Rahnella spp. are commonly isolated from onion (Allium cepa) bulbs and are weakly pathogenic.

AIMS: Bacterial decays of onion bulbs have serious economic consequences for growers, but the aetiologies of these diseases are often unclear. We aimed to determine the role of Rahnella, which we commonly isolated from bulbs in the United States and Norway, in onion disease. METHODS AND RESULTS: Isolated bacteria were identified by sequencing of housekeeping genes and/or fatty acid methyl ester analysis. A subset of Rahnella spp. strains was also assessed by multilocus sequence analysis (MLSA); most onion strains belonged to two clades that appear closely related to R. aquatilis. All tested strains from both countries caused mild symptoms in onion bulbs but not leaves. Polymerase chain reaction primers were designed and tested against strains from known species of Rahnella. Amplicons were produced from strains of R. aquatilis, R. victoriana, R. variigena, R. inusitata and R. bruchi, and from one of the two strains of R. woolbedingensis. CONCLUSIONS: Based on binational testing, strains of Rahnella are commonly associated with onions, and they are capable of causing mild symptoms in bulbs. SIGNIFICANCE AND IMPACT OF THE STUDY: While Rahnella strains are commonly found within field-grown onions and they are able to cause mild symptoms, the economic impact of Rahnella-associated symptoms remains unclear.

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.

Eop1 from a Rubus strain of Erwinia amylovora functions as a host-range limiting factor.

Strains of Erwinia amylovora, the bacterium causing the disease fire blight of rosaceous plants, are separated into two groups based on host range: Spiraeoideae and Rubus strains. Spiraeoideae strains have wide host ranges, infecting plants in many rosaceous genera, including apple and pear. In the field, Rubus strains infect the genus Rubus exclusively, which includes raspberry and blackberry. Based on comparisons of limited sequence data from a Rubus and a Spiraeoideae strain, the gene eop1 was identified as unusually divergent, and it was selected as a possible host specificity factor. To test this, eop1 genes from a Rubus strain and a Spiraeoideae strain were cloned and mutated. Expression of the Rubus-strain eop1 reduced the virulence of E. amylovora in immature pear fruit and in apple shoots. Sequencing the orfA-eop1 regions of several strains of E. amylovora confirmed that forms of eop1 are conserved among strains with similar host ranges. This work provides evidence that eop1 from a Rubus-specific strain can function as a determinant of host specificity in E. amylovora.

First Report of Enterobacter Bulb Decay of Onions Caused by Enterobacter cloacae in New York.

During the summer of 2010, onions (Allium cepa L.) of several cultivars growing in muck-land soils in Orange, Genesee, Orleans, and Oswego counties of New York exhibited leaf dieback and bulb decay consistent with disease symptoms caused by Enterobacter cloacae as described previously (1,3,4). Isolations of bacteria from symptomatic tissues and muck soil were made using onion extract medium (OEM), which contains extracts of autoclaved onions, salts, and inhibitors of fungi and gram-positive bacteria. Some presumptive strains of E. cloacae were isolated; 5 from symptomatic onions growing in Genesee County, 2 from muck-land soil, and 27 from bulbs stored for ~2.5 months in a farm storage facility in Oswego County. Tentative identification was based on colony morphology (convex, cream-color colonies, 2 to 3 mm in diameter following incubation at 28°C for 1 day on OEM), which was similar to the morphology of reference strains of E. cloacae ATCC 23355, ATCC 13047, and strain 310 (gift of H. F. Schwartz, which was derived from reference 4; personal communication). Strains were gram-negative rods, negative for oxidase and indole, positive for nitrate reductase and catalase; produced acid from glucose aerobically and anaerobically. Also, all strains produced PCR products from the 16S-23S internal transcribed spacer (ITS) DNA region of the predicted sizes using primers T5A and T3B designed for identification of E. cloacae (2). The growth of eight of the isolated strains and strains ATTC 23355 and 310 were evaluated on several carbon sources with RapiD 20E test strips (bio Mérieux, Inc, Durham, NC). All strains were positive for ß-d-galactosidase, ornithine decarboxylase, utilization of citrate and malonate, and production of acetoin. Hydrolysis of esculin by ß-glucosidase differed among the eight. All strains were negative for lysine decarboxylase, urease, para-phenylalanine deaminase, indole, and oxidase. All produced acid from arabinose, xylose, rhamnose, cellobiose, melibiose, saccharose, trehalose, raffinose, and glucose; no strains produced acid from adonitol. These characteristics are consistent with published data for E. cloacae. Surface-disinfested onion bulbs and sets were inoculated with 50 to 100 µl of bacterial suspensions containing ~108 CFU/ml, injected with hypodermic needles and syringes, and incubated at 37°C for 2 weeks. Bisected onions revealed dry brown discoloration in each of the four bulbs and sets that had been inoculated with each presumptive strain. Symptoms were indistinguishable from those apparent in onions inoculated with the authentic strains mentioned. Strains recovered on OEM were identified as E. cloacae based on the stated biochemical properties and analysis of the 16S rRNA gene amplified by PCR as above. The sequence of the amplicon from the isolated strains was identical to that of reference strains ATCC 23355 and 310. Amplicon sequences of the 16S rRNA gene of New York strains Ecl3, Ecl6, and Ecl7 were deposited in GenBank as JF832951, JF832952, and JF832953, respectively. The strains were accessioned as ATCC BAA-2271, ATCC BAA-2272, and ATCC BAA-2273, respectively. To our knowledge, this is the first published report of E. cloacae causing Enterobacter bulb decay of onion in New York. References: (1) A. L. Bishop and R. M. Davis. Plant Dis. 74:692, 1990. (2) M. M. Clementino et al. J. Clin. Microbiol. 39:3865, 2004. (3) B. K. Schroeder and L. J. du Toit. Plant Dis. 93:323, 2009. (4) H. F. Schwartz and K. Otto. Plant Dis. 84:808, 2000.

