# Fighting for Survival at the Stomatal Gate

Maeli Melotto, Brianna Fochs, Zachariah Jaramillo, Olivier Rodrigues

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Maeli Melotto, Brianna Fochs, Zachariah Jaramillo, Olivier Rodrigues. Fighting for Survival at the Stomatal Gate. Annual Review of Plant Biology, 2024, 75, pp.551-5577. 10.1146/annurev-arplant-070623-091552. hal-04995590

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*Annual Review of Plant Biology*

Fighting for Survival at the
Stomatal Gate

Maeli Melotto,<fnref n="1" /> Brianna Fochs,<fnref n="1" />,<fnref n="2" />

Zachariah Jaramillo,<fnref n="1" />,<fnref n="2" /> and Olivier Rodrigues<fnref n="3" />

Annu. Rev. Plant Biol. 2024. 75:551–77

The *Annual Review of Plant Biology* is online at
plant.annualreviews.org

https://doi.org/10.1146/annurev-arplant-070623-091552

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use, distribution, and reproduction in any medium,
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Keywords

guard cell signaling, stomatal immunity, effector-triggered immunity, pattern-triggered immunity, systemic stomatal immunity, human pathogens

Abstract

Stomata serve as the battleground between plants and plant pathogens. Plants can perceive pathogens, inducing closure of the stomatal pore, while pathogens can overcome this immune response with their phytotoxins and elicitors. In this review, we summarize new discoveries in stomata–pathogen interactions. Recent studies have shown that stomatal movement continues to occur in a close-open-close-open pattern during bacterium infection, bringing a new understanding of stomatal immunity. Furthermore, the canonical pattern-triggered immunity pathway and ion channel activities seem to be common to plant–pathogen interactions outside of the well-studied *Arabidopsis–Pseudomonas* pathosystem. These developments can be useful to aid in the goal of crop improvement. New technologies to study intact leaves and advances in available omics data sets provide new methods for understanding the fight at the stomatal gate. Future studies should aim to further investigate the defense–growth trade-off in relation to stomatal immunity, as little is known at this time.

<footnote n="1">¹Department of Plant Sciences, University of California, Davis, California, USA;
email: melotto@ucdavis.edu</footnote>

<footnote n="2">²Plant Biology Graduate Group, University of California, Davis, California, USA</footnote>

<footnote n="3">³Unité de Recherche Physiologie, Pathologie et Génétique Végétales, Université de Toulouse,
INP-PURPAN, Toulouse, France</footnote>
## Contents

<table><tr><td>1. INTRODUCTION</td><td>552</td></tr><tr><td>2. THE ROLE OF STOMATA IN PLANT RESISTANCE TO PATHOGENS</td><td>553</td></tr><tr><td>2.1. Signaling During Phytobacterium-Triggered Stomatal Closure via Flagellin Perception</td><td>555</td></tr><tr><td>2.2. Phytobacterium-Triggered Stomatal Closure After Lipopolysaccharide and Plant Elicitor Peptide Perception</td><td>557</td></tr><tr><td>2.3. Signaling During Stomatal Closure Triggered by Fungal Pathogens</td><td>557</td></tr><tr><td>2.4. Stomatal Opening Prevents Phytobacterium Proliferation in the Apoplast</td><td>560</td></tr><tr><td>2.5. Systemic Stomatal Defense</td><td>560</td></tr><tr><td>3. STOMATAL SUSCEPTIBILITY TO PHYTOPATHOGENS</td><td>562</td></tr><tr><td>3.1. Novel Regulators of Stomatal Opening During Bacterial Invasion</td><td>562</td></tr><tr><td>3.2. The Role of Stomata in Controlling Water Availability to Phytobacteria</td><td>563</td></tr><tr><td>3.3. Fungus-Derived Molecules that Highjack Guard Cell Metabolism</td><td>564</td></tr><tr><td>4. STOMATA-HUMAN PATHOGEN INTERACTIONS</td><td>564</td></tr><tr><td>5. STOMATAL IMMUNITY, ABIOTIC STRESS, AND PLANT GROWTH</td><td>565</td></tr><tr><td>6. RECENT TECHNOLOGICAL ADVANCES TO STUDY GUARD CELL RESPONSE TO PATHOGENS</td><td>567</td></tr><tr><td>6.1. Using Molecular Sensors and Reporters to Understand Guard Cell Response to Elicitors</td><td>567</td></tr><tr><td>6.2. Available Omic Data Sets to Further Explore Stomata-Pathogen Interactions</td><td>567</td></tr><tr><td>6.3. Imaging and Artificial Intelligence for Large-Scale Stomatal Defense Phenotyping</td><td>568</td></tr></table>

# 1. INTRODUCTION

Plants have developed sophisticated defense strategies to prevent, or at least reduce, pathogen infection of their internal tissues. Beyond preexisting, passive defense mechanisms such as a thick cuticle, trichomes, and reduced stomatal density, the activation of immune responses is also effective in warding off highly virulent pathogens. One such induced immune response is the quick closure of stomatal pores upon pathogen recognition and subsequent activation of a signaling cascade in guard cells (82). This process reduces the internalization of most pathogens into leaves, restricting their ability to proliferate in the apoplast. Once in contact with leaves, however, some pathogens can activate their virulence mechanisms, such as the production of phytotoxins and activation of the type-three secretion system (TTSS) to overcome stomatal immunity. Interestingly, stomata and bacterial pathogens can continue to interact after the initial invasive stage of infection, leading to a close-open-close-open pattern of stomatal movement (138), which continuously alters the physiology of both the plant and the pathogen throughout their fight for survival (Figure 1).

Over the last two decades, our understanding of stomata-pathogen interactions has increased significantly due to extremely detailed studies of the *Arabidopsis thaliana*–*Pseudomonas syringae* pathosystem and the wealth of resources developed for studying stomata because of their tremendous importance for plant growth and their potential as targets for agricultural improvement (26). In more recent years, this knowledge has been translated into some crop plant systems and recent technological advances, such as remote sensing and canopy imaging, which will soon allow for field studies to assess stomatal immunity for crop improvement.

Stomatal immunity:
guard cell immune
responses against
microbial invasion
## 2. THE ROLE OF STOMATA IN PLANT RESISTANCE TO PATHOGENS

Stomatal immunity is initiated by the recognition of microbe- and herbivore-associated molecular patterns (MAMPs and HAMPs) (68) or host-derived damage-associated molecular patterns (DAMPs) by membrane-localized pattern recognition receptors (PRRs) (156). This recognition

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*(Caption appears on following page)*
<caption>**Figure 1** (*Figure appears on preceding page*)</caption>

