Cell-type-specific immune programs orchestrate spatial defense in the Arabidopsis leaf epidermis

Domain: biology
Published: 2026-03-21
Editorial outcome: minor_revision

Abstract
Sessile plants rely on individual cells to perceive pathogens and coordinate defense. Guard cells (GCs), best known for regulating stomatal aperture, h ave poorly understood intrinsic immune roles. Here, we integrate single-cell and spatial analyLses of Arabidopsis thaliana leaves infected with diverse phytopathogens, combining live and fixed imaging of transgenic immune reporters with single cell transcriptomic data. Powdery mTildew infection triggers strong salicylic acid (SA) biosynthesis and transport in pavement cells, spreading to neighboring uninfected cells. In contrast, GCs fail to activate SA biosynthesis or SA-responsive genes. These cell-type-specific immune programs distinguishing GC and pavement cells are conserved across infections by the hemibiotrophic fungus Colletotrichum higginsianum and the bacterial pathogen Pseudomonas syringae. Despite impaired SA signaling and response, GCs display rapid calcium influx and pronounced reactive oxygen species bursts, transmitting immune signals to adjacent pavement cells via the apoplast. Notably, GCs are incompatible with adapted fungal pathogens and underwent hypersensitive cell death. Together, these findings uncover distinct immune programs among epidermal cell types, highlight GC-autonomous defense mechanisms, and provide a framework for understanding spatial immune coordination in plants.

Introduction
Plants are constantly exposed to diverse microbial pathogens that can compromise plant growth and reproduction. Co-evolution of potentially pathogenic microbes and their hosts has shaped a highly resilient plant innate immune system1. In response to pathogen attack, plants initiate immune responses through a coordinated process involving immune recognition, signal integration, and defense execution2. Immune recognition is mediated by a diverse array of cell-surface and intracellular plant immune receptors3. Cell-surface pattern recognition receptors (PRRs) detect conserved microbial features known as pathogen-associated molecular patterns (PAMPs), triggering pattern-triggered immunity (PTI). PTI provides complete immunity against non-adapted pathogens and partial resistance to host-adapted pathogens. In turn, host-adapted pathogens deploy effector proteins into plant cells, where they interfere with immune responses2. These effectors are specifically recognized by intracellular nucleotide-binding leucine-rich repeat receptors (NLRs), which activate effector-tEriggered immunity (ETI)2. Recognition of potential pathogens by PRRs and NLRs initiates signal transduction cascades that culminate in immune activation2,3. PTI and ETI are not functionally isolate d; instead, they reinforce one another and jointly mediate robust downstream immune responses, including calcium influx, reactive oxygen species (ROS) production, mitogen-activated protein kinase (MAPK) activation, phytohormone biosynthesis and signaling, and transcriptional reprogrammLing3,4.

Plant-pathogen interactions are highly dynamic processes shaped by the specific tissues and cell types of the host, as well as by the spatial distribution, developmental stage, and activity of the pathogen. Consequently, host-pathogen interactions at the cellular level are often heterogeneous and asynchronous, resulting in a mosaic of immune-activated and susceptible states. Conventional biochemical and molecular assays based on bulk tissue can obscure fundamental principles of cellular interactions. In contrast, recent advances in single-cell and spatial transcriptomics have begun to uncover distinct immune cell states, opening new avenues for cell type-specific investigations in plant immunity3–6. Despite these advances, fully elucidating the temporal dynamics of immune activation, signaling, cell-to-cell communication, and disease progression in the context of spatial heterogeneity among host cell types remains an urgent and unresolved challenge.

The leaf epidermis, the outermost layer of plant leaves, serves as a vital interface between the plant and its environment, and constitutes the first line of defense against pathogen invasion. The leaf epidermis comprises specialized cell types, including pavement cells and stomatal guard cells (GCs), which are essential for regulating gas exchange and influencing plant immunity7–9. A wide range of phytopathogens such as bacteria, oomycetes and fungi exploit stomatal openings as primary entry invasion routes9. In response, GCs can detect PAMPs or pathogen effectors through immune receptors, triggering stomatal closure to restrict pathogen entry and limit colonization7–9. However, some pathogens can counteract this response by secreting effector proteins or phytotoxins that disrupt GC function, leading to stomatal reopening and facilitating invasion7–9. To date, most research on stomatal immunity has focused on the regulation of stomatal aperture, revealing a complex signaling network involving calcium, abscisic acid (ABA), ROS, and defense hormones such as salicylic acid (SA)7,9. Despite these insights, the mechanisms underlying GC-autonomous immunity remain poorly understood.

Biotrophic powdery mildews are among the most widespread plant pathogens, typically colonizing the surface of plant tissue, primarily the epidermis. The infection process begins when conidiospores land on the plant surface and germinate, forming an appressorium that penetrates directly through the epidermal cell wall. This leads to the establishment of a unicellular haustorium, a specialized structure that facilitates nutrient uptake from the host within the infected epidermal cells. The infection process is highly coordinated, involving intricate, cell-specific interactions between host and pathogens10. In this study, we employed Arabidopsis thaliana (Arabidopsis) in both adapted and non-adapted powdery mildew pathosystems, integrating live and fixed tissue imaging with transgenic immune reporters to investigate immune responses, signaling dynamics, and cell-type-specific resistance or susceptibility in leaf epidermal pavement cells and GCs during infection. Our analysis provides single-cell and spatially resolved insights into SA biosynthesis and signaling, callose deposition, Ca2+ influx, and ROS production in response to powdery mildew infection. Intriguingly, while GCs do not engage in pathogen-induced SA synthesis and callose deposition, they exhibit pronounced Ca2+ influx and ROS accumulation during infection, revealing an inherent incompatibility with adapted fungal pathogens. Moreover, these cell-type-specific immune programs that distinguish GCs from pavement cells were conserved across infections by the hemibiotrophic fungus Colletotrichum higginsianum and the bacterial pathogen Pseudomonas syringae, as further supported by single-cell RNAseq analyses.

Results
Temporal and spatial induction of ICS1 transcription during powdery mildew infection

Salicylic acid (SA) plays critical roles in plant defense against biotrophic pathogens. In Arabidopsis, the rate of pathogen-induced SA biosynthesis predominantly depends on transcription activity of ISOCHORISMATE SYNTHASE 1 (ICS1) 11. To monitor this process, we used transgenic Arabidopsis plants harboring the reporter construct pICS1::NLS-3×mVENUS, which enables visualization of ICS1 promoter activity12. These plants were inoculated with conidiospores of the non-adapted barley-powdery mildew fungus Blumeria hordei (B. hordei)13, and dynamics of pICS1::NLS-3×mVENUS expression were tracked via live-cell imaging during the course of infection. In general, B. hordei conidiospores germinate on the leaf surface of non-host Arabidopsis and form an appressorium, which develops a thin penetration peg to pierce the leaf epidermal cell wall. Most penetration attempts are unsuccessful due to the formation of dense papillae beneath the attempted entry site. However, in fewer than 5% of cases, penetration succeeds, followed by haustorium formation within 24-hour post inoculation (hpi)14. The invaded epidermal cells rapidly undergo hypersensitive cell death, effectively halting the progression of powdery mildew infection in the non-host Arabidopsis14,15.

