO16, 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.

Plant-associated microbiota as a source of antagonistic bacteria against the phytopathogen Erwinia amylovora.

Control of bacterial plant diseases is a major concern, as they affect economically important species and spread easily, such as the case of fire blight of rosaceous caused by Erwinia amylovora. In the search for alternatives to the use of agrochemicals and antibiotics, this work presents a screening of natural bacterial antagonists of this relevant and devastating phytopathogen. We recovered bacterial isolates from different plant tissues and geographical origins and then selected those with the strongest ability to reduce fire blight symptoms ex vivo and remarkable in vitro antagonistic activity against E. amylovora. None of them elicited a hypersensitivity reaction in tobacco leaves, most produced several hydrolytic enzymes and presented other biocontrol and/or plant growth-promoting activities, such as siderophore production and phosphate solubilization. These isolates, considered as biocontrol candidates, were identified by 16S rRNA sequencing as Pseudomonas rhizosphaerae, Curtobacterium flaccumfaciens, Enterobacter cancerogenus, Pseudomonas azotoformans, Rosenbergiella epipactidis and Serratia plymuthica. This is the first time that the last five bacterial species are reported to have biocontrol potential against E. amylovora.

Enzymatic Thioamide Formation in a Bacterial Antimetabolite Pathway.

6-Thioguanine (6TG) is a DNA-targeting therapeutic used in the treatment of various cancers. While 6TG was rationally designed as a proof of concept for antimetabolite therapy, it is also a rare thioamide-bearing bacterial natural product and critical virulence factor of Erwinia amylovorans, plant pathogens that cause fire blight. Through gene expression, biochemical assays, and mutational analyses, we identified a specialized bipartite enzyme system, consisting of an ATP-dependent sulfur transferase (YcfA) and a sulfur-mobilizing enzyme (YcfC), that is responsible for the peculiar oxygen-by-sulfur substitution found in the biosynthesis of 6TG. Mechanistic and phylogenetic studies revealed that YcfA-mediated 6TG biosynthesis evolved from ancient tRNA modifications that support translational fidelity. The successful in vitro reconstitution of 6TG thioamidation showed that YcfA employs a specialized sulfur shuttle that markedly differs from universal RNA-related systems. This study sheds light on underexplored enzymatic C-S bond formation in natural product biosynthesis.

Loss-of-Function Mutations in the Dpp and Opp Permeases Render Erwinia amylovora Resistant to Kasugamycin and Blasticidin S.

Extensive use of the antibiotic streptomycin to control fire blight disease of apples and pears, caused by the enterobacterial plant pathogen Erwinia amylovora, leads to the development of streptomycin-resistant strains in the United States and elsewhere. Kasugamycin (Ksg) has been permitted to be used as an alternative or replacement to control this serious bacterial disease. In this study, we investigated the role of two major peptide ATP-binding cassette transporter systems in E. amylovora, the dipeptide permease (Dpp) and oligopeptide permease (Opp), in conferring sensitivity to Ksg and blasticidin S (BcS). Minimum inhibitory concentration and spot dilution assays showed that the dpp deletion mutants exhibited slightly enhanced resistance to Ksg in rich medium, whereas the opp mutant exhibited slightly enhanced resistance to Ksg in minimal medium and BcS in rich medium. Deletion of both dpp and opp conferred a higher level of resistance to Ksg in both rich and minimal media, whereas deletion of opp alone was sufficient to confer high level of resistance to BcS in minimal medium. In addition, bioinformatic analysis combined with reverse transcription-quantitative polymerase chain reaction showed that the Rcs phosphorelay system negatively regulates opp expression and the rcsB mutant was more sensitive to both Ksg and BcS in minimal medium as compared with the wild type. An electrophoresis motility shift assay further confirmed the direct binding of the RcsA/RcsB proteins to the promoter region of the opp operon. However, neither the Dpp nor the Opp permeases contributed to disease progress on immature pears, hypersensitive response on tobacco leaves, or exopolysaccharide amylovoran production. These results suggested that Ksg and BcS employ the Dpp and Opp permeases to enter E. amylovora cells and the Dpp and Opp permeases act synergistically for illicit transport of antibiotics.

An Erwinia amylovora yjeK mutant exhibits reduced virulence, increased chemical sensitivity and numerous environmentally dependent proteomic alterations.

