A proteinaceous elicitor of the plant defense reaction known as the hypersensitive response was isolated from Erwinia amylovora, the bacterium that causes fire blight of pear, apple, and other rosaceous plants. The elicitor, named harpin, is an acidic, heat-stable, cell-envelope-associated protein with an apparent molecular weight of 44 kilodaltons. Harpin caused tobacco leaf lamina to collapse and caused an increase in the pH of bathing solutions of suspension-cultured tobacco cells. The gene encoding harpin (hrpN) was located in the 40-kilobase hrp gene cluster of E. amylovora, sequenced, and mutated with Tn5tac1. The hrpN mutants were not pathogenic to pear, did not elicit the hypersensitive response, and did not produce harpin.

Communicated by Steven D. Tanksley, Cornell University, Ithaca, NY, November 18, 1997 (received for review June 25, 1997 ABSTRACTThe ''disease-specific'' (dsp) region next to the hrp gene cluster of Erwinia amylovora is required for pathogenicity but not for elicitation of the hypersensitive reaction. A 6.6-kb apparent operon, dspEF, was found responsible for this phenotype. The operon contains genes dspE and dspF and is positively regulated by hrpL. A BLAST search revealed similarity in the dspE gene to a partial sequence of the avrE locus of Pseudomonas syringae pathovar tomato. The entire avrE locus was sequenced. Homologs of dspE and dspF were found in juxtaposed operons and were designated avrE and avrF. Introduced on a plasmid, the dspEF locus rendered P. syringae pv. glycinea race 4 avirulent on soybean. An E. amylovora dspE mutant, however, elicited a hypersensitive reaction in soybean. The avrE locus in trans restored pathogenicity to dspE strains of E. amylovora, although restored strains were low in virulence. DspE and AvrE are large (198 kDa and 195 kDa) and hydrophilic. DspF and AvrF are small (16 kDa and 14 kDa) and acidic with predicted amphipathic ␣ helices in their C termini; they resemble chaperones for virulence factors secreted by type III secretion systems of animal pathogens.Erwinia amylovora causes fire blight of apple, pear, and other rosaceous plants and elicits plant defense responses in nonhost plants. Required for these interactions are the clustered bacterial hrp genes, encoding regulatory proteins (ref. 1; Z.M.W., B. J. Sneath, and S.V.B., unpublished data), a large set of proteins broadly conserved among plant and animal pathogens and constituting a type III secretion pathway (known as the ''Hrp pathway'' in phytopathogenic bacteria; refs. 2 and 3), and at least two proteins secreted via the Hrp pathway (4, 5). hrp genes, present in all Gram-negative necrogenic plant pathogens, were discovered by transposon mutagenesis of Pseudomonas syringae pathovars and were named for the ''hypersensitive reaction'' (HR) and ''pathogenicity'' (reviewed in ref. 6). The HR is a manifestation of plant defense characterized by rapid necrosis at the site of pathogen ingress.Pathogen avirulence (avr) genes (for a review see ref.7) generate signals that trigger defense responses leading to disease resistance in plants with corresponding resistance (R) genes. Typically, avr genes are isolated by expressing a cosmid library from one pathogen in another pathogen and screening for narrowed host range. avr genes traditionally have been considered as negative determinants of host specificity at the race-cultivar level, but some, including the avrE locus from the bacterial speck pathogen Pseudomonas syringae pathovar (pv.) tomato (8), may restrict host range at the pathovar-species or species-species level (9, 10). Many avr genes, including avrE, are hrp regulated. avrE and avrPphE (11) are physically linked to hrp genes. Only a few avr genes (such as avrE), however, play detectable roles in pathogen fitness...

Seven hrp loci that are essential for the hypersensitive reaction elicited by Erwinia amylovora were transcriptionally fused with a derivative of transposon TnS, containing the promoterless Escherichia coli 1-glucuronidase reporter gene. The seven hrp fusions were used to monitor expression of the hrp loci in vitro and in planta. No significant expression was detected in rich medium for any of the fusions. However, five of them were expressed highly in planta and in inducing medium that contains mannitol, salts, and 5 mM (NH4)2SO4. Expression of these five hrp loci is regulated by ammonium, nicotinic acid, complex-nitrogen sources, certain carbon sources, temperature, and pH. Under well-defined conditions, i.e., in inducing medium, no specific plant components were required for transcriptional activation of the hrp loci. The high levels of expression detected in vitro were comparable to those determined during the developme * of the hypersensitive reaction in tobacco. Differential levels of expression of the hrp loci occurred in host and nonhost plants. In pear, a host plant, expression of the hip loci was delayed and greatly reduced compared with expression in tobacco leaves, a nonhost.

