Bacterial Pathogens in Plants Life Up Against the WalI

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The Plant Cell, Vol. 8, 1683-1698, October 1996 O 1996 American Society of Plant Physiologists Bacterial Pathogens in Plants: Life up against the WalI James R. Alfano and Alan Collmer' Department of Plant Pathology, Cornell University, Ithaca, New York 14853-4203 INTRODUCTION Higher plants contain potentially vast sources of nutrients for the myriad bacterial species in their environment, and most bacteria are small enough to pass through stomates and other natural openingsinto the apoplast-th
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  The Plant Cell, Vol. 8, 1683-1698, October 1996 O 1996 American Society of Plant Physiologists Bacterial Pathogens in Plants: Life up against the WalI James R. Alfano and Alan Collmer' Department of Plant Pathology, Cornell University, Ithaca, New York 14853-4203 INTRODUCTION Higher plants contain potentially vast sources of nutrients forthe myriad bacterial species in their environment, and mostbacteria are small enough to pass hrough stomates and othernatural openingsinto the apoplast-the anteroom for theseriches. However, surprisingly few bacteria raid the nutrientstores of living plant cells, apparently because the metabolicintimacy involved n parasitism requires the work of specialists.Of these specialists, some in the Rhizobiaceae produce hyper-trophies that are genetically engineered or developmentallytricked into providing an undefended, nutritive niche in rootcortical tissues and rhizospheres (see Long, 1996; Sheng andCitovsky, 1996, in this issue), whereas others, mostly Gram-negative bacteria in the Pseudomonadaceae and Enterobac-teriaceae, specialize in colonizing the apoplast.It is the apoplastic colonizers that are the common patho-gens that produce the rots, spots, wilts, cankers, and blightsafflicting virtually all crop plants, and their relationship withthe host is defined by two features. They spend their parasiticlife up against the wall of plant cells, in the intercellular spacesof various plant organs or in the xylem, and they are nec-rogenic-able to cause the death of plant cells. Their abilityto multiply and then sooner or later to kill plant cells dependson secreted enzymes that degrade the wall or on moleculesthat pass through it. This review addresses our progress inunderstanding this molecular traffic and how it may enablenecrogenic bacterial pathogens to colonize the apoplast.The present picture of pathogenesis has been strongly de-termined by three developments. The first was the discoverythat bacteria elicited the defense-associated hypersensitiveresponse (HR) in plants during incompatible interactions. TheHR was first observed as a rapid localized collapse of tobaccoleaf tissue after infiltration of high numbers of bacterial patho-gens that are host specific for other plant species (Klement,1963; Klement et ai., 1964). Because the ability to elicit theHR is a unique attribute of the necrogenic pathogens and thesebacteria can avoid or suppress its elicitation in their hosts, theHR phenomenon appears central to bacterial pathogenicityand host specificity and has attracted much attention (Klement,1982; Goodman and Novacky, 1994; see also Dangl et al., 1996;Hammond-Kosack and Jones, 1996, in this issue). The sec-ond development was the application of the molecular tools Towhom correspondence should be addressed.of transposon mutagenesis, broad-host-rangecosmid vectors,and marker-exchange mutagenesis o identify and manipulatebacterial genes that have a readily scored phenotype whenmutated, conjugated nto a related strain, or expressed n Esch-erichia coli. These approaches have yielded a large inventoryof hrp (hypersensitive [esponse and pathogenicity) and avr (eirulence) genes that directly relate o the HR puzzle as we!l as numerous other genes associatedwith pectic enzyme, toxin,and extracellular polysaccharide EPS) production. Rather thandetail this inventory (which may be fundamentally ncomplete;see below), we use representative components to develop amodel for bacterial plant pathogenesis that is based on thevery recent third development-the discovery that the hrp genes encode a protein secretion system, shared in plant andanimal pathogens, that has the potential to transfer virulenceproteins into eukaryotic host cells.The necrogenic bacteria have diverse pathogenic personal-ities with a bewilderingarray of symptoms and host specificities.The growing evidence that the hrp genes are ubiquitous inthese pathogens, controlling early (and generally essential)interactions with plants, provides a unifying entry point for ex-ploring bacterial phytopathogenicity. Hence, after introducingthe representative pathogens, we explore the dynamic opera-tion of the Hrp system and then turn briefly to factors suchas toxins, EPS, and pectic enzymes that affect the full devel-opment of plant disease. MODEL PATHOGENS