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329 Determinants of pathogenicity and avirulence in plant pathogenic bacteria Alan Collmer Many plant pathogenic bacteria possess a conserved protein secretion system that is thought to transfer Avr (avirulence) proteins, with potential activities in both parasitism and defense elicitation, into plant cells. avr genes may be acquired horizontally by these bacteria, and avr gene compositions are highly variable. In the past year, heterologous expression experiments have revealed that the products of avr genes can be interchanged among different genera of bacteria with retention of secretion, pathogenicity, and avirulence activities, suggesting mechanisms for rapid coevolution of these parasites with changing plant hosts. Addresses Department of Plant Pathology, Cornell University, Ithaca, NY 14853-4203, USA; e-mail: [email protected] Current Opinion in Plant Biology 1998, 1:329–335 http://biomednet.com/elecref/1369526600100329 Current Biology Ltd ISSN 1369-5266 Abbreviations Avr avirulence HR hypersensitive response hrp hypersensitive response and pathogenicity pv pathovar R resistance Yop Yersinia outer protein Introduction The most common bacterial pathogens of plants colonize the apoplast, and from this location outside the walls of living cells they incite a variety of diseases in most cultivated plants [1]. The majority of these pathogens are Gram-negative bacteria in the genera Erwinia, Pseudomonas, Xanthomonas, and Ralstonia. Most are host-specific and will elicit the hypersensitive response (HR) in nonhosts. The HR is a rapid, programmed death of plant cells in contact with the pathogen. Some of the defense responses associated with the HR are localized at the periphery of plant cells at the site of bacterial contact, but what actually stops bacterial growth has not been established [2,3,4••]. Pathogenesis in host plants, in contrast, involves prolonged bacterial multiplication, spread to surrounding tissues, and the eventual production of macroscopic symptoms characteristic of the disease. Although these bacteria are diverse in their taxonomy and pathology, they all possess hypersensitive response and pathogenicity (hrp) genes which direct their ability to elicit the HR in nonhosts or to be pathogenic (and parasitic) in hosts [5•]. The hrp genes encode a type III protein-secretion system that appears to be capable of delivering Avr (avirulence) proteins across the walls and plasma membranes of living plant cells [6•]. The hrp genes encode a type III protein secretion system that appears to be capable of delivering Avr proteins across the walls and plasma membranes of living plant cells [6•]. Bacterial type III protein secretion systems are characterized by an ability to inject effector proteins into host cells, a membrane translocation apparatus containing several flagellum biogenesis homologs, and a lack of processed amino-terminal signal peptides on the secreted proteins. The Avr proteins are so named because they can betray the parasite to the resistance (R) gene-encoded surveillance system of plants, thereby triggering the HR [7•,8]. But Avr proteins also appear to be key to parasitism in ‘compatible’ host plants, where the parasite proteins are undetected and the HR is not triggered. Thus, bacterial avirulence and pathogenicity are inter-related phenomena and explorations of HR elicitation are furthering our understanding of parasitic mechanisms. Proteins delivered by the Hrp system are not the only molecular weapons in pathogen arsenals; toxins, phytohormones, and enzymes that degrade the plant cell wall often contribute significantly to the full expression of symptoms [1]. The Hrp system and its protein traffic, however, appear to underlie basic parasitism, and this article will focus on that aspect of pathogenesis. The succession of publications in 1996 providing evidence that Avr proteins indeed function inside plant cells following delivery by the Hrp system has been extensively reviewed [5•–7•,9,10•,11•,12,13,14•]. This article will focus on more recent reports concerning the operation and ubiquity of Hrp systems, novel extracellular Hrp proteins, and the secretion, virulence functions, and potential interchangeability of Avr proteins. Hrp systems Type III protein secretion systems are present in bacterial pathogens of both animals and plants, and are exemplified by the type III system of Yersinia spp. [15,16•]. These animal pathogens are primarily extracellular parasites, and their Yops (Yersinia outer proteins) are secreted and translocated directly into host cells in a contact-dependent manner [16•]. A similar host-contact dependency may operate in most plant pathogenic bacteria. Nine of the hrp genes are universal components of type III secretion systems, and these have been renamed hrc (HR and conserved) and given the last-letter designation of their Yersinia homolog (with the exception of hrcV) [17]. The Hrc proteins enable protein movement across the bacterial