Stomatal Defense a Decade Later

Plants and microbes have long coevolved in a constant battle to overcome the mechanisms of defense and attack from both sides. Plants have developed means to prevent pathogen attack by hampering invasion of plant tissues and actively warding off pathogen colonization. On the other hand, pathogens have evolved strategies to mask their presence and/or evade host defenses. Plant-microbe interaction starts with molecular recognition of each other, leading to a cascade of signaling events with the final output of plant resistance or susceptibility to the pathogen. In this molecular war, epidermis of plants is the first barrier that pathogens need to overtake. Natural openings on the leaf surface, such as stomata, provide an entry site to pathogens. Plants have evolved a mechanism to close stomata upon sensing microbe-associated molecular patterns (MAMPs). This mechanism is known as stomatal defense. A decade has passed since the discovery of stomatal defense, and the field has expanded considerably with significant understanding of the basic mechanisms underlying the process. Here, we give a perspective of these findings and their implications in the understanding of plant-microbe interactions. It has been long recognized that infection of plants by foliar pathogens involves pathogen penetration into inner tissues, a niche conducive for living, where they obtain water and nutrients from internal cells. Routes for pathogen penetration into the leaves include stomatal pores, hydathodes, and wounds (either accidental or direct breaching of the cuticle by the pathogen or its vector). The vast majority of contemporary experiments designed to understand mechanisms of pathogenesis in plants has relied on inoculation by artificial wounding or direct infiltration of inocula into the apoplast. Although these are valuable approaches to dissect plant diseases, they preclude thorough understanding of a key step for the establishment of disease: pathogen internalization into the host plant. In the last decade, we came to realize how dynamic and complex this entry process is. It requires active, inducible responses on both the host and the pathogen, as well as specific environmental conditions at the time of penetration. Perhaps these strict requirements contribute to the fact that widespread diseases are rare events in nature. A large number of pathogens use the stomatal pore as a site for penetration into inner leaf tissues. In fact, some pathogens, such as the bacterium Xanthomonas campestris pv armoraciae (Hugouvieux et al., 1998), the oomycete Plasmopara viticola (Allègre et al., 2007), and species of the fungus Puccinia (Shafiei et al., 2007; Grimmer et al., 2012) are specialized to internalize into leaves only through stomata. Earlier observations provided some clues that stomatal closure might diminish bacterial disease severity in a biologically relevant context. For instance, reduced number of lesions developed on dark- or abscisic acid (ABA)-treated tomato (Solanum lycopersicum) plants after inoculation with X. campestris pv vesicatoria (Ramos and Volin, 1987). Furthermore, direct comparison of disease severity after surface-inoculation and apoplastic-infiltration in this pathosystem revealed that bacterium penetration through stomata may be a control point for disease progression (Ramos and Volin, 1987). Characterization of either bacterial or plant mutants also provided indications for possible stomatal control of bacterial infection. The coronatine (COR)-deficient mutant of Pseudomonas syringae pv tomato (Pst) causes less disease when inoculated on the leaf surface than when inoculated directly into the apoplast of Arabidopsis (Arabidopsis thaliana) or tomato (Mittal and Davis, 1995). Similarly, the flagellin-sensitive2 (fls2) mutant of Arabidopsis, which lacks the receptor for bacterial flagellin, is more susceptible than the wild-type plant only when surface-inoculated with Pst DC3000 (Zipfel et al., 2004). These previous studies set the foundation for a direct demonstration that guard cells surrounding the stomatal pore can sense microbes and close the pore, a process that is now known as stomatal defense or stomatal immunity (Melotto et al., 2006; Sawinski et al., 2013). A number of recent reviews have discussed topics ranging from signaling networks that regulates stomatal defense to mechanisms of endogenous and exogenous signal 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 thermoimaging technology in crop breeding programs and precision agriculture to assess pathogen-induced stomatal movement has been highlighted (Ishimwe et al., 2014; Singh et al., 2016). Furthermore, stomatal closure in response to fungus- and plant-derived