The Gram-negative bacterium Erwinia amylovora causes fire blight, an economically important disease of apples and pears. Elongation factor P (EF-P) is a highly conserved protein that stimulates the formation of the first peptide bond of certain proteins and facilitates the translation of certain proteins, including those with polyproline motifs. YjeK and YjeA are two enzymes involved in the essential post-translational ß-lysylation of EF-P at a conserved lysine residue, K34. EF-P, YjeA and YjeK have been shown to be essential for the full virulence of Escherichia coli, Salmonella species and Agrobacterium tumefaciens, with efp, yjeA and yjeK mutants having highly similar phenotypes. Here, we identified an E. amylovora yjeK::Tn5 transposon mutant with decreased virulence in apple fruit and trees. The yjeK::Tn5 mutant also showed pleiotropic phenotypes, including reduced growth in rich medium, lower extracellular polysaccharide production, reduced swimming motility and increased chemical sensitivity compared with the wild-type, whilst maintaining wild-type level growth in minimal medium. All yjeK::Tn5 mutant phenotypes were complemented in trans with a plasmid bearing a wild-type copy of yjeK. Comprehensive, quantitative proteomics analyses revealed numerous, environmentally dependent changes in the prevalence of a wide range of proteins, in higher abundance and lower abundance, in yjeK::Tn5 compared with the wild-type, and many of these alterations could be linked to yjeK::Tn5 mutant phenotypes. The environmental dependence of the yjeK::Tn5 mutant proteomic alterations suggests that YjeK could be required for aspects of the environmentally dependent regulation of protein translation. YjeK activity may be critical to overcoming stress, including the challenging host environment faced by invading pathogenic bacteria.

The RNA-binding protein CsrA plays a central role in positively regulating virulence factors in Erwinia amylovora.

The GacS/GacA two-component system (also called GrrS/GrrA) is a global regulatory system which is highly conserved among gamma-proteobacteria. This system positively regulates non-coding small regulatory RNA csrB, which in turn binds to the RNA-binding protein CsrA. However, how GacS/GacA-Csr system regulates virulence traits in E. amylovora remains unknown. Results from mutant characterization showed that the csrB mutant was hypermotile, produced higher amount of exopolysaccharide amylovoran, and had increased expression of type III secretion (T3SS) genes in vitro. In contrast, the csrA mutant exhibited complete opposite phenotypes, including non-motile, reduced amylovoran production and expression of T3SS genes. Furthermore, the csrA mutant did not induce hypersensitive response on tobacco or cause disease on immature pear fruits, indicating that CsrA is a positive regulator of virulence factors. These findings demonstrated that CsrA plays a critical role in E. amylovora virulence and suggested that negative regulation of virulence by GacS/GacA acts through csrB sRNA, which binds to CsrA and neutralizes its positive effect on T3SS gene expression, flagellar formation and amylovoran production. Future research will be focused on determining the molecular mechanism underlying the positive regulation of virulence traits by CsrA.

Characterization and regulation of the resistance-nodulation-cell division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora.

BACKGROUND: The Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. In order to survive and multiply in a host, microbes must be able to circumvent the toxic effects of antimicrobial plant compounds, such as flavonoids and tannins. E. amylovora uses multidrug efflux transporters that recognize and actively export toxic compounds out of the cells. Here, two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump. RESULTS: To find the substrate specificities of the two efflux systems, we overexpressed the transporters in a hypersensitive mutant lacking the major RND pump AcrB. Both transporters mediated resistance to several flavonoids, fusidic acid and novobiocin. Additionally, MdtABC mediated resistance towards josamycin, bile salts and silver nitrate, and MdtUVW towards clotrimazole. The ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock was reduced. Quantitative RT-PCR analyses revealed that the expression of the transporter genes was induced during infection of apple rootstock. The polyphenolic plant compound tannin, as well as the heavy metal salt tungstate was found to induce the expression of mdtABC. Finally, the expression of the mdtABC genes was shown to be regulated by BaeR, the response regulator of the two-component system BaeSR, a cell envelope stress response system that controls the adaptive responses to changes in the environment. CONCLUSIONS: The expression of MdtABC and MdtUVW is induced during growth of E. amylovora in planta. We identified the plant polyphenol tannin as inducer of mdtABC expression. The reduced ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock suggests that the efflux pumps are involved in resistance to plant antimicrobials, maybe including flavonoids, which were identified as substrates of both pumps. Furthermore, we found that the mdtABC operon belongs to the regulon of the two-component regulator BaeR suggesting a role of this RND transporter in the cell envelope stress response of E. amylovora.

Alternative sigma factor RpoN and its modulation protein YhbH are indispensable for Erwinia amylovora virulence.