Complete genome sequence of the plant pathogen Erwinia amylovora strain ATCC 49946.

Erwinia amylovora causes the economically important disease fire blight that affects rosaceous plants, especially pear and apple. Here we report the complete genome sequence and annotation of strain ATCC 49946. The analysis of the sequence and its comparison with sequenced genomes of closely related enterobacteria revealed signs of pathoadaptation to rosaceous hosts.

First Report of Bulb Disease of Onion Caused by Pantoea ananatis in New York.

In winter 2007, disease symptoms were observed in stored yellow onion bulbs (Allium cepa) grown in New York (NY) in 2006. Similar symptoms were observed in bulbs produced in 2007, 2008, and 2009. Symptoms were associated with one to three bulb scales near the midsection. Infected scales were light brown to brown, not macerated, and lacking foul odors typical of onion bulbs infected with Burkholderia cepacia. Onion grower-packers located in Orange County, NY were concerned that onion lots were rejected following grading by inspectors who cut bulbs to check market quality. Extent of the problem statewide is not currently clear. Isolation attempts were made from symptomatic tissues onto nutrient agar plates (3), with incubation for 24 h at 26 to 28°C, and PA-20 (2), a semiselective medium for the isolation of Pantoea ananatis, with similar incubation for 4 to 6 days. Most strains that grew on PA-20 were gram negative and yellow pigmented with dark centers. Isolated strains were tentatively identified as P. ananatis on the basis of growth on PA-20, a positive indole and negative oxidase test, positive tests for catalase, fermentation of glucose, Voges-Proskauer, and citrate utilization; negative for phenylalanine deaminase, urease, nitrate reductase, methyl red tests, and hypersensitive response induction in tobacco. The BIOLOG (Hayward, CA) system indicated that all presumptive strains of P. ananatis utilized d-mannose, d-cellobiose, d-melibiose, l-inositol, d-arabinose, cellulose, glycerol, d-arabitol, and sucrose, but not glycogen, N-acetyl-d-galactosamine, malonic acid, l-fucose, or xylitol. Strains of P. ananatis recovered from diseased onions in Georgia (GA) (1) were included in all tests as positive controls. We used PCR primers suggested by R. D. Gitaitis (University of Georgia): PanITS1 (5'-GTC TGA TAG AAA GAT AAA GAC-3') and AS2b (5'-TTC ATA TCA CCT TAC CGG CGC-3'). Together, they amplify the 16S-23S rDNA internal transcribed spacer region of 398 bp; the nucleotide sequences of six NY and three GA strains are identical to each other and 99.3% identical to P. ananatis LMG 20103 (GenBank CP001875) and 93.3% identical to P. stewartii (AJ311838). Pathogenicity tests were done in onion leaves. For inoculation, strains were grown on nutrient agar for 24 h and bacterial suspensions of ~108 CFU/ml were prepared in sterile water. Tips of healthy, greenhouse-grown onion leaves were cut and inoculum was applied to the cut surfaces with cotton swabs. Plants were incubated in a greenhouse for up to 2 weeks. Plants mock inoculated with water were symptomless. Bacteria were recovered from all lesions induced by artificial inoculation with the presumptive strains of P. ananatis. Recovered bacteria had characteristics of P. ananatis. Pathogenic strains from NY and GA produced off-white lesions that extended the length of the leaf, which was consistent with previous studies of the pathogenicity of P. ananatis (1). On the basis of microbiological and molecular analyses and pathogenicity tests, 14 NY strains, each isolated from a different diseased bulb, were identified as P. ananatis. To our knowledge, this is the first published report of P. ananatis causing a disease of onion in New York. References: (1) R. D. Gitaitis et al. USA Crop Prot. 21:983, 2002. (2) T. Goszczynska et al. J. Microbiol. Methods. 64:22, 2006. (3) N. W. Shaad et al, eds. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2000.