AND INTERACTIONS Key characteristics of severa1 model Gram-negative phyto-pathogens are shown in Table 1. These bacteria are ali capableof causing necrosis, but their necrogenic aggressivenessvaries. Brute-force, necrotrophic pathogens rapidly kill paren-chymatous tissues during active pathogenesis, whereasstealthy, biotrophic pathogens characteristically multiply n hosttissues for some period before causing any necrosis (Collmerand Bauer, 1994). The HR is elicited by the biotrophic patho-gens during incompatible interactions with nonhosts, but Erwinia chrysanthemi mutants with a reduced pectolytic ca-pacity can also elicit a typical HR that is independent of hostrange (Bauer et al., 1994). Strains in Xanfhomonas campes- tris and Pseudomonas syringae are assigned to pathovars  1684 The Plant Cell Table 1. Model Necrogenic Gram-Negative Phytopathogens ~ ~~~~~ Phenotype of hrp Phenotype ofHost Range; Typical (Type 111 Secretion) Type II SecretionPathogen Model Hosts Diseases Mutantsa Mutantsb Other Disease FactorsNecrotrophicErwinia carotovora Wide; potato,Saintpauliaand E. chrysanthemiC tobacco seedlings,Biotrophic E. amylovora E. stewartiiRalstoniasolanacearumeRosaceae; appleand pearMaizeSolanaceae; tomatoand tobacco Soft rotsFire blightStewart’s wilt WiltsHR-d; No maceration Pectic infectivity enzymes;reduced but siderophores;wild-type autoinductionmacerationHrp-dWts-dHrp-Xanthomonas lndividually narrow; Foliar spots Hrp-campestris pathovars pepper, tomato, andbrassicas blightsPseudomonas syringae lndividually narrow; Foliar spots Hrp-pathovars tomato, andArabidopsis, blightslegumesNot knownNot knownVirulence reduced Virulence reducedNot knownEPS; harpinEPS; autoinductionEPS; volatilesignal andglobalregulationAvr proteins;global regulation Avr proteins;toxins - a Harpins are the only proteins directly shown to travel via this pathway; evidence for Avr protein traffic is discussed in the text. The virulencephenotype reflects the collective contribution of all proteins traveling the pathway. For references, see Bauer et al. (1994) regarding E. chrysanthemitype 111 mutants and those in Bonas (1994) for all other bacteria.Virtually all plant cell wall-degrading enzymes travel this pathway. For references, see Kang et al. (1994) and those in Salmond (1994). C E. chrysanthemi has been the model for the Hrp system; E. carotovora for autoinduction.HR- denotes loss of HRelicitation activity in these bacteria; Hrp- denotes loss of HRand parasitismlpathogenicity; Wts- denotes lack of water- soaked lesions. e Synonyms are Pseudomonas solanacearum and Burkholderia solanacearum (Yabuuchi et al., 1992, 1995).based on host specificity and associated phenotypic charac-teristics and sometimes to races within pathovars based oninteractions with differential cultivars of the host. For exam-ple, X. campestris pv campestris causes black rot of crucifers,and /? syringae pv glycinea causes bacterial blight of soybean,but both elicit the HR in tobacco. Table 1 also highlights theimportance of two protein secretion pathways in the virulence of these bacteria and indicates other specific factors that arediscussed below.Much research has focused on the differing interactions be-tween plants and biotrophic pathogens (compatible andincompatible) and nonpathogens. These interactions are sum- marized in Figure 1. The HR is the most dramatic of theseresponses, and several additional observations are importantin considering its nature. First, the macroscopically observ-able HR requires high levels of bacteria (>5 x 106 ells/mL)because it results from single bacteria eliciting death in sin-gle plant cells in a one-to-one manner, and a threshold leve1of individual cell deaths is required for tissue death (Turnerand Novacky, 1974). Second, HR elicitation appears to requirecontact between plant and bacterial cells that are both meta-bolically active and synthesizing new proteins (Holliday et al., 1981; lement, 1982). lthough tissue collapse and death maynot occur until 12 o 36 hr postinoculation, antibiotic treatmentexperiments suggest that bacteria may deliver the HR elicita-tion signal within a few minutes of contact (Huynh et al., 1989). Third, the HR appears to represent programmed cell death(He et al., 1993; ietrich et al., 1994; reenberg et al., 1994), but the signal transduction events and mechanisms underly-ing this process are still unknown (see Dangl et al., 1996, nthis issue).Although several plant responses are consistently associatedwith incompatible interactions and the HR (Figure l), heir ac-tua1 roles are not clear. For example, the data are either lacking  Bacterial Pathogens in Plants 1685 or conflicting regarding (1) the causal relationship between ac-tive oxygen generation and HR elicitation (Levine et al., 1994;Glazener et al., 1996; see also Hammond-Kosack and Jones,1996, in this issue), (2) the relationship between the HR andthe XR (K+ efflux/H+ influx exchange Lesponse; Atkinson,1993; He et