In Erwinia amylovora, ECF (extracytoplasmic functions) alternative sigma factor HrpL regulates the transcription of hrp (hypersensitive response and pathogenicity)-type III secretion system (T3SS) genes by binding to a consensus sequence known as the hrp box in hrp gene promoters. In turn, the expression of hrpL has been proposed to be positively controlled by alternative sigma factor 54 (σ(54)) (RpoN) and HrpS, a member of the σ(54) enhancer-binding proteins (EBPs). However, the function of RpoN has not been characterized genetically in E. amylovora. In this study, we investigated the role of RpoN, a nitrogen limitation sigma factor, and its modulation protein YhbH, a novel ribosome-associated protein, in E. amylovora virulence. Our results showed that mutations in hrpS, hrpL, rpoN and yhbH, but not yfiA and rmf3, resulted in a nonpathogenic phenotype on immature pear fruits and apple shoots. Consistently, the expression of T3SS genes, including hrpL, dspE, hrpN and hrpA, was barely detected in hrpS, hrpL, rpoN and yhbH mutants. These mutants were also not capable of eliciting a hypersensitive response (HR) on tobacco; however, the overexpression of hrpL using an inducible promoter rescued the HR-eliciting abilities of these mutants. These results suggest that a sigma factor cascade exists in the regulatory networks of E. amylovora and regulates important virulence factors. On the basis of this study and previously reported data, a model is proposed for the regulation of T3SS in E. amylovora.

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

RpoN is a σ(54) 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.

HopX1 in Erwinia amylovora functions as an avirulence protein in apple and is regulated by HrpL.

Fire blight is a devastating disease of rosaceous plants caused by the Gram-negative bacterium Erwinia amylovora. This pathogen delivers virulence proteins into host cells utilizing the type III secretion system (T3SS). Expression of the T3SS and of translocated and secreted substrates is activated by the alternative sigma factor HrpL, which recognizes hrp box promoters upstream of regulated genes. A collection of hidden Markov model (HMM) profiles was used to identify putative hrp boxes in the genome sequence of Ea273, a highly virulent strain of E. amylovora. Among potential virulence factors preceded by putative hrp boxes, two genes previously known as Eop3 and Eop2 were characterized. The presence of functionally active hrp boxes upstream of these two genes was confirmed by ß-glucuronidase (GUS) assays. Deletion mutants of the latter candidate genes, renamed hopX1(Ea) and hopAK1(Ea), respectively, did not differ in virulence from the wild-type strain when assayed in pear fruit and apple shoots. The hopX1(Ea) deletion mutant of Ea273, complemented with a plasmid overexpressing hopX1(E)(a), suppressed the development of the hypersensitivity response (HR) when inoculated into Nicotiana benthamiana; however, it contributed to HR in Nicotiana tabacum and significantly reduced the progress of disease in apple shoots, suggesting that HopX1(Ea) may act as an avirulence protein in apple shoots.

Genomics of iron acquisition in the plant pathogen Erwinia amylovora: insights in the biosynthetic pathway of the siderophore desferrioxamine E.

Genomics has clarified the biosynthetic pathway for desferrioxamine E critical for iron acquisition in the enterobacterial fire blight pathogen Erwinia amylovora. Evidence for each of the individual steps and the role of desferrioxamine E biosynthesis in pathogen virulence and cell protection from host defenses is presented. Using comparative genomics, it can be concluded that desferrioxamine biosynthesis is ancestral within the genera Erwinia and Pantoea.

Secretion and translocation signals and DspB/F-binding domains in the type III effector DspA/E of Erwinia amylovora.

DspA/E is a type III effector of Erwinia amylovora, the bacterial pathogen that causes fire blight disease in roseaceous plants. This effector is indispensable for disease development, and it is translocated into plant cells. A DspA/E-specific chaperone, DspB/F, is necessary for DspA/E secretion and possibly for its translocation. In this work, DspB/F-binding sites and secretion and translocation signals in the DspA/E protein were determined. Based on yeast two-hybrid assays, DspB/F was found to bind DspA/E within the first 210 amino acids of the protein. Surprisingly, both DspB/F and OrfA, the putative chaperone of Eop1, also interacted with the C-terminal 1059 amino acids of DspA/E; this suggests another chaperone-binding site. Secretion and translocation assays using serial N-terminal lengths of DspA/E fused with the active form of AvrRpt2 revealed that at least the first 109 amino acids, including the first N-terminal chaperone-binding motif and DspB/F, were required for efficient translocation of DspA/E, although the first 35 amino acids were sufficient for its secretion and the presence of DspB/F was not required. These results indicate that secretion and translocation signals are present in the N terminus of DspA/E, and that at least one DspB/F-binding motif is required for efficient translocation into plant cells.