inner and outer membranes independently of the general protein export (Sec) pathway [18]. In contrast to the Hrc proteins, the Hrp proteins may be peripheral components 330 Host–microbe interactions of the Hrp secretion system and are more likely to perform type III secretion functions that are extracellular and specific to protein transfer across the plant cell wall and plasma membrane (discussed below). The genes encoding type III secretion systems are usually clustered, and the emerging concept that genes with related functions in virulence are often grouped on plasmids or in horizontally acquired pathogenicity islands has important implications throughout pathogenic microbiology [19,20,21••]. Some pathogenicity islands govern key steps in pathogenesis, such as the entry of Salmonella into epithelial cells, and differences in codon usage and GC content between genes in the island and those in the rest of the genome provide part of the evidence that these islands are obtained by horizontal transfer from other bacteria. There is some evidence for horizontal acquisition of hrp gene clusters in plant pathogenic bacteria, and the hrp cluster in Ralstonia solanacearum is carried on a megaplasmid [1]. The finding of a plasmid-borne hrp gene cluster in Erwinia herbicola pv. gypsophilae suggests that virulence may be acquired readily by plant-associated bacteria [22•]. E. herbicola is a common epiphyte that is usually benign, but strains classified as E. herbicola pv. gypsophilae cause galls on gypsophila and, like many plant pathogenic bacteria, can elicit the HR in tobacco. A 150 kb plasmid carries phytohormone biosynthetic genes and hrp genes, and the latter are required for both gall formation and HR elicitation [22•]. The clustering of genes with related functions is also consistent with the ability of some cloned hrp clusters to enable nonpathogens like Escherichia coli to elicit the HR. This has been reported for cosmids pHIR11 from Pseudomonas syringae pv. syringae, pCPP430 from Erwinia amylovora [1], pPPY430 from P. syringae pv. phaseolicola [23], and pCPP2156 from Erwinia chrysanthemi [24••]. Although these cosmids support heterologous HR elicitation, they do not enable E. coli to become pathogenic. The basis for HR elicitation is best understood with pHIR11. The cosmid carries a 25 kb set of hrp genes that is intact and functional, as revealed by DNA sequencing and the ability to direct secretion of the HrpZ harpin (a protein of unknown function that appears to be targeted to the plant cell wall, as discussed below) [6•]. The cosmid also carries, adjacent to the hrp cluster, the hrmA gene, which is avr-like in producing an avirulence phenotype when expressed in a tobacco pathogen and in being lethal when heterologously expressed inside tobacco cells [25•]. The concept that the minimal requirement for bacterial elicitation of the HR is a functional Hrp system and an avr gene whose product is recognized by the R-gene surveillance system of the test plant is supported by experiments in which the HR is observed only when an appropriate, heterologous avr gene is supplied in trans of the hrp+ cosmid [23,24••,26,27]. Hrp regulation The hrp genes are expressed when bacteria are inoculated into plants or are growing in apoplast-mimicking minimal media but not usually in complex bacteriological media [5•]. The Hrp regulatory systems in plant pathogenic bacteria can be divided into two groups, which correspond also to differences in hrp cluster composition [6•]. In the group I Hrp systems of Erwinia and Pseudomonas, hrp operons are activated by HrpL, a sigma factor [5•,13]. In contrast, hrp transcription in the group II Hrp systems of Xanthomonas (HrpX) and Ralstonia (HrpB) is activated by an AraC homolog [5•]. Upstream activators of these two factors have been described for P. syringae (HrpR and HrpS) [5•,13], Xanthomonas campestris pv. vesicatoria (HrpG) [28], and R. solanacearum (PrhA) [29••]. The recent discovery of PrhA is particularly significant because this putative outer membrane protein, which appears to act at the top of the Hrp regulatory hierarchy, is required specifically for induction of hrp genes in the presence of plant cells and for full virulence in Arabidopsis [29••]. In the host-promiscuous pathogen E. carotovora, production of the hrpN-encoded harpin is activated by the quorum (cell density) sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone, and negatively regulated by RsmA, which are two global regulators that similarly control exoenzyme production [30•,31]. Although the ability to alter hrp expression through genetic manipulation or appropriate media has been experimentally useful, our knowledge of Hrp regulation is still fundamentally incomplete regarding the inventory of regulatory components, regulation in planta, and the presumed contact-dependent activation of Avr protein transfer. Extracellular Hrp proteins Two classes of extracellular Hrp proteins have now been defined — harpins and pilins. Harpins are