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Role of Stomata in PlantInnate Immunity andFoliar Bacterial DiseasesMaeli Melotto,1 William Underwood,2,3

and Sheng Yang He2

1Department of Biology, University of Texas at Arlington, Texas, 76019;email: melotto@uta.edu2Department of Energy Plant Research Laboratory, Michigan State University,East Lansing, Michigan 48824; email: hes@msu.edu3Present address: Energy Biosciences Institute, University of California-Berkeley,Berkeley, California 94720

Annu. Rev. Phytopathol. 2008. 46:101–22

The Annual Review of Phytopathology is online atphyto.annualreviews.org

This article’s doi:10.1146/annurev.phyto.121107.104959

Copyright c© 2008 by Annual Reviews.All rights reserved

0066-4286/08/0908-0101$20.00

Key Words

coronatine, plant hormones, guard cell, plant defense, innateimmunity, Pseudomonas syringae

AbstractPathogen entry into host tissue is a critical first step in causing infection.For foliar bacterial plant pathogens, natural surface openings, such asstomata, are important entry sites. Historically, these surface openingshave been considered as passive portals of entry for plant pathogenicbacteria. However, recent studies have shown that stomata can play anactive role in limiting bacterial invasion as part of the plant innate im-mune system. As a counter-defense, the plant pathogen Pseudomonassyringae pv. tomato DC3000 uses the virulence factor coronatine to ac-tively open stomata. In nature, many foliar bacterial disease outbreaksrequire high humidity, rain, or storms, which could favor stomatal open-ing and/or bypass stomatal defense by creating wounds as alternativeentry sites. Further studies on microbial and environmental regulationof stomatal closure and opening could fill gaps in our understanding ofbacterial pathogenesis, disease epidemiology, and microbiology of thephyllosphere.

101

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FurtherANNUALREVIEWS

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Phyllosphere: leafsurfaces or totalabove-ground surfacesof a plant. In thisreview, phyllospherealso includes the leafinterior

Epiphytes: organismsthat grow on thesurface of a living plant

Endophytes:organisms that growinside a living plant

Stomata: microscopicpores in the epidermisof aerial parts of aplant essential for gasexchange and watertranspiration

INTRODUCTION

The phyllosphere of terrestrial plants providesone of the most important niches for microbialinhabitation (15, 72). The phyllosphere com-prises both the surface and interior of a leaf.The leaf interior is composed mostly of meso-phyll cells, in which the bulk of photosynthate ismade: some vascular tissues (veins); and a large,air-filled intercellular space (apoplast) betweenmesophyll cells (Figure 1). Because of its closeproximity to mesophyll cells, the leaf apoplast isthought to contain abundant nutrients, whereasnutrients on the leaf surfaces are much morelimited and spatially heterogeneous, mostlyleaked out from the apoplast through naturalsurface openings and wounds (72). Numerousmicroorganisms, both pathogenic and sapro-phytic, can survive and even proliferate to var-ious degrees as epiphytes on the plant surfacewithout causing disease. Strict epiphytes com-plete their life cycles on the surface of plants andgenerally do not (or are unable to) colonize theleaf apoplast (6, 7). In contrast, foliar pathogens,such as Pseudomonas syringae, can multiply ag-gressively in the apoplast as pathogenic endo-phytes, ultimately leading to disease produc-tion under favorable conditions (47). Entry intoplant tissue is likely a critical first step in the es-tablishment of foliar infection. To gain access tothe intercellular spaces and internal leaf tissues,pathogens must cross the surface cuticle andepidermis. Many fungal plant pathogens havethe ability to directly penetrate the epidermisusing cuticle- and cell wall–degrading enzymes,mechanical force, or both (78). Bacteria, how-ever, cannot directly penetrate the leaf epider-mis and must enter leaf tissues by alternativemeans.

There are a number of natural surface open-ings through which bacterial pathogens havebeen observed to enter the plant: these in-clude (a) hydathodes: water-exuding pores atthe edges of leaves (e.g., Xanthomonas campestrispv. campestris), (b) stomata: openings in the aerialpart of plants that control gas exchange and wa-ter transpiration between the plant interior andthe environment (e.g., Pseudomonas syringae),

(c) nectarthodes: nectar-secreting pores at thepoint of emergence of the styles and stamens(e.g., Erwinia amylovora), and (d ) lenticels: res-piration pores in stems and roots (E. carotovoravar. atroseptica). Among natural openings, stom-ata dominate in number in the aerial part of theplant and therefore represent one of the mostimportant routes for the entry of foliar bacterialpathogens. In addition to natural openings, bac-teria can also reach the plant interior throughsurface wounds caused by insects, mechanicalor frost damage, hail, wind-blown sand, rain-storms, or damage caused by lateral root for-mation, breakage of trichomes, or abscission ofleaves (leaf scars).

It has been widely assumed that natural sur-face openings and wounds are passive ports forbacterial entry, where bacteria lack active mech-anisms for gaining entry, and plants similarlylack active mechanisms for preventing entry.However, recent evidence suggests that entryof bacteria into leaf tissue through stomata ismore complex and dynamic than the simpleact of swimming into the leaf through passiveopenings (77). In this review, we discuss the re-cent findings on this topic, focusing mainly onthe foliar pathogen Pseudomonas syringae. Webegin with a brief overview of the lifestyle ofP. syringae before presenting an in-depth discus-sion of the role of stomata in bacterial infectionand host defense.

PSEUDOMONAS SYRINGAE—A MODEL FOLIAR PATHOGEN

Basic Infection Cycle

The species P. syringae is composed of strainsthat collectively infect hundreds of plant speciesand cause disease symptoms ranging from leafspots to stem cankers. Different strains ofP. syringae are known for their diverse andhost-specific interactions with plants. A spe-cific strain may be assigned to one of about50 pathovars based on its limited host rangeat the plant species level (35), and then furtherassigned to a race based on differential inter-actions among cultivars of a plant species. The

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a b

def

c

Figure 1A simplified diagram of the infection cycle of Pseudomonas syringae. (a) A diagram of healthy plant leaves.(b) Bacterial cells on a leaf surface, illustrating aggregation of some bacteria near a trichome. (c) Bacteriapenetrating open stomate. (d ) Cross-section of a leaf showing bacteria colonizing the plant apoplast.(e) Extensive multiplication of bacteria in the apoplast of a leaf. ( f ) Visible disease-associated necrosisand chlorosis.

infection cycle of P. syringae is typical for a fo-liar pathogenic bacterium. In the field, there aremultiple sources of P. syringae inocula, includinginfected seeds, diseased plants, and/or asymp-tomatic alternative hosts or even nonhost plants(47). In a successful disease cycle, P. syringaestrains generally live two lifestyles that are spa-tially and temporally interconnected: an initial,largely epiphytic phase upon the arrival on thesurface of a healthy plant and a subsequent,largely endophytic phase in the apoplast (6) (seeFigure 1 for a conceptual illustration). Underfavorable environmental conditions (e.g., heavyrain, high humidity, moderate temperature),P. syringae can multiply very aggressively in asusceptible host plant, resulting in the appear-ance, within several days, of disease symptoms(e.g., water-soaking in the apoplast of infectedtissues, localized tissue necrosis, and various tis-sue discolorations). It has been suggested that

some strains of P. syringae may be primarilyepiphytes and cause disease only accidentally(46, 47).