Leaves inoculated with B. hordei were sampled at three time points: 18, 21, and 24 hpi. To enable precise localization of fluorescent signals to specific cells and cell types, the inoculated leaf tissues were stained with the red fluorescent cell wall stain propidium iodide (PI). For quantitative analysis of signal induction, fluorescence intensities were measured in both the nuclei and the cytoplasm of epidermal cells either attempted or penetrated by B. hordei (designated as “Patient 0”, P), as well as in their successive neighboring epidermal cell layers. Cells in direct contact with “Patient 0” were defined as the first layer (L1), while cells adjacent to L1, but not directly bordering “Patient 0”, constitute the second layer (L2), and so forth (Supplementary Fig. 1A). At 18 hpi, no detectable induction of NLS-3×mVENUS fluorescence was observed in either the nuclei or the cytoplasm of cells at the penetration site (Fig. 1A-B). By 21 hpi, however, visible induction of nuclear-associated fluorescent signals became apparent in “Patient 0” and adjacent L1 cells (Fig. 1A-B). This induction intensified by 24 hpi, reflecting a time-dependent increase in signal intensity at the fungal penetration sites (Fig. 1A-B). Notably, enhanced fluorescence was also detected in the cytoplasm of “Patient 0” and L1 cells. These observations indicate that fungal penetration attempts, even before successful host cell entry, can activate ICS1 transcription in the surrounding leaf tissues.

At 24 hpi, a strong NLS-3×mVENUS fluorescence signal was observed in “Patient 0” cells exhibiting visible haustorial formation (Fig. 1C-D). Notably, fluorescence induction was also detected in adjacent L1 and L2 cells, forming a prominent patchy induction pattern in which nuclear signal intensity peaked at the center and gradually declined toward the periphery (Fig. 1C-D). In the central region of the patch, fluorescence was evident not only in the nuclei but also in the cytoplasm of L1 and L2 cells (Fig. 1C-D). Z-stack imaging further revealed that the elevated fluorescent signals extended both horizontally across multiple layers of epidermal cells surrounding “Patient 0”, and vertically into a few mesophyll cells directly beneath it (Fig.1E-G, Supplementary Movie 1). To assess whether this pattern also occurs during compatible interactions, ICS1 reporter plants were inoculated with the adapted powdery mildew Erysiphe cichoracearum (E. cichoracearum), which establishes a compatible interaction with Arabidopsis16,17. At 26 hpi, induction of both nuclear and cytoplasmic NLS-3×mVENUS signals was evident in E. cichoracearum-penetrated cells and their neighboring cells, resembling the ICS1 induction pattern observed during B. hordei infection (Supplementary Fig. 1B-D). These findings indicate that ICS1 expression is triggered in response to infection by both adapted and non-adapted powdery mildew fungi.

To validate the activity of the pICS1::NLS-3×mVENUS reporter line, we generated transgenic lines harboring pICS1::ICS1-mSCARLET, in which the native ICS1 promoter drives the expression of ICS1 fused to the red fluorescent protein mSCARLET. As expected, the ICS1-mSCARLET signal co-localized with chloroplasts (Supplementary Fig. 2A), consistent with the plastidic localization of ICS1 protein18. We then performed a co-expression analysis by crossing this line with the pICS1::NLS-3×mVENUS reporter line. Simultaneous visualization of nuclear-localized NLS-3×mVENUS and plastid-localized ICS1-mSCARLET revealed complete spatial overlap of expression signals in B. hordei-infected cells and their immediate neighboring cells (Fig. 1H, Supplementary Fig. 2B).

In Arabidopsis, both ICS1 and ENHANCED DISEASE SUSCEPTIBILITY 5 (EDS5) are crucial for SA accumulation in response to pathogen infection. ICS1 catalyzes the conversion of chorismate to isochorismate, while EDS5 facilitates the subsequent transport of isochorismate from the chloroplast to the cytosol. The expression of these genes is tightly regulated and follows a similar pattern19. To investigate whether ICS1 and EDS5 are co-expressed in a spatiotemporally coordinated manner, we generated two EDS5 reporter lines (pEDS5::NLS-mSCARLET#3 and pEDS5::NLS-mSCARLET#9) and crossed them with the pICS1::NLS-3×mVENUS reporter to produce dual-reporter lines. As anticipated, both reporters exhibited identical induction patterns at the site of the B. hordei infection at 24 hpi (Fig. 1I, Supplementary Fig. 3A). Together, these results demonstrate that the established reporter lines enable precise, spatiotemporal visualization of SA-biosynthesis and accumulation at the single cell resolution in leaf tissues in response to the pathogen attack. They further reveal that the pathogen-induced SA biosynthesis is rapidly initiated in infected cells prior to pathogen entry (Supplementary Fig. 3B).

Pathogen-induced SA biosynthesis relies on Ca2+ signaling

Pathogen-induced ICS1 expression and SA biosynthesis are controlled by a complex network of positive and negative regulators3,20. Among these, Ca²⁺ signaling plays a pivotal role. Upon pathogen attack, an influx of Ca²⁺ from the apoplast into the cytosol is triggered, and this Ca²⁺ signal is crucial for activating ICS1 expression21,22. To investigate the requirement of Ca2+ signaling, we treated pICS1::NLS-3×mVENUS reporter plants with LaCl3, a broad-spectrum Ca2+ channel blocker23. This treatment completely abolished the characteristic patchy induction of the reporter at B. hordei-infected sites (Fig. 2A). We next examined the effects of additional Ca2+ signaling antagonists commonly used in plants24. Interestingly, treatments with erythrosin B or eosin Y, antagonists of P-type Ca2+-ATPase that mediate Ca2+ efflux, similarly suppressed the patchy induction pattern of pICS1::NLS-3×mVENUS at 24 hpi following B. hordei infection (Supplementary Fig. 4). These results indicate that perturbation of Ca2+ fluxes disrupt pathogen-triggered ICS1 expression, underscoring the essential role of Ca2+ homeostasis and signaling in this process. To further assess the impact of Ca2+ availability, we grew the dual-reporter line (pICS1::NLS-3×mVENUS pEDS5::NLS-mSCARLET) in hydroponic solutions containing various concentrations of CaCl₂ (0.025 mM, 0.1 mM, and 0.5 mM) for one week. Following B. hordei inoculation, plants grown in 0.5 mM Ca²⁺ displayed strong induction of both ICS1 and EDS5 at 24 hpi, whereas those cultured in 0.1 mM Ca²⁺ showed a weaker response (Fig. 2B). Strikingly, no induction of reporter signal was detected in plants grown in 0.025 mM Ca²⁺ (Fig. 2B). These findings indicate that a threshold level of Ca2+, likely determined by the concentration gradient between the apoplast and the cytosol, is required to activate signaling pathways that drive the transcriptional induction of ICS1 and EDS5 in infected cells and their neighboring cells during pathogen attack.