A diagram of a temporal pattern of stomatal movement during *Pseudomonas syringae* pv. tomato DC3000 infection of *Arabidopsis*. Arrows indicate the transition from one state to another with novel mechanisms depicted on the right. (a) Cross-section of the leaf blade showing the apoplast with water (*blue circles*), mesophyll cells (*green circles*), and a pair of guard cells that form the stomata. (b) Upon bacterial perception, guard cells undergo PTI responses, causing stomatal closure (9, 63, 81, 124, 151). (c) The bacterium overcomes stomatal immunity through the production of COR followed by downstream JAZ/MYC-dependent signaling (81, 91), in addition to other mechanisms (82), resulting in stomatal opening. (d) Apoplastic bacterial cells release the type-three secretion effector proteins AvrE and HopM1, closing the stomata via guard cell-localized ABA signaling, which increases water content within the apoplast (*blue circles*) and proliferation of bacteria (45, 110, 139), while CLF can epigenetically modify the ABA pathway (139). (e) *Arabidopsis* fights against the water soaking through the SCREW/NUT system. This process promotes the activity of ABI1/ABI2, inhibiting stomatal closure through the reduction of OST1 activity and subsequently SLAC1 activity. The apoplast is then dehydrated, thereby restricting bacterial growth (29, 73). (f) In the later stages of Pst DC3000 infection, the pathogen enters a necrotrophic phase, causing plant cell death (*yellow tissues*) and promoting population growth and dispersal of pathogens (107, 152). Abbreviations: ABA, abscisic acid; ABI, ABA INSENSITIVE; BAK1, BRI1-ASSOCIATED RECEPTOR KINASE; CLF, CURLY LEAF; COI, CORONATINE-INSENSITIVE 1; COR, coronatine; flg22, 22-amino acid peptide derived from flagellin; FLS2, FLAGELLIN-SENSING 2; JA, jasmonate; JAZ, JASMONATE-ZIM DOMAIN; MPK, MITOGEN-ACTIVATED PROTEIN KINASE; MYC, basic helix-loop-helix transcription factor; NUT, PLANT SCREW UNRESPONSIVE RECEPTOR; OST1, OPEN STOMATA 1; Pst, *Pseudomonas syringae* pv. tomato; PTI, pattern-triggered immunity; SCREW, SMALL PHYTOCYTOKINES REGULATING DEFENSE AND WATER LOSS; SERK, SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE; SLAC1, SLOW ANION CHANNEL-ASSOCIATED 1. Figure adapted from images created with BioRender.com.

induces complex signaling events in the guard cell known as pattern-triggered immunity (PTI) (149), in which phytohormones play a central role (38) (see the sidebar titled The Action of Hormones During Stomata-Pathogen Interactions).

New evidence suggests that the initial stomatal closure is not the only role of stomata in resistance to pathogens. Interestingly, at the postinvasive stage of infection, stomatal opening through the recognition of phytocytokines can also be a mechanism of resistance, as it causes a decrease in apoplastic water availability for the pathogen (73). Furthermore, bacterial pathogens can induce a systemic change in stomatal density and immunity, as discussed below. Stomatal movement

**Phytocytokines:**

plant peptides released
into the apoplast
during pathogen
infection

THE ACTION OF HORMONES DURING STOMATA–PATHOGEN INTERACTIONS

Hormone signaling and crosstalk are also essential for the control of stomatal immunity and counterdefense by the pathogen (1, 8, 38, 82, 140). In particular, abscisic acid (ABA), salicylic acid (SA), and jasmonoyl-L-isoleucine (JA-Ile) are key players in the process: ABA and SA are positive regulators and JA-Ile is a negative regulator of stomatal immunity. However, auxin, ethylene, and cytokinins can affect the action of the above hormones, creating a complex network of interactions, depending on the presence or absence of other stressors (e.g., drought and light) (38, 141), thereby affecting stomatal movement output. A newly discovered positive regulator of stomatal immunity is melatonin, a hormone present in animals and vascular plants. Melatonin is perceived by the putative receptor PHYTOMELATONIN RECEPTOR 1 (PMTR1) at the plasma membrane, inducing stomatal closure through RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD)-triggered reactive oxygen species (ROS) production and Ca²⁺ influx into the guard cell and acting downstream of FLAGELLIN-SENSING 2/BRI1-ASSOCIATED RECEPTOR KINASE (FLS2/BAK1)-dependent flagellin perception (137, 145). Our knowledge of the hormonal control of stomatal immunity is still fragmented, and further spatiotemporal analyses of hormonal signaling at the single-cell level need to be explored to dissect the underlying mechanisms.
(opening and closure) is important for many aspects of plant resistance against pathogens, ranging
from single-cell, short-term responses to whole-organ, systemic long-term responses.

**2.1. Signaling During Phytobacterium-Triggered Stomatal Closure via Flagellin Perception**

To activate PTI, the plant apoplastic β-galactosidase BGAL1 hydrolyzes the bacterium flagellin, releasing the epitope flg22, inhibiting bacterial movement, and activating leucine-rich repeat receptor kinase (LRR-RK) FLAGELLIN-SENSING 2 (FLS2)-mediated stomatal immunity (10) (Figure 2).

*Arabidopsis* FLS2 recognizes bacterial flg22 and, with the coreceptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), phosphorylates BOTRYTIS-INDUCED KINASE 1 (BIK1) (63, 124, 151). The L-type lectin receptor kinase-V.2 (LecRK-V.2) and LecRK-VII.1 can interact with the FLS2/BAK1 PRR complex and activate the guard cells' Ca²⁺-permeable cation channels during stomatal closure (147). BIK1 is involved in the activation of OPEN STOMATA 1 (OST1)/SNF1-RELATED PROTEIN KINASE 2.6 (SnRK2.6), subsequently activating the plasma membrane ion channels SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) and SLAC1 HOMOLOG 3 (SLAH3) (39). A screening of transfer DNA (T-DNA) insertion mutants of *Arabidopsis* allowed the identification of the LRR-RK STRESS INDUCED FACTOR 2 (SIF2) required for stomatal closure in response to *P. syringae* pv. tomato (Pst), flg22, and abscisic acid (ABA) treatments (12). SIF2 physically interacts with FLS2/BAK1 and SLAC1 and activates SLAC1 by transphosphorylation of the SLAC1 N-terminal domain (12). The ion fluxes induced by SLAC1 lead to the activation of outward channels, such as GATED OUTWARDLY-RECTIFYING K⁺ CHANNEL (GORK) (2, 32), and subsequent induction of osmotic-driven water efflux from the guard cell through the PLASMA MEMBRANE INTRINSIC PROTEIN 2;1 (PIP2;1) aquaporin, leading to stomatal closure. The regulation of PIP2;1 by OST1 is crucial for evoking ABA- and flg22-induced stomatal closure through the transport of H₂O₂ and water (36, 106). Following a single-particle fluorescence of GFP-PIP2;1 with variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), it was observed that flg22 induces a dynamic, cell type-specific regulation of PIP2;1, thereby controlling water transport during *Arabidopsis* defense responses (16).

As the connection between reactive oxygen species (ROS) and Ca²⁺ signaling with flg22- and ABA-induced stomatal closure has been discussed in detail recently (54, 73, 82, 99, 107), here we describe only recent advances in this area. It has been shown that, in addition to activating SLAC1 and SLAH3 through OST1, BIK1 also activates the RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) to trigger a ROS burst (51, 63) and activates calcium channels, such as cyclic nucleotide-gated channels (CNGC2 and CNGC4) (129) and OSCA1.3 (128), inducing Ca²⁺ influx into the guard cell cytoplasm. While CNGCs are involved in many aspects of plant physiology (48), including activation of Ca²⁺ flux during ABA-induced stomatal closure (126), the Ca²⁺ channels OSCA1.3 and OSCA1.7 are expressed specifically in the plasma membrane of the guard cells (53, 128). Interestingly, BIK1-dependent phosphorylation of OSCA1.3/OSCA1.7 specifically occurs during stomatal immune responses (128), suggesting a decoupling of ABA and flg22 signaling for Ca²⁺ influx in the guard cell and a stimulus-specific calcium signature (148). The involvement of CNGCs in flg22-induced stomatal closure and the coordination of OSCAs and CNGCs for calcium transport remain unresolved.