In terms of the mode of pathogene-sis, P. syringae may be best described as ahemibiotrophic pathogen, because the most ag-gressive phase of population increase occurs inthe absence of apparent host cell death. How-ever, at the late stage of pathogenesis (oftenafter bacteria have almost reached their peakpopulation in the apoplast), host cells die andinfected tissues show extensive necrosis. Thispathogenesis mode seems distinct from thatof strictly biotrophic pathogens, which obtainnutrients from living host cells without caus-ing host cell death, or of strictly necrotrophicpathogens, which kill host cells as the mainstrategy of obtaining nutrients, causing ex-tensive host cell death during early stages ofinfection.

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Pst: Pseudomonassyringae pv. tomato

Epiphytic vs Endophytic Lifestyles

The fact that P. syringae has evolved two dis-tinct lifestyles (epiphytic vs endophytic) begsthe question of how P. syringae (and other fo-liar bacterial pathogens) makes the transitionfrom the epiphytic phase to the endophyticphase during a successful infection cycle. Thisis clearly one of the most outstanding questionsin bacterial disease epidemiology, yet we stillhave little understanding of the process. Theplant surface is generally thought to be a hos-tile environment for microorganisms with lim-ited nutrients, drastic and rapid variations intemperature and humidity even during a sin-gle day, direct exposure to damaging UV ir-radiation, and competition and/or antagonismfrom other epiphytes (6, 72, 101). Studies withmolecular biosensors have provided insights onthe real, microscale environment that bacte-ria live in as epiphytes (72). It seems that theleaf surface is a very heterogeneous space inwhich a few nutrient-rich spots are present.Preferred niches are found along the veins, tri-chomes, and stomata (47, 82). Bacteria that landon less favorable areas of the leaf have to evolvemechanisms to cope with the harsh environ-ment. Some bacteria avoid the stress in theleaf surface by creating microenvironments toprotect themselves. For instance, some Pseu-domonas strains alter the leaf surface propertiesby producing surfactants that increase the wet-ness of the leaf surface (19, 50). In addition, epi-phytes frequently live in large, heterogeneousaggregates and biofilms on the leaf surface, pre-sumably to better resist environmental stressessuch as desiccation (25, 81, 82).

Because P. syringae and other foliarpathogens can multiply very aggressively in theleaf apoplast, one might ask why P. syringae doesnot just quickly enter the plant and multiplyas endophytes, instead of evolving a separateepiphytic lifestyle. The plant interior wouldpresumably shield bacteria from many of thesurface-associated stresses. As this questionis intimately related to the discussion of theentry process, we consider several possibleevolutionary forces that may have driven the

development and maintenance of a distinctepiphytic lifestyle in foliar pathogenic bacteriain spite of their high multiplication potentialin the apoplast.

First, the plant interior may present anotherharsh environment for bacteria. Many sapro-phytic and human pathogenic bacteria can mul-tiply significantly on the plant surface (6, 15);however, they generally do not seem to beable to multiply inside the leaf apoplast (57;W. Underwood & S.Y. He, unpublished data).It is likely that the apoplast contains higherconcentrations of defense compounds and/orunique defense mechanisms not encounteredon the leaf surface, because the surface cuti-cle and epidermis provide formidable physicalbarriers that would reduce diffusion of chemi-cals to the plant surface. In support of this no-tion, several recent studies show that the leafsurface of cutin-defective mutant Arabidopsisplants seems to contain higher concentrationsof antifungal compounds (10, 20, 106). Nev-ertheless, it is now well documented that vir-ulent P. syringae and presumably other foliarpathogens have evolved virulence effectors toevade or suppress host defenses in the apoplast(23, 53, 88). Therefore, although the apoplastmay be a hostile environment and inhabitablefor saprophytic and other nonphytopathogenicbacteria, it seems unlikely that host defense inthe apoplast would be the primary reason whyP. syringae and other adapted foliar pathogensneeded to evolve and maintain an epiphyticlifestyle.

The second possible reason for maintainingan epiphytic lifestyle could be that the notion ofnutrient abundance in the plant apoplast may beincorrect. Theoretical calculations by Hancock& Huisman (42) suggest that there is a sufficientamount of nutrition in the apoplast to supporthigh-density growth of bacteria without activetransport of sugars from host cells. Consistentwith such calculations, P. syringae pv. tomato (Pst)DC3000 can grow to very high numbers inthe wash fluid of the Arabidopsis leaf apoplast,almost approaching those in rich media(Figure 2). However, as preparation of apoplastwash fluids involves infiltration of water into the

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apoplast followed by low-speed centrifugation,it is possible that these retrievable apoplast nu-trients are normally sequestered in locations notaccessible by bacteria. One of the early diseasesymptoms commonly associated with foliar in-fections by pseudomonads and xanthomonadsis water-soaking in the apoplast of infected tis-sues (30, 52), although it is not yet clear whetherapoplast water-soaking is a bacterial strategy forretrieving nutrients or simply a side effect ofhost cell damage caused by infection. Even ifthe bulk of apoplast nutrients is not accessi-ble to nonphytopathogenic bacteria, it seemslikely that P. syringae and other adapted foliarpathogens must have evolved effective virulencemechanisms to obtain nutrients from the hostapoplast.

If neither host defense nor nutrient avail-ability provides a definitive explanation for theneed of foliar pathogens to evolve and maintainan extended epiphytic lifestyle, a third possi-ble reason could be that entry sites on the leafsurface are limited. How limited are the poten-tial entry sites on the plant surface? In leaves, asmentioned above, stomata dominate in numberand represent an important surface opening forbacterial pathogens to enter the plant. The den-sities of stomata vary among plant species andare regulated developmentally and in responseto environmental changes (64, 116). There maybe up to 300 stomata per mm2 on the leaf sur-faces, and the stomatal pores may occupy up to2% of the total leaf surface area (64, 87, 116).Therefore, despite their abundance, stomataaccount for only a minor portion of the leafsurface; the chance of a bacterial immigrant ar-riving directly on top of a natural opening, suchas a stomate, is small. The density of stomata onthe upper leaf (where bacteria are most likely toland) is generally significantly lower than thaton the lower surface of the leaf. Furthermore,stomata open only during the day, to uptakeCO2 for photosynthesis; they close at night toreduce water transpiration. Thus, during peri-ods of a disease cycle, the only niche availablefor many individuals of a P. syringae populationmight be the surface of a plant, even thoughthe plant apoplast may ultimately provide a bet-

Water Luria-Bertanibroth

1.E+10

1.E+09

1.E+08

1.E+07

1.E+06

1.E+05

1.E+04Intercellularwash fluid

hrp-inducingminimal medium

Figure 2Pst DC3000 populations 8 h after transferring 1.2 × 106 CFU/ml bacteria inwater, Luria-Bertani broth, intercellular wash fluid, and hrp-inducing minimalmedium. Data points are means and standard deviations of three replicatecultures.