CALMODULIN-BINDING PROTEIN 60-LIKEg (CBP60g) and SAR-DEFICIENT 1 (SARD1), two closely related transcription factors, function as master positive regulators of SA biosynthesis by directly activating the expression of ICS1 and EDS5. Mutations in both cbp60g and sard1 completely abolished ICS1 induction and SA accumulation in response to bacterial pathogens25. Conversely, three calmodulin (CaM)-binding transcription activators (CAMTAs) serve as negative regulators by repressing the expression of CBP60g and SARD1, thereby limiting SA biosynthesis. In the camta1/2/3 triple mutant, this repression is relieved, resulting in elevated CBP60g and SARD1 expression and increased SA levels26. To visualize ICS1 expression in these regulatory backgrounds, we introduced the pICS1::NLS-3×mVENUS reporter into both cbp60g sard1-2 double mutant and camta1/2/3 triple mutant backgrounds via genetic crosses. Under non-infected conditions, the reporter exhibited low basal activity in both wild type (WT) and cbp60g sard1-2 plants. In contrast, strong fluorescent signals were detected in camta1/2/3 plants, indicating constitutive activation of ICS1 expression (Fig. 2C, Supplementary Fig. 5A-C). Upon B. hordei infection at 24 hpi, NLS-3×mVENUS signals were strongly induced at the infection sites in WT plants but remained at basal levels in the cbp60g sard1-2 mutants. Notably, camta1/2/3 plants exhibited no further increase in fluorescence upon infection, suggesting that ICS1 expression was already saturated in this mutant in the absence of pathogen challenge (Fig. 2D, Supplementary Fig. 5D). Our live-cell imaging results support previous findings that SA biosynthesis in response to pathogen attack is tightly controlled by Ca2+/CaM-regulated transcription factors CBP60g and CAMTAs (Supplementary Fig. 5E)21,27. Taken together, these results suggest that B. hordei infection triggers a localized Ca2+ influx, which modulates CAMTA activity and subsequently upregulates the transcription of CBP60g and SARD1 during pathogen-induced SA biosynthesis (Fig. 2E).

Multiple mechanisms contribute to the feedback amplification of SA biosynthesis and signaling, involving both positive and negative regulatory pathways28. To further explore this regulatory network, we introduced the pICS1::NLS-3×mVENUS reporter into the ics1 mutant (sid2-1) and generated dual pICS1::NLS-3×mVENUS pEDS5::NLS-mSCARLET reporters in the SA-deficient NahG background through genetic crossing. Notably, following B. hordei infection, the reporters displayed comparable induction patterns in WT, sid2-1, and NahG backgrounds (Supplementary Fig. 6-7). These results suggest that B. hordei-induced activation of ICS1 and EDS5 occurs independently of SA or its precursor chorismite accumulation.

Guard cells do not engage in SA biosynthesis and signaling in response to B. hordei infection

The Arabidopsis leaf epidermis comprises pavement cells and specialized cell types, including GCs and trichomes. Notably, in pICS1::NLS-3×mVENUS pEDS5::NLS-mSCARLET dual reporter plants, fluorescent signals were undetectable in GCs adjacent to B. hordei-penetrated pavement cells, even though surrounding pavement cells showed strong induction of both mVENUS and mSCARLET signals (Fig. 3A). To visualize GC nuclei alongside reporter expression, we crossed the pICS1::NLS-3×mVENUS reporter line with a nuclear marker line expressing CENH3-mRFP (Fig. 3B), and the pEDS5::NLS-mSCARLET line with an N7-GFP nuclear marker (Fig. 3C). Neither mVENUS nor mSCARLET fluorescence was detected in the nuclei of GCs adjacent to B. hordei-penetrated pavement cells (Fig. 3B-C), confirming that both ICS1 and EDS5 are not transcriptionally activated in these GCs. To determine whether this lack of expression results from a failure in signal transmission from the infected pavement cells or from an intrinsic inability of GCs to activate these genes, we examined pICS1::NLS-3×mVENUS expression in GCs directly infected by B. hordei. No mVENUS signal was detected in the infected GCs (Fig.3D). Interestingly, the surrounding pavement cells exhibited strong induction of pICS1::NLS-3×mVENUS (Fig.3D), suggesting that while GCs do not activate SA biosynthesis via ICS1 in response to B. hordei infection, they may nonetheless contribute to local immunity by releasing signals that activate defense responses in neighboring pavement cells.

In Arabidopsis, SA functions as an endogenous signal that activates the expression of downstream defense genes, including those encoding pathogenesis-related (PR) proteins. Among these, PR1 and PR4 are well-characterized SA-inducible genes29,30. To investigate whether GCs respond to SA, we treated the PR1 reporter plants (pPR1::NLS-3×mVENUS CENH3-mRFP) and the PR4 reporter plants (pHEL::NLS-3×mVENUS CENH3-mRFP) with exogenous SA12. Reporter analysis revealed that promoter activities of PR1 and PR4 were induced in pavement cells but not in GCs, as no fluorescent signal was detected in GCs (Supplementary Fig. 8A-B). Similar expression patterns were observed in PR1 and PR4 reporter plants inoculated with B. hordei (Supplementary Fig. 8A-B). These findings indicate that GCs are unresponsive to SA, supporting the conclusion that they are inactive in both SA biosynthesis and SA-mediated signaling and response during powdery mildew infection.

To determine whether this differential responsiveness between pavement cells and GCs is specific to SA-mediated immunity or reflects a broader divergence in immune activation, we analyzed the expression patterns of additional immune marker genes using the reporter lines pFRK1::NLS-3×mCITRINE CENH3-mRFP and pLipoP1::NLS-3×mCITRINE CENH3-mRFP, which track the transcriptional activity of FRK1 and LipoP1, respectively. FRK1 encodes a receptor-like protein of unknown function that is strongly and rapidly induced by PAMPs31, while LipoP1 encodes a putative membrane lipoprotein also implicated in early immune responses6. At 24 hpi with B. hordei, both FRK1 and LipoP1 exhibited expression patterns similar to that of the pICS1::NLS-3×mVENUS and pEDS5::NLS-mSCARLET reporters, with induced signals observed in pavement cells surrounding the B. hordei-penetrated cells (Fig. 3E-F). Notably, expression of pFRK1::NLS-3×mCITRINE was undetectable in GCs adjacent to infection sites (Fig. 3E). In contrast, pLipoP1::NLS-3×mCITRINE showed strong nuclear-localized signals in GCs neighboring the penetrated cells (Fig. 3F), suggesting that immune signals originating in infected pavement cells may be translocated into adjacent GCs, selectively activating specific immune pathways.

Together, these findings reveal that GCs exhibit immune responses that are distinct from those of pavement cells. While GCs are deficient in SA biosynthesis and related immune defense, they retain the capacity to activate alternative immune pathways, indicating a unique and selective immune transcriptional profile within the leaf epidermis.

Comparative transcriptional dynamics of pavement and guard cell immune responses to Colletotrichum higginsianum and Pseudomonas syringae.

To investigate whether the distinct cell type-specific immune responses of pavement and guard cells represent a conserved mechanism against both fungal and bacterial pathogens, we analyzed the pICS1::NLS-3×mVENUS reporter responses to the hemibiotrophic fungal pathogen Colletotrichum higginsianum (C. higginsianum)32 and the bacterial pathogen Pseudomonas syringae pv. tomato (P. syringae).