elicitors (e.g. chitin, chitosan, and oligogalacturonic acid) and stomatal opening in response to fungal metabolites (e.g. fusicoccin) has been extensively reviewed (Grimmer et al., 2012; Arnaud and Hwang, 2015; Murata et al., 2015). We refer readers to these excellent reviews. Here, we focus on significant advances toward a mechanistic understanding of stomatal defense and the impact of this discovery on the study of plant-bacterial interactions. Stomata open and close daily, reflecting the internal circadian rhythm of plants. However, bacteria can trigger stomatal closure under bright daylight (Melotto et al., 2006; Gudesblat et al., 2009; Schellenberg et al., 2010; Roy et al., 2013), suggesting that stomatal guard cells can perceive bacteria and trigger a signaling cascade that overrides the natural circadian rhythm of stomatal movement. Bacterium-triggered stomatal closure is a fast response (<1 h) and the basic mechanism underlying this process includes the following. Plant perception of bacteria begins with the recognition MAMPs by cognate pattern recognition receptors (PRRs). The most widely studied example of such recognition is flagellin perception by the FLS2 receptor in Arabidopsis. Although flagellin recognition has a prominent role in stomatal defense during the Arabidopsis-P. syringae pv tomato DC3000 interaction (Zeng and He, 2010), the existence of other MAMP-PRR pairs that function in stomatal defense is likely. For instance, stomata of the fls2 mutant still close in response to lipopolysaccharide and Escherichia coli O157:H7 (Melotto et al., 2006). However, tools to characterize the importance of other MAMP-PRR are not completely developed as in many cases either the MAMP or the PRR is not known. The l-type lectin receptor kinases have been implicated in Arabidopsis stomatal response to Pst (Desclos-Theveniau et al., 2012, Singh et al., 2012); however, the cognate ligand(s) have not been described. Thus, the contribution of this bacterial recognition system to stomatal defense cannot be fully assessed. Similarly, purified lipopolysaccharide from various bacterial strains trigger stomatal closure (Melotto et al., 2006), but the role of its potential cognate receptor LIPOPOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (Ranf et al., 2015) has not been described yet. Another characterized MAMP-PRR pair is the elongation factor Tu (EF-Tu) and EF-Tu RECEPTOR (Zipfel et al., 2006). Purified elf26, an EF-Tu-derived peptide, closes the stomatal pore in the Arabidopsis ecotypes Col-0 and Ws4 (Desikan et al., 2008); however, the EF-Tu receptor mutant efr-1 still retains the wild-type stomatal closure phenotype in response to P. syringae (Zeng and He, 2010). It is probably due to the response triggered by other MAMPs, such as flagellin. It has been noted that elf peptides from different bacteria differ in their potency in inducing stomatal closure. Zeng and He (2010) observed that elf18, an EF-Tu-derived peptide from E. coli, is more potent than that of Pst in closing the stomatal pore of Col-0. Although additional experimentation in a biologically relevant context is still needed, emerging evidence suggests that, in principle, Arabidopsis guard cells may respond to different bacterial species at various degrees in part depending on the natural variations of bacterial MAMPs. Since 2006, there have been extensive efforts by various groups to elucidate the signaling cascade that occurs downstream of bacterial recognition. This signaling cascade is largely mediated by (1) secondary messengers such as reactive oxygen species (ROS), nitric oxide (NO), and calcium; (2) regulators of innate immune response such as MPK3, MPK4, and MPK6; and (3) plant hormones. While ROS/NO production and [Ca2+]cyt oscillation have been documented in guard cells after MAMP recognition (Melotto et al., 2006; Desclos-Theveniau et al., 2012; Arnaud and Hwang, 2015), biosynthesis and accumulation of hormones in the guard cell have not been fully demonstrated due to technical impediments to directly quantify hormone concentration in this specialized cell type. Only recently, a fluorescence resonance energy transfer-based reporter system, ABAleons, has been developed in Arabidopsis that enables temporal and spatial mapping of ABA concentration changes in response to various cues (Waadt et al., 2014). The research community would really benefit if similar real-time, in planta reporter systems are available for other hormones that play a role in stomatal defense (see Outstanding Questions). Nonetheless, guard cells do respond to plant hormones. Pharmacological and genetic 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 water loss through the leaves. However, the role of this hormone in