Steven V. Beer

Unified nomenclature for broadly conserved hrp genes of phytopathogenic bacteria

Genes of plant-pathogenic bacteria controlling hypersensitive response (HR) elicitation and pathogenesis were designated ‘hrp’ by Lindgren et al. in 1986 (J Bacteriol 168: 512–522). hrp genes have been characterized in several species of the four major genera of Gramnegative plant pathogens, Erwinia, Pseudomonas, Ralstonia (a new proposed genus including Pseudomonas solanacearum) and Xanthomonas. To date, hrp genes have been found mainly in large clusters, and they have been shown to be conserved physically and, in many cases, functionally among different bacteria. Hybridization studies and genetic analyses have revealed the presence of functional hrp genes even in species that are not typically observed to elicit an HR, such as Erwinia chrysanthemi and Erwinia stewartii, suggesting that hrp genes may be common to all Gram-negative plant pathogens, possibly excluding Agrobacterium spp. Current knowledge of hrp genes has been reviewed by Bonas (1994, Curr Top Microbiol Immunol 192: 79–98) and by Van Gijsegem et al. (1995, In Pathogenesis and Host–Parasite Specificity in Plant Diseases: Histopathological, Biochemical, Genetic and Molecular Basis. Volume 1. (Kohmoto et al., eds); Oxford: Pergamon Press, pp. 273–292). The nucleotide sequences of four hrp gene clusters, those of Ralstonia solanacearum (previously P. solanacearum) (Genin et al., 1992, Mol Microbiol 6: 3065–3076; Gough et al., 1992, Mol Plant–Microbe Interact 5: 384–389; Gough et al., 1993, Mol Gen Genet 239: 378–392; Van Gijsegem et al., 1995, Mol Microbiol 15: 1095–1114), Erwinia amylovora (Bogdanove et al., 1996, J Bacteriol 178: 1720– 1730; Wei and Beer, 1993, J Bacteriol 175: 7958–7967; Wei and Beer, 1995, J Bacteriol 177: 6201–6210; Wei et al., 1992, Science 257: 85–88; S. V. Beer, unpublished), Pseudomonas syringae pv. syringae (Huang et al., 1992, J Bacteriol 174: 6878–6885; Huang et al., 1993, Mol Plant–Microbe Interact 6: 515–520; Huang et al., 1995, Mol Plant–Microbe Interact 8: 733–746; Lidell and Hutcheson, 1994, Mol Plant–Microbe Interact 7: 488–497; Preston et al., 1995, Mol Plant–Microbe Interact 8: 717–732; Xiao et al., 1994, J Bacteriol 176: 1025–1036), and Xanthomonas campestris pv. vesicatoria (Fenselau et al., 1992, Mol Plant–Microbe Interact 5: 390–396; Fenselau and Bonas, 1995, Mol Plant–Microbe Interact 8: 845–854; U. Bonas, unpublished), have been largely determined. These clusters each contain more than twenty genes, many of which encode components of a novel proteinsecretion pathway designated ‘type III’. It has been shown directly that various extracellular proteins involved in pathogenesis and defence elicitation by plantpathogenic bacteria utilize this pathway (Arlat et al., 1994, EMBO J 13: 543–553; He et al., 1993, Cell 73: 1255–1266; Wei and Beer, 1993, ibid.), and the pathway is known to function in the export of virulence factors from the animal pathogens Salmonella typhimurium, Shigella flexneri, and Yersinia entercolitica, Yersinia pestis, and Yersinia pseudotuberculosis (for reviews, see Salmond and Reeves, 1993, Trends Biochem Sci 18: 7–12; and Van Gijsegem et al., 1993, Trends Microbiol 1: 175– 180). Nine type III secretion genes are conserved among all four of the plant pathogens listed above and among the animal pathogens. Based on sequence analysis and some experimental evidence, they are believed to encode one outer-membrane protein, one outer-membrane-associated lipoprotein, five inner-membrane proteins, and two cytoplasmic proteins, one of which is a putative ATPase. All of the predicted gene products, except the outer-membrane protein, show significant similarity to components of the flagellar biogenesis complex (for reviews see Blair, 1995, Annu Rev Microbiol 49: 489–522; and Bischoff and Ordal, 1992, Mol Microbiol 6: 23–28). We herein refer to the hrp-encoded type III pathway as the ‘Hrp pathway’. Because hrp genes have been characterized independently in diverse plant-pathogenic bacteria, hrp gene nomenclature differs in different species, and it is not always consistent even within the same organism. Different designations are used for homologous genes, and, even worse, the same designation is used for different genes in different organisms. For example, hrpI of E. amylovora is homologous with hrpC2 of X. campestris pv. vesicatoria and hrpO of R. solanacearum, and the homologue in P. syringae pv. syringae appears in the literature both as hrpI and as hrpJ2. Also, ‘hrpN ’ in R. solanacearum designates a secretion-pathway gene, whereas in E. amylovora, ‘hrpN ’ designates the gene encoding the elicitor harpin. Furthermore, in many bacteria the number of known hrp genes approaches 26. In anticipation of exhausting the alphabet, some authors chose to designate hrp genes with a letter and a number, creating the potential for confusion of distinct genes with alleles of the same gene. For hrp gene researchers, the current nomenclature is at best inconvenient; for other scientists, it is bewildering. Another problem exists: accumulation of knowledge about the structure of hrp loci has outpaced the accumulation of Molecular Microbiology (1996) 20(3), 681–683