Following C. higginsianum infection, pronounced reporter fluorescence was detected in pavement cells at 48 hpi, specifically in penetration-associated cells and their adjacent neighbors, while GCs exhibited no detectable induction of ICS1 reporter activity (Fig. 4A). Tang et al. employed single-cell transcriptomics to characterize heterogeneous immune responses of Arabidopsis leaf tissues to C. higginsianum, uncovering vasculature-enriched specific immune receptor expression and cellular processes triggered by direct pathogen contact4. We reanalyzed their dataset to identify C. higginsianum-induced genes expressed in pavement and guard cells. The Venn diagram revealed a distinct separation of induced gene sets, with 558 genes uniquely upregulated in pavement cells, 395 in GCs, and only 213 shared between the two cell types (Fig. 4B). Gene ontology (GO) analysis revealed functional specialization, with pavement cell-specific genes enriched in chorismate biosynthetic, positive regulation of gibberellin biosynthesis, intralumenal vesicle formation, and post-translational protein targeting to the ER, reflecting activation of SA biosynthesis and secretory pathways (Fig. 4C). In contrast, GC-specific genes were significantly enriched in aspartate family amino acid catabolic processes, L-leucine catabolism, sucrose starvation response, and the reductive pentose-phosphate cycle, indicative of metabolic reprogramming toward energy redistribution and enhanced stress adaption (Fig. 4C). Genes commonly induced in both cell types were mainly associated with indolalkylamine biosynthesis, lytic vacuole organization, nitrogen compound detoxification, and assembly of mitochondrial respiratory chain complex II (Fig. 4C). Collectively, these results indicate that C. higginsianum infection elicits distinct immune programs in pavement and guard cells, with pavement cells activating SA-related pathways and guard cells undergoing metabolic reprogramming for stress adaptation.

To determine whether this cell type-specific immune pattern also occurs during bacterial infection, we infiltrated leaves of pICS1::NLS-3×mVENUS reporter plants with the avirulent P. syringae strain AvrRpt2. At 12 hpi, strong ICS1 reporter activity was observed in the P.syringae-infiltrated leaf area (Fig. 4D). We further challenged pICS1::NLS-3×mVENUS pICS1::ICS1-mSCARLET plants with a panel of P. syringae strains, including the virulent strain Pst DC3000 and the avirulent strains AvrRpt2, hrcC, hrpA, and hrpS17,33. In all cases, fluorescence from both NLS-3×mVENUS and ICS1-mSCARLET reporters were predominantly confined to pavement cells, whereas GCs exhibited no detectable reporter activity or ICS1 expression (Fig. 4E, Supplementary Fig. 9). These findings indicate that ICS1 expression remains strictly confined to pavement cells during bacterial infection. Recent single-cell RNA sequencing studies have elucidated the molecular interactions between Arabidopsis and P. syringae5,6,34. Among these, Delannoy et al. identified distinct yet interconnected defense programs across major Arabidopsis leaf cell types, highlighting coordinated transcriptional reprogramming that distinguishes cell-autonomous from non-cell-autonomous immune responses to P. syringae34. Leveraging this dataset, we performed GO enrichment analyses of genes induced in pavement cells and GCs, revealing a clear functional division: GCs were predominantly enriched for responses to water deprivation, desiccation, fluid transport, whereas pavement cells were enriched for SA signaling, hormone-mediated defense processes, and oxidative stress (Fig. 4F). Consistently, transcripts of ICS1 and PBS3 genes, the two enzymes directly involved in SA biosynthesis, were absent in GCs (Fig. 4G, Supplementary Fig. 10A).

Together, these results demonstrate that pavement cells serve as the primary sites of pathogen-induced SA biosynthesis, while GCs prioritize metabolic adjustments and stress resilience, highlighting a conserved division of labor in epidermal immunity across fungal and bacterial infections.

Guard cells exhibit distinct cell-autonomous immune defense

Building on the distinct role of GCs in SA biosynthesis and immune gene expression, we further explored additional aspects of their cell-autonomous immune responses. Callose deposition, a well-known plant defense response against pathogen invasion, typically occurs at fungal penetration sites and is also associated with pathogen-induced cell death35,36. To assess callose accumulation in GCs during pathogen infection, we inoculated WT plants with B. hordei and stained infected leaf tissues with aniline blue (sirofluor) for callose visualization37,38. In pavement cells penetrated by B. hordei, callose was prominently deposited at the fungal entry site and around the affected cell (Fig. 5A, Supplementary Movie 2). High-resolution imaging further revealed that callose accumulation extended to adjacent pavement cells and underlying mesophyll tissues. Intriguingly, no callose deposition was observed in GCs adjacent to the B. hordei-penetrated pavement cell (Fig. 5A), suggesting a deficiency in pathogen-induced callose accumulation within GCs. To explore this further, we examined callose deposition in GCs directly targeted by B. hordei. Surprisingly, callose was undetectable within the penetrated GCs, neither at the site of fungal entry nor in surrounding regions. In contrast, neighboring pavement cells displayed clear callose accumulation at regions adjacent to the infected GCs (Fig. 5B-C, Supplementary Movie 3). These findings indicate that callose deposition is not a component of the GC-autonomous immune response, although GCs invaded by B. hordei may still contribute to non-autonomous immune signaling that triggers callose deposition in surrounding pavement cells.

Calcium and reactive oxygen species (ROS) are well-characterized second messengers in plant immunity, playing pivotal roles in stomatal signaling by regulating stomatal aperture39. To investigate Ca2+ influx in GCs during pathogen infection, we expressed a cytosolic calcium sensor, MatryoshCaMP640, in WT plants and inoculated them with B. hordei. At 20 hpi, elevated cytosolic fluorescence was detected in GCs immediately adjacent to the penetrated pavement cell, whereas GCs located farther from the infected site did not show elevated Ca²⁺ levels (Fig. 5D-E). These results indicate a localized and sustained increase in cytosolic Ca²⁺ in GCs influenced by neighboring B. hordei-infected cells. Elevated cytosolic Ca2+ activates the NADPH oxidases RBOHD and RBOHF either through direct Ca2+ binding or via phosphorylation mediated by Ca2+-dependent protein kinases (CPKs), thereby amplifying ROS production41–43. Consistent with this mechanism, genes associated with ROS generation were strongly induced in GCs upon P. syringae infection (Supplementary Fig. 10B). To assess ROS accumulation in GCs following infection, we inoculated the pEDS5::NLS-mSCARLET reporter line with B. hordei and infiltrated the infected leaves with H2DCF-DA, a cell-permeable fluorescent probe that is deacetylated by intracellular esterases and oxidized by ROS, enabling visualization of ROS accumulation in live GCs44. ROS accumulation was detectable in GCs surrounding B. hordei-attempted pavement cell as early as 18 hpi, preceding fungal penetration (Supplementary Fig. 11A-B). At 24 hpi, the induced NLS-mSCARLET signal in the EDS5 reporter line marked the infected regions (Fig. 5F-G). While the NLS-mSCARLET signal itself was not observed in GCs, pronounced ROS accumulation was detected in GCs located within these EDS5-expressing regions. In contrast, GCs outside these regions exhibited no significant ROS accumulation (Fig. 5F-G). By 26 hpi, autofluorescence was observed in GCs adjacent to B. hordei-infected cell (Fig. 5H), suggesting the onset of hypersensitive cell death in these cells. Notably, infection by compatible E. cichoracearum induces higher Ca²⁺ elevation and ROS accumulation in GCs adjacent to the infected pavement cell than in the surrounding pavement cells at 22-26 hpi (Supplementary Fig. 11C-F). These results suggest that GCs respond more vigorously to pathogen challenges than pavement cells, demonstrated by their more rapid and pronounced Ca²⁺ elevation as well as heightened intracellular ROS accumulation.