Another parallel pathway downstream of the cell surface receptors mentioned above is the mitogen-activated protein kinase (MPK) cascade that delivers and amplifies a diverse array of extracellular stimuli through activation by protein phosphorylation (123). Recently, the *Arabidopsis*
<figure><img src="image_5.png" /><figcaption>Figure 2</figcaption></figure>

A simplified model with novel components of stomatal immunity. (①) BGAL1 cleaves flagellin, releasing flg22 peptides (10). (②) flg22 is recognized via FLS2/BAK1 associated with LecRKs, activating calcium channels (147). (③) FLS2 and BAK1 phosphorylate BIK1 (63, 124, 151) and activate the MPK cascade (123). VLN3 is phosphorylated by MPK3/MPK6, leading to the reorganization of actin bundles (157). (④) BIK1 activates RBOHD, which triggers ROS production and changes in the ion fluxes (51, 54, 63). BIK1 phosphorylates OSCA1.3/OSCA1.7, inducing Ca²⁺ influx (53, 54, 128). (⑤) BIK1 activates OST1, which subsequently induces the ion channels SLAC1 and SLAH3 (39). Ion fluxes driven by SLAC1 activate GORK (2, 32) and subsequently the induction of PIP2;1 that is directly regulated by OST1 (36, 106). PIP2;1 also facilitates the inward transport of H₂O₂ (106). (⑥) SLAC1 can also be activated by the receptor-like kinase SIF2, which physically associates with FLS2/BAK1 (12). Solid and dashed arrows indicate direct and indirect connections, respectively. Abbreviations: BAK1, BRI1-ASSOCIATED RECEPTOR KINASE; BGAL1, β-GALACTOSIDASE1; BIK1, BOTRYTIS-INDUCED KINASE1; flg22, 22-amino acid peptide derived from flagellin; FLS2, FLAGELLIN-SENSING 2; GORK, GATED OUTWARDLY-RECTIFYING K⁺ CHANNEL; LecRK, L-TYPE LECTIN RECEPTOR KINASE; MKK, MITOGEN-ACTIVATED PROTEIN KINASE KINASE; MPK, MITOGEN-ACTIVATED PROTEIN KINASE; OST1, OPEN STOMATA 1; P, phosphorylation; PIP2;1, PLASMA MEMBRANE INTRINSIC PROTEIN 2;1; RBOHD, RESPIRATORY BURST OXIDASE HOMOLOG D; ROS, reactive oxygen species; SIF2, STRESS INDUCED FACTOR 2; SLAC1, SLOW ANION CHANNEL-ASSOCIATED 1; SLAH3, SLAC1 HOMOLOG 3; VLN3, VILLIN3. Figure adapted from images created with BioRender.com.
VILLIN3 (VLN3) protein was identified as a substrate of MPK3 and MPK6 that is important for actin bundle reorganization (157), linking perception of the pathogen to actin dynamics required for stomatal closure (61). In tomato, a new plasma membrane-localized receptor-like cytoplasmic kinase (RLCK) called FLS2/FLS3-interacting receptor-like cytoplasmic kinase 1 (FIR1) is also involved in flagellin-triggered PTI signaling. Interestingly, *fir1* loss-of-function mutants are impaired in hallmark immune responses induced by flg22 and a second epitope of flagellin called flgII-28, including stomatal immunity against Pst DC3000, but these mutants show a normal MPK phosphorylation cascade (121). Collectively, these findings in two different plant systems point to the existence of several pathways leading to stomatal immunity that may or may not converge at specific steps.

**Elicitors:** biological compounds that activate plant immunity

## 2.2. Phytobacterium-Triggered Stomatal Closure After Lipopolysaccharide and Plant Elicitor Peptide Perception

Bacterial pathogens have several PAMPs that trigger stomatal immunity, although most are not as well studied as flagellin. Lipopolysaccharides (LPSs), major components of the outer membrane of Gram-negative bacteria that consist of lipid A, core oligosaccharide, and O-polysaccharide, are recognized by the receptor/coreceptor CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) in rice (*Oryza sativa*) (20) (**Figure 3**). In *Arabidopsis*, LPS is perceived by a sensitive system that includes the recognition of its component medium chain 3-hydroxy fatty acids (mc-3-OH-FAs) by LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) (57, 102, 103).

LPS recognition also triggers immune responses through ROS production and a rapid elevation of Ca²⁺ concentration in the cytoplasm (103). However, unlike flg22, LPS triggers a weak early ROS burst and a strong late ROS burst, possibly because the lipid A moiety of LPS provokes a conserved second ROS burst from chloroplasts (118). Because an LPS- and lipid A-induced second ROS burst partially occurs in *lore* mutants, it is possible that LPS activates multiple pathways during plant immunity (118). Nonetheless, the *delt1-2 Arabidopsis* plants carrying a mutation in the residue 919 of the RBOHD C terminus have impaired LPS-triggered stomatal immunity (64). The molecular mechanisms underlying the regulation of RBOHD at this residue remain unknown.

Pathogen attack can also lead to the release of molecules that can contribute to plant immunity (149). One such class of molecules is comprised of plant elicitor peptides (Peps) that are perceived by the receptors PEPR1 and PEPR2, inducing stomatal closure that is mechanistically linked to the ion transport activity of SLAC1 and SLAH3 but independent of OST1 regulation (154). In *Arabidopsis* guard cells, another secreted peptide, PAMP-INDUCED PEPTIDE 1 (PIP1), which is a phytocytokine derived from the proteolysis of prePIP1 (44), binds to its receptor, RECEPTOR-LIKE KINASE 7 (RLK7), to activate stomatal immunity against Pst DC3000 (44) (**Figure 3**). The PIP1-RLK7 pair amplifies the FLS2 signaling pathway, contributes to the expression of precursors and receptors as a positive feedback loop, and induces a ROS burst (44). In addition, a reverse genetics approach and guard cell electrophysiology experiments connected PIP1-RLK7-induced stomatal closure with kinase activity by BAK1, OST1, and MPK3/MPK6, as well as induction of ROS and Ca²⁺ signaling (119). These results point to an overlap of molecular signaling during DAMP-, phytocytokine-, and flg22-induced stomatal closure.

## 2.3. Signaling During Stomatal Closure Triggered by Fungal Pathogens

Some fungal pathogens can enter the plant by directly penetrating the leaf surface with specialized structures, while others are able to infect tissues through the stomatal pore. A major component of the fungal cell wall is chitin, which acts as a PAMP recognized by the PRRs LYSINE
<figure><img src="image_6.png" /><figcaption>Figure 3</figcaption></figure>

A simplified model for stomatal closure upon perception of the bacterial LPS and Peps. (①) mc-3-OH-FAs, constituents of LPS Lipid A, are recognized by the receptor LORE in *Arabidopsis* (57), and LPS is recognized by CERK1 in rice (20), triggering a weaker ROS response via RBOHD (64) and rapid increase in Ca²⁺ concentration in the cytoplasm (93). After the initial weak ROS response, there is a stronger ROS response in the chloroplast (44, 118). (②) PEP1 and PEP2 are perceived by the receptors PEPR1 and PEPR2, inducing stomatal closure through the activation of SLAC1 and SLAH3 (156). BIK1 binds to PEPR1/PEPR2 (156) and phosphorylates OSCA1.3/OSCA1.7, inducing Ca²⁺ influx (53, 54, 128). (③) PIP1, another Pep, associates with the receptors RLK7 and BAK1 and induces gene expression (119). Additionally, PIP1-RLK7 induces stomatal closure through an MPK signaling cascade; OST1; and the activation of ROS, Ca²⁺ signaling, and SLAC1 (119). Solid and dashed arrows indicate direct and indirect connections, respectively. Abbreviations: BAK1, BRI1-ASSOCIATED RECEPTOR KINASE; BIK1, BOTRYTIS-INDUCED KINASE 1; CERK1, CHITIN ELICITOR RECEPTOR KINASE 1; FLS2, FLAGELLIN-SENSING 2; LORE, LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION; LPS, lipopolysaccharide; mc-3-OH-FA, medium chain 3-hydroxy fatty acid; MPK, MITOGEN-ACTIVATED PROTEIN KINASE; OST1, OPEN STOMATA 1; P, phosphorylation; Peps, plant elicitor peptides; PEPR, PEP1 RECEPTOR; PIP1, PAMP-INDUCED PEPTIDE 1; RBOHD, RESPIRATORY BURST OXIDASE HOMOLOG D; RLK7, RECEPTOR-LIKE KINASE 7; ROS, reactive oxygen species; SLAC1, SLOW ANION CHANNEL-ASSOCIATED 1; SLAH3, SLAC1 HOMOLOG 3. Figure adapted from images created with BioRender.com.