glycine-rich proteins that lack cysteine, are secreted in culture when the Hrp system is expressed, and possess heat-stable HR elicitor activity when infiltrated into the leaves of tobacco and several other plants [1]. Mutation of the prototypical hrpN harpin gene in E. amylovora strain Ea321 strongly diminishes HR and pathogenicity phenotypes [32•], but mutation of the hrpZ harpin gene in various P. syringae strains has little or no effect on Hrp phenotypes [33,34•]. The natural function of harpins and the basis for their ability to elicit an apparent programmed cell death when artificially introduced into the apoplast of plants is unknown. Two lines of evidence, however, point to a site of action in the plant cell wall. First, purified P. syringae harpin binds to cell walls and has biological activity only with walled cells [35]. Second, HrpW, another harpin discovered in both E. amylovora and P. syringae, has an amino-terminal half that is harpin-like but a carboxy-terminal half that is homologous to a newly defined class of pectate lyases found in fungal and bacterial pathogens [32•,34•]. Elicitor activity resides in the harpin Determinants of pathogenicity and avirulence in plant pathogenic bacteria Collmer domain, and the pectate lyase domain, although lacking enzymatic activity, binds specifically to pectate [34•]. The second class of extracellular Hrp proteins is represented by the P. syringae HrpA pilin, which is a subunit of an Hrp pilus that is 6–8 nm in diameter and is formed on bacteria in an Hrp-dependent manner [36••]. The Hrp pilus is required for pathogenicity and elicitation of the HR, and a similar structure is important for T-DNA transfer in Agrobacterium tumefaciens [37]. Whether these structures promote the transfer of bacterial macromolecules into plant cells by serving as conduits, guides, or attachment factors is not known. Avr proteins as swappable, secreted, virulence factors A current model for plant–bacterium interaction and coevolution based on Hrp delivery of Avr proteins into plant cells (Figure 1) proposes firstly that Avr-like proteins are the primary effectors of parasitism, secondly that conserved Hrp systems are capable of delivering many, diverse Avr-like proteins into plant cells, and thirdly that genetic changes in host populations that reduce the parasitic benefit of an effector protein or allow its recognition by the R-gene surveillance system will lead to a proliferation of complex arsenals of avr-like genes in coevolving bacteria [1]. There are still many gaps in this picture. For example, the physical transfer of Avr proteins into plant cells has never been observed, the virulence functions of ‘Avr’ proteins are unknown, and it is likely that previous searches for avr genes in various bacteria have yielded incomplete inventories of the genes encoding effector proteins. Recent progress, however, has been made in each area. Avr proteins had not previously been reported outside of the cytoplasm of living P. syringae and Xanthomonas spp. cells [8,23], but it now appears that the Hrp systems of Erwinia spp. can secrete Avr proteins in culture. A homolog of the P. syringae pv. tomato avrE gene has been found in E. amylovora and designated dspA in strain CFBP1430 and dspE in strain Ea321 [38••,39••]. The dsp (disease specific) genes are required for the pathogenicity of E. amylovora, but not for HR elicitation. A protein of the size expected for DspA is secreted in a Hrp- and DspB-dependent manner by CFBP1430 (DspB is a potential chaperone required for DspA secretion) [38••]. Specific antibodies were used to demonstrate unambiguously that DspE is efficiently secreted in a Hrp-dependent manner by strain Ea321 [40••]. Nothing is known of the localization or expected site of action of AvrE. There is strong evidence, however, that the site of action of the P. syringae AvrB and AvrPto proteins is inside plant cells (e.g. see [10•]), and both proteins have now been found to be secreted by an E. chrysanthemi Hrp system functioning heterologously in E. coli [24••]. This secretion is Hrp-dependent, and E. coli cells carrying the E. chrysanthemi hrp genes also elicit an avrB-dependent HR 331 in appropriate test plants. A strong implication of this work is that E. chrysanthemi, which is a host-promiscuous soft-rot pathogen, also carries avr-like genes. The ability of the cloned E. chrysanthemi Hrp system to secrete P. syringae Avr proteins should promote searches for additional avr-like genes by providing an assay that can be performed in culture and is independent of the requirement for test plants that happen to have a corresponding R gene, and it will enable direct investigation of Avr targeting signals and secretion mechanisms. For example, chaperone-independent targeting information in two Yop proteins has been shown to reside in the mRNA encoding the amino terminus of the protein [41••]. The involvement of similar signals in Avr secretion is suggested by the need for continued protein (but not mRNA) synthesis in