Update on Stomatal Defense

Stomatal Defense a Decade Later1[OPEN]

Maeli Melotto*, Li Zhang, Paula R. Oblessuc, and Sheng Yang He*

Department of Plant Sciences, University of California, Davis, California 95616 (M.M., P.R.O.); Department ofEnergy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824 (L.Z., S.Y.H.);Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824 (L.Z., S.Y.H.); and PlantResilience Institute, Howard Hughes Medical Institute-Gordon and Betty Moore Foundation, Michigan StateUniversity, East Lansing, Michigan 48824 (S.Y.H.)

ORCID IDs: 0000-0001-6021-9803 (M.M.); 0000-0003-3112-7373 (L.Z.); 0000-0002-4977-3317 (P.R.O.); 0000-0003-1308-498X (S.Y.H.).

Plants and microbes have long coevolved in a con-stant battle to overcome themechanisms of defense andattack from both sides. Plants have developed means toprevent pathogen attack by hampering invasion ofplant tissues and actively warding off pathogen colo-nization. On the other hand, pathogens have evolvedstrategies to mask their presence and/or evade hostdefenses. Plant-microbe interaction starts with molec-ular recognition of each other, leading to a cascade ofsignaling events with the final output of plant resis-tance or susceptibility to the pathogen. In thismolecularwar, epidermis of plants is the first barrier that patho-gens need to overtake. Natural openings on the leafsurface, such as stomata, provide an entry site topathogens. Plants have evolved a mechanism to closestomata upon sensing microbe-associated molecularpatterns (MAMPs). This mechanism is known as sto-matal defense. A decade has passed since the discoveryof stomatal defense, and the field has expanded con-siderably with significant understanding of the basicmechanisms underlying the process. Here, we give aperspective of these findings and their implications inthe understanding of plant-microbe interactions.

It has been long recognized that infection of plantsby foliar pathogens involves pathogen penetration intoinner tissues, a niche conducive for living, where theyobtain water and nutrients from internal cells. Routesfor pathogen penetration into the leaves include sto-matal pores, hydathodes, and wounds (either acciden-tal or direct breaching of the cuticle by the pathogenor its vector). The vast majority of contemporary

experiments designed to understand mechanisms ofpathogenesis in plants has relied on inoculation by ar-tificial wounding or direct infiltration of inocula into theapoplast. Although these are valuable approaches todissect plant diseases, they preclude thorough under-standing of a key step for the establishment of disease:pathogen internalization into the host plant. In the lastdecade, we came to realize how dynamic and complexthis entry process is. It requires active, inducible re-sponses on both the host and the pathogen, as well asspecific environmental conditions at the time of pene-tration. Perhaps these strict requirements contribute tothe fact that widespread diseases are rare events innature.

A large number of pathogens use the stomatal poreas a site for penetration into inner leaf tissues. In fact,

1 This research topic in theM.M. lab and S.Y.H. lab is supported bygrants from the U.S. National Institute of Allergy and Infectious Dis-ease (5R01AI068718, M.M. and S.Y.H.), the U.S. Department ofAgriculture–National Institute of Food and Agriculture (2015-67017-23360, M.M. and S.Y.H.), the Center for Produce Safety(CPF43206, M.M.), University of California Davis-FAPESP SPRINT(award 40747474, M.M.), the National Science Foundation (IOS-1557437, S.Y.H.), and the Gordon and Betty Moore Foundation(GBMF3037, S.Y.H.).

* Address correspondence to melotto@ucdavis.edu and hes@msu.edu.

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some pathogens, such as the bacterium Xanthomonascampestris pv armoraciae (Hugouvieux et al., 1998), theoomycete Plasmopara viticola (Allègre et al., 2007), andspecies of the fungus Puccinia (Shafiei et al., 2007;Grimmer et al., 2012) are specialized to internalize intoleaves only through stomata. Earlier observations pro-vided some clues that stomatal closure might diminishbacterial disease severity in a biologically relevantcontext. For instance, reduced number of lesions de-veloped on dark- or abscisic acid (ABA)-treated tomato(Solanum lycopersicum) plants after inoculation with X.campestris pv vesicatoria (Ramos and Volin, 1987). Fur-thermore, direct comparison of disease severity aftersurface-inoculation and apoplastic-infiltration in thispathosystem revealed that bacterium penetrationthrough stomata may be a control point for diseaseprogression (Ramos and Volin, 1987). Characterizationof either bacterial or plant mutants also provided in-dications for possible stomatal control of bacterial in-fection. The coronatine (COR)-deficient mutant ofPseudomonas syringae pv tomato (Pst) causes less diseasewhen inoculated on the leaf surface than when inocu-lated directly into the apoplast of Arabidopsis (Arabi-dopsis thaliana) or tomato (Mittal and Davis, 1995).Similarly, the flagellin-sensitive2 (fls2) mutant of Arabi-dopsis, which lacks the receptor for bacterial flagellin, ismore susceptible than the wild-type plant only whensurface-inoculated with Pst DC3000 (Zipfel et al., 2004).These previous studies set the foundation for a directdemonstration that guard cells surrounding the stomatalpore can sense microbes and close the pore, a processthat is now known as stomatal defense or stomatal im-munity (Melotto et al., 2006; Sawinski et al., 2013).