MOTIF (LysM), RECEPTOR KINASE 5 (LYK5), and CERK1 (11, 69, 71, 125). Similar to the FLS2-triggered signaling cascade upon perception of bacterial flagellin, chitin also induces stomatal closure when perceived by the LYK5-CERK1-PROBABLE SERINE/THREONINE-PROTEIN KINASE-LIKE 27 (PBL27) receptor complex followed by phosphorylation of SLAH3 by PBL27 (72). Furthermore, chitin oligosaccharide perception leads to Ca²⁺ influx and activation of SLAC1 in *Arabidopsis* guard cells (146). Chitin treatment also induces the
<figure><img src="image_7.png" /><figcaption>Figure 4</figcaption></figure>

A model of stomatal closure after chitin and chitosan perception to reduce fungal infection of the apoplast. (①) Chitin is recognized by LYK5 and CERK1 (11, 69, 71, 125) associated with PBL27 (72). PBL27 then phosphorylates SLAH3 (70). (②) A functional RAN1 leads to the production of ROS and NO (122). Ca²⁺ influx and SLAC1 activation also occur upon chitin perception (146). (③) BIK1 binds to CERK1 (120) and phosphorylates OSCA1.3/OSCA1.7, inducing Ca²⁺ influx (53, 54, 128). (④) A derivative of chitin, chitosan, also induces stomatal closure via components of the salicylic acid signaling pathway (ISC2 and NPR1) (98) and calcium-dependent cell death (146). Solid and dashed arrows indicate direct and indirect connections, respectively. Abbreviations: BIK1, BOTRYTIS-INDUCED KINASE 1; CERK1, CHITIN ELICITOR RECEPTOR KINASE 1; GC, guard cell; ISC1, ISOCHROMATE SYNTHASE 1; LYK5, LYSM-CONTAINING RECEPTOR-LIKE KINASE 5; NO, nitric oxide; NPR1, NONEXPRESSER OF PR GENES 1; P, phosphorylation; PBL27, PROBABLE SERINE/THREONINE-PROTEIN KINASE-LIKE 27; RAN1, RESPONSE TO ANTAGONIST 1; ROS, reactive oxygen species; SLAC1, SLOW ANION CHANNEL-ASSOCIATED 1; SLAH3, SLAC1 HOMOLOG 3. Figure adapted from images created with BioRender.com.

expression of *Arabidopsis Ras-RELATED NUCLEAR PROTEIN 1* (*AtRAN1*), a gene preferentially expressed in guard cells that encodes a small G protein. *Atran1* mutants have deficient ROS and nitric oxide (NO) accumulation, suggesting that a functional AtRAN1 is needed for stomatal closure and chitin-induced resistance to both Pst DC3000 and *Sclerotinia sclerotiorum* (122) (Figure 4).

A deacylated derivative of chitin, chitosan, is another component of the fungal cell wall that is recognized and induces stomatal closure associated with a functional salicylic acid (SA) signaling pathway through ISOCHROMATE SYNTHASE 1 (ISC1) and NONEXPRESSER OF PR GENES 1 (NPR1) (98). Chitosan can induce guard cell death in a Ca²⁺-dependent manner to reduce fungal entry into the plant apoplast (146) (Figure 4). These results further support a conserved mechanism for flg22- and chitin-induced signaling pathways in *Arabidopsis*. The role of chitosan in stomatal immunity has also been validated in chickpea, an economically important crop plant. Priming chickpea seedlings with chitosan induces greater ROS and NO production in
both wilt-resistant (WR-315) and wilt-susceptible (JG-62) cultivars when challenged with *Fusarium oxysporum*. A greater amount of ROS and NO causes an increase in extracellular adenosine triphosphate (ATP), leading to a stronger stomatal immunity response (87). Supporting the notion of ATP potentiation of stomatal immunity, ATP can also act as a DAMP upon recognition by its receptor DOESN'T RESPOND TO NUCLEOTIDES 1 (DORN1) and subsequent phospvoactivation of RBOHD (13). This process is linked with bacterium-induced stomatal closure in *Arabidopsis* (13).

New plant factors that contribute to stomatal immunity have been discovered in economically important crops. A newly discovered fungal PAMP, the nonproteinaceous *F. oxysporum* cell wall extract (FoCWE), induces typical plant immune responses, including stomatal closure, in both *Arabidopsis* and cotton (4). How FoCWE is recognized by the plant remains to be determined. In wheat, the major gene *Stb16q* confers resistance to the fungus *Zymoseptoria tritici* and is required for stomatal closure when the fungal hyphae attempt to penetrate the stomatal pore (6). *Stb16q* encodes a cysteine-rich receptor kinase that possibly recognizes carbohydrates released during the fungal infection to initiate wheat immune responses (113). Expression of the grape *SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (SERK3)/BAK1* gene restores stomatal immunity in the *bak1* mutant of *Arabidopsis* (65), suggesting functional orthology between the encoded proteins. In potato, the nonspecific lipid transfer protein 10 (StLTP10; also known as a pathogenesis-related protein) interacts with and relocates the ABA receptor PYR1-LIKE 4 (PYL4) to the cell membrane, positively regulating stomatal closure during *Phytophthora infestans* infection via a ROS and MPK cascade (132).

## 2.4. Stomatal Opening Prevents Phytobacterium Proliferation in the Apoplast

At the postinvasion stage of infection, some phytobacteria such as *P. syringae* and *Xanthomonas* species can cause extensive water soaking in the apoplast, a typical early symptom (9–24 h postinfection) that promotes proliferation not only of phytopathogens but also of other endophytic symbionts (33, 97, 142). Interestingly, a new mechanism of plant resistance against water soaking has been recently discovered in which the plant opens the stomatal pore to resume gas and water exchange and prevent pathogen colonization of the apoplast (reviewed in 130). Briefly, MAMP perception by PRRs can induce the production of plant peptides (43, 127) including SMALL PHYTOCYTOKINES REGULATING DEFENSE AND WATER LOSS 1 (SCREW1) and SCREW2. *SCREW1* and *SCREW2* are highly expressed in guard cells and mesophyll cells in response to flg22, and their encoded proteins are perceived by the receptor kinase PLANT SCREW UNRESPONSIVE RECEPTOR (NUT) (73). SCREW and NUT, also known as CTNIPs and HAESEA-LIKE 3 (HLS3), respectively (104), inhibit both flg22- and ABA-induced stomatal closure. More precisely, this peptide-receptor pair increases the activity of ABA INSENSITIVE 1 (ABI1) and ABI2 phosphatases, thereby reducing OST1 activity and SLAC ion transport activity. This process leads to stomatal opening and promotes apoplastic water loss (73) (Figure 1d,e).