al., 1994), and (3) the role n defense of antimicrobialphytoalexins (Long et al., 1985; Pierce and Essenberg, 1987;Glazebrook and Ausubel, 1994) and pathogenesis-related pro-teins (see Ryals et al., 1996, in this issue). However, the XR may be particularly important n compatible (disease-causing)interactions because alkalinization of the apoplast has beenshown to foster both sucrose eakage rom plant cells and bac-teria1 multiplication (Atkinson and Baker, 1987a, 1987b).As suggested by the different response patterns outlinedin Figure 1, the fate of plant-bacterium interactions may bedetermined very early after inoculation. When considering pos-sible determinative factors, it is useful to keep in mind thatcompatible pathogens, which appear to be able to suppressrapid, “weak defense responses, can promote the growth ofnonpathogens, whereos coinoculation of compatible and in-compatible pathogens results in incompatibility unless thecompatible pathogen has been given a substantial head start(Young, 1974; Klement, 1982; Jakobeket al., 1993). Of course,a critical decision in the interaction is whether or not the HRis triggered, and much of the remainder of this article con-cerns the bacterial factors involved in HR elicitation. A Bacterial Growth THE HRP SYSTEM UNDERLYING BASlCPATHOGENICITY hrp Genes The ability of the necrogenic phytopathogens to elicit the HRresides in hrp genes, which were first found in F! syringae pvsyringae and /? syringae pv phaseolicola by identifying Tn5transposon mutants that grew normally in minimal media butfailed to elicit the HR in nonhost tobacco or cause diseaseor multiply in host bean (Niepold et al., 1985; Lindgren et al.,1986). Thus, hrp mutants behave essentially like nonpatho-gens in plants. hrp genes are clustered and are likely to occurwithin “pathogenicity islands” containing supporting virulencegenes (e.g., Lorang and Keen, 1995). The hrp clusters of F! s. syringae 61 and E. amy/ovora Ea321, carried on recombinantcosmids pHIR11 and pCPP430, respectively, enable nonpatho-genic bacteria such as F! fluorescens and E. coli to elicit theHR (but not disease) in tobacco and many other plants (Huanget al., 1988; Beer et al., 1991).lnitial DNA sequencing of the hrp clusters of Ralsroniasolanacearum GM11000, X. c. vesicatoria 85-10, and F! s. syrin-gae 61 revealed homologies with components of the virulenceprotein (Yop) secretion system in animal pathogenic Yersinia.spp (Fenselau et al., 1992; Gough et al., 1992; Huang et al., B lnteraction Plant Responses HR P AOll XR DR Compatibleno yes nolncompatible Yes no --- Nonpathogenic no no . Yesnogradual delayedrapid,rapid strongrapid,no weak Figure 1. Typical lnteractions between Compatible and lncompatible Biotrophic Pathogens, or Nonpathogens, and Plants. (A) Generalized bacterial population dynamics graphically relate the potential o elicit necrosis and the ability o multiply in plants, and they showthat multiplication ceases upon actual development of the necrosis associated with either the HR or disease lesions (Klement, 1982). (B) lnteraction classes are defined by the differing bacterial growth patterns and by the suites of plant responses. HR is further described in thetext.P denotes the development of lesions and other symptoms that accompany pathogenesis. AOll denotes a sustained generation of active oxygen that occurs 1.5 to 3 hr after inoculation (AO1 sia brief nonspecific response mmediately after inoculation; reviewed in Baker and Orlandi, 1995; see also Dangl et al., 1996; Hammond-Kosack and Jones, 1996, in this issue). XR denotes a K+ efflux/H+ nflux that occurs simultaneously with AOll in incompatible nteractions reviewed in Atkinson, 1993). DR denotes the expression of a variety of defense-response genes, particu-larly those directing the synthesis of phenylpropanoid pathway enzymes and their phytoalexin products, which occurs rapidly (within 6 hr) exceptin compatible interactions, where it can be delayed for severa1 days (Jakobek and Lindgren, 1993; Meier and Slusarenko, 1993).  1686 The Plant Cell1992), thereby establishing he existence of the conserved ”type 111” secretion system n Gram-negative bacteria (Salmond andReeves, 1993; Van Gijsegem et al., 1993). The near comple-tion of the hp cluster sequences n these three phytopathogensand in E, amylovora Ea321 has revealed that the homologieswith type 111 protein secretion system components in animalpathogenic Yersinia, Shigella, and Salmonella spp are exten-sive (Huang et al., 1993; Lidell and Hutcheson, 1994; Fenselauand Bonas, 1995; H.C. Huang et al., 1995; Preston et al., 1995;Van Gijsegem et al., 1995; Bogdanove et al., 1996b). This hasled to nomenclatura1 hanges and refinement of the hrp geneconcept (Bogdanove et al., 1996a). The nine hrp genes thatare broadly conserved in plant and animal pathogens havebeen redesignated as hrc (hypersensitive response and con-served) and given the last letter assignment of their Yersiniaysc (vop gecretion) homologs. The hrp genes, and particu-larly the hrc subset, are now considered to be fundamentallyinvolved in type 111 protein secretion in phytopathogenicbacteria.The type 111 secretion system appears to have been acquiredby horizontal transfer in a variety of pathogenic bacteria(Groisman and Ochman, 1993; Barinaga, 1996). Within thephytopathogens, comparisons of hrp gene sequences(Bogdanove et al., 1996b), hrp gene arrangements Fenselauand Bonas, 1995; H.