A number of recent reviews have discussed topicsranging from signaling networks that regulates stomataldefense to mechanisms of endogenous and exogenoussignal integration in guard cells (Arnaud and Hwang,2015; Murata et al., 2015; Cotelle and Leonhardt, 2016;Lee et al., 2016). In addition, the use of thermoimagingtechnology in crop breeding programs and precisionagriculture to assess pathogen-induced stomatal move-ment has been highlighted (Ishimwe et al., 2014; Singhet al., 2016). Furthermore, stomatal closure in response tofungus- and plant-derived elicitors (e.g. chitin, chitosan,and oligogalacturonic acid) and stomatal opening inresponse to fungal metabolites (e.g. fusicoccin) has beenextensively reviewed (Grimmer et al., 2012; Arnaud andHwang, 2015; Murata et al., 2015). We refer readers tothese excellent reviews. Here, we focus on significantadvances toward a mechanistic understanding of sto-matal defense and the impact of this discovery on thestudy of plant-bacterial interactions.

BACTERIUM-TRIGGERED STOMATAL CLOSURE

Stomata open and close daily, reflecting the internalcircadian rhythm of plants. However, bacteria cantrigger stomatal closure under bright daylight (Melottoet al., 2006; Gudesblat et al., 2009; Schellenberg et al.,

2010; Roy et al., 2013), suggesting that stomatal guardcells can perceive bacteria and trigger a signaling cas-cade that overrides the natural circadian rhythm ofstomatal movement. Bacterium-triggered stomatalclosure is a fast response (,1 h) and the basic mecha-nism underlying this process includes the following.

Recognition of Bacteria

Plant perception of bacteria begins with the recog-nitionMAMPs by cognate pattern recognition receptors(PRRs). The most widely studied example of such rec-ognition is flagellin perception by the FLS2 receptor inArabidopsis. Although flagellin recognition has a prom-inent role in stomatal defense during the Arabidopsis-P.syringae pv tomato DC3000 interaction (Zeng and He,2010), the existence of other MAMP-PRR pairs thatfunction in stomatal defense is likely. For instance,stomata of the fls2 mutant still close in response tolipopolysaccharide and Escherichia coli O157:H7(Melotto et al., 2006). However, tools to characterize theimportance of other MAMP-PRR are not completelydeveloped as in many cases either the MAMP or thePRR is not known. The L-type lectin receptor kinaseshave been implicated in Arabidopsis stomatal responseto Pst (Desclos-Theveniau et al., 2012, Singh et al., 2012);however, the cognate ligand(s) have not been de-scribed. Thus, the contribution of this bacterial recog-nition system to stomatal defense cannot be fullyassessed. Similarly, purified lipopolysaccharide fromvarious bacterial strains trigger stomatal closure(Melotto et al., 2006), but the role of its potential cog-nate receptor LIPOPOLIGOSACCHARIDE-SPECIFICREDUCED ELICITATION (Ranf et al., 2015) has notbeen described yet. Another characterizedMAMP-PRRpair is the elongation factor Tu (EF-Tu) and EF-TuRECEPTOR (Zipfel et al., 2006). Purified elf26, anEF-Tu-derived peptide, closes the stomatal pore in theArabidopsis ecotypes Col-0 and Ws4 (Desikan et al.,2008); however, the EF-Tu receptor mutant efr-1 stillretains the wild-type stomatal closure phenotype inresponse to P. syringae (Zeng and He, 2010). It is prob-ably due to the response triggered by other MAMPs,such as flagellin. It has been noted that elf peptides fromdifferent bacteria differ in their potency in inducingstomatal closure. Zeng and He (2010) observed thatelf18, an EF-Tu-derived peptide from E. coli, is morepotent than that of Pst in closing the stomatal pore ofCol-0. Although additional experimentation in a bio-logically relevant context is still needed, emerging evi-dence suggests that, in principle, Arabidopsis guardcells may respond to different bacterial species at vari-ous degrees in part depending on the natural variationsof bacterial MAMPs.