ABA: abscisic acid

ter niche for P. syringae. The limited entry siteswould require a successful bacterial pathogento adhere and move toward natural openings orwounds. Indeed, adherence and motility havebeen shown to be important for surface infec-tion by P. syringae and other foliar pathogens(5, 41, 43, 90, 95). Furthermore, movement to-ward natural openings and wounds would prob-ably occur only if the leaf surface is wet, whichmay be one reason why foliar disease outbreaksoften follow periods of rain and high humidity(47).

STOMATAL RESPONSETO BACTERIA

Stomata are composed of a pair of special-ized epidermal cells referred to as guard cells(Figure 3). Stomata regulate gas exchange be-tween the plant and environment and controlof water loss by changing the size of the stom-atal pore. This stomatal movement is affectedby several environmental stimuli, such as rela-tive humidity, CO2 concentration, and light in-tensity. The plant hormone abscisic acid (ABA)plays a central role in guard cell signaling, lead-ing to stomatal closure during drought stress.The ABA signal in guard cells is transducedthrough a complex network of signaling events,

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PAMPs or bacteria

TobaccoTomato

30

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Water

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Figure 3(a) Light-conditioned leaf epidermis (top pictures) showing mostly open stomata and the same leaf epidermisafter 1 h exposure to purified PAMP (100 ng/ul LPS; tomato leaf epidermis) or live bacteria (1 × 10e8CFU/ml of P. syringae pv. tabaci; tobacco leaf epidermis). (bottom pictures). Note that most stomatal pores areclosed after exposure to bacteria or PAMPs. (b) Stomatal response in light-exposed epidermal peels ofArabidopsis leaves exposed to water, DC3000 (1 × 10e7 CFU/ml), or DB29 (1 × 10e7 CFU/ml). DB29 is acoronatine-biosynthesis-defective (cor) mutant (17). Stomatal response to this cor mutant is similar toresponse to DC3118, another cor mutant (74, 77).

NO: nitric oxide

Pathogen-associatedmolecular pattern(PAMP): sometimescalled microbe-associated molecularpattern (MAMP),referring to hostimmune response-eliciting moleculescommon to manyclasses of microbes,such as bacterialflagellin

SA: salicylic acid

including the production of compounds such asnitric oxide (NO) and H2O2, signaling interme-diaries such as the guard cell-specific OST1 ki-nase, changes in cytosolic Ca2+ concentrations,and Ca2+ oscillations (31, 51, 84, 96, 97). Thesignaling events triggered by ABA ultimatelylead to regulation of ion channels, such as theguard cell outwardly rectifying potassium chan-nel GORK1, which modulates ion efflux fromthe guard cells (48). Ion efflux from the guardcells also drives the efflux of water and results ina change in guard cell turgor that causes closureof the stomatal pore. For a more comprehensiveoverview of guard cell signaling networks, thereader is directed to two recent reviews (31, 51).

Until recently, it was thought that stomatawere a strictly passive port of entry, suggest-ing that bacteria could freely invade throughopen stomata. However, Melotto and col-leagues (77) found that Arabidopsis stom-ata close in response to live bacteria andpurified pathogen/microbe-associated molec-ular patterns (PAMPs/MAMPs). Bacterium-induced stomatal closure requires PAMP sig-

naling and homeostasis of the defense hormonesalicylic acid (SA), and it is connected to signal-ing regulated by ABA in the guard cell.

Basic Observations

Melotto et al. (77) used light-adapted Arabidop-sis plants, in which 70%–80% of stomata in theleaves are open, for their experiments. Whenleaves or epidermal peels were exposed to a sus-pension of Pst DC3000, a virulent pathogen oftomato and Arabidopsis (54, 114), a marked re-duction in the number of open stomata was ob-served within 1 to 2 h of incubation (Figure 3).In contrast, the number of open stomata re-mained virtually the same in leaves incubatedwith water. The Pst DC3000-induced closingof stomata was transient. After 3 h of incuba-tion, most stomata (Figure 3b) reverted to theopen state.

Because the phyllosphere is also colo-nized by other microorganisms, including hu-man pathogenic bacteria (11, 72, 85), Melottoand colleagues (77) conducted experiments

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with Escherichia coli O157:H7, a humanpathogenic bacterium commonly associatedwith vegetable-based food poisoning (91).Again, stomatal closure was observed within 2 hof incubation. However, in this case, stomataclosure persisted for the duration of the experi-ment (4 to 8 h). These results demonstrate thatstomata actively close as an initial response toboth plant and human pathogenic bacteria, andPst DC3000 has evolved a mechanism (or mech-anisms) to reopen stomata 3 h after incubationwith plant leaves or epidermal peels.

The ability of both human and plantpathogenic bacteria to induce stomatal closurewithin the first hour of contact with plant tissuesuggests that guard cells can sense conservedbacterial molecules. PAMPs are such molecules,and they are best known for their ability to stim-ulate innate immunity in plants and animals (37,102). Indeed, flg22 (a biologically active pep-tide derived from flagellin; 3, 123) and LPS(lipopolysaccharide) (120) cause stomatal clo-sure in the wild-type Col-0 plant (77), suggest-ing that stomatal closure is part of the PAMP-triggered innate immune response.