## 2.5. Systemic Stomatal Defense

The systemic acquired resistance (SAR) phenomenon is an effective mechanism to decrease secondary infection by a broad range of pathogens (30). SAR signaling molecules travel via the plant vasculature from pathogen-infected tissues to distal tissues where they are perceived and cellular defense responses are activated (50) (Figure 5). As stomatal movement in leaves can be modulated by the perception of drought stress in plant roots (154) or leaf-to-leaf communication of stimuli such as light, heat, and wounding (21), one could expect that stomatal immunity could also be part of SAR. Indeed, inoculation of *Arabidopsis* leaves with Pst DC3000 induces the closure
<figure><img src="image_8.png" /><figcaption>Figure 5</figcaption></figure>

A diagram depicting systemic stomatal defense. (Yellow circle) Upon initial pathogen perception, stomata start to close. A systemic acquired resistance (SAR) signal travels through the plant vasculature to distal areas to activate systemic stomatal defense (30, 50). (Red circle) In response to the SAR signal, distal leaves close their stomata to prevent further infection (76, 114). (Blue circle) Newly developed leaves may have a reduction in stomatal density (24, 75, 78, 86). Figure adapted from images created with BioRender.com.

of stomata in distant leaves (18) in an NPR1-dependent process (37). Multiomic analyses of Pst DC3000-primed guard cells revealed the regulation of multiple aspects of SAR (18). Further characterization of *Arabidopsis* T-DNA mutants led to the identification of DEFECTIVE IN INDUCED RESISTANCE 1 (DIR1) as required for guard cell SAR (19). DIR1 is a nonspecific lipid transfer protein (nsLTP) involved in long-distance signaling during SAR (76). Supporting these studies in *Arabidopsis*, a potato nsLTP functions in stomatal immunity as mentioned above (132), and, in tomato, treatment with chitosan induces systemic stomatal closure associated with ethylene and a superoxide radical ($O_2^−$) (17). Furthermore, exogenous application of an SA analog, acibenzolar-S-methyl (ASM), locally and systemically protects crucifers against *Pseu- domonas cannabina* invasion through the stomatal pore (116). These findings have been reviewed by Sakata & Ishiga (114). Thus, systemic stomatal defense can be activated by similar mechanisms previously associated with SAR during basal plant immune responses (30, 50).
Stomatal density is known to be associated with plant resistance and susceptibility to pathogens. For instance, resistant clones of rubber tree have a lower stomatal density than their susceptible counterparts (75), and higher stomatal density in poplar also contributes to disease susceptibility (78). A notable outcome of plant infection by pathogens is the systemic modulation of stomatal density (Figure 5). It has been observed that only *Nicotiana tabacum* and *Arabidopsis* plants susceptible to *Tobacco mosaic virus* (TMV) and *Turnip vein-clearing virus* (TVCV), respectively, have decreased stomatal density in systemic leaves (86). Similarly, Pst DC3000, flg22, and SA induce a reduction of stomatal density in newly formed *Arabidopsis* leaves (24). Furthermore, this systemic response requires PAMP perception through FSL2, SA biosynthesis and signaling via ISC1 and NPR1, and the nsLTP AZALEIC ACID INDUCED 1 (AZI1), but not DIR1 or ABA (24). Increased stomatal density has been correlated with increased penetration rates of Pst DC3000 into *Arabidopsis* leaves (24).

Due to the importance of reducing stomatal density as a part of SAR to decreasing secondary pathogen infection, it would be extremely valuable to improve the understanding of how SAR signals affect stomatal immunity and improve crop resilience. The exciting finding that volatile esters synthesized by alcohol acyltransferases are induced by Pst DC3000 in tomato leaves triggering stomatal closure in a broad range of plants (74) can also offer potential applications in crop protection. In addition, guard cell-specific, large-scale quantitative omic approaches may uncover the underlying mechanisms (18, 93) and pave the way toward this ambitious goal.

## 3. STOMATAL SUSCEPTIBILITY TO PHYTOPATHOGENS

The initial perception of the pathogen and the subsequent signaling events in guard cells that lead to a fast stomatal closure can be counteracted by some pathogens. Bacterial phytotoxins and effectors, which are generally induced upon pathogen contact with plants (58, 91), are notorious for reopening the stomatal pore and allowing the bacterium to gain access to the leaf apoplast. Recently, the action of coronatine (COR) and the antagonism between jasmonate (JA) and SA have been confirmed in cabbage and oat plants during *P. cannabina* pv. *alisalensis* interactions with stomata (115), underscoring the conserved phenomenon in eudicot and monocot systems. The molecular details of the strategies used by these pathogens (including modulation of plant hormonal pathways, degradation of regulatory proteins, and changes in ion channel activities, among others) revealed an intricate mechanism of pathogens interfering with stomatal immunity. We would like to direct the reader to previous comprehensive reviews of this topic for details (82, 141). Below, the most recent advances in the field are highlighted. In particular, it has become evident that, beyond the stomatal closure and reopening observed in the first hours after contact with pathogens, stomatal defense and counterdefense continue on until the later stages of leaf colonization (Figure 1).

### 3.1. Novel Regulators of Stomatal Opening During Bacterial Invasion

SA and JA signaling pathways in guard cells are known to be positive and negative regulators of stomatal closure in *Arabidopsis* (90). The balance between these signaling pathways seems to be regulated at the transcriptional level by NAC DOMAIN-CONTAINING PROTEINS (NACs) (82). More recently, another main player was identified: the WRKY family of transcriptional factors that may act as positive and negative regulators of plant immune responses in leaves, among other whole-plant processes (14). In the *Xanthomonas campestris* pv. *vesicatoria* pepper pathosystem, the type-three secretion effector *Xanthomonas* outer protein S (XopS) prevents bacterium-induced stomatal closure by binding to and stabilizing the CaWRKY40 transcription factor. Notably, CaWRKY40 binds to the promoter of *CaPR1*, an SA-responsive gene, and CaJAZ8, a
JASMONATE ZIM-domain protein that represses the JA signaling, causing a reduction in the expression of these target genes (100). These findings provide a link between the action of a bacterial effector to directly modulate the balance between SA and JA signaling to the bacterium's advantage. However, unlike CaWRKY40 of pepper, WRKY22 and WRKY25 of *Nicotiana benthamiana* are positive regulators of plant immune responses (101). CRISPR-Cas9 knockouts of their orthologs in tomatoes compromise stomatal immunity and render the plants more susceptible to Pst DC3000. Whether this bacterium controls SIWRKY22/SIWRKY25 is not yet known.

The *Xanthomonas* outer protein C2 (XopC2) was identified as a new regulator of stomatal opening in rice. This effector of *X. oryzae* pv. *oryzicola* is a kinase that can phosphorylate and suppress OSK1, a component of the Skp1-Cullin-F-box ubiquitin ligase (134). Interestingly, phosphorylated OSK1 has increased affinity to binding with the rice jasmonoyl-L-isoleucine (JA-Ile) receptor OsCOI1b (an ortholog of CORONATINE INSENSITIVE 1 of *Arabidopsis*), causing enhanced degradation of the OsJAZ8 protein. Similar to the action of AtJAZ2 in stomatal immunity (34), the perception of JA-Ile and the degradation of a JAZ transcriptional repressor activate the JA signaling, leading to increased stomatal susceptibility to bacterial invasion. Bacteria can also overcome stomatal immunity through other mechanisms. For example, *X. oryzae* pv. *oryzicola* can overcome stomatal defenses by secreting the XopAP effector (70). This effector is a lipase that controls the pH of lysosomes through binding to vacuolar H<sup>+</sup> pyrophosphatase, a process that inhibits vacuolar acidification, leading to stomatal opening (70).