Downstream Signaling

Since 2006, there have been extensive efforts by var-ious groups to elucidate the signaling cascade that

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occurs downstream of bacterial recognition. This sig-naling cascade is largely mediated by (1) secondarymessengers such as reactive oxygen species (ROS),nitric oxide (NO), and calcium; (2) regulators of innateimmune response such as MPK3, MPK4, and MPK6;and (3) plant hormones. While ROS/NO productionand [Ca2+]cyt oscillation have been documentedin guard cells after MAMP recognition (Melotto et al.,2006; Desclos-Theveniau et al., 2012; Arnaud andHwang, 2015), biosynthesis and accumulation of hor-mones in the guard cell have not been fully demon-strated due to technical impediments to directlyquantify hormone concentration in this specialized celltype. Only recently, a fluorescence resonance energytransfer-based reporter system, ABAleons, has beendeveloped in Arabidopsis that enables temporal andspatial mapping of ABA concentration changes in re-sponse to various cues (Waadt et al., 2014). The researchcommunity would really benefit if similar real-time, inplanta reporter systems are available for other hor-mones that play a role in stomatal defense (see Out-standing Questions). Nonetheless, guard cells dorespond to plant hormones. Pharmacological and ge-netic evidence supports that ABA and salicylic acid(SA) are positive regulators, while (+)-7-iso-jasmonoyl-L-Ile (JA-Ile) is a negative regulator of stomatal defense(see below).ABA has long been recognized to induce stomatal

closure under drought stress, thereby minimizing waterloss through the leaves. However, the role of this hor-mone in Arabidopsis defense against P. syringae differsdepending on the stage of infection. At the postinvasivestage of the disease,ABAenhances plant susceptibility viasuppression of both callose deposition and SA-mediatedplant resistance (de Torres-Zabala et al., 2007; Ton et al.,2009). However, at the preinvasion stage, ABA promotesresistance to bacterial infection as it favors stomatal de-fense (Inoue and Kinoshita, 2017; Eisenach and deAngeli, 2017; Vialet-Chabrand et al., 2017). For instance,purified MAMP and live Pst DC3000 do not inducestomatal closure in the ABA-deficient aba3-1 mutant(Melotto et al., 2006). Similarly, the notabilis mutantof tomato, which lacks a functional ABA biosynthesisenzyme, 9-cis-epoxycarotenoid dioxygenase, is alsocompromised in Pst DC3000-induced stomatal clo-sure (Du et al., 2014). Furthermore, the core signalingcomponents of the ABA pathway that lead to sto-matal closure in Arabidopsis (Joshi-Saha et al., 2011)are involved in Pst-triggered stomatal closure. Inparticular, these components include (1) pyrabactinresistance1/pyrabactin resistance1-like/regulatory com-ponents of ABA receptors; (2) protein phosphatase2CA; (c) OPEN STOMATA1 (OST1); (4) the ABAsignaling-related secondary messengers ROS, NO,Ca2+, and G-protein a-subunit; and (5) the membranechannels SLOW ANION CHANNEL-ASSOCIATED1(SLAC1) and K+ channels (Melotto et al., 2006; Zhanget al., 2008; Montillet et al., 2013; Lim et al., 2014; GuzelDeger et al., 2015; Sierla et al., 2016). Thus, current ex-perimental evidence suggests a prominent role of ABA

signaling in stomatal defense (Inoue and Kinoshita,2017; Eisenach and de Angeli, 2017; Vialet-Chabrandet al., 2017). However, the specific step of ABA biosyn-thesis or signaling needed for stomatal defense is notclear. Montillet et al. (2013) suggested that flg22 andABA converge at the SLAC1 level, while Guzel Degeret al. (2015) provided evidence that these pathwaysconverge at the OST1 level that is upstream of SLAC1.An ABA-independent pathway in the early signalingleading to stomatal defense has also been proposedbased on the fact that high concentration of flg22 (10 mM)closes the stomatal pore of ost-1 epidermal peels, and100 nM flg22 does not activate OST1 on Arabidopsis cellsuspension (Montillet et al., 2013; Montillet and Hirt,2013). Now that the ABAleons reporter system is avail-able (Waadt et al., 2014), the question as towhether ABAconcentration changes in response to bacterial elicitorscan be addressed at a single-cell resolution in planta (seeOutstanding Questions).

The immune signal SA is also required for stomataldefense, as evidenced by the fact that ics1, eds5/sid1/scord3 (two SA synthesis mutants), and npr1 (an SAsignaling mutant) are defective in stomatal defense(Melotto et al., 2006; Zeng andHe, 2010; Zeng et al., 2011).Furthermore, the SA-responsive genes ICS1, EDS1, andPAD4 are induced in guard cellswithin 1 h after exposureto flg22 (Zheng et al., 2015). Direct measurement of SAconcentration in guard cell is currently not possible dueto current technical challenges. The twomain factors thathave prevented the success of this measurement are that(1) a large amount of isolated guard cells (. 150 mg)would be needed to quantify SA using the standardHPLC method (X.-y. Zheng and X. Dong, personalcommunication), and (2) SA synthesis seems to be rap-idly induced in guard cells during stomatal defense (,1 h;Zheng et al., 2015). The current procedure to isolate guardcells cannot be completed in ,2 h (Obulareddy et al.,2013). While it is clear that SA can induce stomatal clo-sure, it is yet to be determinedwhether SA is produced inthe guard cells per se or transported from other cell typesduring stomatal defense.