As mentioned above, one difference be-tween stomatal response to the humanpathogen E. coli O157:H7 and to the plantpathogen Pst DC3000 is that stomata reopenafter approximately 3 h of incubation with PstDC3000, but not with E. coli O157:H7. Melottoet al. (77) suspected that Pst DC3000, but notE. coli O157:H7, may have evolved a naturalvirulence mechanism that can counter PAMP-induced stomatal closure. Pst DC3000 containstwo well-characterized virulence factors: thehrc/hrp gene-encoded type III secretion system(TTSS), which delivers numerous effector pro-teins into the host cell (1, 9, 38, 45, 70), and thephytotoxin coronatine (COR) (17, 74). It wasfound that a COR-deficient mutant could notreopen closed stomata, whereas a hrc mutantwas not affected in the ability to reopen stom-ata (77). Furthermore, green fluorescent pro-tein (GFP)-labeled Pst DC3000 bacteria wereable to reach the upper surface of Arabidopsisepidermal peels when placed underneath,whereas fewer GFP-labeled cor− bacteria were

LPS:lipopolysaccharide

hrp: hypersensitiveresponse andpathogenicity

Type III secretionsystem (TTSS): aspecialized proteinsecretion system usedby Gram-negativebacteria to injecteffector proteins intoplant or animal hostcells; composed ofapproximately 20proteins located in thebacterial or host cellwall/membrane

Coronatine (COR):a phytotoxin producedby several pathovars ofPseudomonas syringaeconsisting ofcoronafacic acid(CFA), an analog ofmethyl jasmonic acid(MeJA), andcoronamic acid(CMA), whichresembles1-aminocyclopropane-1-carboxylic acid(ACC), a precursor toethylene

Plant innate immunesystem: a complexdefense networkconsisting of twobranches; the firstrecognizes moleculescommon to manyclasses of microbes,such as bacterialflagellin, and thesecond responds tospecific pathogenvirulence factors

found on the upper surface of the epidermalpeels (77). Taken together, these results showthat one of the virulence factors involved in sup-pressing stomatal defense in this bacterium iscoronatine.

These initial observations indicate thatstomata are not merely passive ports for bacte-rial entry. Guard cells could perceive live bac-teria including P. syringae and E. coli and closea significant percentage of stomata, suggest-ing that plants have evolved mechanisms to re-duce the entry of bacteria as an integral partof the plant innate immune system. Becausenot all stomata are closed in response to bacte-ria or PAMPs (77), the apparently nonrespon-sive and/or dead open stomata would providea route for bacterial entry, but only at a basallevel. In the case of the virulent plant pathogenPst DC3000, bacteria produce the diffusible vir-ulence factor COR to reopen closed stomata,thereby significantly increasing the number ofsites for bacterial invasion (Figure 4).

Signaling Components duringStomatal Response to Bacteriaand PAMPs

How do stomatal guard cells sense bacteriaand PAMPs? Currently our understanding ofthis process is at an infant stage. Plants sensePAMPs via leucine-rich repeat (LRR) recep-tors (4, 9, 44), such as the flagellin receptorFLS2 (21, 37). In addition to flagellin, otherhighly conserved bacterial molecules such asRNA-binding motif RNP-1, elongation factorTu, and LPS also elicit hallmark responses ofPAMP-induced innate immune response (33,63, 120, 122). Melotto and colleagues (77)found that the flg22 PAMP failed to induce theclosure of stomata in epidermal peels of the Ara-bidopsis fls2 flagellin receptor mutant (37), sug-gesting that guard-cell perception of flg22 re-quires the cognate PAMP receptor. Both flg22and LPS induced the production of nitric oxide(NO) in guard cells of wild-type stomata within10 min. In addition, Nω-nitro-l-arginine (l-NNA), an inhibitor of nitric oxide synthase, ef-fectively prevented stomatal closure caused by

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PAMPs Effectors

COR

COR

PAMPs

PAMPsCOR

PAMPs PAMPs

EffectorsCOR

1 h after bacterial exposure

PAMPs

PAMPs

PAMPs

PAMPs PAMPs

Epidermis

Mesophyll cells

GC

GC

GC

GC

GC

aetamots desolCetamots nepO

b

c

GC

Most stomata are closed, allowing only a basal level of bacterial entry

3 h after exposure to Pst DC3000

Most stomata are open, allowing increased bacterial entry

Figure 4A diagram depicting stomata as entry sites for bacterial invasion. (a) A cross-sectional view of leaf epidermis and mesophyll cellsshowing that stomata, formed by pairs of guard cells (GC), in light-adapted Arabidopsis leaves are mostly fully open. (b) Upon exposureto bacteria, guard cells perceive PAMPs and many stomata close within 1 h. Because not all stomata are closed, those “nonresponsive”open stomata (e.g., stomate on the left) may provide a route for bacterial entry at a basal level. Dashed arrows indicate diffusion ofPAMPs on the epidermal surface or in the intercellular space (apoplast) between mesophyll cells, which can also perceive PAMPs andactivate additional host defenses. (c) In the case of the virulent plant pathogen Pst DC3000, 3 h after infection bacteria producediffusible COR in the apoplast and/or on the plant surface to reopen closed stomata (e.g., middle stomate), thereby increasing thenumber of sites for bacterial invasion. Pst DC3000 bacteria also inject TTSS effectors into mesophyll cells to suppress host defensesand to release nutrients. COR and TTSS effectors have overlapping functions in the suppression of host defenses in the apoplast and inthe promotion of disease symptoms (see text). COR also induces disease susceptibility systemically in leaves (24; see text). This figure isadapted from Underwood et al. (110) with permission of the publisher.

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flg22, LPS, and E. coli O157:H7, suggesting thatNO is also required for PAMPs and bacteria toclose stomata.

In addition to the PAMP signal transductionpathway, SA and ABA were found to be requiredfor stomatal response to bacteria or PAMPs.Neither flg22 nor LPS induce stomatal clo-sure in the ABA biosynthetic mutant aba3-1 (68)or ABA signaling mutant ost1-2 (84). Similarly,stomata in SA-deficient Arabidopsis nahG (27)or eds16 (115) plants do not respond to PAMPs(77). The defect in PAMP/bacterium-triggeredstomatal closure in SA-deficient plants is in-teresting, as previous studies have shown thatSA is a potent inducer of stomatal closure (66,75, 83). Taken together, these results suggestmechanistic connections between PAMP sig-naling, SA homeostasis, and ABA homeosta-sis and signaling in guard cell response tobacteria.

The requirement of SA and ABA forPAMP/bacterium-triggered stomatal closuredoes not mean necessarily that the SA orABA signaling network lies downstream ofthe PAMP signaling network, with the PAMPreceptors at the top of a signaling cascade. Itis equally possible that PAMP, SA, and ABAsignaling networks function in parallel, butare interconnected through specific branches,in the guard cell. A defect in the SA orABA signaling network may cause pathwayperturbation in the guard cell that indirectlycompromises PAMP signaling. In addition, italso remains to be determined whether percep-tion of PAMPs by immune receptors such asFLS2 leads to increased biosynthesis of SA andABA or, simply, whether PAMP signaling inthe guard cell requires a basal level of SA andABA. Clearly, a more comprehensive analysisusing additional signaling mutants is necessaryto understand the connections between variousbranches of SA, ABA, and PAMP signalingnetworks in the guard cell. Current studiesof plant defenses are mostly based on mixedcell types (i.e., whole leaves). It is possiblethat stomatal guard cells have unique signalingproperties that may require cell type-specificanalyses.