planta for Avr signal delivery, which would be consistent with a cotranslational secretion process [23]. The biochemical activities or parasite-promoting functions of Avr proteins remain unclear, although several of those known make measurable contributions to virulence [8]. Members of the AvrBs3 family in Xanthomonas spp. are targeted to the plant cell nucleus [10•,42], and some of these have been shown recently to redundantly direct the production of watersoaking symptoms associated with virulence [43]. AvrD (P. syringae pv. tomato) directs the synthesis of syringolide elicitors of the HR [8]; AvrBs2 (X. campestris pv. vesicatoria) shows similarity to A. tumefaciens agrocinopine synthase, which enables crown gall tumors to produce a specialized carbon source for utilization by A. tumefaciens [44]; and AvrRxv (X. campestris pv. vesicatoria) is a homolog of AvrA (Salmonella typhimurium) and YopJ (Yersina spp.), proteins which travel the type III pathway in animal pathogens and trigger apoptosis in macrophages [45,46]. This last observation has led to the suggestion that avr–R gene interactions may occur also in animal pathogenesis [47•]. The primary sequences of the P. syringae Avr proteins reveal little about their potential function, but interestingly, when heterologously expressed in plants, three of them have produced necrosis in test plants lacking the cognate R gene [26,48•,49]. A key question is whether this results from interaction of abnormally high levels of the bacterial protein with plant virulence targets or from interaction with cross-reacting R-gene products. Further evidence suggesting that some avr genes in P. syringae are beneficial to the bacteria in host plants was found in recent studies of avrD and avrPphE; highly conserved, nonfunctional alleles of these genes have been retained in pathogens whose hosts would recognize the functional Avr product [48•,50•]. Avr-like genes may function heterologously to support pathogenesis as well as HR elicitation. The pathogenicity of an E. amylovora dspE mutant can be restored (at least partially) by a plasmid carrying the P. syringae avrE locus, suggesting that DspE and AvrE have similar functions 332 Host–microbe interactions Figure 1 avr Horizontal avr transfer with other pathogens? Hypervariable region with many avr genes Conserved hrp/hrc gene cluster Region with hrpW and additional avr genes Avr proteins IM OM } N RSTUV J C Hrc proteins in Hrp (type III) secretion apparatus HrpA pilus CW ? HrpZ HrpW } Harpins Conclusions PM Plant R-gene directed surveillance system Plant susceptibility targets } Parasitism [39••]. That dspE is essential for E. amylovora pathogenicity, whereas avrE contributes only quantitatively to the virulence of P. syringae pv. tomato [51], suggests that there is less redundancy in the E. amylovora virulence system. This would be consistent with a more recent acquisition of the Hrp system by E. amylovora and/or with a slower coevolution with its perennial hosts [39••]. The heterologous functioning of P. syringae avr genes in E. amylovora and E. chrysanthemi suggests that Hrp+ bacteria in the field may be able to ‘sample’ a buffet of avr-like genes from diverse sources during their coevolution with changing plant populations. Many avr genes have been thought to be potentially mobile because of their presence on plasmids [7•,8]. Recent observations with P. syringae highlight the apparent mobility of avr genes. Several P. syringae avr genes are linked with transposable elements or phage sequences ([52]; Kim JF et al., unpublished data), and the hrp clusters in different strains of P. syringae, although conserved in themselves, are bordered by a hypervariable region enriched in avr genes and mobile DNA elements (JR Alfano, AO Charkowski, A Collmer, unpublished data). Avr proteins e.g. AvrPto-Pto HR and defense Current Opinion in Plant Biology Proposed model for bacterial pathogenicity and coevolution with plants, which is based on the injection by a conserved Hrp (type III) secretion system of horizontally interchangeable bacterial Avr-like proteins. A typical Pseudomonas syringae strain is depicted with many avr genes linked to the hrp/hrc gene cluster in a region containing mobile genetic elements and also carried on plasmids. The Hrp secretion apparatus is capable of delivering the products of avr genes introduced from other pathovars or even other genera of plant pathogenic bacteria. Widely conserved Hrc proteins are core components of a secretion apparatus that translocates Avr proteins across the bacterial inner membrane (IM) and outer membrane (OM). Extracellular Hrp proteins such as the HrpA pilus protein and possibly the HrpZ and HrpW harpins are proposed to contribute to the subsequent transfer of Avr proteins across the plant cell wall (CW) and plasma membrane (PM). Inside plant cells, the recognition of a single Avr protein by the R-gene surveillance system triggers the hypersensitive response (HR) and plant defenses that lead to resistance. Avr