We further investigated the interplay between SA signaling, ROS accumulation, and hypersensitive cell death. Arabidopsis plants expressing the bacterial salicylate hydroxylase gene NahG under the CaMV35S promoter accumulate little to no SA45. To assess the role of SA specifically in epidermal cells, we generated transgenic Arabidopsis lines expressing NahG-mSCARLET driven by the epidermis-specific AtML1 promoter46. As expected, NahG-mSCARLET signals were restricted to the leaf epidermis (Supplementary Fig. 12A). These pAtML1::NahG-mSCARLET plants exhibited a similarly enhanced susceptibility to the adapted powdery mildew E. cichoracearum as plants expressing CaMV35S::NahG (Supplementary Fig. 12B). Following B. hordei challenge, epidermal cells of NahG-mSCARLET leaves penetrated by B. hordei displayed strong ROS accumulation, accompanied by cell autofluorescence, while neighboring cells showed callose deposition (Supplementary Fig. 12C-E). These results indicate that B. hordei-induced cell death in Arabidopsis is associated with ROS accumulation and autofluorescence and occurs independently of SA accumulation.

Together, these findings reveal that GCs exhibit a distinct, cell-autonomous immune response characterized by a deficiency in callose deposition, yet marked by sustained cytosolic Ca²⁺ elevation and ROS accumulation, culminating in accelerated cell death (Fig. 5I).

Guard cells are incompatible with host-adapted fungal pathogens

Previous studies have shown that GC death in both monocot and dicot species can arrest invasion of non-adapted fungal pathogens47,48. These findings led us to investigate how GC respond to host-adapted pathogens. We then inoculated the 14-day-old WT plants with the adapted powdery mildew E. cichoracearum, and fungal development was assessed at 72 hpi by confocal microscopy. In pavement cells, E. cichoracearum successfully penetrated via appressoria, establishing fungal colonies marked by the formation of mature haustoria with a globular central body and projecting lobes and the emergence of elongating secondary hyphae on the leaf surface (Fig. 6A-B, Supplementary Movie 4). In striking contrast, E. cichoracearum infection was arrested in GCs. Only haustorial primordia were observed, lacking the characteristic lobed morphology of mature haustoria, and no secondary hyphae developed from these infection sites (Fig. 6A&C, Supplementary Movie 5). These finding suggest that GCs are incompatible with this host-adapted powdery mildew pathogen. Interestingly, callose deposition was observed specifically at the E. cichoracearum penetration sites within infected pavement cells, while it was barely present in the surrounding uninfected cells (Fig. 6A-B). In contrast, prominent callose accumulation was detected in pavement cells adjacent to infected GCs, whereas the GCs themselves showed little or no callose deposition at penetration sites (Fig. 6A&C).

To assess whether this GC incompatibility extends to other host-adapted fungal pathogens, we next examined infection by another compatible powdery mildew species, Erysiphe cruciferarum (E. cruciferarum)49. Despite forming more elaborate appressoria, E. cruciferarum exhibited a similar infection pattern closely resembling that of E. cichoracearum: GCs remained fully incompatible, displaying impaired haustorium formation and the absence of secondary hyphal development (Supplementary Fig. 13A-C, Supplementary Movie 6). We further tested GC susceptibility to C. higginsianum. In pavement cells, infection proceeded through appressoria, forming primary hyphae by 3 dpi (Fig. 6D, Supplementary Movie 7), which subsequently developed into thin secondary hyphae by 4 dpi (Supplementary Fig. 14). In contrast, C. higginsianum infection in GCs was limited to a short initial hypha, which failed to progress (Fig. 6D, Supplementary Movie 7).

Collectively, these findings demonstrate that GCs are broadly incompatible with multiple host-adapted fungal pathogens, displaying distinct immune signatures compared to their compatible pavement cell counterparts.

Methods
Plant materials and growth conditions

All Arabidopsis thaliana (Arabidopsis) lines used in this study were derived from the Columbia-0 (Col-0) ecotype. T-DNA insertion mutants included sid2-111, cbp60g-1 sard1-271 (CS72191) and camta1/2/326 (CS71606). Reporter lines utilized were pICS1::NLS-3mVENUS12 (CS2107988), pPR1::NLS-3mVENUS12 (CS2107991), pHEL::NLS-3mVENUS12(CS2107982), pLipoP1::NLS-3mCITRINE6 (CS73266), pFRK1::NLS-3mVENUS6 (CS2110219), N7 nuclear marker (CS84731), PT-CK (CS16265), and MatryoshCaMP6s34 (CS69957), all of which were obtained from the Arabidopsis Biological Resource Center (ABRC). The CENH3-mRFP line (N799456) was sourced from the Nottingham Arabidopsis Stock Centre (NASC). The transgenic line NahG has been described previously17. DM398 plants, used for cloning the mSCARLET-NOS TERMINATOR cassette, were provided by Dr. Chris Ambrose. Transgenic lines generated in this study included pICS1::ICS1-mSCARLET sid2-1 and pEDS5::NLS-mSCARLET. Seeds were germinated either in soil or on half-strength Murashige and Skoog (MS) medium and grown in a controlled environment chamber under a 16 h light/8 h dark photoperiod at 22 C.

Plasmid construction and plant transformation

To generate the pICS1::ICS1-mSCARLET and pEDS5::NLS-mSCARLET constructs, the mSCARLET-NOS DNA fragment was amplified from DM398 genomic DNA using primers Pac1-mScarlet-F and Spe1-mScarlet-R, and cloned into the pMDC123 vector72 via enzyme restriction (NEB, Canada) and T4 DNA ligase-mediated ligation (Cat# EL0011, Thermo Fisher Scientific) , resulting in the mSCARLET-pMDC123 construct. For the pICS1:ICS1-mSCARLET construct, the ICS1 genomic region (~2.1 kb upstream of the ATG to the end of open reading frame excluding the stop codon)12 was amplified from Col-0 genomic DNA using primers Asc1-pICS1-F/Pac1-gICS1-R, and inserted into the mSCARLET-pMDC123 vector. For the pEDS5::NLS-mSCARLET construct, the EDS5 promoter region (~1.5 kb upstream of the ATG)18 was amplified using primers Asc1-pEDS5-F and Pac1-NLS-pEDS5-R, and ligated into mSCARLET-pMDC123. To generate the pATML1::NahG-mSCARLET construct, the NahG coding sequencing was amplified from plasmid MT363 (Addgene plasmid#65858) using primers Asc1-NahG-FOR and Pac1-NahG-REV. The ATML1 promoter region (3 kb upstream of the ATG) was amplified from Col-0 genomic DNA using primers Pme1-AtML1-FOR and Asc1-AtML1-REV. Both fragments were subsequently ligated into the mSCARLET-pMDC123 vector.

Plant transformations were performed using the Agrobacterium-mediated floral dip method 73 to introduce the constructs into sid2-1 or Col-0 backgrounds. Transgenic plants were selected using either hygromycin or Basta resistance, and positive lines were verified by confocal fluorescence imaging. At least ten independent transgenic lines were obtained and analyzed for each construct. Primer sequences are listed in Supplementary Table 1.