COR-Mediated Suppressionof Stomata-Based Defense

The discovery that COR is required for re-opening stomata by Pst DC3000 represents thefirst identification of a bacterial virulence factorthat suppresses stomatal closure (77). COR isa nonhost-specific polyketide toxin consistingof two distinct structural components, coron-afacic acid (CFA) and coronamic acid (CMA);it is produced by at least five different pathovarsof P. syringae: pvs. tomato, glycinea, atropurpurea,maculicola, and morsprunorum (8). The leavesof many plants develop chlorosis when treatedwith purified COR (8). In addition, COR isrequired for full virulence of COR-producingP. syringae strains on Arabidopsis, tomato, andsoybean, as it promotes bacterial growthand contributes to symptom development(Figure 5) (16–18, 80, 121). Although CORis one of the most extensively studied bacte-rial phytotoxins, the precise molecular mech-anisms through which COR acts to promotebacterial virulence and disease development arenot yet clear. The possibility that COR couldsuppress an early defense response during Pst

Wild-typebacterium

Coronatine-defectivemutant

Figure 5Tomato leaves 5 days after dip-inoculation with1 × 107 CFU/ml suspension of the wild-typebacterium Pst DC3000 (left) and the coronatine-defective mutant Pst DC3118 (right). Whereas PstDC3000 caused necrotic lesions with diffusechlorosis, the coronatine-defective mutant bacteriacaused only some lesions and no chlorosis.

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Jasmonates ( JA): agroup of lipid-derivedplant hormonesinvolved in plantsexual reproductionand defense againstinsects and somepathogens; synthesizedfrom linolenic acid bythe octadecanoidpathway

DC3000 infection of Arabidopsis and tomato wassuggested more than a decade ago. Mittal &E Davis (80) first noticed that cor− mutantbacteria achieved population densities sim-ilar to those of wild-type Pst DC3000 inArabidopsis leaves inoculated by infiltratingbacterial suspension (1 × 106 CFU·ml−1) di-rectly into the leaf intercellular spaces, butcaused fewer disease symptoms, including lesschlorosis and necrosis compared to inoculationwith wild type. However, when bacteria (1 ×108 CFU·ml−1) were dip-inoculated onto thesurface of plant leaves, cor− mutant bacteriaachieved significantly lower population densi-ties than wild-type Pst DC3000 and were unableto cause disease symptoms.

A similar study was conducted with P.syringae pv. glycinea (Psg), a cool-weatherpathogen of soybean. In this pathogen, pro-duction of COR is temperature-dependent.COR production peaks at a temperature of18◦C, whereas at 28◦C no COR productioncan be detected (18). When spray-inoculatedonto soybean plants, bacteria derived fromcultures grown at 18◦C achieved significantlyhigher population densities and caused moresevere disease symptoms than bacteria cul-tured at 28◦C (18). However, when bacteriawere artificially infiltrated into the leaf inter-cellular spaces, no effect of the preinocula-tion temperature of bacterial cultures was ob-served. Additionally, COR-deficient mutants ofPsg PG4180 were severely reduced in multipli-cation (∼10,000-fold less than wild type) wheninoculated by spraying. When inoculated by in-filtration, however, the COR-deficient mutantbacteria achieved population levels only five-to tenfold lower than those of wild type. Theseresults again suggest that COR plays an impor-tant role early in the infection process, likely bypromoting entry of bacteria into leaf tissues.

The demonstrated ability of COR to sup-press stomatal closure therefore offers one pos-sible explanation for these earlier observations.COR also plays roles in the late stages of the in-fection process, including suppression of localand systemic host defenses and promotion ofdisease symptoms (24, 62, 110, 112). As disease

progresses, Pst DC3000 heavily colonizes thesubstomatal cavity (14), and it is possible thatCOR-induced stomatal opening is also impor-tant for the release of bacteria to the leaf surfaceto facilitate subsequent spread to nearby healthyplants.

How Might COR SuppressBacterium/PAMP-TriggeredStomatal Closure?

COR has long been hypothesized to be a struc-tural mimic of jasmonates ( JA), possibly bybinding to the same or similar receptors. In par-ticular, COR is most similar to JA conjugatedto the amino acid isoleucine ( JA-Ile; Figure 6)(61, 99). COR also seems to have functionalsimilarities to JA (34, 39, 60, 113). COR inducesa number of responses that are also induced bytreatment with JA, including induction of pro-tease inhibitors and phytoalexins and regula-tion of JA-responsive transcripts (89, 104, 107,111, 113, 121). Identification of the Arabidop-sis JA/COR perception mutant coi1 (coronatineinsensitive 1), which is insensitive to both JAand COR, further suggests a similar mode ofaction (58, 117). The COI1 gene encodes the

CFA CMA

C

O

O

O

CO2H

CO2H

NH

C

O

NH

COR

JA-Ile

Figure 6The chemical structures of JA-Ile and coronatine aresimilar. Drawn with the help of Dr. Barbara Kunkel.

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F-box subunit of the SCFCOI1 ubiquitin ligase,which is involved in the ubiquitin-conjugationpathway of protein degradation (28, 109, 119).The coi1 and jai1 (jasmonic acid insensitive 1)mutants of Arabidopsis and tomato show en-hanced resistance to Pst DC3000 (34, 58, 121)and the ability of COR to inhibit stomatal clo-sure is dependent on the COI1 gene (77). How-ever, the reduction in bacterial multiplicationin coi1 and jai1 plants cannot be fully explainedby a lack of COR function. This reductionis much greater than that observed after in-oculation with cor− mutant bacteria, suggest-ing that other virulence factors produced byPst DC3000 also target COI1/JAI1-dependentpathways to promote susceptibility. Consistentwith this hypothesis, several studies suggest thatCOR and TTSS effector proteins have over-lapping functions and act synergistically to in-duce JA signaling (107, 121). Thus, althoughCOR has a unique role in suppressing stom-atal closure (77), its contribution in later stagesof infection could be potentially masked by thefunctions of some TTSS effectors under certainconditions, as hrp genes and TTSS effectors arealso important for bacterial multiplication andsymptom production (26, 71).

Although COR likely mimics JA-Ile in ex-erting its virulence function, Suhita and col-leagues (100) showed that exogenous applica-tion of JA or methyl JA (MeJA) alone causedstomatal closure. However, the authors did notexamine a possible effect of JA or MeJA onABA-induced stomatal closure. In our own ex-periments we found that high concentrations(>20 μM) of JA and MeJA (Sigma Co.) causedsignificant stomatal closure. However, lowerconcentrations (e.g., 2 μM) of JA and MeJA didnot; instead, like COR, they effectively inhib-ited ABA from closing stomata (M. Melotto &S.Y. He, unpublished data). These results sug-gest a potentially bimodal effect of exogenousJA and MeJA on stomatal response, dependingon concentrations used.