Several derivatives of jasmonates (JAs) are natu-rally present in plants (Staswick and Tiryaki, 2004),some of which are biologically active for regulatingJA-associated biological responses (Thines et al., 2007;Chini et al., 2007; Yan et al., 2007). In particular, meth-ylated JA (MeJA) has been used extensively to elucidateJA-dependent responses. Although some studies haveprovided evidence that MeJA closes the stomatal pore(Suhita et al., 2004; Munemasa et al., 2007; Desclos-Theveniau et al., 2012; Hua et al., 2012; Yan et al.,2015), this could not be verified by other researchgroups (Speth et al., 2009; Montillet et al., 2013;Savchenko et al., 2014). This inconsistency might beexplained by the fact that an endogenous ABA thresh-old is needed for MeJA-induced stomatal closure(Hossain et al., 2011). It is possible that plant growthconditions used in these studies resulted in differentbasal ABA levels, which could influence the differentstomatal responses observed by these groups. ABA

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concentration in the plant is known to be highly de-pendent on the air relative humidity (Okamoto et al.,2009). It is also possible that the recently proposedlink between ABA and JA responses by the modulationof MYC2 transcriptional activity via PLY6 ABA re-ceptor (Aleman et al., 2016) and the JASMONATEZIM-DOMAIN12 (JAZ12) degradation via three RINGligase KEEP ONGOING in an ABA-dependent manner(Pauwels et al., 2015) may contribute to this issue.Nonetheless, conclusions drawn solely from pharma-cological evidence can be confounded by the fact thatthe chemical applied (e.g. MeJA) may be further me-tabolized in the plant and the functional output (i.e.stomatal closure) is a pleiotropic effect of multiplestimuli. At the moment, this alternative has not beenexplored for MeJA-induced stomatal closure. Contrarytowhat has been observed forMeJA, the role of JA-Ile asa negative regulator of stomatal defense is stronglysupported in the literature. Evidence includes (1) COR,which is a molecular mimic of JA-Ile (Zhao et al., 2003;Staswick and Tiryaki, 2004), induces stomatal opening,and repress pathogen-associated molecular pattern-triggered stomatal closure (Mino et al., 1987; Melottoet al., 2006; Zhang et al., 2008; Montillet et al., 2013;Panchal et al., 2016b); (2) JA-Ile, but not jasmonic acid,induces stomatal opening with the same potency asCOR in Ipomea tricolor (Okada et al., 2009); (3) thecoronatine insensitive1 (coi1) mutant of Arabidopsis thatlacks the functional receptor for both COR and JA-Ile(Sheard et al., 2010) has constitutively smaller stomatalaperture than the wild-type plant (Panchal et al.,2016a).

Functional Output

The outcome of stomatal defense is a reduction ofpathogen penetration into the plant. Reduced pathogenentry into the plant diminishes the severity of foliardiseases or, in the case of human pathogens, reducedleafy vegetable contamination, as will be discussedbelow. In the context of a plant-microbe interaction,stomatal closure or opening appears to depend on thestrength of the opposing signals from the plant and themicrobe, which could vary depending on the specificplant-microbe combination. For instance, using thesame experimental setup and environmental condi-tions, Panchal et al. (2016a) found that high relativehumidity negatively affects stomatal defense againstP. syringae, whereas Roy et al. (2013) demonstratedthat two human pathogens, E. coli O157:H7 andSalmonella enterica serovar Typhimurium SL1344,could still induce significant stomatal closure under highrelative humidity. Furthermore, increasing the con-centration of flg22 (Felix et al., 1999) could override theeffect of high humidity on the opening of the stomatalpore (Roy et al., 2013), illustrating that the relativestrength of the opposing signals contribute to the finaloutput. Thus, a detailed description of the experi-mental setup could facilitate the comparison and

interpretation of results from various stomatal bio-assays (Chitrakar and Melotto, 2010; Montano andMelotto, 2017).

MECHANISMS OF BACTERIUM COUNTERDEFENSEAT STOMATA

If stomatal defense is a natural form of disease re-sistance, one would expect that highly adapted patho-gens may have evolved virulence mechanisms tocounter stomatal defense. Indeed, several pathogensare able to overcome stomatal defense using secretedvirulence factors (Fig. 1). Unlike many fungi andoomycetes that can directly penetrate the leaf epider-mis and viruses that are injected into the leaves bytheir insect vectors, many bacteria rely only onwounds or natural openings to colonize leaves. It istherefore understandable that developing mecha-nisms to open the stomatal pore may be particularlyimportant for these pathogens to enter the apoplast.Production of phytotoxins and the type III secretionsystem have emerged as important factors to overcomestomatal defense by bacterial pathogens. The chemicalnature and mode of action of these molecules vary asdiscussed below.