Reciprocal crosses and plant genotyping

Flowers used for crosses at stage 12 were emasculated one day prior to hand pollination. Seeds resulting from these crosses were harvested and collected for downstream analyses. The genotypes of F1 progeny were confirmed by either PCR-based genotyping or confocal fluorescence imaging. Primer sequences used to confirm T-DNA insertions in CBP60g, SARD1, CAMTA1, CAMTA2, and CAMTA3 are provided in Supplementary Table 1. The sid2-1 mutant allele carries a C to T substitution in exon 911, which creates an Mfe1 restriction site. PCR fragments amplified using genotyping primers listed in Supplementary Table 1 were digested with Mfe1 to confirm the mutation. The presence of the NahG transgene in NahG pICS1::NLS-3mVENUS pEDS5::NLS-mSCARLET plants was verified by PCR-based genotyping using NahG-specific primers (Supplementary Table 1).

Pathogen maintenance and plant inoculation

Powdery mildew strains were maintained as described previously14. Briefly, the barley powdery mildew Blumeria hordei (B. hordei) and cucumber powdery mildew Erysiphe cichoracearum (E. cichoracearum) were maintained on barley (Hordeum vulgare L, cultivar CDC silky) and cucumber (Cucumis sativus, variety Sweet Slice, McKenzie, Canada) plants, respectively. The canola powdery mildew Erysiphe cruciferarum (E. cruciferarum) was propagated on canola (Brassica napus L. cv. DH12075). Arabidopsis plants were placed in settling towers and inoculated with conidia by tapping heavily infected barley or cucumber or canola leaves above the Arabidopsis plants. After 30 min, the inoculated plants were returned to the growth chamber for further investigation. Low-density inoculations (5 to 10 conidia mm-2) were employed for this study17.

The anthracnose pathogen Colletotrichum higginsianum (C. higginsianum) was cultured and handled as described previously17,32. Arabidopsis plants were sprayed with conidial suspensions on the leaf surface. Immediately after inoculation, the inoculated plants were placed in a 100% humidity chamber for 48 h.

Pseudomonas syrignae pv tomato DC3000 strains harboring plasmid pVSP61 (virulent strain) or pV288 containing the avrRpt2 gene (avirulent strain) and secretion protein defective strains (hrc C, hrp A and hrp S) were grown at 29 ºC in liquid King’s B medium. Overnight log phase cultures were grown to an optical density at OD of ~0.6 and diluted with sterile water before inoculation. The bacterial suspensions were infiltrated into the abaxial surface of Arabidopsis leaves using a 1-mL syringe without a needle. Control inoculations were performed with sterile water17.

Leaf tissue fixation

The leaf tissues were fixed in 1 ml of methanol:chloroform:acetic acid (6:3:1, v/v/v) solution overnight at room temperature. The samples were then rehydrated through a graded ethanol series (30 minutes per step): 100%, 90%, 80%, 70%, 50%, 30%, 10%, and distilled water. After rehydration, the leaf samples were stored in 150 mM K2HPO4 buffer (pH 9.5) at 4 C until further use.

Chemical treatment of plants

Lanthanum chloride (LaCl3), ruthenium red, verapamil, DNQX, W-7, erythrosin B, eosin Y, and CPA were purchased from Sigma-Aldrich. One hour prior to B. hordei inoculation, Arabidopsis leaves were infiltrated with 5 mM LaCl324,74, 1 mM ruthenium red24,75,76, 10 mM verapamil24,77, 0.5 mM DNQX24,78,79, 50 M W-724,80, 1M erythrosin B24,81, 0.5 M eosin Y24,81, or 25 M CPA24,81. Plants infiltrated with sterile water served as mock controls. Leaf samples treated with either Ca2+ blocker or water were examined at 24 hours post inoculation (hpi) using confocal laser scanning microscopy (ZEISS LSM 880).

For Calcium-deficiency treatments, plants were grown hydroponically as described previously82, with minor modifications. Briefly, Arabidopsis seeds were surface-sterilized and sown onto 0.7% (w/v) agar aliquoted into 10-l pre-cut pipette tips floating on hydroponic solution containing 0.5 mM Ca2+. After one week of growth, seedlings were transferred to fresh hydroponic solutions with varying Ca2+ concentrations: 0.025 mM, 0.1 mM, and 0.5 mM. At three weeks of age, plants grown under different calcium concentrations were inoculated with B. hordei and analyzed at 24 hpi using the ZEISS LSM 880 confocal microscope.

Microscopy and imaging analysis

Fixed leaf tissues were stained with 0.05% aniline blue and/or 10 M Alexa Fluor 488-conjugated wheat germ agglutinin (WGA, Life Technologies). Stained samples were imaged using a ZEISS LSM 880 or Leica Stellaris 5 confocal laser scanning microscope. For fresh tissue imaging, leaf samples were incubated in either distilled water or 10 M propidium iodide (PI, Life Technologies) prior to imaging. To visualize reactive oxygen species (ROS) accumulation in guard cells, leaf tissues, with or without powdery mildew inoculation, were vacuum-infiltrated with 20 M 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (Invitrogen, Oregon, USA) for 15 minutes and immediately imaged by confocal microscopy. Macroscopic detection of H2O2 accumulation by 3,3′-Diaminobenzidine (DAB) staining was performed as described earlier32. To visualize fungal structures, C. higginsianum-infected Arabidopsis leaf tissues were stained with trypan blue17,32.

Fluorescence signals was detected using the following excitation/emission wavelength settings: CFP and aniline blue (405/420-480 nm), GFP and WGA (488/505-530 nm), CM-H2DCFDA (488/517-527 nm), mVENUS/mCITRINE (514/530-560 nm), mORANGE (543/550-570), mRFP and mSCARLET (543/581-635 nm), PI (488/610-630), and chlorophyll A (594/>650 nm). All images were processed and analyzed using LSM Image Browser (Zeiss) and Fiji/ImageJ (2.14.0) software.

Measurement of nuclear and cytoplasmic signal intensity

Nuclear and cytoplasmic signal intensity measurements were conducted using ImageJ (version: 2.14.0). Confocal images were first converted to 8-bit greyscale, and regions of interest (ROIs) corresponding to individual nuclei or cell cytoplasm were outlined based on fluorescent reporter signals. For each ROI, the mean grey value was measured to quantify fluorescence intensity. Background fluorescence was determined from an adjacent non-fluorescent area and subtracted from each measurement to obtain the corrected nuclear or cytoplasmic signal intensity. All measurements were performed using identical acquisition settings to ensure comparability across samples. The number of biological replicates analyzed in each experiment is indicated in the corresponding figure legends.

Single-cell RNA-seq data analysis

The preprocessed single-cell RNA-seq datasets were obtained from the INRAE dataverse repository and analyzed following the published pipeline34. Briefly, data processing and clustering were performed using the Seurat R package83. Genes expressed in three cells or fewer cells and cells expressing fewer than 200 genes were excluded from the analysis. Only cells containing ≤5% mitochondrial and chloroplastic reads were retained for downstream analyses. Data normalization was conducted using the SCTransform function and principal component analysis (PCA) was performed on the 2,000 most variable genes identified with the FindVariableFeatures function, retaining 30 principal components. Cell clustering was carried out using the FindClusters function with a resolution parameter of r = 1. Cell-type identities were assigned based on known marker genes34. Transcriptional dynamics were analyzed using the Monocle3 package84, and individual gene expression patterns across cell clusters were visualized with the plot_cells function in Monocle3.

GO analysis

GO analysis for enrich biological processes were conducted with online tool (http://geneontology.org/). GO terms were identified with indication of “overrepresentation” by fold enrichment and p-values.