Recent studies have begun to shed somelight on how COR and JA might act throughSCFCOI1 to perform their functions in the plantcell (22, 108). The COI1 protein shares a

SCF ubiquitin ligase:a type of E3 ubiquitinligase consisting ofSKP1, CULLIN1, andF-box proteins. E3ubiquitin ligasescatalyze conjugation ofubiquitin to targetproteins, which is akey step in initiatingprotein degradationthrough the 26Sproteasome

COI1: coronatineinsensitive 1

TIR1: transportinhibitor response 1

JAZ: jasmonate ZIMdomain protein

high sequence similarity with TIR1 (transportinhibitor response 1), which is the F-boxsubunit of the SCFTIR1 ubiquitin ligase in-volved in the ubiquitination and degradation ofAUX/IAA repressor proteins during plant hor-mone auxin signaling (55). TIR1 and AUX/IAAproteins interact with each other and this inter-action does not require any other plant protein,but is facilitated by auxin, which directly bindsto the TIR1 receptor protein (29, 105). This ex-citing demonstration raises the possibility thatF-box proteins or E3 ubiquitin ligase-substratecomplexes may have a general role in serving asreceptors of other plant hormones and relatedligands. It was shown recently that COI1 inter-acts with JAZ ( jasmonate ZIM-domain) pro-teins in Arabidopsis (22, 108). JAZ proteins arerepressors of the JA signaling pathway and aredegraded by the 26S proteasome in a SCFCOI1-dependent manner (22, 108). We have shownthat COI1 and JAZ proteins interact in yeastcells (i.e., in the absence of any other plant pro-tein), but only in the presence of JA-Ile (108)or COR (M. Melotto & S.Y. He, unpublisheddata). This observation implicates COI1-JAZcomplexes as receptors of JA-Ile and COR inplants (Figure 7).

It is not yet clear how the SCFCOI1-dependent action of COR translates into inhi-bition of stomatal closure. Several studies haveshown strong antagonistic interactions betweenJA signaling and SA- or ABA-mediated sig-naling (2, 16, 36, 65, 69, 73, 93, 98). For in-stance, transcripts associated with SA-mediateddefenses are upregulated in coi1 and jai1 mu-tants and the reduced susceptibility of coi1 toPst DC3000 is dependent on SA accumula-tion (58, 121). Additionally, the multiplica-tion of cor− mutant bacteria is significantlyenhanced in plants defective in SA accumu-lation, such as those expressing the salicylatehydroxylase encoded by nahG, and plants defec-tive in SA biosynthesis, such as the Arabidopsissid2 (eds16-1) mutants (13, 16, 77). Similarly, theArabidopsis ABA biosynthesis mutant aba3-1and the guard cell signaling mutant ost1, allowenhanced multiplication of cor− mutant bacte-ria after surface inoculation (77). Therefore, an

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26S

COI1

Guard cell wall

JA response gene MYC2

Innate immunity responses, including stomatal closure

Pst DC3000

COI1

X

Guard cell

Flagellin, LPS, etc.

SAABAOST1

FLS

2

JAZ

JAZ

Figure 7A model of COR action in the plant cell (possibly all cell types). COR (structure shown) is secreted by PstDC3000 into the plant cell and increases the affinity of the COI1 protein (as part of the SCFCOI1-ubiquitinligase complex; not shown here) toward JAZ repressor and possibly other host proteins (denoted by X). TheSCFCOI1 complex catalyzes ubiquitination of JAZ and other host proteins, which are then degraded throughthe 26S proteasome (denoted as 26S). JAZ proteins normally function as repressors by physically binding totranscriptional activators (such as MYC2) of jasmonate response genes. In the stomatal guard cell, PAMPs(e.g., flagellin and LPS) are perceived by cognate immune receptors (e.g., flagellin receptor FLS2).Perception of PAMPs triggers stomatal closure, which requires SA, ABA, and OST1 kinase. COR-mediatedinhibition of PAMP-triggered stomatal closure and other innate immune responses could be mediated byJAZ and/or X proteins.

attractive hypothesis is that COR exploits theendogenous antagonistic interactions betweenJA, SA, and ABA hormone signaling pathwaysin plants to affect stomatal response. Future re-search should determine whether COR antag-onizes both SA and ABA signaling pathwaysor inhibits only SA or ABA signaling, whichin turn affects the other pathway through vari-ous network connections. Currently, the entiresignaling network in the guard cell is not well

understood, and a systems biology approachwill ultimately be necessary to fully understandthe extremely dynamic nature that character-izes guard cell signaling (69a).

POSSIBLE IMPLICATIONSIN OTHER PATHOSYSTEMS

The discovery of bacterium/PAMP-inducedstomatal closure in Arabidopsis as part of the

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plant innate immune response against bacterialinvasion prompts an important question: Arestomata regulated in other pathosystems? Toanswer this question, Melotto and colleagues(77) investigated the response of tomato stom-ata to Pst DC3000 and LPS. They found thatstomatal movement in tomato was affected sim-ilarly to that in Arabidopsis. However, only a fewpathovars of P. syringae produce COR. There-fore, if stomatal regulation is widespread inplants, other phytopathogenic bacteria likelyemploy distinct virulence factors or lifestyles toovercome or circumvent stomatal closure. P. sy-ringae pv. tabaci, which does not produce COR,induced closure of stomata initially, but was ableto reopen stomata at later time points, similarto Pst DC3000 (77). These results suggest thatthere may be other virulence factors used bysome bacteria to overcome stomatal defense.

The ability to overcome stomatal defense, orlack thereof, may influence pathogenesis strate-gies and the lifestyle of foliar bacteria. Some fo-liar bacteria may not produce virulence factorsthat suppress stomatal closure and thereforemay not be able to actively overcome PAMP-induced stomatal closure. These bacteria wouldprobably have to wait for favorable environ-mental conditions under which stomatal de-fense is compromised, or rely on wounds, orevolve alternative mechanisms to exploit othernatural openings. Histochemical detection ofa β-glucuronidase (GUS)-expressing strain ofthe vascular pathogen Xanthomonas campestrispv. campestris (Xcc) demonstrated that Xcc entersArabidopsis leaves primarily through hydath-odes, whereas the closely related X. campestrispv. armoraciae enters leaves primarily throughstomata (49), even though Xcc can multiplyand cause disease if pressure-infiltrated throughstomata (92). Although the molecular mecha-nisms underlying this portal of entry preferenceare not yet known, it is tempting to speculatethat Xcc may lack the ability to overcome stom-atal defense and, instead, has evolved the abilityto enter leaves through hydathodes.