-C. Huang et al., 1995; Van Gijsegem etal., 1995; Bogdanove et al., 1996b), and hrp regulatory ele-ments (discussed below) reveal wo groups. Group I contains f! syringae and E. amylovora; group II contains R. solanacea- rum and X. c. vesicaforia. The discrepancy between the hrp gene similarity groups and taxonomic relationships s consis-tent with horizontal acquisition of the system by phytopathogens. The Hrp (Type 111) Protein Secretion Systemand Its Regulation The type 111 secretion pathway is one of at least three distinctpathways hat Gram-negativebacteria use to secrete proteinsacross their inner and outer membranes (Salmondand Reeves,1993). It is unique among these secretion pathways n ts abil-ity to deliver virulence proteins directly nto host cells (Rosqvistet al., 1994; Sory and Cornelis, 1994). In Yersinia, Shigella, andSalmonella spp, it appears that the pathway can direct pro-teins into either the extracellular milieu or host cells. Proteinsthat are secreted into the milieu may regulate the secretionpathway or form extracellular components of the secretion ap- paratus (and may also have a direct role n virulence). Proteinsthat are transferred nto host cells appear to be important vir-ulence factors (reviewed in Galan, 1996). In plant pathogens,harpin proteins are known to be secreted into the milieu bythe Hrp pathway, and there is evidence that Avr proteins aretransferred into plant cells.Eight of the nine Hrc proteins are homologous to proteinsinvolved n the biogenesis of bacterial flagella and the secre-tion of flagellar-specific proteins. This is likely importantbecause the flagellar system supports highly regulated pro-tein secretion events involving ordered translocation of differentproteins, release of measured protein “doses,” and formationof extracellularappendages, all of which may serve the properdelivery of virulence proteins into host cells (Macnab, 1996).Unfortunately or researchers, proteins targeted to the hostvia the type III pathway may elude identification or two rea-sons. First, secretion via this flagellar-derived system isindependent of the general export (Sec) system; hence, theseproteins ack N-terminal signal peptides (or any other sharedfeature yet identified rom their sequences) hat would revealthem as targeted or secretion. Second, the secretion of manyof these proteins does not appear to occur in culture because it is dependent on contact with host cells (Rosqvist et al., 1994;Galan, 1996).Regulation of hrp gene expression offers further clues toHrp function in these bacteria. With the possible exception of the necrotroph E. chrysanfhemi (Collmer et al., 1994), hrp genes are not expressed in rich media (Bonas, 1994). Rather,they are most strongly expressed in various minimal mediathat mimic plant apoplastic luids, particularly media deficientin organic nitrogen Huynh et al., 1989; Arlat et al., 1992; Rahmeet al., 1992; Schulte and Bonas, 1992; Wei et al., 1992b; Xiaoet al., 1992). No plant inducers of the hrp genes have beenidentified, and hrp-dependentelicitation of the HR in nonhostsargues against host-specific hrp gene induction.The genetics of hrp regulation are surprisingly different inbacteria harboring the group I and II Hrp systems. In group I, hrp expression s dependent on HrpL, a member of the ECF(extra cytoplasmic function) family of sigma factors (Xiao andHutcheson, 1994; Xiao et al., 1994; Wei and Beer, 1995). hrpLexpression, although normally dependent on HrpR and HrpS,can be manipulated experimentally to permit useful hyperex-pression of the hrp regulon (Grimm and Panopoulos, 1989;Xiao et al., 1994; Grimm et al., 1995). In the group II system, R. solanacearum hrp expression s dependent on HrpB, a mem-ber of the AraC family of positive activators, and thehomologous HrpX appears to have the same function in Xan-fhomonas spp (Genin et al., 1992; Oku et al., 1995; Wengelnkikand Bonas, 1996). All of these regulatory proteins have beenfound through the Hrp- phenotype of respective mutants, andadditional regulatory genes with more subtle phenotypes ikelyawait discovery. PROTEINS DELIVERED BY THE HRP SYSTEMHarpins Harpins are glycine-rich, cysteine-lacking proteins that aresecreted in culture when the Hrp system is expressed and thatpossess heat-stable HR elicitor activity when infiltrated at rel-atively high concentrations (> 0.1 pM) into he leaves of tobaccoand severa1 other plants. This broad definition can encompass
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