Phytotoxins

COR is a phytotoxin produced by several pathovarsof P. syringae including PstDC3000 (Bender et al., 1999)and can effectively prevent MAMP-triggered stoma-tal closure (Melotto et al., 2006, Zhang et al., 2008,Montillet et al., 2013; Panchal et al., 2016b). A putativesignaling cascade by which COR prevents bacterium-triggered stomatal closure has been elucidated (Fig. 1).As amolecular mimic of JA-Ile, COR promotes physicalinteraction between the F-box protein COI1 and thetranscriptional repressors JAZ, leading to ubiquitina-tion and proteasome-mediated degradation of JAZproteins (Chini et al., 2007; Thines et al., 2007; Yan et al.,2007). Degradation of JAZ proteins derepresses bHLHtranscription factors, such asMYC2,MYC3, andMYC4,leading to activation of downstream transcriptionalresponses (reviewed in Zhang et al., 2017). CORactivation of JA signaling induces the expression ofthree homologous NAC transcription factor genes,ANAC019, ANAC055, and ANAC072, which are thedirect targets of MYC2 (Zheng et al., 2012). Geneticcharacterization of nac null mutants shows that NACsmediate COR-induced stomatal reopening and bacte-rial multiplication in plant tissues by inhibiting theaccumulation of SA. Specifically, these NACs exert aninhibitory effect by repressing the expression of genesinvolved in SA biosynthesis and activating the ex-pression of genes involved in SA metabolism, result-ing in overall depletion of SA in infected plants (Zhenget al., 2012; Gimenez-Ibanez et al., 2017). A very sim-ilar mechanism has also been described in tomato. JAand COR activate the expression of the NAC tomato

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Figure 1. A simplified diagram of microbial virulence factors that manipulateMAMP-induced stomatal closure. NADPH oxidaseRBOHD mediates flg22-induced ROS production and stomatal defense through BIK1-/CDPKs-regulated phosphorylation (Fenget al., 2012; Dubiella et al., 2013; Gao et al., 2013; Kadota et al., 2014; Li et al., 2014). Type III peroxidase also contributes toflg22-induced ROS production (Daudi et al., 2012; O’Brien et al., 2012). MAMP-induced stomatal closure includes accumu-lation of ROS and NO, cytosolic calcium oscillations, activation of S-type anion channels, and inhibition of K+

in channels(Klusener et al., 2002; Melotto et al., 2006; Desikan et al., 2008; Zhang et al., 2008; Zeng and He, 2010; Macho et al., 2012;Montillet et al., 2013). COR induces COI1-JAZ interaction, mediates JAZ degradation, and activates the expression of NAC TFs,which inhibit the expression of ICS1 and induce expression of BSMT1, thereby leading to decreased SA level (Chini et al., 2007;Thines et al., 2007; Yan et al., 2007, 2009; Katsir et al., 2008; Melotto et al., 2008; Sheard et al., 2010; Zheng et al., 2012;Gimenez-Ibanez et al., 2017). COR also may induce stomatal opening through RIN4, which interacts with H+-ATPase AHA1 andAHA2, resulting in induction of K+

in channels (Zhang et al., 2008; Liu et al., 2009). Syringolin A inhibits SA signaling via its

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homolog, JASMONIC ACID2-LIKE. This transcriptionfactor binds to and activates the expression of SAMT1and SAMT2, which encode enzymes that deactivateSA by methylation, thereby suppressing the accumu-lation of SA and promoting stomatal opening (Duet al., 2014).

Interestingly, another member of the NAC family oftranscription factors, ANA032 acts as both a positiveregulator of SA signaling and a negative regulator of JAsignaling (Allu et al., 2016). ANAC032 directly binds tothe promoter of MYC2 (positive regulator of JA sig-naling) and NIMIN1 (negative regulator of SA signal-ing) and concomitantly suppresses their transcriptionwithin 6 h of Pst DC3000 infection (Allu et al., 2016).Accordingly, overexpression of ANAC032 in Arabi-dopsis inhibits COR-dependent reopening of stomata(Allu et al., 2016).

COR has also been shown to trigger cellular responsesthat do not rely on the canonical COI1-JAZ signalingpathway to manipulate guard cell movement. For ex-ample, COR requires RPM1-INTERACTING4 (RIN4), anegative regulator of plant innate immunity, to open thestomatal pore as evidenced by the facts that neither PstDC3000 nor COR was able to reopen stomata of rpm1/rps2/rin4mutants (Liu et al., 2009; Zhou et al., 2015; Leeet al., 2015). The perception of flg22 via the FLS2 receptorleads to phosphorylation of the plasma membrane H+-ATPases and subsequent alkalization of the apoplast,which, along with the induction of ROS via NADPHoxidase RbohD, triggers stomatal closure (Liu et al.,2009; Li et al., 2014). RIN4 interacts with the plasmamembrane H+-ATPases AHA1 and AHA2, leading totheir inhibition and acidification of the apoplast throughhyperpolarization of the plasma membrane and subse-quent induction of inward K+ channels, promoting sto-matal opening (Zhang et al., 2008; Liu et al., 2009). CORwas shown to reverse the inhibitory effects of flg22 onK+

currents to promote stomatal opening (Zhang et al.,2008).