The inability to efficiently enter leaf tissuesthrough stomata may also be a factor in pro-moting the evolution of an enhanced ability

OGA:oligogalacturonic acid

to live epiphytically on leaf surfaces. On theother hand, Pst DC3000 can actively overcomestomatal defense and appears to be highly in-vasive and a good endophyte, but is a relativelypoor epiphytic colonizer of tomato leaf surfaces(14). It will be interesting to determine whetherthere is a correlation between lack of abilityto overcome stomatal defense and enhancedepiphytic fitness. Comparison of the completegenome sequences of Pst DC3000 and P. s. pv.syringae (Pss) B728a, a bean pathogen with awell-known epiphytic phase in the field, revealsseveral intriguing differences that may be re-lated to adaptation to specific lifestyles (32). Asmentioned above, UV irradiation and water de-ficiency are common stresses on the leaf sur-face, and adapted epiphytes presumably haveevolved mechanisms to cope with such stresses(6, 72, 101). Indeed, Pss 728a carries severalcopies of the rulAB operon involved in muta-genic DNA repair and UV resistance, whereasPst DC3000 carries only one rulB gene (32).Furthermore, the regulatory sequence of therulB gene in Pst DC3000 seems distinct fromthat of rulAB operons in Pss B728a, suggestingpossibly different regulation of UV-tolerance-related genes in these two strains. Adapted epi-phytes respond to water deficiency on the leafsurface often by accumulating certain solutesto increase osmotolerance. Pss B728a seems tocontain more genes involved in the produc-tion of osmotolerance-associated solutes thanPst DC3000 (32).

In addition to plant-bacterial interactions,stomatal regulation has also been observed insome plant-fungal interactions. For example,the fungal toxin fusicoccin has long been knownto promote stomatal opening and to antago-nize ABA-induced stomatal closure through ac-tivation of a plasma membrane H+ ATPase; ithas been widely used as a tool for the study ofmany aspects of plant biology (76). Oligogalac-turonic acid (OGA), an elicitor derived fromthe degradation of the plant cell wall by fun-gal cell wall-degrading enzymes, and chitosan,a component of the fungal cell wall, were bothshown to affect stomatal movements in tomato(67). Both OGA and chitosan elicited H2O2

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production in guard cells and inhibited light-induced opening of closed stomata. Addition-ally, OGA accelerated midday stomatal closurein tomato and in Commelina communis pro-moted closure and inhibited opening of stom-ata. Chitosan and yeast elicitor derived fromSaccharomyces cerevisiae were both found to in-duce stomatal closure and activate Ca2+ influxcurrents in a manner similar to ABA in Ara-bidopsis guard cells (59). Oxalic acid is a viru-lence factor of several phytopathogenic fungi,including Sclerotinia sclerotiorum. Guimaraes &Stotz (40) made the interesting observation thatstomatal pores of Vicia faba leaves infected withan oxalate-deficient mutant of S. sclerotiorumwere partially closed, whereas wild-type funguscauses stomatal opening. Furthermore, exoge-nous application of oxalic acid induces stom-atal opening. Open stomata seem to be theexit sites of many fungal hyphae from infectedleaves. Finally, stomata have also been impli-cated in the response to resistance (R) gene-mediated recognition of a fungal defense elici-tor, Avr9. Recognition of the Avr9 protein fromthe fungus Cladosporium fulvum by transgenicNicotiana tabacum plants expressing the Cf-9 Rprotein from tomato led to the activation ofcurrent through outward-rectifying K+ chan-nels and the inactivation of current throughinward-rectifying K+ channels (12). This pat-tern of regulation of cation channels wouldbe predicted to promote stomatal closure. Al-though in general the biological relevance ofthe reported responses of stomata to fungal-derived compounds with respect to plantdefense or fungal invasion is not yet clear,it is possible that stomate-based defense andcounter-defense also occur in some plant-fungal interactions.

CONCLUDING REMARKS

Stomata have been historically regarded as pas-sive ports of bacterial entry into plant leaves.The discovery of stomatal defense and counter-defense in Arabidopsis plants is likely to openup several new areas for research. Some of thequestions of significant importance that remain

to be answered are highlighted under FutureIssues.

Understanding the effects of varying envi-ronmental conditions on the effectiveness ofstomatal defense would be a particularly im-portant area to enhance our understanding ofoutbreaks of foliar bacterial disease in the field.Stomata are required to respond to a num-ber of stimuli, including light, humidity, CO2

concentration, microorganisms, and the circa-dian clock. How these inputs are prioritized byguard cells and the mechanisms of prioritiza-tion are unknown but are interesting and im-portant questions for future study. Note thatsevere outbreaks of bacterial disease in cropplants are often associated with periods of heavyrain or high humidity. These conditions couldcreate wounds and leaf surface wetness thatwould be favorable for bacterial movement. Itis also possible that, under such environmentalconditions, stomatal defense is partially com-promised because high humidity inhibits stom-atal closure (86, 94, 103, 118), therefore al-lowing more bacteria to enter the leaf tissuethrough stomata to promote infection. To pro-mote infection in the laboratory, researcherscommonly use surfactants to reduce surfacetension and expose plants to very high humid-ity for an extended period after surface inocula-tion. Is stomatal defense partially compromisedunder such conditions? Also, the surface struc-tures (e.g., cuticle, trichomes, stomata) in dif-ferent plant species may differ in sensitivity tosurfactant, humidity, and/or mechanical pres-sure. It would be important to determine howstomata in different plant species are affectedby surfactants, humidity, and/or pressure sprayin early stages of infection.

Stomata open during the day and close atnight. In the absence of extensive wounding, fo-liar bacteria may have to infect leaves primarilyduring the day. However, COR was previouslyfound to induce opening of dark-closed stom-ata in broad bean and Italian ryegrass, demon-strating that COR not only blocks closing ofstomata but also can promote opening of closedstomata (79). The ability of COR to promoteopening of dark-closed stomata may provide a

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mechanism for bacterial entry into plantleaves at night when most stomata are closed,which may have significant epidemiologicalimplications.

Because 108 cfu/ml bacteria are necessaryto reproducibly and uniformly induce diseasein Arabidopsis leaves by the surface inoculationmethod (17, 54, 123), Melotto et al. (77) usedthis inoculation dose in most of their exper-iments. In nature, however, epiphytic popu-lations of bacteria are expected to be highlyvariable, even on the same leaf, due to tremen-dous spatial heterogeneity of nutrient availabil-ity and/or leaf surface topology (72). The totalepiphytic bacterial population on the leaf sur-face can be very high, up to 108 cfu/g freshweight (56, 72). Even when the total numberof bacteria is low on a leaf, bacterial concen-

trations at specific sites can be high, especiallyin aggregates (72), which could contribute tothe observed discrete infection sites/lesions onthe same leaf in the field (in contrast to theuniform infection of entire leaves in the lab-oratory). It will be interesting to determinehow the sizes and locations of these bacte-rial communities affect stomatal defense andwhether aggregation of bacteria near stom-ata promotes stomatal aperature by exposingstomata to high concentrations of compoundsthat promote opening or closure. Clearly, muchis to be learned and, it is hoped, future re-search will provide answers to some of theseimportant questions and contribute to advancesand discoveries in the study of bacterial patho-genesis, guard cell signaling, and microbialecology.