Pantoea agglomerans strain EH318 produces two antibiotics that inhibit Erwinia amylovora in vitro.

Pantoea agglomerans (synonym: Erwinia herbicola) strain Eh318 produces through antibiosis a complex zone of inhibited growth in an overlay seeded with Erwinia amylovora, the causal agent of fire blight. This zone is caused by two antibiotics, named pantocin A and B. Using a genomic library of Eh318, two cosmids, pCPP702 and pCPP704, were identified that conferred on Escherichia coli the ability to inhibit growth of E. amylovora. The two cosmids conferred different antibiotic activities on E. coli DH5alpha and had distinct restriction enzyme profiles. A smaller, antibiotic-conferring DNA segment from each cosmid was cloned. Each subclone was characterized and mutagenized with transposons to generate clones that were deficient in conferring pantocin A and B production, respectively. Mutated subclones were introduced into Eh318 to create three antibiotic-defective marker exchange mutants: strain Eh421 (pantocin A deficient); strain Eh439 (pantocin B deficient), and Eh440 (deficient in both pantocins). Cross-hybridization results, restriction maps, and spectrum-of-activity data using the subclones and marker exchange mutants, supported the presence of two distinct antibiotics, pantocin A and pantocin B, whose biosynthetic genes were present in pCPP702 and pCPP704, respectively. The structure of pantocin A is unknown, whereas that of pantocin B has been determined as (R)-N-[((S)-2-amino-propanoylamino)-methyl]-2-methanesulfonyl-s uccina mic acid. The two pantocins mainly affect other enteric bacteria, based on limited testing.

Regulation of hrp genes and type III protein secretion in Erwinia amylovora by HrpX/HrpY, a novel two-component system, and HrpS.

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.

Riboflavin induces disease resistance in plants by activating a novel signal transduction pathway.

ABSTRACT The role of riboflavin as an elicitor of systemic resistance and an activator of a novel signaling process in plants was demonstrated. Following treatment with riboflavin, Arabidopsis thaliana developed systemic resistance to Peronospora parasitica and Pseudomonas syringae pv. Tomato, and tobacco developed systemic resistance to Tobacco mosaic virus (TMV) and Alternaria alternata. Riboflavin, at concentrations necessary for resistance induction, did not cause cell death in plants or directly affect growth of the culturable pathogens. Riboflavin induced expression of pathogenesis-related (PR) genes in the plants, suggesting its ability to trigger a signal transduction pathway that leads to systemic resistance. Both the protein kinase inhibitor K252a and mutation in the NIM1/NPR1 gene which controls transcription of defense genes, impaired responsiveness to riboflavin. In contrast, riboflavin induced resistance and PR gene expression in NahG plants, which fail to accumulate salicylic acid (SA). Thus, riboflavin-induced resistance requires protein kinase signaling mechanisms and a functional NIM1/NPR1 gene, but not accumulation of SA. Riboflavin is an elicitor of systemic resistance, and it triggers resistance signal transduction in a distinct manner.

Harpin induces disease resistance in Arabidopsis through the systemic acquired resistance pathway mediated by salicylic acid and the NIM1 gene.

Harpin, the product of the hrpN gene of Erwinia amylovora, elicits the hypersensitive response and disease resistance in many plants. Harpin and known inducers of systemic acquired resistance (SAR) were tested on five genotypes of Arabidopsis thaliana to assess the role of SAR in harpin-induced resistance. In wild-type plants, harpin elicited systemic resistance to Peronospora parasitica and Pseudomonas syringae pv. tomato, accompanied by induction of the SAR genes PR-1 and PR-2. However, in experiments with transgenic Arabidopsis plants containing the nahG gene which prevents accumulation of salicylic acid (SA), harpin neither elicited resistance nor activated SAR gene expression. Harpin also failed to activate SAR when applied to nim1 (non-inducible immunity) mutants, which are defective in responding to SA and regulation of SAR. In contrast, mutants compromised in responsiveness to methyl jasmonate and ethylene developed the same resistance as did wild-type plants. Thus, harpin elicits disease resistance through the NIM1-mediated SAR signal transduction pathway in an SA-dependent fashion. The site of action of harpin in the SAR regulatory pathway is upstream of SA.

Assessment of Phenotypic Variability in Erwinia stewartii Based on Metabolic Profiles.