Another virulence factor that impacts stomatal de-fense is syringolin A, a peptide toxin produced by P.syringae pv syringae that has a proteasome inhibitoryfunction (Groll et al., 2008). Syringolin A promotesstomatal opening (Schellenberg et al., 2010). Unlike PstDC3000, P. syringae pv syringae does not induce aninitial stomatal closure on either its bean host or Ara-bidopsis (Schellenberg et al., 2010; Panchal et al.,2016a). This finding suggests that this bacterium con-stitutively produces syringolin A and that syringolin

A is a stronger signal than the MAMPs produced by P.syringae pv syringae (see discussion on the strengthof the signal above) and/or that these plants donot recognize P. syringae pv syringae MAMPs effi-ciently. More recently, Misas-Villamil et al. (2013)demonstrated that syringolin A diffuses from the siteof infection and suppresses SA signaling thereby de-creasing immune responses in adjacent tissues. Thismight be the mechanism involved in syringolin A in-hibition of MAMP-induced stomatal closure becausesyringolin A inhibits the proteasome-mediated turn-over of NPR1, an important component of SA signal-ing to induce stomatal defense (Schellenberg et al.,2010).

The phytopathogenic bacterium X. campestris pvcampestris is also capable of interfering with stoma-tal closure induced by MAMP or ABA signaling(Gudesblat et al., 2009). X. campestris pv campestrisdoes so by inducing the production of diffusible sig-nal factor that is involved in bacterium-to-bacteriumsignaling (Gudesblat et al., 2009). How diffusiblesignal factor mediates stomatal reopening upon bac-terial invasion is still unknown. AnotherXanthomonasspecies, X. axonopodis pv citri produces a compound,known as plant natriuretic peptide-like, that can openstomata during plant infection, which correlates withenhanced disease symptoms (Gottig et al., 2008).Plant natriuretic peptide-like controls stomatal ap-erture in a cGMP-dependent manner (Gottig et al.,2008).

Type III Secreted Effectors

In addition to phytotoxins, pathogenic bacteria alsoproduced type-III-secretion-system effectors (T3SEs)that can overcome stomatal defense by either inhibitingMAMP-triggered stomatal closure or actively inducingstomatal opening. The T3SE HopM1 of P. syringae dis-rupts the function of a 14-3-3 protein, GRF8, leading toreduction in MAMP-triggered ROS burst and stomataldefense (Lozano-Durán et al., 2014). However, it isnot clear in this case whether stomatal opening is aconsequence of HopM1-mediated ROS suppression.Likewise, the P. syringae effector HopF2 inhibits flg22-induced ROS and stomatal defense (Hurley et al., 2014).HopF2 is an ADP-ribosyltransferase; however, theADP-ribosyltransferase activity of HopF2 is not re-quired for the ability of HopF2 to disable stomatal

Figure 1. (Continued.)proteasome inhibitor activity (Misas-Villamil et al., 2013). Multiple bacterial effectors HopM1, HopF2, HopX1, AvrB, HopZ1,and XopR could disrupt stomatal defense. HopM1 disrupts the function of GRF8 (a 14-3-3 protein), resulting in reduction ofMAMP-induced ROS production (Lozano- Duran et al., 2014). HopF2 directly targets BAK1, MKK5, and RIN4, resulting in in-hibition of ROS production (Wang et al., 2010; Wilton et al., 2010; Hurley et al., 2014; Zhou et al., 2014). AvrB enhances theinteraction of RIN4 and AHA1, which could promote the interaction between COI1 and JAZs, leading to stomatal opening (Zhouet al., 2015; Lee et al., 2015). HopX1 and HopZ1 induce stomatal opening after PTI-mediated stomatal closure, through a COI1-independent and COI1-dependent manner, respectively (Jiang et al., 2013; Gimenez-Ibanez et al., 2014; Ma et al., 2015). XopRinteracts with BIK1 and suppresses stomatal closure possibly by inhibition of RBOHD (Wang et al., 2016).

