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.avrgenes may be acquired horizontally by these bacteria, andavrgene compositions are highly variable. In the past year, heterologous expression experiments have revealed that the products ofavrgenes 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 Biology1998,1:329–335 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 Yersiniaouter protein
Introduction
The most common bacterial pathogens of plants colo-nize 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
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 solanacearumis carried on a megaplasmid [1]. The finding of a plasmid-bornehrpgene cluster inErwinia herbicolapv.
gypsophilaesuggests that virulence may be acquired readily by plant-associated bacteria [22•].E. herbicolais 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 fromErwinia 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, heterologousavrgene is suppliedin trans
of thehrp+cosmid [23,24••,26,27].
Hrp regulation
Thehrpgenes 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 inhrp 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], andR. 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-homo-serine 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 prototypicalhrpNharpin gene inE. amylovorastrain Ea321 strongly diminishes HR and pathogenicity phenotypes [32•], but mutation of thehrpZ 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 bothE. amylovoraand
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 theP. syringaeHrpA 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 theP. syringaeAvrB and AvrPto proteins is inside plant cells (e.g. see [10•]), and both proteins have now been found to be secreted by anE. chrysanthemiHrp system functioning heterologously in E. coli[24••]. This secretion is Hrp-dependent, andE. colicells carrying the
E. chrysanthemi hrpgenes also elicit anavrB-dependent HR
in appropriate test plants. A strong implication of this work is thatE. chrysanthemi, which is a host-promiscuous soft-rot pathogen, also carries avr-like genes. The ability of the clonedE. chrysanthemiHrp system to secreteP. syringaeAvr 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) synthesisin 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 byA. tumefaciens [44]; and AvrRxv (X. campestris pv.vesicatoria) is a homolog of AvrA (Salmonella typhimurium) and YopJ (Yersinaspp.), 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 interest-ingly, 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•].
Figure 1
Hypervariable region with many avr genes
Conserved hrp/hrc gene cluster
Region with hrpW and additional avr genes
Avr proteins
Avr proteins
avr Horizontal
avrtransfer with other pathogens?
NRSTUV J C
?
}
HrpA pilus
CW
PM
HrpZ HrpW
}
Harpinse.g. AvrPto-Pto
HR and defense Parasitism
Plant susceptibility
targets
Plant R-gene directed surveillance
system
Hrc proteins in Hrp (type III) secretion apparatus IM
OM
}
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 typicalPseudomonas syringaestrain is depicted with manyavrgenes linked to thehrp/hrcgene cluster in a region containing mobile genetic elements and also carried on plasmids. The Hrp secretion apparatus is capable of delivering the products ofavrgenes 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 theR-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.
[39••]. ThatdspEis essential forE. amylovora pathogenic-ity, 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 byE. amylovoraand/or with a slower coevolution with its perennial hosts [39••]. The heterologous functioning of P. syringae avr genes in E. amylovoraandE. chrysanthemisuggests 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 JFet 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).
Conclusions
A fundamental characteristic of the prevalent bacterial plant pathogens in the genera Erwinia, Pseudomonas,
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).
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
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•
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•
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•
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•
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•
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•
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•
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•
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•
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•
34. Charkowski AO, Alfano JR, Preston G, Yuan J, He SY, Collmer A:Pseudomonas syringaepv. 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
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This paper, which is a companion to Kimet al.[32•], additionally reports that HrpW (but not the HrpZ harpin) binds to pectate and that sequences hybridizing with theP. syringae hrpWgene are present inXanthomonas
spp. andR. 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 require-ment of plant metabolic activities, such as protein synthesis, for the lethal action of harpins.
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38. Gaudriault S, Malandrin L, Paulin J-P, Barny M-A:DspA, an essential pathogenicity factor ofErwinia amylovorashowing homology with AvrE ofPseudomonas syringae, is secreted via the Hrp secretion pathway in a DspB-dependent way.Mol Microbiol1997,26:1057-1069.
This important report of anE. amylovorahomolog of theP. 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 byYersiniaspp. [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 ahrp-linked pathogenicity operon,dspEF, of
Erwinia amylovoraand theavrElocus ofPseudomonas syringaepathovar tomato.Proc Natl Acad Sci USA1998,
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 theP. syringae avrElocusin transcan restore pathogenic-ity toE. amylovora dspEmutants. 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.
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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 Bacteriol1998,
180:in press.
Through the use of specific antibodies, this paper provides definitive ev-idence 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 byYersinia enterocolitica.Science
1997,278:1140-1143.
This landmark paper describes evidence signals that target two Yop proteins to the prototypical type III pathway ofYersiniaspp. reside in the cognate mRNAs.
42. Gabriel DW:Targeting of protein signals fromXanthomonasto the plant nucleus.Trends Plant Sci1997,2:204-206.
43. Yang Y, Yuan Q, Gabriel DW:Watersoaking function(s) of XcmH1005 are redundantly encoded by members of the
Xanthomonas avr/pthgene family.Mol Plant–Microbe Interact
1996,9:105-113.
44. Swords KMM, Dahlbeck D, Kearney B, Roy M, Staskawicz BJ:
45. Hardt WE, Galan JE:A secretedSalmonellaprotein with homology to an avirulence determinant of plant pathogenic bacteria.Proc Natl Acad Sci USA1997,94:9887-9892. 46. Monack DM, Mecsas J, Ghori N, Falkow S:Yersiniasignals
macrophages to undergo apoptosis and YopJ is necessary for this cell death.Proc Natl Acad Sci USA1997,94:10385-10390. •
47. Galan JE:‘Avirulence genes’ in animal pathogens?Trends Microbiol.1998,6:3-6.
This provocative commentary highlights the similarities between the hyper-sensitive 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 na¨ıve animals to bacterial pathogens.
•
48. Stevens C, Bennet MA, Athanassopoulos E, Tsiamis G, Taylor JD, Mansfield JW:Sequence variations in alleles of the avirulence geneavrPphE.R2fromPseudomonas syringaepv.phaseolicola
lead to loss of recognition of the AvrPphE protein within bean cells and gain in cultivar specific virulence.Mol Microbiol1998, in press.
A thorough study of the nonfunctional alleles ofavrPphEthat are found in all races ofP. syringaepv.phaseolicola, including the effects of alleles when heterologously expressed in differential bean cultivars.
49. McNellis TW, Mudgett MB, Li K, Aoyama T, Horvath D, Chua N-H, Staskawicz BJ:Glucocorticoid-inducible expression of a bacterial avirulence gene in transgenicArabidopsisinduces hypersensitive cell death.Plant J1998, in press.
•
50. Keith LW, Boyd C, Keen NT, Partridge JE:Comparison of
avrDalleles fromPseudomonas syringaepv.glycinea.Mol Plant–Microbe Interact1997,10:416-422.
This latest chapter in a long series of important studies ofavrD, the only
avrgene directing a known biochemical activity, reveals that multiple races ofP. syringaepv.glycineapossessavrDalleles that are nonfunctional with respect to syringolide elicitor production, but are highly conserved (although the regions flanking them are polymorphic). These results suggest thatavr
genes are both beneficial and highly mobile in plant pathogens.
51. Lorang JM, Shen H, Kobayashi D, Cooksey D, Keen NT:avrAand
avrEinPseudomonas syringaepv.tomatoPT23 play a role in virulence on tomato plants.Mol Plant–Microbe Interact1994,
7:508-515.