One hundred twenty-four bacterial isolates originating from sweet corn or corn flea beetles in the northeastern, midwestern, and mid-Atlantic United States were verified as Erwinia stewartii (Pantoea stewartii subsp. stewartii) and characterized phenotypically by their respiratory response to 91 carbon sources. The unweighted pair group method of averages (UPGMA) was used to construct a dendrogram that revealed homogeneous metabolic profiles at 93% similarity. Two-thirds of the isolates formed 18 separate groups, each sharing the same metabolic profile. One-third of the isolates had distinct metabolic profiles. Most groups shared either isolation source, geographical location, and/or year of isolation. Members of some groups persisted through time and had been isolated from diverse geographical locations. Four representative strains of the proposed Pantoea stewartii subsp. indologenes were also characterized; their metabolic profiles were most similar to those of Erwinia herbicola (Pantoea agglomerans).

HrpW of Erwinia amylovora, a new harpin that contains a domain homologous to pectate lyases of a distinct class.

Harpins, such as HrpN of Erwinia amylovora, are extracellular glycine-rich proteins that elicit the hypersensitive reaction (HR). We identified hrpW of E. amylovora, which encodes a protein similar to known harpins in that it is acidic, rich in glycine and serine, and lacks cysteine. A putative HrpL-dependent promoter was identified upstream of hrpW, and Western blot analysis of hrpL mutants indicated that the production of HrpW is regulated by hrpL. HrpW is secreted via the Hrp (type III) pathway based on analysis of wild-type strains and hrp secretion mutants. When infiltrated into plants, HrpW induced rapid tissue collapse, which required active plant metabolism. The HR-eliciting activity was heat stable and protease sensitive. Thus, we concluded that HrpW is a new harpin. HrpW of E. amylovora consists of two domains connected by a Pro and Ser-rich sequence. A fragment containing the N-terminal domain was sufficient to elicit the HR. Although no pectate lyase activity was detected, the C-terminal region of HrpW is homologous to pectate lyases of a unique class, suggesting that HrpW may be targeted to the plant cell wall. Southern analysis indicated that hrpW is conserved among several Erwinia species, and hrpW, provided in trans, enhanced the HR-inducing ability of a hrpN mutant. However, HrpW did not increase the virulence of a hrpN mutant in host tissue, and hrpW mutants retained the wild-type ability to elicit the HR in nonhosts and to cause disease in hosts.

The hrpC and hrpN operons of Erwinia chrysanthemi EC16 are flanked by plcA and homologs of hemolysin/adhesin genes and accompanying activator/transporter genes.

The hrpC operon of Erwinia chrysanthemi EC16 encodes five genes conserved in Erwinia amylovora and Pseudomonas syringae. Mutagenesis indicated that hrcC is required for elicitation of the hypersensitive reaction in tobacco leaves. The unexpected presence of plcA and homologs of hemolysin/activator genes in the regions flanking the hrcC and hrpN operons is reported.

Erwinia amylovora secretes DspE, a pathogenicity factor and functional AvrE homolog, through the Hrp (type III secretion) pathway.

Erwinia amylovora was shown to secrete DspE, a pathogenicity factor of 198 kDa and a functional homolog of AvrE of Pseudomonas syringae pv. tomato. DspE was identified among the supernatant proteins isolated from cultures grown in an hrp gene-inducing minimal medium by immunodetection with a DspE-specific antiserum. Secretion required an intact Hrp pathway.

Homology and functional similarity of an hrp-linked pathogenicity locus, dspEF, of Erwinia amylovora and the avirulence locus avrE of Pseudomonas syringae pathovar tomato.

The "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 alpha helices in their C termini; they resemble chaperones for virulence factors secreted by type III secretion systems of animal pathogens.

The hrpA and hrpC operons of Erwinia amylovora encode components of a type III pathway that secretes harpin.

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-N17-18-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 Rhizobim 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.

Characterization of Erwinia amylovora strains using random amplified polymorphic DNA fragments (RAPDs).

The genetic diversity among 16 strains of Erwinia amylovora, chosen to represent different host plant origins and geographical regions, was investigated by RAPD analysis. One strain of Erwinia herbicola and one of Agrobacterium vitis were used as outgroups. Ninety-eight different RAPD fragments were produced by polymerase chain reaction amplification with six different 10-mer primers. RAPD banding profiles were found that enabled the Erw. amylovora strains to be distinguished from one another. Cluster analysis based on the number of RAPD fragments shared between strains showed that strains of Erw. amylovora isolated from subfamily Pomoideae formed a single group, whereas two strains from Rubus (subfamily Rosoideae) formed a second group. Two strains isolated from Asian pear on Hokkaido, Japan, formed a third group. Sets of RAPD fragments were identified that enabled each of the two host-range groups and one geographical region (Hokkaido) of Erw. amylovora strains to be unambiguously distinguished from one another and from the outgroups. This study shows that strains of Erw. amylovora exhibit genetic diversity detectable by RAPD analysis, and that molecular and statistical analysis of RAPD fragments can be used both to distinguish between strains and to determine relatedness between them.

Erwinia amylovora secretes harpin via a type III pathway and contains a homolog of yopN of Yersinia spp.

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 or 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.