Erwinia chrysanthemi HarpinEch: an elicitor of the hypersensitive response that contributes to soft-rot pathogenesis

Mutants of the soft-rot pathogen Erwinia chrysanthemi EC16 that are deficient in the production of the pectate lyase isozymes PelABCE can elicit the hypersensitive response (HR) in tobacco leaves. The hrpNEch gene was identified in a collection of cosmids carrying E. chrysanthemi hrp genes by its hybridization with the Erwinia amylovora hrpNEa gene. hrpNEch appears to be in a monocistronic operon, and it encodes a predicted protein of 340 amino acids that is glycine-rich, lacking in cysteine, and highly similar to HrpNEa in its C-terminal half. Escherichia coli DH5 alpha cells expressing hrpNEch from the lac promoter of pBluescript II accumulated HrpNEch in inclusion bodies. The protein was readily purified from cell lysates carrying these inclusion bodies by solubilization in 4.5 M guanidine-HCl and reprecipitation upon dialysis against dilute buffer. HrpNEch suspensions elicited a typical HR in tobacco leaves, and elicitor activity was heat-stable. Tn5-gusA1 mutations were introduced into the cloned hrpNEch and then marker-exchanged into the genomes of E. chrysanthemi strains AC4150 (wild type), CUCPB5006 (delta pelABCE), and CUCPB5030 (delta pelABCE outD::TnphoA). hrpNEch::Tn5-gusA1 mutations in CUCPB5006 abolished the ability of the bacterium to elicit the HR in tobacco leaves unless complemented with an hrpNEch subclone. An hrpNEch::Tn5-gusA1 mutation also reduced the ability of AC4150 to incite infections in witloof chicory leaves, but it did not reduce the size of lesions that did develop. Purified HrpNEch and E. chrysanthemi strains CUCPB5006 and CUCPB5030 elicited HR-like necrosis in leaves of tomato, pepper, African violet, petunia, and pelargonium, whereas hrpNEch mutants did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Erwinia chrysanthemi hrp genes and their involvement in soft rot pathogenesis and elicitation of the hypersensitive response