Arabidopsis defense against P. syringae differs depending on the stage of infection. At the postinvasive stage of the disease, ABA enhances plant susceptibility via suppression of both callose deposition and SA-mediated plant resistance (de Torres-Zabala et al., 2007; Ton et al., 2009). However, at the preinvasion stage, ABA promotes resistance to bacterial infection as it favors stomatal defense (Inoue and Kinoshita, 2017; Eisenach and de Angeli, 2017; Vialet-Chabrand et al., 2017). For instance, purified MAMP and live Pst DC3000 do not induce stomatal closure in the ABA-deficient aba3-1 mutant (Melotto et al., 2006). Similarly, the notabilis mutant of tomato, which lacks a functional ABA biosynthesis enzyme, 9-cis-epoxycarotenoid dioxygenase, is also compromised in Pst DC3000-induced stomatal closure (Du et al., 2014). Furthermore, the core signaling components of the ABA pathway that lead to stomatal closure in Arabidopsis (Joshi-Saha et al., 2011) are involved in stomatal closure. In these components include (1) components of ABA (2) the ABA secondary messengers and and the and (Melotto et al., 2006; et al., et al., et al., 2014; et al., 2015; et al., 2016). Thus, evidence suggests a prominent role of ABA signaling in stomatal defense (Inoue and Kinoshita, 2017; Eisenach and de Angeli, 2017; Vialet-Chabrand et al., 2017). However, the specific step of ABA biosynthesis or signaling for stomatal defense is not et that and ABA at the while et provided evidence that these at the that is of pathway in the signaling leading to stomatal defense has also been on the fact that concentration of closes the stomatal pore of and not on Arabidopsis cell et al., and 2013). that the reporter system is available (Waadt et al., the as to ABA concentration changes in response to bacterial elicitors can be at a in planta (see Outstanding Questions). The immune signal is also for stomatal defense, as by the fact that and signaling are in stomatal defense (Melotto et al., 2006; Zeng and He, 2010; Zeng et al., Furthermore, the and are in guard cells after to et al., 2015). of concentration in guard cell is not possible due to technical The that have the of this are that (1) a large of guard cells would be to quantify the and X. and (2) to be in guard cells during stomatal defense (<1 et al., 2015). The to guard cells cannot be in et al., 2013). While it is that can induce stomatal it is to be is in the guard cells or from other cell during stomatal defense. of are in plants and some of which are biologically for responses et al., 2007; et al., 2007; et al., In has been extensively to elucidate Although some studies have provided evidence that closes the stomatal pore et al., et al., 2007; Desclos-Theveniau et al., 2012; et al., 2012; et al., 2015), this not be by other research groups et al., 2009; et al., et al., 2014). This might be by the fact that an endogenous ABA is for stomatal closure et al., It is possible that plant conditions in these studies in different ABA which the different stomatal responses observed by these ABA concentration in the plant is known to be on the et al., 2009). It is also possible that the ABA and responses by the of via ABA receptor et al., and the via in an et al., 2015) may contribute to this Nonetheless, from evidence can be by the fact that the (e.g. may be in the plant and the functional output stomatal is a of At the this has not been for stomatal closure. to has been observed for the role of as a negative regulator of stomatal defense is in the includes (1) which is a molecular of et al., and stomatal and molecular stomatal closure et al., et al., 2006; et al., et al., et al., (2) but not stomatal opening with the potency as in et al., (3) the coronatine mutant of Arabidopsis that lacks the functional receptor for both and et al., has stomatal than the wild-type plant et al., The of stomatal defense is a of pathogen penetration into the plant. pathogen entry into the plant the severity of foliar diseases in the of pathogens, reduced as be discussed In the context of a plant-microbe stomatal closure or opening to on the of the from the plant and the which depending on the specific plant-microbe For instance, the and environmental et that stomatal defense against P. Roy et demonstrated that pathogens, E. coli O157:H7 and still induce significant stomatal closure under Furthermore, the concentration of et al., the of on the opening of the stomatal pore et al., 2013), that the of the contribute to the final Thus, a of the the comparison and of from various stomatal and 2010; and 2017). stomatal defense is a natural of disease would that pathogens may have evolved mechanisms to stomatal defense. pathogens are to overcome stomatal defense many and that can directly