SUMMARY POINTS

1. Numerous bacteria can survive and multiply as epiphytes on a plant surface withoutcausing disease. Saprophytes complete their life cycles on the surface of plants; foliarpathogens eventually enter the plant and multiply in the apoplast as pathogenic en-dophytes. Bacterial entry into plant tissue is a key step for pathogenesis. Unlike fungalpathogens, bacteria cannot directly penetrate the plant epidermis and must rely on woundand natural openings to colonize their host.

2. Historically, natural surface openings and wounds have been regarded as passive portsof bacterial entry into plant leaves. However, a recent study provides evidence for anactive role of stomata in limiting bacterial infection. Stomatal guard cells have the abilityto perceive live bacteria and close the stomatal pore within 1 h of exposure to bacterialsuspension.

3. Bacterium-induced stomatal closure appears to be part of the plant innate immune systemand can be activated by bacterial PAMPs such as the flagellin peptide flg22. Stomatal clo-sure requires PAMP signaling and SA and ABA homeostasis; it is connected to signalingregulated by ABA in the guard cell.

4. The foliar pathogenic bacterium P. syringae pv. tomato DC3000 produces the virulencefactor COR to suppress stomatal closure and promote bacterial entry through stomata.COR, a molecular mimic of JA-Ile, likely modulates the JA signaling pathway in theplant to antagonize the SA and/or ABA signaling that promotes stomatal closure.

5. The discovery of stomatal defense and counter-defense in Arabidopsis plants is likelyto open up new areas for research. Such research should contribute to advances anddiscoveries in the study of bacterial pathogenesis, guard cell signaling, and microbialecology.

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FUTURE ISSUES

1. Disease epidemiology: Do the environmental conditions that modulate stomatal closureand opening (e.g., humidity, light, temperature, circadian rhythm) influence the effec-tiveness of stomatal defense? If so, does possible failure of stomatal defense under certainenvironmental conditions (especially rain and high humidity) contribute to disease out-breaks in the field? The sizes of stomatal openings vary greatly among plant species. Dothe different stomatal sizes impact the effectiveness of stomatal defense?

2. Pathogen lifestyles: Different strains of pathogenic bacteria seem to have different dura-tions of the epiphytic phase before initiating endophytic infection. Is there a correlationbetween a long epiphytic phase and a lack of anti-stomate virulence factors? In otherwords, do pathogenic bacteria prefer to colonize the surface of the plant or are theyforced to stay there until environmental conditions allow massive invasion?

3. Stomatal defense across plant species: Do all plants have stomatal defense mechanisms?Melotto et al. (77) reported that stomatal movement in response to LPS and Pst DC3000in tomato are similar to that in Arabidopsis. In the future, it would be important to examinethis response in other plants and other bacterial and/or fungal diseases.

4. Phyllosphere ecology: How do the sizes of microbial communities in the phyllosphereinfluence stomatal defense? How does stomatal defense impact the distribution of mi-crobial communities on the plant surface and inside the plant?

5. Stomate-targeted virulence factors: How many bacterial and fungal virulence factorstarget stomatal defense? COR is produced by only a few P. syringae pathovars and ifstomatal defense is widespread among plant species, foliar bacterial pathogens couldhave evolved other virulence factors or developed alternative lifestyles (e.g., extendedepiphytic phase) or infection modes (e.g., through other types of natural openings) toovercome or circumvent stomatal defenses. What are the mechanisms by which othervirulence factors inactivate stomatal defense?

DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity of thisreview.

ACKNOWLEDGMENTS

We thank K. Bird for assistance in preparation of this paper, and Wiley-Blackwell Publishing Ltd.for permission to use the content of the reference Underwood et al. (110). Supported by grantsfrom the U.S. Department of Energy and National Institutes of Health.

LITERATURE CITED

1. Alfano JR, Collmer A. 2004. Type III secretion system effector proteins: double agents inbacterial disease and plant defense. Annu. Rev. Phytopathol. 42:385–414

2. Anderson JP, Badruzsaufari E, Schenk PM, Manners JM, Desmond OJ, et al. 2004. Antago-nistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulatesdefense gene expression and disease resistance in Arabidopsis. Plant Cell 16:3460–79

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Annual Review ofPhytopathology

Volume 46, 2008Contents

The Phenotypic Expression of a Genotype: Bringing Muddy Bootsand Micropipettes TogetherRoger Hull � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

The Origin of Ceratocystis fagacearum, the Oak Wilt FungusJennifer Juzwik, Thomas C. Harrington, William L. MacDonald,

and David N. Appel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �13

The Powdery Mildews: A Review of The World’s Most Familiar(Yet Poorly Known) Plant PathogensDean A. Glawe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �27

Plants as a Habitat for Beneficial and/or Human Pathogenic BacteriaHeather L. L. Tyler and Eric W. Triplett � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �53

The Origins of Plant Pathogens in Agro-EcosystemsEva H. Stukenbrock and Bruce A. McDonald � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �75

Role of Stomata in Plant Innate Immunity and Foliar Bacterial DiseasesMaeli Melotto, William Underwood, and Sheng-Yang He � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 101

Models of Fungicide Resistance DynamicsFrank van den Bosch and Christopher A. Gilligan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 123

Siderophores in Fungal Physiology and VirulenceHubertus Haas, Martin Eisendle, and Gillian Turgeon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 149

Breaking the Barriers: Microbial Effector Molecules SubvertPlant ImmunityVera Gohre and Silke Robatzek � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 189

Yeast as a Model Host to Explore Plant Virus-Host InteractionsPeter D. Nagy � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 217

Living in Two Worlds: The Plant and Insect Lifestylesof Xylella fastidiosaSubhadeep Chatterjee, Rodrigo P.P. Almeida, and Steven Lindow � � � � � � � � � � � � � � � � � � � � � � � � 243

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Identification and Rational Design of Novel Antimicrobial Peptidesfor Plant ProtectionJose F. Marcos, Alberto Muñoz, Enrique Pérez-Payá, Santosh Misra,

and Belén López-García � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 273

Direct and Indirect Roles of Viral Suppressors of RNA Silencingin PathogenesisJuan A. Díaz-Pendón and Shou-Wei Ding � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 303

Insect Vector Interactions with Persistently Transmitted VirusesSaskia A. Hogenhout, El-Desouky Ammar, Anna E. Whitfield,

and Margaret G. Redinbaugh � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 327

Plant Viruses as Biotemplates for Materials and TheirUse in NanotechnologyMark Young, Debbie Willits, Masaki Uchida, and Trevor Douglas � � � � � � � � � � � � � � � � � � � � � � 361

Epidemiological Models for Invasion and Persistence of PathogensChristopher A. Gilligan and Frank van den Bosch � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 385

Errata

An online log of corrections to Annual Review of Phytopathology articles may be found athttp://phyto.annualreviews.org/

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