hrpL of Erwinia amylovora Ea321 encodes a 21.7-kDa regulatory protein, similar to members of the ECF (extra cytoplasmic functions) subfamily of eubacterial RNA polymerase factors. hrpL is a single-gene operon in complementation group VI of the E. amylovora hrp gene cluster. Its product is required by Ea321 to elicit the hypersensitive response (HR) and to cause disease. HrpL controls the expression of five independent hrp loci, including hrpN, which encodes harpin, a proteinaceous elicitor of the HR. hrpL is environmentally regulated, and its expression is affected by hrpS, another regulatory gene of the hrp gene cluster of E. amylovora. pCPP1078, a multicopy plasmid carrying hrpL, is able to restore HR-eliciting ability to hrpS mutants. A conserved motif was identified upstream of the hrpI and hrpN operons, which are transcriptionally regulated by hrpL. This conserved motif shares a high degree of similarity with other biochemically defined or putative ECF-dependent promoter sequences, including sequences upstream of Streptomyces coelicolor dagA P2, Pseudomonas aeruginosa algD, Pseudomonas syringae pv. syringae 61 hrpZ, and P. syringae pv. tomato avrD. In spite of the similarity between the hrpL genes of E. amylovora and P. syringae 61, no functional cross-complementation was observed.Erwinia amylovora is a devastating plant-pathogenic bacterium that causes the fire blight disease of pear, apple, and many other rosaceous plants (1). The bacterium infects blossoms, leaves, succulent shoots, and immature fruit of susceptible hosts. Symptoms include water soaking and discoloration of affected tissue, followed by necrosis (40). Under laboratory conditions, E. amylovora elicits the hypersensitive response (HR) in many higher plants (18, 37), which is characterized by the rapid, localized death of tissues infiltrated with high concentrations of bacterial cells (Ն10 7 CFU/ml) (16, 17). The genes responsible for pathogenicity and the HR, designated hrp genes (20), are clustered in E. amylovora Ea321 on an approximately 40-kb cloned fragment of DNA (4). The hrp gene cluster of E. amylovora is particularly well expressed in Escherichia coli, and it confers on it and other nonpathogenic bacteria the ability to elicit the HR. A 25-kb region of the 40 kb of DNA cloned in cosmid pCPP430 is necessary both for induction of the HR and for pathogenicity by E. amylovora (4). Eight putative transcription units have been defined in the 25-kb region by ␤-glucuronidase (GUS) activity assay of hrp::Tn5gusA1 gene fusions, genetic analysis of complementation, and recent DNA sequence analysis (3a, 42).On the basis of genetic and biochemical characterization of mutants in the eight transcription units, the hrp genes of E. amylovora can be classified into three groups: (i) structural genes, e.g., hrpN, which encodes harpin, a cell surface-associated proteinaceous elicitor of the HR and a pathogenicity determinant (43); (ii) secretion genes that consist of a secindependent (type III [31]) secretory apparatus, e.g., HrpI, an LcrD homolog (27...

Two novel regulatory components, hrpX and hrpY, of the hrp system of Erwinia amylovora were identified. The hrpXY operon is expressed in rich media, but its transcription is increased threefold by low pH, nutrient, and temperature levels--conditions that mimic the plant apoplast. hrpXY is autoregulated and directs the expression of hrpL; hrpL, in turn, activates transcription of other loci in the hrp gene cluster (Z.-M. Wei and S. V. Beer, J. Bacteriol. 177:6201-6210, 1995). The deduced amino -acid sequences of hrpX and hrpY are similar to bacterial two-component regulators including VsrA/VsrD of Pseudomonas (Ralstonia) solanacearum, DegS/DegU of Bacillus subtilis, and UhpB/UhpA and NarX/NarP, NarL of Escherichia coli. The N-terminal signal-input domain of HrpX contains PAS domain repeats. hrpS, located downstream of hrpXY, encodes a protein with homology to WtsA (HrpS) of Erwinia (Pantoea) stewartii, HrpR and HrpS of Pseudomonas syringae, and other delta54-dependent, enhancer-binding proteins. Transcription of hrpS also is induced under conditions that mimic the plant apoplast. However, hrpS is not autoregulated, and its expression is not affected by hrpXY. When hrpS or hrpL were provided on multicopy plasmids, both hrpX and hrpY mutants recovered the ability to elicit the hypersensitive reaction in tobacco. This confirms that hrpS and hrpL are not epistatic to hrpXY. A model of the regulatory cascades leading to the induction of the E. amylovora type III system is proposed.