proteins are also proposed to interact with putative susceptibility targets that produce unknown changes in plant metabolism favoring growth of the parasite in the apoplast. The collective contribution of several Avr-like proteins appears to be necessary for parasitism, whereas a single Avr protein is sufficient for betrayal to the defense system. A fundamental characteristic of the prevalent bacterial plant pathogens in the genera Erwinia, Pseudomonas, Xanthomonas, and Ralstonia is the possession of a conserved type III protein secretion pathway and a variable collection of genes encoding Avr (effector) proteins that appear to be injected into plant cells via this pathway. As is consistent with emerging concepts related to pathogenicity islands, some avr genes are linked to hrp gene clusters, and certain hrp/avr ensembles are functional in that they enable nonpathogens to elicit the HR in appropriate test plants. The evidence that Avr proteins can be delivered by the Hrp system into plant cells remains indirect, but it is strengthened by the demonstration that Avr proteins can travel the Hrp pathway [24••]. This general model demands both rigorous proof and answers to several new questions. How has the type III system been adapted in plant pathogens to permit the transfer of effector proteins across the plant cell wall? What signals target effector proteins to the Hrp pathway and then to specific locations on or in plant cells? What is the inventory of effector proteins delivered into plant cells? What do these effector proteins do in plant cells to promote parasite growth in the apoplast? What genetic mechanisms underlie the high degree of variability in avr gene composition of these pathogens? To what degree is host specificity controlled by differences in Hrp secretion mechanisms, Hrp regulation, the virulence functions of Avr-like effector proteins, the distribution of interacting avr and R genes, or the operation of ancillary virulence systems? What similarities will emerge between the effector proteins and host targets of plant and animal pathogens? The answers to these questions will further elucidate the determinants of pathogenicity and avirulence and lead to a deeper Determinants of pathogenicity and avirulence in plant pathogenic bacteria Collmer understanding of the nature of bacterial parasitism and plant defense. Acknowledgements I thank James R Alfano, David W Bauer, Steven V Beer, Amy O Charkowski, Wen-Ling Deng, Derrick E Fouts, and Jihyun F Kim for critical review of this manuscript. Work in my laboratory was supported by grants MCB-9631530 from National Science Foundation (NSF) and 97-35303-4488 from the National Research Initiative Competitive Grants Program/USDA (United States Department of Agriculture). 333 11. • Baker B, Zambryski P, Staskawicz B, Dinesh-Kumar SP: Recognition and signaling in plant–microbe interactions. Science 1997, 276:726-733. A concisely presented big picture of plant–microbe interactions. 12. Mudgett MB, Staskawicz BJ: Protein signaling via type III secretion pathways in phytopathogenic bacteria. Curr Opin Microbiol 1998, 1:109-114. 13. Hutcheson SW: The hrp-encoded protein export systems of Pseudomonas syringae and other plant pathogenic bacteria and their role in pathogenicity. In Plant–Microbe Interactions, vol 3. Edited by Stacey G, Keen NT. New York: Chapman and Hall, Inc.; 1997:145-179. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1. 2. 3. Alfano JR, Collmer A: Bacterial pathogens in plants: life up against the wall. Plant Cell 1996, 8:1683-1698. Brown I, Mansfield J, Bonas U: hrp genes in Xanthomonas campestris pv. vesicatoria determine ability to suppress papilla deposition in pepper mesophyll cells. Mol Plant–Microbe Interact 1995, 8:825-836. Young SA, Wang X, Leach JE: Changes in the plasma membrane distribution of rice phospholipase D during resistant interactions with Xanthomonas oryzae pv. oryzae. Plant Cell 1996, 8:1079-1090. Bestwick CS, Brown IR, Bennett MHR, Mansfield JW: Localization of hydrogen peroxide accumulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv. phaseolicola. Plant Cell 1997, 9:209-221. This important study reveals that hydrogen peroxide, which has potential defense-signaling and antimicrobial activities, accumulates in the plant cell wall at the site of contact with bacteria expressing the hypersensitive response and pathogenicity (Hrp) system. 14. He SY: Hrp-controlled interkingdom protein transport: learning • from flagellar assembly? Trends Microbiol 1997, 5:489-495. A concise overview emphasizing the similarities between the Hrp and flagellar systems. This is based on the observation that eight of the Hrc proteins have homologs involved in flagellar-specific biogenesis and secretion and that both systems produce extracellular (but quite different) surface appendages. 15. 16. • Cornelis GR, Wolf-Watz H: The Yersinia Yop regulon: a bacterial system for subverting eukaryotic cells. Mol Microbiol 1997, 23:861-867. A highly accessible overview of the prototypical type III secretion system. 