the leaf epidermis and that are into the leaves by their many bacteria only on wounds or natural openings to leaves. It is that mechanisms to open the stomatal pore may be for these pathogens to the apoplast. of and the system have as to overcome stomatal defense by bacterial pathogens. The and of of these as discussed A of that stomatal closure. production and stomatal defense through et al., 2012; et al., et al., et al., 2014; et al., 2014). also to production et al., 2012; et al., stomatal closure includes accumulation of and of and of in et al., et al., 2006; et al., et al., Zeng and He, 2010; et al., 2012; et al., 2013). and the of which the of and induce of thereby leading to et al., 2007; et al., 2007; et al., 2009; et al., et al., et al., 2010; et al., 2012; et al., 2017). also may induce stomatal opening through which with and in of in et al., et al., 2009). A signaling via its et al., 2013). bacterial and stomatal defense. the function of in of production et al., 2014). directly and in of production et al., 2010; et al., 2010; et al., 2014; et al., 2014). enhances the interaction of and which the interaction and leading to stomatal opening et al., 2015; Lee et al., 2015). and induce stomatal opening after stomatal through a and et al., et al., 2014; et al., 2015). with and stomatal closure by of et al., 2016). is a by of P. syringae Pst DC3000 et al., and can prevent stomatal closure (Melotto et al., 2006, et al., et al., et al., A signaling cascade by which stomatal closure has been a molecular of promotes interaction the and the leading to and of et al., 2007; et al., 2007; et al., of such as and leading to of downstream responses in et al., 2017). of signaling the of factor and which are the direct of et al., of mutants that stomatal and bacterial in plant tissues by the accumulation of these an by the of involved in biosynthesis and the of involved in in of in plants et al., 2012; et al., 2017). A similar mechanism has also been described in and the of the tomato This factor to and the of and which that by thereby the accumulation of and stomatal opening (Du et al., 2014). of the of as both a positive regulator of signaling and a negative regulator of signaling et al., 2016). directly to the of regulator of and regulator of and their of Pst DC3000 infection et al., 2016). of in Arabidopsis of stomata et al., 2016). has also been to trigger responses that do not on the signaling pathway to guard cell movement. For requires a negative regulator of plant innate to open the stomatal pore as by the that Pst DC3000 to stomata of mutants et al., 2009; et al., 2015; Lee et al., 2015). The perception of via the FLS2 receptor to of the and of the with the of via stomatal closure et al., 2009; et al., 2014). with the and leading to their and of the apoplast through of the and of stomatal opening et al., et al., 2009). to the of on to stomatal opening et al., Another factor that stomatal defense is a peptide by P. syringae pv syringae that has a function et al., A promotes stomatal opening et al., 2010). Pst P. syringae pv syringae not induce an stomatal closure on either its host or Arabidopsis et al., 2010; et al., This suggests that this bacterium A and that A is a signal than the MAMPs by P. syringae pv syringae (see on the of the signal and/or that these plants do not P. syringae pv syringae MAMPs recently, et demonstrated that A from the site of infection and signaling thereby immune responses in tissues. This might be the mechanism involved in A of stomatal closure A the of an of signaling to induce stomatal defense et al., 2010). The bacterium X. campestris pv campestris is also of with stomatal closure by MAMP or ABA signaling et al., 2009). X. campestris pv campestris by inducing the production of signal factor that is involved in signaling et al., 2009). signal factor stomatal upon bacterial invasion is still Another Xanthomonas X. pv a known as plant that can open stomata during plant which with disease et al., Plant stomatal in a et al., In to bacteria also that can overcome stomatal defense by either stomatal closure or actively inducing stomatal The of P. syringae the function of a leading to in and stomatal defense et al., 2014). However, it is not in this stomatal opening is a of the P. syringae and stomatal defense et al., 2014). is an however, the of is not for the of to stomatal defense, suggesting the existence of of et al., 2014). The from Xanthomonas pv also stomatal however, the mechanism et al., 2016). of stomatal opening through to the P. syringae stomatal opening requires the signaling pathway et al., 2015). with which to the of the et al., 2015). This may an signal that promotes the interaction and to and In addition, of and in Arabidopsis