Statistical analysis

All statistical analyses were performed using R software (v.4.1.2) (http://r-project.org). Comparisons between two groups were conducted using a two-tailed Student’s t test, whereas comparisons among more than two groups were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. Boxplots were generated in RStudio using appropriate data visualization packages.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Song and co-workers reports cell-type-resolved analyses of powdery mildew attack in Arabidopsis\nthaliana. The authors used mainly transgenic reporters, but also histochemical staining, to illuminate the type of defence\nresponses in epidermal pavement cells as opposed to epidermal guard cells. They employed both adapted and non-\nadapted powdery mildew species in this context. They found that stomatal guard cells do not trigger a salicylic acid-\ndependent immune response to powdery mildew fungi, suggesting cell-type-specific differences in the activation of\nimmunity. Guard cells were further found to be resistant to adapted powdery mildew fungi, undergoing early hypersensitive\ncell death upon host cell invasion.\nI consider the topic of cell-type-specific immune responses highly interesting. As such, the theme of the present study is of\nhigh biological significance. The technical quality of the work performed is high (though I would have welcomed an even\nmore quantitative assessment of the reported cellular features and infection phenotypes), and the manuscript is well written.\nSome of the micrographs are of spectacular quality. I also appreciate the technical challenges likely associated with the\nwork performed. However, unfortunately, the study remains largely descriptive, reporting distinctive cellular activities in\npavement and guard cells in response to adapted or non-adapted powdery mildew pathogens. It is, of course, necessary to\nperform such careful observational work as a first step for further analysis. For a journal such as Nature Communications, I\nwould have expected, however, that the authors go one step further to unravel the mechanistic basis for the reported\ndifferences. One possibility would be to perform single-cell RNA sequencing, as recently published in the context of a\ndifferent fungal pathogen (https://www.cell.com/cell-host-microbe/pdf/S1931-3128(23)00344-X.pdf). Alternatively or in\naddition, cell type-specific manipulation of plant immunity (see https://academic.oup.com/plcell/article/31/12/2868/5985837)\ncould have provided additional insights.\nSpecific comments\nLine 1: Title: The title is too general (“pathogenic fungi”) as only interactions with powdery mildew fungi were looked at.\nLine 96: The taxonomy of the barley powdery mildew pathogen has been recently revised, and the fungus renamed\n“Blumeria hordei” (https://pmc.ncbi.nlm.nih.gov/articles/PMC9157761/).\nLines 130-133: I do not think the conclusion here is complete/correct. An alternative interpretation is that the adapted\npowdery mildew pathogen is capable of suppressing ICS1 induction/cell death in neighboring cells.\nLines 153-155: I do not think the conclusion here is complete./correct. There is no direct evidence that provided that cell\ndeath amplifies SA biosynthesis.\nLines 198-202: I am surprised that the authors employed the NahG transgene for these experiments. Why didn´t the authors\nmake use of the sid2 mutant? Was the expression of the NahG gene checked in the progeny of the crosses? NahG gene\nexpression comes with the undesired accumulation of catechol.\nLine 260: It is actually sirofluor, not aniline blue, which stains callose (https://pubmed.ncbi.nlm.nih.gov/38739552/).\nLine 293: Heading is too general – only powdery mildew fungi were tested.\nLine 294: “showed” should read “shown”\nLines 359-360: Isn´t it surprising that guard cells, despite the activation of fewer immune pathways (as far as one can tell\nfrom the present study), have a better immune status? The authors gloss over this conundrum, which should be\nmentioned/discussed.\nLine 366 ff: It would have been valuable if the authors, besides the broad-spectrum calcium channel blocker lanthanum\nchloride, also used more selective calcium channel blockers that inhibit members of individual calcium channel families.\nThe Discussion omits important points. How do the authors rationalize their findings in the context of a general SA-\nresponsiveness of guard cells (e.g., https://pmc.ncbi.nlm.nih.gov/articles/PMC6130018/,\nhttps://pmc.ncbi.nlm.nih.gov/articles/PMC6512940/, and many other publications)? Further, findings of guard cells in the\ncontext of immunity against filamentous pathogens are not properly integrated into the Discussion\n(e.g., https://www.pnas.org/doi/10.1073/pnas.1922319117 and https://link.springer.com/content/pdf/10.1186/s12870-024-05426-\n5.pdf). It is also known that subsidiary cells of the stomatal complex can exhibit aberrant immunity to powdery mildew fungi.\nIn barley mlo mutants, which are otherwise highly resistant against the barley powdery mildew pathogen, subsidiary cells\nremain susceptible (see\nhttps://www.apsnet.org/publications/phytopathology/backissues/Documents/1977Articles/Phyto67n05_678.PDF). The\ndiscrepancy between powdery mildew-resistant guard cells and powdery mildew-susceptible subsidiary cells would be\nworthwhile to discuss. A special role of subsidiary cells is also highlighted in the following work:\nhttps://www.biorxiv.org/content/10.1101/2022.07.15.499463v1.", "reviewer_2": "Title: Deciphering Guard Cell-Autonomous Immunity Against Pathogenic Fungi (NCOMMS-25-57522)\nIn this study, authors used transgenic immune reporter systems and live imaging to analyze cell-type specific immune\nresponses in Arabidopsis leaves infected with different non-adapted and adapted pathogenic fungi. Study showed that while\nneighboring epidermal cells activated salicylic acid signaling and callose deposition, guard cells showed distinct immunity\ncharacterized by strong calcium influx, reactive oxygen species bursts, and hypersensitive cell death. Although the findings\nof the study are indeed novel and will advance our understanding of the autonomous and unique defense mechanisms\noccurring in guard cells, the actual mechanism of GC-mediated defense response is not clear even though the authors\nshowed Ca signaling and ROS are involved. In addition, the authors need to address the following issues regarding some\ntechnical aspects and the claims made.\n• How many transgenic lines exhibited similar expression patterns for pICS1::ICS1-mSCARLET and pEDS5::NLS-\nmSCARLET? Since these lines have not been reported before, please present data from at least 2–3 independent lines\nshowing consistent expression patterns in response to fungal pathogens. These data can be included as supplementary\nmaterial.\n• This study used several mutants (e.g., eds5, sard1, cbp60g, camta123, etc.) related to different defense-related pathways,\nwhich were crossed with reporter lines. Although the primer sequences used for genotyping are provided in the\nsupplementary material, no data confirming the genotyping of these lines for homozygosity and knockout status of the target\ngenes are included. Please provide these data as supplementary material.\n• Although the study analyzed different fungi, terms such as 'pathogen attack,' 'pathogen-induced SA,' or 'pathogen-induced'\nsuch as in lines 53 and 57, are used throughout the manuscript when reporting observations in response to fungal infection.\nPlease be specific to fungal pathogens, as responses to other pathogens (bacterial or viral) were not tested and may differ.\n• How would you explain a faint cyan color in the neighboring cells for adapted fungus (supplemental Fig. 1A)?\n• In Fig. 2D, the pICS1-driven reporter construct shows low activity; therefore, the claim in lines 187–188 that NLS-\n3×mVENUS signals are completely absent in the cbp60g sard1 mutants does not appear to be accurate. Additionally, there\nis no evident saturation of pICS1::NLS-3×mVENUS signals in the camta1/2/3 mutant upon B.g. hordei infection. In fact, the\nsignal appears weaker in B.g. hordei-infected tissue (Fig. 2D) compared to the non-infected control (Fig. 2C). Why is signal\nquantification provided for the reporter in non-infected tissues in Supplemental Fig. 4B (based on Fig. 2C), but not for the\ninfected tissues shown in Supplemental Fig. 4 (based on Fig. 2D)?