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defense, suggesting the existence of another biochemi-cal activity of HopF2 (Hurley et al., 2014). The XopReffector from Xanthomonas oryzae pv oryzae strainPXO99A also inhibits flg22-induced stomatal closure;however, the mechanism remains elusive (Wang et al.,2016).Induction of stomatal opening through plasma

membrane polarizationwas attributed to the P. syringaeeffector AvrB. AvrB-induced stomatal opening re-quires the canonical JA signaling pathway (Zhouet al., 2015). AvrB interacts with RIN4, which leadsto the activation of the plasma membrane H+-ATPase AHA1 (Lee et al., 2015). This may poten-tially generate an unknown signal that promotesthe interaction between COI1 and JAZ proteins to de-grade JAZ and enhance JA signaling. In addition, AvrBinduces MYC-mediated expression of ANAC019,ANAC055, and ANAC072 genes in Arabidopsis and thetomato NAC homolog JASMONIC ACID2-LIKE gene torepress SA responses (Du et al., 2014; Zhou et al., 2015;Gimenez-Ibanez et al., 2017). Therewith, like COR,AvrB appears to regulate stomatal opening throughmanipulating the SA-JA antagonism. Likewise, theHopZ1 effector from P. syringae pv syringae (which doesnot produce COR) also induces JAZ protein degrada-tion. HopZ1 possesses acetyltransferase activity, di-rectly interacts with JAZ proteins, and promotes JAZacetylation and degradation to activate JA signalingin soybean and Arabidopsis (Jiang et al., 2013).Moreover, HopZ1 can induce stomatal opening afterflg22-mediate stomatal closure in Arabidopsis (Maet al., 2015). Finally, the HopX1 Cys protease effectorfrom P. syringae pv tabaci (which also does not pro-duce COR) can interact with and promote degrada-tion of JAZ proteins, in a COI1-independent manner,resulting in stomatal opening (Gimenez-Ibanez et al.,2014). These findings indicate that induction of JAsignaling response is a common target for effectors toovercome stomatal defense and provide insights on avariety of mechanisms that lead to JAZ degradationand JA response in plants.

Unknown Bacterial Factors

The human pathogen Salmonella enterica serovarTyphimurium SL1344 can migrate to open stomata oflettuce to access internal leaf cells and colonize theapoplast (Kroupitski et al., 2009). A recent study showsthat SL1344, like Pst DC3000, causes a transient sto-matal closure (Roy et al., 2013). The mechanisms un-derlying stomatal closure and reopening mediated bySL1344 are not understood, but it appears that not onlyplant pathogens, but also some human pathogens mayhave evolved mechanisms to modulate plant stomatalmovements as part of their colonization strategy of thephyllosphere (Roy et al., 2013; Melotto et al., 2014). Assuch, the study of stomatal defense may have implica-tions beyond plant diseases.

ENVIRONMENTAL CONDITIONS THAT INFLUENCESTOMATAL DEFENSE

As several abiotic environmental conditions alsopromote stomatal closure (e.g. darkness, low humid-ity, low temperature, high CO2), onewould expect thatthese conditions may prevent pathogen penetrationinto leaves, unless the pathogen has evolved the abilityto override the effect of these environmental stimuli.For instance, at night, most land plants close theirstomata, which could potentially decrease pathogeninfection. Indeed, the COR-deficient mutant PstDC3118 colonizes leaf apoplast less efficiently in thedark as compared to plants inoculated under light(Panchal et al., 2016b). This finding suggests that, sim-ilar to MAMPs, darkness could effectively diminishP. syringae penetration into leaves. The mechanismunderlying this process has begun to be elucidated. Thetranscription factors CIRCADIANCLOCKASSOCIATED1(CCA1) and LATE ELONGATEDHYPOCOTYL (LHY)are two key regulators of the circadian clock inArabidopsis. Disruption of the normal clock activityby either knocking out both genes (double mutantcca1-1 lhy-20) or by overexpressing either of them re-sults in plants that are no longer able to properly closestomata in response to dark or P. syringae (Zhang et al.,2013). Zhang et al. (2013) also observed that these ge-netically engineered plants are more susceptible toP. syringae at day 3 after both spray- and pressure-inoculation. The COR-producing Pst DC3000, how-ever, opens dark-closed stomata and efficiently invadesthe leaf at night (Panchal et al., 2016b).

Alternatively, there are environmental conditions(e.g. light, high humidity) that promote stomatal opening,and pathogens might take advantage and penetrateleaves in these situations. Indeed, high humidity com-promises PstDC3118-triggered stomatal closure (Panchalet al., 2016a). High humidity also decreases guard cellsensitivity toflg22,ABA, and SA (Roy et al., 2013; Panchalet al., 2016a) by simultaneously inducing JA signaling andrepressing SA signaling in guard cells (Panchal et al.,2016a). Interestingly, SA accumulates to higher levels atnight and JA accumulates to higher levels during the dayin Arabidopsis (Goodspeed et al., 2012; Grundy et al.,2015; Zheng et al., 2015; Inoue and Kinoshita, 2017;Eisenach and de Angeli, 2017; Vialet-Chabrand et al.,2017). It is tempting to speculate that the endogenousfluctuation of these plant hormones within a 24-h periodcould contribute to dark-induced stomatal closure andlight-induced stomatal opening.