and the tomato to responses (Du et al., 2014; et al., 2015; et al., 2017). to stomatal opening through the the from P. syringae pv syringae not also directly with and promotes and to signaling in and Arabidopsis et al., 2013). can induce stomatal opening after stomatal closure in Arabidopsis et al., 2015). the from P. syringae pv also not can with and of in a in stomatal opening et al., 2014). These findings that of signaling response is a for to overcome stomatal defense and provide on a of mechanisms that lead to and response in plants. The pathogen can to open stomata of to internal leaf cells and the apoplast et al., 2009). A recent study that Pst causes a stomatal closure et al., 2013). The mechanisms underlying stomatal closure and mediated by are not but it that not only plant pathogens, but also some pathogens may have evolved mechanisms to plant stomatal as part of their of the et al., et al., 2014). the study of stomatal defense may have implications plant environmental conditions also stomatal closure (e.g. would that these conditions may prevent pathogen penetration into the pathogen has evolved the to the of these environmental For instance, at most plants close their stomata, which pathogen infection. the mutant Pst leaf apoplast less in the as to plants inoculated under et al., This suggests that, similar to MAMPs, diminish P. syringae penetration into leaves. The mechanism underlying this process has to be The and are key regulators of the circadian in Arabidopsis. of the by either both mutant or by either of in plants that are to close stomata in response to or P. syringae et al., 2013). et also observed that these plants are more susceptible to P. syringae at after both and The Pst however, stomata and the leaf at et al., there are environmental conditions (e.g. that stomatal and pathogens might and leaves in these Pst stomatal closure et al., also guard cell to and et al., et al., by inducing signaling and signaling in guard cells et al., to at and to during the in Arabidopsis et al., 2012; et al., 2015; et al., 2015; and Kinoshita, 2017; Eisenach and de Angeli, 2017; Vialet-Chabrand et al., 2017). It is to that the endogenous of these plant hormones a contribute to stomatal closure and stomatal not E. coli stomatal closure et al., 2013). It is possible that P. syringae have evolved of MAMPs (e.g. that are less potent in stomatal closure to from E. coli O157:H7 (Zeng and He, 2010). this is the is to overcome P. stomatal but not E. coli stomatal closure. It is to that pathogen penetration into leaves through stomata not only on the pore but also on the pathogen on the leaf For instance, movement of bacterial pathogens on the leaf surface can be by toward signaling in 2012; and 2013). may have on the accumulation and of these on bacterial movement on the leaf It has long been observed that infection of plant tissues stomatal leading to stomata in leaves. of the on this that with has stomatal on both and leaf as to the control and of stomatal to be a response et al., 2016). et that of stomatal and only in and but not in and of stomatal and with in plant leaf and water loss in et al., 2016). A possible infection and stomatal may be a in both and Arabidopsis et al., et al., 2009). as an of the et al., and of this in Arabidopsis in number of stomata in and resistance to a close et al., 2009). of Pst DC3000 or also stomatal in Arabidopsis et al., 2015), where stomata in similar to has been observed by et and plant kinases of the RECEPTOR and also known as et al., 2015). These have in stomatal responses and 2015; et al., 2015), is also a signaling of plant immunity et al., The Arabidopsis mutant stomatal and than the wild-type Col-0 and is also susceptible to the fungus et al., 2016). The molecular mechanisms by which pathogens stomatal and how this process is to stomatal defense are to be (see Outstanding Questions). At this it is that bacterial pathogens can stomatal movement to their However, the of stomatal and patterns by via movement or bacteria via are strategies that disease by these pathogens still need has been toward understanding the mechanisms of stomatal defense in the decade, to from many It is now that MAMP perception and signal is an and innate function of stomatal guard cells. that as the first of plant stomata they would have to both and it is that both and have in the of plant stomata, their and the of the guard cell signaling It may be that the discovery of the defense function of stomata and a understanding of the signal pathway involved in stomatal defense, we might have completely the many of stomata. to be stomatal defense and its with other of stomata in the microbe-associated molecular pattern coronatine pattern recognition receptor