In addition to stomatal immunity being disarmed by pathogen-derived effectors and toxins, specific plant factors can also lead to defective stomatal immunity. For instance, the activation of the immune receptor nucleotide-binding leucine-rich repeat protein (NLR) named SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1 (SNC1) inhibits stomatal defense through the repression of ABA signaling in a cell-autonomous manner (143), even though it enhances apoplastic defense (155). These studies provided interesting insights into the spatial separation of stomata- and apoplastic-based immunity in *Arabidopsis*. However, stimuli that may induce SNC1 functions remain to be explored.

Another example of a plant receptor leading to defective stomatal immunity was recently reported in tomato. Tomato Atypical Receptor Kinase 1 (TARK1), a membrane-localized pseudokinase, has been implicated in the signaling pathway triggered by *P. syringae* pv. *tomato* and elicitors of biotic stress in tomato plants. Removing TARK1 via CRISPR-Cas9 knockout prevents stomatal reopening by Pst DC3000, whereas overexpressing TARK1 makes plants hypersensitive to bacterium invasion through the stomata independently of COR (40). Guzman et al. (40) also reported that the stomata of these transgenic plants respond normally to light and ABA, addressing a prominent question in the field of stomatal physiology: To what extent does biotic and abiotic stimulus-activated signaling in guard cells overlap? The identification of these unique signaling components is important to minimize pleiotropic effects for targeted crop improvement.

## 3.2. The Role of Stomata in Controlling Water Availability to Phytobacteria

Many phytobacteria induce apoplastic water soaking that is typically visible around 24 h postinvasion, depending on the environmental conditions and bacterial titers, which precedes the appearance of extensive disease symptoms at later phases of plant infection (3, 7). Answers to how water soaking comes about and whether the plants can control the water availability to the invading pathogens have been largely elusive. Interestingly, recent research has elucidated the role of stomatal movement at this early postinvasion stage and how it is modulated by both the pathogenic bacterium and the plant.

**Stomatal susceptibility:**
the failure of guard cells to stop pathogen infection

**Pseudokinases:**
catalytically deficient kinase variants
Studying infection of rice by *X. oryzae* pv. *oryzae*, Zhang et al. (152) demonstrated that stomatal closure via ABA is essential for susceptibility in the apoplast. They observed that rice mutants with constitutively open stomata were more resistant to that pathogen than the wild-type plant, which is possibly associated with lower water potential in the leaf apoplast (152). Three years after this study, this possibility was also validated in the *Arabidopsis-Pseudomonas* pathosystem. It was demonstrated that after invading the apoplast, Pst DC3000 uses two effector proteins (AvrE and HopM1) to induce stomatal closure, thereby increasing water content in the apoplast (45, 110). Mechanistically, AvrE and HopM1 work synergistically to modulate ABA biosynthesis, transport, and signaling in guard cells (Figure 1d). Interestingly, a loss-of-function mutation in CURLY LEAF (CLF), which epigenetically controls ABA-associated transcription factor genes of the NAC family, yields plants with high ABA levels, enhanced stomatal closure, and excessive apoplastic water soaking conducive to Pst DC3000 growth (139). It turns out that this secondary stomatal closure is also transient. *Arabidopsis* plants fight back by restricting the water flow to the infection site and reopening the stomatal pore, causing the apoplast to dehydrate and increase resistance against bacterial colonization (29, 73). Together, these findings illustrate the continuous struggle between plants and pathogens for water in the apoplast.

The sequences of events over 24 h of plant-pathogen interactions, including MAMP-triggered stomatal closure (1–2 h), COR- and effector-induced stomatal opening (3–4 h), effector-induced closure (6–24 h), and SCREW-NUT-based stomatal opening (>24 h), highlight the battles between plants and pathogens at the ABA signaling field (Figure 1). It is noteworthy that other hormones also participate in these battles (85). Prolonged water soaking could lead to decreased ABA signaling and stomatal opening. In the end, however, Pst DC3000 wins the war against the *Arabidopsis* ecotype Col-0 possibly because it employs additional virulence mechanisms such as modulation of auxin signaling for proliferation in the apoplastic space (23) and has evolved a necrotrophic phase that includes induced cell death (109) beyond 24 h postinvasion of the apoplast when liquid water in the apoplast may not be as relevant for bacterium survival (Figure 1f).

These excellent studies put more pieces together to solve the stoma-phytobacterium interaction puzzle and illustrate the importance of the timing of events. They also set the stage for answering further questions: When exactly and in which specific cells are effectors delivered, what are the roles of other hormones in the process, and what triggers bacterium switching to the necrotrophic phase? Now more than ever, it is crucial to develop reporters for the biosynthesis and signaling of ABA, SA, JA, COR, effectors, and other signaling molecules to provide the spatial and temporal resolution necessary to answer the questions above in planta.

### 3.3. Fungus-Derived Molecules that Highjack Guard Cell Metabolism

Guard cell death can decrease fungal infection through the stomatal pore (146). Interestingly, in the *P. infestans*-potato pathosystem, the fungus prevents the death of guard cells by scavenging H₂O₂ and NO, inducing starch degradation and triacylglycerol breakdown, conditions that induce stomatal opening (144). Overexpression of two potential *P. infestans* effectors (PITG_11755 and PITG_15152) in potato leaves mimics the fungus-induced stomatal opening. Thus, it is highly likely that the fungus uses these effectors to allow its spores to exit through open stomata and infect other plants.

## 4. STOMATA-HUMAN PATHOGEN INTERACTIONS

Many epiphytic microbes, harmful to the plant or not, such as human pathogens, can potentially penetrate open stomatal pores and live in the apoplast (22, 27). One aspect of stomatal function is the ability of the guard cell to recognize and respond to the presence of bacterial pathogens of
humans (31), which is not particularly surprising given that PRRs recognize molecular features conserved in whole classes of microbes, including nonphytopathogens. For instance, flg22 and elf8 were first defined in *Pseudomonas aeruginosa* (28) and *Escherichia coli* (56), respectively. This feature has a major implication for the issue of crop safety that affects the agriculture and food industries. For instance, recent analyses of major human disease outbreaks caused by *E. coli* indicated that most originated in leafy green production fields (77). This suggests that pathogenic *E. coli* can survive in leaves for a long time, escaping standard sanitation procedures used for freshly consumed produce (79). Internalized bacteria can be protected from produce sanitizers. Indeed, cellular appendages of the bacterium *E. coli* O157:H7, such as pili, flagella, and TTSS, are required for its colonization of the stomatal cavity where it can survive in the plant after sanitation (117). However, based on a mutant analysis, *Salmonella* flagellin does not seem to be required for the bacteria to penetrate the tomato apoplast through the stomatal pore (150).

It has been shown that enterohemorrhagic *E. coli* O157:H7 can induce stomatal closure in *Arabidopsis* (81) and leafy greens such as lettuce, basil, cilantro, and spinach (111). A plant with compromised stomatal immunity, such as the *ost1-2* mutant of *Arabidopsis*, is more prone to human pathogen contamination than the respective wild type (112). Furthermore, an *Arabidopsis* mutant screen allowed the identification of novel proteins involved in stomatal immunity, specifically against human pathogens. In particular, SA biosynthesis and signaling through ICS1 and NPR1 are required for stomatal immunity against *E. coli* O157:H7, the nontyphoid pathogen *Salmonella enterica* serovar Typhimurium, and the phytobacterium Pst DC3118, while the EXO70H4 protein is only required for stomatal immunity against the human pathogens (88) **(Figure 6)**. Activation of *Arabidopsis* and lettuce immune responses by these two human pathogens has been confirmed by transcriptomic analysis that also revealed common and unique aspects of these plant-human pathogen interactions (47, 88).