17. Bogdanove AJ, Beer SV, Bonas U, Boucher CA, Collmer A, Coplin DL, Cornelis GR, Huang H-C, Hutcheson SW, Panopoulos NJ, Van Gijsegem F: Unified nomenclature for broadly conserved hrp genes of phytopathogenic bacteria. Mol Microbiol 1996, 20:681-683. 18. Charkowski AO, Huang H-C, Collmer A: Altered localization of HrpZ in Pseudomonas syringae pv. syringae hrp mutants suggests that different components of the type III secretion pathway control protein translocation across the inner and outer membranes of Gram-negative bacteria. J Bacteriol 1997, 179:3866-3874. 19. Lawrence JG, Roth JR: Selfish operons: horizontal transfer may drive the evolution of gene clusters. Genetics 1996, 143:18431860. 20. Groisman EA, Ochman H: Pathogenicity islands: bacterial evolution in quantum leaps. Cell 1996, 87:791-794. 4. •• 5. Lindgren PB: The role of hrp genes during plant-bacterial • interactions. Annu Rev Phytopathol 1997, 35:129-152. A comprehensive review of Hrp systems, their genetics, component proteins, regulation, interactions with Avr (avirulence) proteins. The discussion of the role of the Hrp system in the suppression of plant defenses is particularly important. 6. • Alfano JR, Collmer A: The type III (Hrp) secretion pathway of plant pathogenic bacteria: trafficking harpins, Avr proteins, and death. J Bacteriol 1997, 179:5655-5662. A review of Hrp systems emphasizing type III secretion functions and the delivery of Avr (avirulence) proteins into plant cells. Contains a useful comparison of group I and II hrp gene arrangements and a table of Hrc (hypersensitive response, conserved) protein homologs in Yersinia, Salmonella, Shigella spp. Finlay B, Falkow S: Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 1997, 61:136-169. 21. •• Hacker J, Blum-Oehler G, Muhldorfer I, Tschape H: Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol Microbiol 1997, 23:1089-1097. A definitive overview of pathogenicity islands, which deals exclusively with animal pathogens and focuses on Pais (pathogenicity islands) encoding type III secretion systems. 7. Vivian A, Gibbon MJ: Avirulence genes in plant-pathogenic • bacteria: signals or weapons? Microbiology 1997, 143:693-704. An excellent review and the most current general overview of avr genes. The article has many good insights, for example, in the conceptually useful division of avr genes into two groups: P. syringae-type and avrBs3-like. Nizan R, Barash I, Valinsky L, Lichter A, Manulis S: The presence of hrp genes on the pathogenicity-associated plasmid of the tumorigenic bacterium Erwinia herbicola pv. gypsophilae. Mol Plant–Microbe Interact 1997, 10:677-682. A description of an exciting new Hrp system that may provide a useful case study for portable pathogenicity. 8. Leach JE, White FF: Bacterial avirulence genes. Annu Rev Phytopathol 1996, 34:153-179. 23. 9. Van den Ackerveken G, Bonas U: Bacterial avirulence proteins as triggers of plant defense resistance. Trends Microbiol 1997, 5:394-398. 10. • Bonas U, Van den Ackerveken G: Recognition of bacterial avirulence proteins occurs inside the plant cell: a general phenomenon in resistance to bacterial diseases? Plant J 1997, 12:1-7. A particularly accessible and insightful account of the evidence that Avr proteins act inside plant cells following delivery by the Hrp system. 22. • 24. •• Puri N, Jenner C, Bennet M, Stewart R, Mansfield J, Lyons N, Taylor J: Expression of avrPphB, an avirulence gene from Pseudomonas syringae pv. phaseolicola, and the delivery of signals causing the hypersensitive reaction in bean. Mol Plant–Microbe Interact 1997, 10:247-256. Ham JH, Bauer DW, Collmer A: A cloned Erwinia chrysanthemi Hrp (type III protein secretion) system functions in Escherichia coli to deliver Pseudomonas syringae Avr signals to plant cells and to secrete Avr proteins in culture. Proc Natl Acad Sci USA 1998, in press. This paper describes the technically useful discovery of an Hrp system that functions in E. coli to secrete heterologous Avr (avirulence) proteins, 334 Host–microbe interactions it demonstrates that the two P. syringae Avr proteins with the strongest evidence for a site of action inside plant cells actually travel the Hrp pathway. 25. • Alfano JR, Kim H-S, Delaney TP, Collmer A: Evidence that the Pseudomonas syringae pv. syringae hrp-linked hrmA gene encodes an Avr-like protein that acts in a hrp-dependent manner within tobacco cells. Mol Plant–Microbe Interact 1997, 10:580-588. The functional cluster of P. syringae pv. syringae hrp genes carried on cosmid pHIR11 has provided a useful system for exploring the minimal requirements for bacterial elicitation of the hypersensitive response (HR). This work characterizes hrmA, the avr-like component of the system that is essential for HR elicitation in tobacco. It shows that DNA hybridizing with hrmA is lacking from the tobacco pathogen, P. syringae pv. tabaci, that expression of hrmA in this pathogen renders it avirulent, and that expression of the gene inside tobacco cells is lethal. 