Molecular signature of differential virulence in natural isolates of Erwinia amylovora.

ABSTRACT Erwinia amylovora, the causal agent of fire blight, is considered to be a genetically homogeneous species based on physiological, biochemical, phylogenetic, and genetic analysis. However, E. amylovora strains exhibiting differential virulence are isolated from nature. The exopolysaccharide amylovoran and type III secretion system (T3SS) are two major yet separate virulence factors in E. amylovora. The objective of this study was to investigate whether there is a correlation between E. amylovora virulence and levels of virulence gene expression. Four wild-type strains (Ea1189, Ea273, Ea110, and CFBP1430), widely used in studies of E. amylovora pathogenesis, have been analyzed and compared. E. amylovora strains Ea273 and Ea110 elicited higher severity of disease symptoms than those of Ea1189 and CFBP1430 on apple cv. Golden Delicious and G16 apple root stock plants but not on susceptible Gala plants. In addition, Ea273 and Ea110 elicited severe hypersensitive responses within shorter periods of time at lower inoculum concentrations than those of Ea1189 and CFBP1430 on tobacco plants. Further molecular analyses have revealed that amylovoran production and expression of both amylovoran (amsG) and T3SS (dspE and hrpL) genes were significantly higher in Ea273 and Ea110 than those in Ea1189 and CFBP1430. Other phenotypes such as swarming motility in these four strains also differed significantly. These results indicate that E. amylovora strains of different origin can be divided into subgroups based on molecular signatures of virulence gene expression. Therefore, these molecular signatures may be used to differentiate E. amylovora strains, which may have taxonomical and evolutionary implications.

The PmrAB system in Erwinia amylovora renders the pathogen more susceptible to polymyxin B and more resistant to excess iron.

The PmrAB system is a two-component regulatory system that responds to extracellular iron and acidic pH. The role of the PmrAB system in Erwinia amylovora remains unknown so far. Our results showed that the pmrAB mutants were more resistant to strong acidic conditions than the wild type (WT) strain. The survival rate of the pmrAB mutants was much higher than that of WT when treated with polymyxin B. However, pmrAB mutants were more sensitive to extracellular iron than WT strain. These results demonstrated that the PmrAB system in E. amylovora renders the pathogen more susceptible to polymyxin B and acidic pH, but more resistance to excess iron.

Functional analysis of the N terminus of the Erwinia amylovora secreted effector DspA/E reveals features required for secretion, translocation, and binding to the chaperone DspB/F.

DspA/E is a type III secreted effector protein required for pathogenicity in the apple and pear pathogen Erwinia amylovora, and DspB/F is a small chaperone protein involved in DspA/E secretion. While the secretion and translocation signals of many type III secretion effector proteins in human enteric pathogens have been characterized extensively, relatively little is known about the translocation requirements of many effectors in plant pathogens, including large DspE-like proteins. In this study, we report a functional analysis of the N terminus of DspE. The minimal requirements for secretion, translocation, and chaperone binding were characterized. Translocation assays using an adenylate cyclase (CyaA) reporter indicated that the first 51 amino acids of DspE were sufficient for translocation and that 150 amino acids were required for optimal translocation levels. The minimal translocation signal corresponded with the requirements for secretion into culture media. Mutations of conserved regions in amino acids 2 through 10 and 31 through 40 were found to influence translocation levels of an N-terminal DspE-CyaA fusion. Yeast two-hybrid and in-vitro pull-down assays revealed a chaperone-binding site within amino acids 51 through 100 of DspE and binding to DspF in this region was disrupted by specific mutations. However, neither disruption of the chaperone-binding domain nor deletion of the dspF gene had a significant impact on translocation levels of N-terminal DspE-CyaA fusions. Our results indicate that the minimal translocation signal of DspE is not coincident with the signal for DspF binding and that translocation of the N terminus of DspE is not dependent on the N-terminal DspF-binding domain.

Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by gacA.