Although there is a large overlap between *Arabidopsis* responses (e.g., SA signaling and PTI) to plant and human pathogens (31, 80), it has become evident that each of the bacteria studied also induces unique responses in the plant. Intriguingly, S. Typhimurium shows chemotaxis toward open stomata (55) and, unlike *E. coli* O157:H7, can reopen the stomatal pore in a manner similar to that of Pst DC3000 (112), albeit via modulation of alternative metabolic pathways in the plant (108). Several *Salmonella* genomic regions required for overcoming lettuce stomatal immunity were identified by genetic screening, including the *Salmonella* pathogenicity islands (SPI-1 and SPI-2) that harbor TTSSs and other regions encoding membrane transporters (49, 84) **(Figure 6)**. These studies laid the genetic foundation required for understanding the underlying molecular mechanisms for stomata-human pathogen interaction, a nascent field that holds great promise for decreasing edible leaf contamination and improving crop safety.

## 5. STOMATAL IMMUNITY, ABIOTIC STRESS, AND PLANT GROWTH

In addition to controlling microbial invasion, stomatal pores allow the assimilation of CO₂, regulate transpiration, and function in water flow through the plant. The physiological functions of the remarkably specialized guard cells have been nicely reviewed by Lawson & Matthews (60). Thus, stomata are at the intersection between abiotic and biotic stresses, which relates to plant resilience under environmental stress.

Considering the wide range of biological functions controlled by stomata, one could speculate that plant responses to pathogens through the regulation of stomatal movement, patterning, and density could result in synergisms with plant responses to abiotic stresses, particularly drought (95). Mounting evidence suggests that abiotic and biotic stimulus-triggered stomatal movements share some molecular mechanisms, including, but not limited to, the involvement of hormones,

*Salmonella pathogenicity island:* region within the *Salmonella* genome that encodes determinants of *Salmonella's* interaction with and virulence on a given host
<figure><img src="image_9.png" /><figcaption>Figure 6</figcaption></figure>

A model for human pathogen-stomata interaction. (a) *Salmonella enterica* serovar Typhimurium (*red cells*) and *Escherichia coli* O157:H7 (*yellow cells*) induce stomatal closure. These human pathogens are recognized by the plant via FLS2 (22, 27) and possibly other receptors, resulting in stomatal closure (31). The signaling for stomatal closure requires OST1 (112), SA biosynthesis via ICS1 (88), NPR1, and EXO70H4 (88).

(b) *Salmonella* Typhimurium induces stomatal reopening (108, 112), and several genomic regions are required in the process (*question marks*), including the *Salmonella* pathogenicity islands SPI-1 and SPI-2 (49, 84). The mechanisms have not yet been elucidated. Solid and dashed arrows indicate direct and indirect connections, respectively. Abbreviations: EXO70H4, EXOCYST SUBUNIT EXO70 FAMILY PROTEIN H4; FLS2, FLAGELLIN-SENSING 2; ICS1, ISOCHROMATE SYNTHASE 1; NPR1, NONEXPRESSER OF PR GENES 1; OST1, OPEN STOMATA 1; SA, salicylic acid; SPI, *Salmonella* pathogenicity island. Figure adapted from images created with BioRender.com.

ROS production, and Ca²⁺ signaling (38, 67, 107, 135). However, enhanced drought tolerance and immunity could cause trade-offs with plant growth. A recent study nicely illustrated these concepts in the *Brassica oleracea–X. campestris* pv. *campestris* pathosystem, in which stomatal closure during infection contributed to a reduction in plant biomass due to elevated ABA concentration during pathogen resistance and a limitation of gas exchange with the environment (131).

Uncovering the regulatory mechanisms controlling stomatal movement in response to stress along with growth trait evaluations would enable strategies to decouple undesirable trade-offs. For instance, analysis of *Arabidopsis* mutants defective in the *BIG* locus uncovered its function in promoting stomatal immunity (153). Interestingly, *big-1* mutants not only show disrupted stomatal closure in response to Pst DC3000 (153) and elevated CO₂ (41) but also have smaller rosettes
compared to the wild-type plant (153), suggesting that BIG may regulate multiple pathways that
may be spatiotemporally separated.

It should be noted that studying trade-offs or synergisms linked to plant growth and plant responses to abiotic and biotic stress is very important for both agricultural and natural systems. However, this is a complicated topic to explore because these interactions are largely controlled by environmental factors and depend on numerous features such as plant age, plant species, tissue types, and the intensity and chronology of the exposure to stresses. One of the challenges will be to move from large-scale association studies to a mechanistic understanding of these phenomena, allowing for a fine-tuned, spatiotemporal characterization of molecular signaling with the potential for metabolic engineering strategies toward crop improvement.

# 6. RECENT TECHNOLOGICAL ADVANCES TO STUDY GUARD CELL RESPONSE TO PATHOGENS

The significant advances in the field of stomata–pathogen interactions were only possible with the advances in technologies to study metabolism at the single-cell resolution. Traditional characterization of mutants continues to be extremely valuable. However, additional approaches are required to fully study guard cell response to pathogens in planta and in real time without the typical pleiotropic effects of the mutations. The development of sensors and reporters along with advanced microscopy for live-cell imaging of intact leaves; the ability to isolate viable guard cell protoplast for cell-specific omics studies; and the recent breakthroughs in remote sensing, canopy spectroscopy, modeling, and artificial intelligence are invaluable to advance this area of research. Below are some examples of the significant findings enabled by these advanced technologies.

## 6.1. Using Molecular Sensors and Reporters to Understand Guard Cell Response to Elicitors

In recent years, useful reporter systems have been developed. For instance, roGFP2, which works by redox-dependent fluorescence, allows the evaluation of guard cell redox responses to chloroplast inhibitors and ABA (52, 96). R-GECO1-mTurquoise is a genetically encoded calcium indicator (GECI) that improves upon the existing GECI R-GECO1 by the addition of the mT-fluorescent protein. This modification allows for more accurate measurements of [Ca²⁺]cyt over time. This reporter has recently been used to study the guard cell Ca²⁺ response to ABA and flg22 (25, 46). CapHensor is another reporter used to study the ions in guard cells. CapHensor is a dual reporter that performs simultaneous ratio imaging for both Ca²⁺ and H⁺ (pH) within the guard cells, as well as other pollen tubes and mesophyll cells. It has been proven to be an effective method to measure guard cells’ specific Ca²⁺ and pH responses to ABA, flg22, and H₂O₂ (62). While these reporters probably have not been used in plant–pathogen interactions, they could be useful in future approaches to further understand guard cell response to specific pathogens during stomatal immunity.

## 6.2. Available Omic Data Sets to Further Explore Stomata–Pathogen Interactions

Many omic data sets and analyses published within recent years, including transcriptomics, proteomics, and metabolomics, as well as some variations of these platforms (e.g., phosphoproteomics), are excellent resources to advance the understanding of metabolic pathways associated with stomatal immunity, as well as guard cell physiology in general.