26. Gopalan S, Bauer DW, Alfano JR, Loniello AO, He SY, Collmer A: Expression of the Pseudomonas syringae avirulence protein AvrB in plant cells alleviates its dependence on the hypersensitive response and pathogenicity (Hrp) secretion system in eliciting genotype-specific hypersensitive cell death. Plant Cell 1996, 8:1095-1105. 27. Pirhonen MU, Lidell MC, Rowley DL, Lee SW, Jin S, Liang Y, Silverstone S, Keen NT, Hutcheson SW: Phenotypic expression of Pseudomonas syringae avr genes in E. coli is linked to the activities of the hrp-encoded secretion system. Mol Plant–Microbe Interact 1996, 9:252-260. hypersensitive response and to bind to pectate. J Bacteriol 1998, in press. This paper, which is a companion to Kim et al. [32•], additionally reports that HrpW (but not the HrpZ harpin) binds to pectate and that sequences hybridizing with the P. syringae hrpW gene are present in Xanthomonas spp. and R. solanacearum. Both papers discriminate the lethal activity of active pectate lyases and harpins on the basis of the requirement of plant metabolism for the lethal action of harpins. Both papers discriminate the lethal activity of active pectate lyases and harpins on the basis of the requirement of plant metabolic activities, such as protein synthesis, for the lethal action of harpins. 35. 36. •• Roine E, Wei W, Yuan J, Nurmiaho-Lassila E-L, Kalkkinen N, Romantschuk M, He SY: Hrp pilus: an hrp-dependent bacterial surface appendage produced by Pseudomonas syringae pv. tomato DC3000. Proc Natl Acad Sci USA 1997, 94:3459-3464. This breakthrough paper reports that hrpA encodes a subunit for a Hrp pilus and that the pilus is required for Hrp phenotypes. 37. 28. Wengelnik K, Van den Ackerveken G, Bonas U: HrpG, a key hrp regulatory protein of Xanthomonas campestris pv. vesicatoria is homologous to two-component response regulators. Mol Plant–Microbe Interact 1996, 9:704-712. 29. •• Marenda M, Brito B, Callard D, Genin S, Barberis P, Boucher C, Arlat M: PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells. Mol Microbiol 1998, 27:437-453. This breakthrough study provides evidence for the specific induction of hrp genes in the presence of co-cultivated plant cells and for a novel regulatory component involved in receiving plant signals. The paper describes the discovery of the prhA gene, prhA mutant phenotypes in virulence and hypersensitive response assays, phylogenetic and structural relationships of PrhA and known TonB-dependent receptors, and the effects of prhA mutation and plant cell co-cultivation on the expression of various hrp operons. 30. • Mukherjee A, Cui Y, Liu Y, Chatterjee AK: Molecular characterization and expression of the Erwinia carotovora hrpNEcc gene, which encodes an elicitor of the hypersensitive reaction. Mol Plant–Microbe Interact 1997, 10:462-471. A particularly thorough study of the environmental and regulatory factors affecting expression of a harpin gene, which reveals overlapping control of harpin and exoenzyme production by two global regulators. 31. Cui Y, Madi L, Mukherjee A, Dumenyo CK, Chatterjee AK: The RsmA– mutants of Erwinia carotovora subsp. carotovora strain Ecc71 overexpress hrpNEcc and elicit a hypersensitive reaction-like response in tobacco leaves. Mol Plant–Microbe Interact 1996, 9:565-573. 32. • Kim JF, Beer SV: HrpW of Erwinia amylovora, a new harpin that is a member of a proposed class of pectate lyases. J Bacteriol 1998, in press. This discovery of a second harpin gene linked to the E. amylovora hrp gene cluster is particularly significant because HrpW carries a domain that is a member of a newly defined class (III) of pectate lyases, and the ability of hrpW to restore full hypersensitive response elicitation activity to an E. amylovora hrpN mutant suggests that both harpins have similar functions. 33. 34. • Alfano JR, Bauer DW, Milos TM, Collmer A: Analysis of the role of the Pseudomonas syringae pv. syringae HrpZ harpin in elicitation of the hypersensitive response in tobacco using functionally nonpolar deletion mutations, truncated HrpZ fragments, and hrmA mutations. Mol Microbiol 1996, 19:715728. Charkowski AO, Alfano JR, Preston G, Yuan J, He SY, Collmer A: Pseudomonas syringae pv. tomato secretes a protein via the Hrp (type III) pathway that has domains similar harpins and pectate lyases and the capacity to elicit the plant Hoyos ME, Stanley CM, He SY, Pike S, Pu X-A, Novacky A: The interaction of HarpinPss with plant cell walls. Mol Plant–Microbe Interact 1996, 9:608-616. Fullner KJ, Lara JC, Nester EW: Pilus assembly by Agrobacterium T-DNA transfer genes. Science 1996, 273:11071109. 38. •• Gaudriault S, Malandrin L, Paulin J-P, Barny M-A: DspA, an essential pathogenicity factor of Erwinia amylovora showing homology with AvrE of Pseudomonas syringae, is secreted via the Hrp secretion pathway in a DspB-dependent way. Mol Microbiol 1997, 26:1057-1069. This important report of an E. amylovora homolog of the P. syringae avrE gene has particularly detailed information on the regulation of these dsp genes and provides evidence suggesting that DspB is a chaperone required for DspA secretion. Customized chaperones are required for the secretion of Yop effector proteins by Yersinia spp. [16•], but their importance in the secretion of Avr-like proteins is unclear. 