The xylem-limited, insect-transmitted bacterium Xylella fastidiosa causes Pierce's disease in grapes through cell aggregation and vascular clogging. GacA controls various physiological processes and pathogenicity factors in many gram-negative bacteria, including biofilm formation in Pseudomonas syringae pv. tomato DC3000. Cloned gacA of X. fastidiosa was found to restore the hypersensitive response and pathogenicity in gacA mutants of P. syringae pv. tomato DC3000 and Erwinia amylovora. A gacA mutant of X. fastidiosa (DAC1984) had significantly reduced abilities to adhere to a glass surface, form biofilm, and incite disease symptoms on grapevines, compared with the parent (A05). cDNA microarray analysis identified 7 genes that were positively regulated by GacA, including xadA and hsf, predicted to encode outer membrane adhesion proteins, and 20 negatively regulated genes, including gumC and an antibacterial polypeptide toxin gene, cvaC. These results suggest that GacA of X. fastidiosa regulates many factors, which contribute to attachment and biofilm formation, as well as some physiological processes that may enhance the adaptation and tolerance of X. fastidiosa to environmental stresses and the competition within the host xylem.

The luxS gene is involved in AI-2 production, pathogenicity, and some phenotypes in Erwinia amylovora.

Erwinia amylovora causes fire blight of apple, pear, and other members of the Rosaceae family. The enzyme LuxS catalyzes the last step in the production of autoinducer-2 (AI-2), a molecule implicated with quorum sensing in many bacterial species. It is now well recognized that LuxS also plays a central role in sulfur metabolism and in the activated methyl cycle, which is responsible for the generation of S-adenosyl-L-methionine. A research paper has reported that luxS is not involved with quorum sensing in Er. amylovora, but in our study, Er. amylovora strain NCPPB1665 (Ea1665) produced luxS-dependent extracellular AI-2 activity. Additionally, the maximal AI-2 activity occurred during late-exponential and early-stationary growth phases and diminished during the stationary phase. The luxS mutant of Ea1665 was constructed, and the phenotypes of a defined luxS mutant have been characterized. Inactivation of luxS in Ea1665 impaired motility, extracellular polysaccharide (EPS) production, and tolerance for hydrogen peroxide, and reduced virulence on pear leaves.

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.

HrpN of Erwinia amylovora functions in the translocation of DspA/E into plant cells.

The type III secretion system (T3SS) is required by plant pathogenic bacteria for the translocation of certain bacterial proteins to the cytoplasm of plant cells or secretion of some proteins to the apoplast. The T3SS of Erwinia amylovora, which causes fire blight of pear, apple and other rosaceous plants, secretes DspA/E, which is an indispensable pathogenicity factor. Several other proteins, including HrpN, a critical virulence factor, are also secreted by the T3SS. Using a CyaA reporter system, we demonstrated that DspA/E is translocated into the cells of Nicotiana tabacum'Xanthi'. To determine if other T3-secreted proteins are needed for translocation of DspA/E, we examined its translocation in several mutants of E. amylovora strain Ea321. DspA/E was translocated by both hrpW and hrpK mutants, although with some delay, indicating that these two proteins are dispensable in the translocation of DspA/E. Remarkably, translocation of DspA/E was essentially abolished in both hrpN and hrpJ mutants; however, secretion of DspA/E into medium was not affected in any of the mentioned mutants. In contrast to the more virulent strain Ea273, secretion of HrpN was abolished in a hrpJ mutant of strain Ea321. In addition, HrpN was weakly translocated into plant cytoplasm. These results suggest that HrpN plays a significant role in the translocation of DspA/E, and HrpJ affects the translocation of DspA/E by affecting secretion or stability of HrpN. Taken together, these results explain the critical importance of HrpN and HrpJ to the development of fire blight.

The hrpN gene of Erwinia amylovora stimulates tobacco growth and enhances resistance to Botrytis cinerea.

Erwinia amylovora is a member of the harpin proteins that induces pathogen resistance and hypersensitive cell death in plants. To obtain tobacco plants displaying a hypersensitive response, the hrpN gene from Erwinia amylovora was cloned into vector pMJC-GB under the control of the rice cytochrome promoter and transfected into tobacco. Southern hybridization with a hrpN probe revealed that the gene was present in one copy in the transgenic plants. In addition, hrpN transcripts could be detected in transgenic plants but not in wild-type tobacco. The wild type gave 75 products in RAPD analysis with 12 primers while the transgenic plants gave 73, suggesting that hrpN gene had been integrated into the transgenic plant genomic DNA. The distribution of cell cycle phases in the wild type and transgenic plants was G0-G1: 71.25%, G2-M: 20.41%, S: 8.33%, while in transgenic plant was G0-G1: 54.95%, G2-M: 43.82%, S: 10.23%. The sizes of stomata and guard cells on transgenic leaves were similar to those of the wild type, but the epidermal cells were clearly smaller. The transgenic plants showed accelerated growth and development as well as enhanced resistance to Botrytis cinerea.