Type III secretion functions in flagellar biosynthesis and in export of virulence factors from several animal pathogens, and for plant pathogens, it has been shown to be involved in the export of elicitors of the hypersensitive reaction. Typified by the Yop delivery system of Yersinia spp., type III secretion is sec independent and requires multiple components. Sequence analysis of an 11.5-kb region of the hrp gene cluster of Erwinia amylovora containing hrpI, a previously characterized type III gene, revealed a group of eight more type III genes corresponding to the virB or lcrB (yscN-to-yscU) locus of Yersinia spp. A homolog of another Yop secretion gene, yscD, was found between hrpI and this group downstream. Immediately upstream of hrpI, a homolog of yopN was discovered. yopN is a putative sensor involved in host-cell-contact-triggered expression and transfer of protein, e.g., YopE, to the host cytoplasm. In-frame deletion mutagenesis of one of the type III genes, designated hrcT, was nonpolar and resulted in a Hrp ؊ strain that produced but did not secrete harpin, an elicitor of the hypersensitive reaction that is also required for pathogenesis. Cladistic analysis of the HrpI (herein renamed HrcV) or LcrD protein family revealed two distinct groups for plant pathogens. The Yersinia protein grouped more closely with the plant pathogen homologs than with homologs from other animal pathogens; flagellar biosynthesis proteins grouped distinctly. A possible evolutionary history of type III secretion is presented, and the potential significance of the similarity between the harpin and Yop export systems is discussed, particularly with respect to a potential role for the YopN homolog in pathogenesis of plants.Erwinia amylovora causes fire blight, an often devastating disease of apple, pear, and other rosaceous plants. In nonhost plants, such as tobacco plants, E. amylovora elicits the rapid, localized necrosis known as the hypersensitive reaction (HR). The HR is an active process (34) that is correlated with plant defense responses. Several genes required by plant-pathogenic bacteria both for elicitation of the HR and for pathogenicity have been characterized. These genes are designated hrp for hypersensitive response and pathogenicity (52). Large clusters of hrp genes have been cloned from E. amylovora, Pseudomonas solanacearum, Xanthomonas campestris, and Pseudomonas syringae. In large part, these clusters were discovered to be physically and functionally conserved (7, 50,74,84). The hrp gene cluster of E. amylovora was cloned from strain Ea321 in the cosmid pCPP430 and found to confer on Escherichia coli and other nonplant pathogens and saprophytes the ability to elicit an HR in tobacco plants (10) and to enable bacterial multiplication and induction of electrolyte leakage and necrosis in apple leaf segments (88). Harpin, an extracellular elicitor of the HR and a pathogenicity determinant, was identified as a product of the hrpN gene located within this cluster (82). Complementation analysis of transposon insert...

HrpI, a 78-kDa protein, functions in the secretion of harpin, a proteinaceous elicitor of the hypersensitive response from Erwinia amylovora. The predicted amino acid sequence of HrpI is remarkably similar to that of LcrD of Yersinia species, the first member of a recently described protein family. Other proteins of the family are MixA from Shigella flexneri, InvA from Salmonella typhimurium, FlhA from Caulobacter crescentus, HrpI from Pseudomonas syringae pv. syringae, HrpO from Pseudomonas solanacearum, and HrpC2 from Xanthomonas campestris pv. vesicatoria. Cells of E. amylovora containing mutated hrpI genes or cells of Escherichia coli containing the cloned hrp gene cluster with mutated hrpI produce but do not export harpin. When similar cells with functional hrpI genes were grown at 25 degrees C, but not at 37 degrees C, harpin was exported to the culture supernatant. Direct evidence that HrpI is involved in the secretion of a virulence protein has been offered. Two other loci of the hrp gene cluster are involved in the regulation of harpin, and four other loci also are involved in the secretion of harpin. Since harpin and other proteins likely to be secreted by the LcrD family of proteins lack typical signal peptides, their secretion mechanism is distinct from the general protein export pathway.

A 6.2-kb region of DNA corresponding to complementation groups II and III of the Erwinia amylovora hrp gene cluster was analyzed. Transposon mutagenesis indicated that the two complementation groups are required for secretion of harpin, an elicitor of the hypersensitive reaction. The sequence of the region revealed 10 open reading frames in two putative transcription units: hrpA, hrpB, hrcJ, hrpD, and hrpE in the hrpA operon (group III) and hrpF, hrpG, hrcC, hrpT, and hrpV in the hrpC operon (group II). From promoter regions of the hrpA, hrpC, and hrpN operons, sequences similar to those of the HrpL-dependent promoters of Pseudomonas syringae pathovars were identified with a consensus sequence of 5-GGAAC-N 17-18 -CACTNAA-3. The protein products of seven genes, hrpA, hrcJ, hrpE, hrpF, hrpG, hrcC, and hrpV, were visualized with a T7 polymerase/ promoter expression system. HrcC, HrcJ, and HrpT sequences contained potential signal peptides, and HrcC appeared to be envelope associated based on a TnphoA translational fusion. Comparison of deduced amino acid sequences indicated that many of the proteins are homologous to proteins that function in the type III protein secretion pathway. HrcC is a member of the YscC-containing subgroup in the PulD/pIV superfamily of outer membrane proteins. HrcJ is a member of a lipoprotein family that includes YscJ of Yersinia spp., MxiJ of Shigella flexneri, and NolT of Rhizobium fredii. Additional similarities were detected between HrpB and YscI and between HrpE and YscL. HrcJ and HrpE were similar to flagellar biogenesis proteins FliF and FliH, respectively. In addition, HrpA, HrpB, HrcJ, HrpD, HrpE, HrpF, and HrcC showed various degrees of similarity to corresponding proteins of P. syringae. Comparison of hrp clusters with respect to gene organization and similarity of individual proteins confirms that the hrp systems of E. amylovora and P. syringae are closely related to each other and distinct from those of Ralstonia (Pseudomonas) solanacearum and Xanthomonas campestris. Possible implications of extensive similarities between the E. amylovora and P. syringae hrp systems in pathogenesis mechanisms are discussed.