Unlike the bacterial pathogens that typically cause the hypersensitive response (HR) in plants, Erwinia chrysanthemi has a wide host range, rapidly kills and macerates host tissues, and secretes several isozymes of the macerating enzyme pectate lyase (Pel). PelABCE- and Out- (secretion-deficient) mutants were observed to produce a rapid necrosis in tobacco leaves that was indistinguishable from the HR elicited by the narrow-host-range pathogens E. amylovora Ea321 and Pseudomonas syringae pv. syringae 61. E. amylovora Ea321 hrp genes were used to identify hybridizing cosmids in a cosmid library of E. chrysanthemi EC16 DNA in Escherichia coli. A 16-kb BamHI fragment in one of these cosmids, pCPP2030, hybridized with E. amylovora hrp genes and was mutagenized with Tn10mini-kan. The mutations were introduced into the PelABCE- mutant CUCPB5006 by marker exchange. Two of the resultant hrp::Tn10mini-kan mutations were found to abolish the ability of CUCPB5006 to cause any necrosis in tobacco leaves unless complemented with pCPP2030. These two mutations were also marker-exchanged into the genome of wild-type strain AC4150. Analysis of DNA sequences flanking the hrp-2::Tn10mini-kan insertion revealed the mutated gene to be similar to a gene in E. amylovora Ea321 hrp complementation group VIII and to P. s. pv. syringae 61 hrpX. Neither of the hrp::Tn10mini-kan mutations affected the production or secretion of pectic enzymes by AC4150 or CUCPB5006. However, the hrp mutations reduced the ability of both AC4150 and CUCPB5006 to incite successful infections in witloof chicory leaves.(ABSTRACT TRUNCATED AT 250 WORDS)

Pantoea agglomerans Strain EH318 Produces Two Antibiotics That Inhibit Erwinia amylovora In Vitro

ABSTRACT 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 ofE. amylovora. The two cosmids conferred different antibiotic activities on E. coli DH5α 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-succinamic acid. The two pantocins mainly affect other enteric bacteria, based on limited testing.

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.

Downstream Divergence of the Ethylene Signaling Pathway for Harpin-Stimulated Arabidopsis Growth and Insect Defense

Ethylene (ET) signal transduction may regulate plant growth and defense, depending on which components are recruited into the pathway in response to different stimuli. We report here that the ET pathway controls both insect resistance (IR) and plant growth enhancement (PGE) in Arabidopsis (Arabidopsis thaliana) plants responding to harpin, a protein produced by a plant pathogenic bacterium. PGE may result from spraying plant tops with harpin or by soaking seeds in harpin solution; the latter especially enhances root growth. Plants treated similarly develop resistance to the green peach aphid (Myzus persicae). The salicylic acid pathway, although activated by harpin, does not lead to PGE and IR. By contrast, PGE and IR are induced in both wild-type plants and genotypes that have defects in salicylic acid signaling. In response to harpin, levels of jasmonic acid (JA) decrease, and the COI1 gene, which is indispensable for JA signal transduction, is not expressed in wild-type plants. However, PGE and IR are stimulated in the JA-resistant mutant jar1-1. In the wild type, PGE and IR develop coincidently with increases in ET levels and the expression of several genes essential for ET signaling. The ET receptor gene ETR1 is required because both phenotypes are arrested in the etr1-1 mutant. Consistently, inhibition of ET perception nullifies the induction of both PGE and IR. The signal transducer EIN2 is required for IR, and EIN5 is required for PGE because IR and PGE are impaired correspondingly in the ein2-1 and ein5-1 mutants. Therefore, harpin activates ET signaling while conscribing EIN2 and EIN5 to confer IR and PGE, respectively.

Alternative disease control agents induce resistance to blue mold in harvested 'red delicious' apple fruit.