39. •• Bogdanove AJ, Kim JF, Wei Z, Kolchinsky P, Charkowski AO, Conlin AK, Collmer A, Beer SV: Homology and functional similarity of a hrp-linked pathogenicity operon, dspEF, of Erwinia amylovora and the avrE locus of Pseudomonas syringae pathovar tomato. Proc Natl Acad Sci USA 1998, 95:1325-1330. This important report characterizes the dspE and dspF genes of E. amylovora, compares the completed sequences of AvrE and DspE, and demonstrates that the P. syringae avrE locus in trans can restore pathogenicity to E. amylovora dspE mutants. This evidence that Hrp-dependent effector loci can heterologously support pathogenicity across bacterial genera has important implications for the evolution of plant pathogenic bacteria because it suggests that pathogens can recruit from each other in the development of their virulence factor arsenals. 40. •• Bogdanove AJ, Bauer DW, Beer SV: Erwinia amylovora secretes DspE, a pathogenicity factor and functional AvrE homolog, through the Hrp (type III secretion) pathway. J Bacteriol 1998, 180:in press. Through the use of specific antibodies, this paper provides definitive evidence for the Hrp-dependent secretion of DspE, an Avr-like protein, by E. amylovora. 41. •• Anderson DM, Schneewind O: An mRNA signal for the type III secretion of Yop proteins by Yersinia enterocolitica. Science 1997, 278:1140-1143. This landmark paper describes evidence signals that target two Yop proteins to the prototypical type III pathway of Yersinia spp. reside in the cognate mRNAs. 42. Gabriel DW: Targeting of protein signals from Xanthomonas to the plant nucleus. Trends Plant Sci 1997, 2:204-206. 43. Yang Y, Yuan Q, Gabriel DW: Watersoaking function(s) of XcmH1005 are redundantly encoded by members of the Xanthomonas avr/pth gene family. Mol Plant–Microbe Interact 1996, 9:105-113. 44. Swords KMM, Dahlbeck D, Kearney B, Roy M, Staskawicz BJ: Spontaneous and induced mutations in a single open reading frame alter both virulence and avirulence in Xanthomonas campestris pv. vesicatoria avrBs2. J Bacteriol 1996, 4661-4669. Determinants of pathogenicity and avirulence in plant pathogenic bacteria Collmer 45. Hardt WE, Galan JE: A secreted Salmonella protein with homology to an avirulence determinant of plant pathogenic bacteria. Proc Natl Acad Sci USA 1997, 94:9887-9892. 46. Monack DM, Mecsas J, Ghori N, Falkow S: Yersinia signals macrophages to undergo apoptosis and YopJ is necessary for this cell death. Proc Natl Acad Sci USA 1997, 94:10385-10390. 47. Galan JE: ‘Avirulence genes’ in animal pathogens? Trends • Microbiol. 1998, 6:3-6. This provocative commentary highlights the similarities between the hypersensitive response in plants and infection-limiting inflammatory responses in animals, the presence of the AvrRxv/YopJ/AvrA family of effector proteins in both plant and animal pathogens, and the potential therapeutic importance of defense systems involving specific responsiveness of naı̈ve animals to bacterial pathogens. 48. • Stevens C, Bennet MA, Athanassopoulos E, Tsiamis G, Taylor JD, Mansfield JW: Sequence variations in alleles of the avirulence gene avrPphE.R2 from Pseudomonas syringae pv. phaseolicola lead to loss of recognition of the AvrPphE protein within bean cells and gain in cultivar specific virulence. Mol Microbiol 1998, in press. A thorough study of the nonfunctional alleles of avrPphE that are found in all races of P. syringae pv. phaseolicola, including the effects of alleles when heterologously expressed in differential bean cultivars. 49. 335 McNellis TW, Mudgett MB, Li K, Aoyama T, Horvath D, Chua N-H, Staskawicz BJ: Glucocorticoid-inducible expression of a bacterial avirulence gene in transgenic Arabidopsis induces hypersensitive cell death. Plant J 1998, in press. 50. • Keith LW, Boyd C, Keen NT, Partridge JE: Comparison of avrD alleles from Pseudomonas syringae pv. glycinea. Mol Plant–Microbe Interact 1997, 10:416-422. This latest chapter in a long series of important studies of avrD, the only avr gene directing a known biochemical activity, reveals that multiple races of P. syringae pv. glycinea possess avrD alleles that are nonfunctional with respect to syringolide elicitor production, but are highly conserved (although the regions flanking them are polymorphic). These results suggest that avr genes are both beneficial and highly mobile in plant pathogens. 51. Lorang JM, Shen H, Kobayashi D, Cooksey D, Keen NT: avrA and avrE in Pseudomonas syringae pv. tomato PT23 play a role in virulence on tomato plants. Mol Plant–Microbe Interact 1994, 7:508-515. 52. Hanekamp T, Kobayashi D, Hayes S, Stayton MM: Avirulence gene D of Pseudomonas syringae pv. tomato may have undergone horizontal gene transfer. FEBS Lett 1997, 415:4044.