Transcriptomic profiling of *Arabidopsis* leaves inoculated with Pst DC3000 mutants (*hopM1*⁻, *avrE1*⁻, and *hopM1*/*avrE1*⁻) revealed functional redundancy in the ABA biosynthesis and
signaling pathways, and it also linked water soaking induced by the pathogen to HopM1- and AvrE1-dependent signaling pathways and ABA biosynthesis (110). The transcriptome of the *Arabidopsis* mutant *smd3b-1* revealed that impaired stomatal defense against Pst DC3000 can also be attributed to the regulation of messenger RNA splicing (35). Simultaneous transcriptome profiling of both the plant host and the microbial invader has also yielded significant understanding of the timing and potential mechanisms underlying the interaction between these organisms. Such data sets are available for *Arabidopsis* and lettuce interactions with *S. enterica* and *E. coli* O157:H7, which have deepened our understanding of stomatal immunity in response to nonpathogen microbes, in addition to providing an interesting contrast as to how *S. enterica* overcomes stomatal defense, whereas *E. coli* O157:H7 does not (47). Because guard cells comprise a very small percentage of the total cells within the leaf, it is important to understand which functions are guard cell-specific by running omic analyses on isolated guard cells (89). As such, two recent studies created an inventory of potential NO- or redox-sensitive guard cell proteins. Using *cysTMTRAQ* on proteins isolated from guard cells, it was possible to identify proteins, specifically lipid transfer proteins (LTPs), that have a relevant role in immune-elicited guard cells (5). *iodoTMTRAQ* quantitative proteomic analysis of isolated guard cells also revealed novel NO-responsive proteins in *flg22-challenged Arabidopsis* (59). Furthermore, a dual proteomic and phosphoproteomic analysis in response to Pst DC3000 allowed the identification of many differentially regulated proteins and phosphoproteins involved in a wide array of responses linked to stomatal immunity, such as ABA, SA, calcium, and ROS signaling pathways (93). A recent proteomic data set comparing guard cells with the mesophyll cells in *Arabidopsis* enabled deciphering of cell type-specific phosphorylation cascades via OST1 (133).

Metabolomic analysis methods and protocols have been significantly improved in recent years. For example, isotope labeling allows for tracing the origin of biomolecules to either the plant or pathogen (92). Untargeted metabolomic studies have provided insight into the plant-pathogen metabolome, including the function of AtDIR1 in guard cell-specific SAR (18, 19) and volatile organic compounds produced in response to virulent and avirulent pathogens (74). Untargeted metabolomic studies on *Sclerotium* spp. on common bean and *Dendrobium catenatum* shed some light on how the pathogen uses the stomata to enter the plant (105, 138). A recent review highlighting guard cell metabolism sets the baseline for understanding how the guard cell metabolome could be reprogrammed by different microbes (60).

The use of multiomic approaches, that now can be done simultaneously using the same sample (18), is particularly relevant for nonmodel organisms, for which genetic resources are not readily available and may be intractable to genetic manipulation. Using proteomics with metabolomics, it was possible to examine the stomatal immunity of *Sclerotium rolfsii* in pepper and *F. oxysporum* in chickpea (66, 87). Comparative analyses with these data sets could allow for the identification of shared pathways of stomatal immunity in a broad range of plant-pathogen interactions.

## 6.3. Imaging and Artificial Intelligence for Large-Scale Stomatal Defense Phenotyping

The analysis of stomatal response to stimuli in whole plants is very useful to understanding the dynamic behavior of stomatal movement in a biologically relevant context. In addition to being useful for stomatal mutant screening (41, 108, 136), infrared thermal imaging of leaf surfaces can capture such dynamic behaviors over time due to the correlation between leaf temperature and stomatal conductance (42). Furthermore, nondestructive methods of studying stomatal phenotypes on a large scale could be valuable to understanding stomatal immunity in crop fields and orchards in real time. Recent studies have taken advantage of this technology to measure stomatal traits by capturing images of leaves in their native growing conditions, and then using an
artificial intelligence system that detects, counts, and measures stomatal phenotypes (15, 94). This automated high-throughput system allows for rapid, inexpensive, and quantitative measurements with a high level of accuracy [e.g., >98% precision for approximately 9,000 individual stomatal apertures of citrus (15)] that would otherwise be impossible to accomplish with traditional methods. Thus, the advancement of imaging and automated processing technologies will enable researchers to answer crucial biological questions in the field of stomata–pathogen interactions in agricultural and natural systems.

## SUMMARY POINTS

1. Leaf infection by foliar pathogens that internalize into tissues through stomatal pores and cause apoplastic water soaking results in a temporal open-close-open-close-open pattern of stomatal movement.

2. Stomatal response to pathogen infection can span from a local, fast response to a long-term systemic response in existing leaves and change stomatal density in newly formed leaves after infection.

3. The canonical pattern-triggered immunity pathway and the modulation of ion channels in the guard cell membrane emerged as common components of the stomatal response to microbes of diverse lifestyles and multiple elicitors.

4. A major question in the field has been the potential to translate the mechanistic understanding of stomata–pathogen interactions, achieved with the study of *Arabidopsis–Pseudomonas* interaction, into crop improvement. Although this achievement may be a little farther in the future, steps toward this goal have been taken in a few crop plants such as pepper, tomato, rice, and wheat.

5. Human pathogens vectored by plants utilize stomata in parallel with the internalization mechanisms used by plant pathogens. This phenomenon is particularly important for plant–*Salmonella enterica* interactions through a yet-to-be-discovered mechanism(s).

6. Stomatal immunity can occur as a trade-off with plant growth, but molecular intersections of these pathways have been identified that could be used to decouple this effect.

7. State-of-the-art technologies, including sensors, multiomics, and high-throughput imaging coupled with artificial intelligence for canopy analyses, are available to study stomata–pathogen interactions, enabling new discoveries in the field.

8. Future advances could include ionomic studies, as ions play a significant and important role in stomatal movement. Many ions in the guard cell, outside of potassium, calcium, and chloride, are understudied (83), and a recent ionome analysis during plant–pathogen interactions (9) could be adapted for guard cell–specific experiments.

## FUTURE ISSUES

1. Guard cells are extremely sensitive to experimental conditions, which may have contributed to contradictory findings among different research groups. Furthermore, there seems to be redundant functionality among pathways controlling stomatal movement, which might explain seeming contradictions in the literature.
2. Although some progress has been made toward understanding how stomata-microbe interactions affect plant responses to abiotic stresses and plant growth, our current knowledge of connections among these metabolic processes is still limited.

3. Very little about stomatal metabolism is known outside of a few model systems. Research into the regulatory foundations of the multiple functions of stomata and the points of crosstalk will not only advance basic science but also enable strategies for enhanced crop resilience.

## DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

## ACKNOWLEDGMENTS

Our program is currently funded by grants from the US Department of Agriculture, National Institute of Food and Agriculture (USDA-NIFA); the NIFA Hatch (CA-D-PLS-2327-H); the California Leafy Greens Research Board; various initiatives at the College of Agriculture and Environmental Sciences at the University of California, Davis; and fellowships and scholarships to support students such as the Plant Sciences Graduate Student Research awards, Henry A. Jastro Research awards, and the NIFA-National Need Fellowship programs (NIFA-NNF #2021-38420-34070). The authors thank members of the laboratory (David Bridges, Madeleine MacConnell, Maria de los Angeles Miccono, Gabrielle Rossidivito, Ho-Wen Yang, and Jirachaya Yeemin) for providing useful comments on the manuscript.

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<footnote n="6">6. Identified
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immunity using the
nonmodel organism
wheat.</footnote>
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