Curiously, high humidity does not compromiseE. coli O157:H7-induced stomatal closure (Roy et al.,2013). It is possible that P. syringae have evolved vari-ants of MAMPs (e.g. elf18) that are less potent in trig-gering stomatal closure compared to those from E. coliO157:H7 (Zeng and He, 2010). If this is the case, highhumidity is sufficient to overcome P. syringae-triggeredstomatal closure, but not E. coli O157:H7-triggeredstomatal closure.

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It is important to note that pathogen penetration intoleaves through stomata depends not only on the porebeing open, but also on the pathogen behavior on theleaf surface. For instance, directional movement ofbacterial pathogens on the leaf surface can be assistedby chemotaxis toward signalingmolecules (reviewed inVorholt, 2012; Melotto and Kunkel, 2013). Relativehumidity may have great influence on the accumula-tion and diffusion of these signals and, consequently,on directional bacterial movement on the leaf surface.

STOMATAL DEVELOPMENT AND IMMUNITY

It has long been observed that viral infection of planttissues alters normal stomatal development, leading tofewer stomata in infected leaves. One of the earliestreports on this phenomenon shows that sugar beetinfected with Beet yellow virus has lower stomataldensity on both upper and lower leaf surfaces ascompared to the mock control (Hall and Loomis,1972). Virus-induced reduction of stomatal densityseems to be a systemic response (Murray et al., 2016).Additionally, Murray et al. (2016) reported that sys-temic reduction of stomatal index and density onlyoccurred in two compatible interactions, Nicotianatabacum-Tobacco mosaic virus (TMV) and Arabidopsis-Turnip vein-clearing virus, but not in two incompatibleinteractions, namely, Nicotiana glutinosa-TMV andChenopodium quinoa-TMV. Reduction of stomatal in-dex and density correlated with reduction in plant leaftranspiration and water loss in TMV-inoculated N.tabacum (Murray et al., 2016). A possible link between

viral infection and stomatal development may be amicrotubule-associated protein, MPB2C, found inboth N. tabacum and Arabidopsis (Kragler et al., 2003;Ruggenthaler et al., 2009). MPB2C was identified asan interactor of the TMV-movement protein (Kragleret al., 2003) and overexpression of this protein inArabidopsis results in increased number of stomataorganized in clusters and resistance to Oilseed rapemosaic virus, a TMV close relative (Ruggenthaler et al.,2009).

Interestingly, overexpression of PstDC3000 effectors,AvrPto or AvrPtoB, also impairs stomatal patterningin Arabidopsis (Meng et al., 2015), where stomata occurin clusters similar to what has been observed byRuggenthaler et al. (2009). AvrPto and AvrPtoB tar-get several plant kinases including members of theSOMATIC EMBRYOGENESIS RECEPTOR KINASE(SERK) protein family, including SERK1, SERK2, andBAK1 (Brassinosteroid insensitive1-Associated Ki-nase, also known as SERK3; Meng et al., 2015). Thesethree SERK proteins have redundant functions inregulating stomatal development responses (Cheungand Wu, 2015; Meng et al., 2015), whereas BAK1 isalso a common signaling component of plant immu-nity (Chinchilla et al., 2007). The Arabidopsis bak1-5mutant shows higher stomatal index and densitythan the wild-type Col-0 and is also susceptible to thePlectosphaerella cucumerina BMM fungus (Jordá et al.,2016). The molecular mechanisms by which patho-gens manipulate stomatal development and how thisprocess is linked to stomatal defense are beginning tobe elucidated (see Outstanding Questions). At thismoment, it is clear that bacterial pathogens can ma-nipulate stomatal movement to their own benefit.However, whether the interference of stomatal den-sity and patterns by viruses via movement protein orbacteria via T3SEs are strategies that promote dis-ease by these diverse pathogens still need furtherinvestigation.

CONCLUSION

There has been impressive progress made towardunderstanding the mechanisms of stomatal defense inthe past decade, thanks to contributions from manylaboratories. It is clear now that MAMP perceptionand signal transduction is an important and innatefunction of stomatal guard cells. One could imaginethat as the first lineage of plant stomata appeared, theywould have to “deal with” both biotic and abioticstresses. Therefore, it is conceivable that both bioticand abiotic signals have had substantial contributionsin shaping the evolution of plant stomata, possiblyincluding their shape, density, and the remarkablecomplexity of the guard cell signaling network. It maybe said that without the discovery of the defensefunction of stomata and a deep understanding of thesignal transduction pathway involved in stomataldefense, we might have never completely appreciated

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the many sophisticated behaviors of stomata. Muchneeds to be learned about stomatal defense and itscross talk with other functions of stomata in the com-ing decade!ReceivedDecember 6, 2016; acceptedMarch 22, 2017; publishedMarch 24, 2017.

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Mechanisms Underlying Stomatal Defense

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