ABSTRACT Alternative control agents, including UV-type C (254 nm) irradiation, yeasts antagonistic to fungal growth, chitosan and harpin, were evaluated for their ability to induce resistance in cv. Red Delicious apple fruit against postharvest blue mold caused by Penicillium expansum. Freshly harvested and controlled atmosphere (CA)-stored fruit were treated with these agents at different doses and concentrations or with paired combinations of the agents. Treated fruit were inoculated with P. expansum 24, 48, or 96 h following treatment, and stored at 24 degrees C in the dark. The fruit were evaluated for development of disease every 2 days for 14 days by measuring the diameter of lesions that formed. The area under the disease progress curve (AUDPC) was calculated and analyzed statistically. All treatments were effective in reducing the AUDPC; UV-C was most effective, followed by harpin, chitosan, and the yeasts, respectively. Regardless of treatment, fresh fruit were more responsive to treatments than CA-stored fruit. There was a clear time-dependent response of the fruit to the treatments, in which treatments applied 96 h before inoculation provided the best results. In a few situations, the combinations of agents did provide an additive effect, but no synergistic effects were detected. Moreover, disease severity in fruit treated by any combination was markedly better than that in the controls. Although the combinations of treatments was overall less effective than the single treatments, they did provide significant reductions of the progress of disease in comparison with the controls. Because the fungus did not come into contact with any of the control agents, this study showed conclusively that the agents studied were able to induce resistance in the fruit rather than merely inhibit the pathogen directly. It also showed, for the first time, that harpin is able to induce resistance in harvested apple fruit. The use of these control agents may minimize the costs of control strategies and reduce the risks associated with the excessive use of fungicides in harvested apple fruit.

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.

Harpin-elicited hypersensitive cell death and pathogen resistance require the NDR1 and EDS1 genes

Abstract Plants sprayed with harpin, a bacterial protein that induces hypersensitive cell death (HCD), develop systemic acquired resistance (SAR) without macroscopic necrosis. HCD sometimes accompanies the development of resistance conferred by resistance ( R ) genes. In Arabidopsis , some R genes require one or both of the signalling components NDR1 and EDS1 for function. This study addresses whether HCD, NDR1 and EDS1 are required for induction of SAR by harpin. When Arabidopsis and tobacco leaves were sprayed with harpin, microscopic hypersensitive response (micro-HR) lesions developed. Systemic expression of PR genes and the development of resistance were accompanied by micro-HR, except in the ndr 1-1 mutant, in which harpin induced micro-HR without the development of resistance or expression of the PR -1 gene. Cell death and resistance did not occur following treatment with harpin in plants that could not accumulate salicylic acid. Harpin also failed to induce resistance in Arabidopsis eds 1-1 mutants. Therefore, harpin-induced resistance seems to develop concomitantly with cell death and resistance requires NDR 1 and EDS 1.

The Hrp pathogenicity island of Erwinia amylovora and identification of three novel genes required for systemic infection

SUMMARY Sequence analysis of the region bordering the hrp/dsp gene cluster of Erwinia amylovora strain Ea321, which causes fire blight, revealed characteristics of pathogenicity islands (PAIs). Included are genes for a phage integrase, a tRNA(Phe), several orthologues of genes of YAPI, a PAI of Yersinia pseudotuberculosis, and several putative virulence genes with HrpL-dependent promoter motifs. The island is designated the Hrp PAI of E. amylovora. It is comprised of a chromosomal region of c. 62 kb with 60 open reading frames (ORFs). Comparison of the Hrp PAI of E. amylovora with those of four closely related bacteria showed that orfB, a homologue of avrBsT of Xanthomonas campestris pv. vesicatoria, and orfA, its putative chaperone gene, are present only in the Hrp PAI of E. amylovora. As regions flanking the hrp/dsp gene cluster are quite diverse, addition and deletion may have occurred during divergent evolution of the five bacteria. Among ORFs of the PAI of Ea321, three new HrpL-dependent genes were identified. Because they are required for full virulence in apple, they were designated hsvC, hsvB and hsvA (hrp-associated systemic virulence). They encode a homologue of an amidinotransferase for phaseolotoxin biosynthesis and homologues of a nikkomycin-biosynthetic protein of Pseudomonas syringae.