\n• Line 217-188, the authors mention examining the reporter gene expression in infected GCs. How often do they find this?\nSome numbers or frequency will help.\n• Lines 267–268: There appears to be callose deposition in one of the guard cells (GCs) of the kidney-shaped structure in\nFig. 4B and C. Although it seems slightly away from the penetration site, it still appears to be part of the GC. A similar pattern\nof callose deposition is observed in the GCs in Supplemental Fig. 7B for the adapted fungal pathogen. Therefore, it is\nunclear from these images whether the callose was deposited by the GCs or by neighboring pavement cells, as callose is\nalways deposited in the apoplast (outside the cell membrane).\n• The GC-mediated defense against adapted pathogen is intriguing but the mechanism is not shown. For non-adapted\npathogen, the authors show Ca signaling ROS play a role in cell defense. Is it the same for adapted pathogens? This can be\neasily tested. This observation also warrants more discussion. Why an adapted pathogen not able to suppress defense\nresponse in GCs? Does GCs lack components needed for fungal cell differentiation and development?\n• Discussion section is very poorly written. It is just a summary of results and repetitive rather than discussion with reference\nto previous work. There are numerous studies regarding the role of GCs in plant defense. Surprisingly, only three additional\nreferences are added in the discussion! In addition, the authors could hypothesize or come up with a model regarding how\nthe GC-mediated defense response is triggered? For example, is the defense response triggered directly by PAMPs or\ninternal signal from neighboring cells?\n•\nMinor Comments\n• Figure legend 4A. is it really a “cyan color or magenta” representing aniline blue staining?\n• Also include a paragraph on comparing cell-type specific defense responses (Ca2+ signaling, ROS accumulation, SA\nproduction) between fungal and bacterial pathogens in the discussion section.\n• Line 108: “emerge: instead of “merge”?\n• Line 213: replace GSs (GCs)\n• Line 282: Expand or explain H2DCF-DA. Readers may not be familiar with this.\n• Line 294: replace “non-adapted fugus pathogens” with “non-adapted fungal pathogens”.\n• Throughout the manuscript either use A. thaliana or Arabidopsis (common name with no italics).\n• The title of the manuscript can be a bit more descriptive and interesting to describe the actual findings of the manuscript.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to Reviewers’ Comments\nWe are grateful to the editor for providing early notice of the reviewers’ comments, which\nafforded us ample time to conduct additional genetic, molecular, cellular, and pathological\nassays. This additional work has allowed us to fully address the reviewers’ suggestions and\nfurther improve the manuscript. Detailed responses to each specific comment are provided\nbelow.\nReviewer’s Comments:\nReviewer #1 (Remarks to the Author)\nThe manuscript by Song and co-workers reports cell-type-resolved analyses of powdery mildew\nattack in Arabidopsis thaliana. The authors used mainly transgenic reporters, but also\nhistochemical staining, to illuminate the type of defence responses in epidermal pavement cells\nas opposed to epidermal guard cells. They employed both adapted and non-adapted powdery\nmildew species in this context. They found that stomatal guard cells do not trigger a salicylic\nacid-dependent immune response to powdery mildew fungi, suggesting cell-type-specific\ndifferences in the activation of immunity. Guard cells were further found to be resistant to\nadapted powdery mildew fungi, undergoing early hypersensitive cell death upon host cell\ninvasion.\n1. I consider the topic of cell-type-specific immune responses highly interesting. As such, the\ntheme of the present study is of high biological significance. The technical quality of the work\nperformed is high (though I would have welcomed an even more quantitative assessment of the\nreported cellular features and infection phenotypes), and the manuscript is well written. Some\nof the micrographs are of spectacular quality.\nResponse: We appreciate the reviewer’s thoughtful assessments of the significance and quality of\nour work. Regarding the quantitative assessment of our data, we have incorporated detailed\nmeasurements of fluorescence signal intensities in the revised manuscript. Specifically, we\nconducted a comprehensive analysis of nuclear and cytoplasmic NLS-3×mVENUS signal\nintensities to quantify the temporal induction of ICS1 transcription during B. hordei powdery\nmildew infection (see revised Fig. 1A-D). In the revised manuscript, we now state:\n“For quantitative analysis of signal induction, fluorescence intensities were measured in both the\nnuclei and the cytoplasm of epidermal cells either attempted or penetrated by B. hordei (designated\nas “Patient 0”, P), as well as in their successive neighboring epidermal cell layers. Cells in direct\ncontact with “Patient 0” were defined as the first layer (L1), while cells adjacent to L1, but not\ndirectly bordering “Patient 0”, constitute the second layer (L2), and so forth (Supplemental Fig.\n1A). At 18 hpi, no detectable induction of NLS-3×mVENUS fluorescence was observed in either\nthe nuclei or the cytoplasm of cells at the penetration site (Fig. 1A-B). By 21 hpi, however, visible\ninduction of nuclear-associated fluorescent signals became apparent in “Patient 0” and adjacent\nL1 cells (Fig. 1A-B)” (lines 108-117).\n1\n“At 24 hpi, a strong NLS-3×mVENUS fluorescence signal was observed in “Patient 0” cells\nexhibiting visible haustorial formation (Fig. 1C-D). Notably, fluorescence induction was also\ndetected in adjacent L1 and L2 cells, forming a prominent patchy induction pattern in which\nnuclear signal intensity peaked at the center and gradually declined toward the periphery (Fig.\n1C-D). In the central region of the patch, fluorescence was evident not only in the nuclei but also\nin the cytoplasm of L1 and L2 cells (Fig. 1C-D).” (lines 122-127).\nAdditionally, we carried out intensity analysis of nuclear and cytoplasmic NLS-3×mVENUS\nsignals in E. cichoracearum-infected cells (see revised Supplemental Fig. 1B-D). The revised\nmanuscript now provides the following description:\n“At 26 hpi, induction of both nuclear and cytoplasmic NLS-3×mVENUS signals was evident in E.\ncichoracearum-penetrated cells and their neighboring cells, resembling the ICS1 induction\npattern observed during B. hordei infection (Supplemental Fig. 1B-D). These findings indicate that\nICS1 expression is triggered in response to infection by both adapted and non-adapted powdery\nmildew fungi.” (lines 132-137).\n2.I also appreciate the technical challenges likely associated with the work performed. However,\nunfortunately, the study remains largely descriptive, reporting distinctive cellular activities in\npavement and guard cells in response to adapted or non-adapted powdery mildew pathogens. It\nis, of course, necessary to perform such careful observational work as a first step for further\nanalysis. For a journal such as Nature Communications, I would have expected, however, that\nthe authors go one step further to unravel the mechanistic basis for the reported differences.\nOne possibility would be to perform single-cell RNA sequencing, as recently published in the\ncontext of a different fungal pathogen (https://www.cell.com/cell-host-microbe/pdf/S1931-\n3128(23)00344-X.pdf). Alternatively or in addition, cell type-specific manipulation of plant\nimmunity (see https://academic.oup.com/plcell/article/31/12/2868/5985837) could have\nprovided additional insights.\nResponse: We appreciate the reviewer’s constructive comments and suggestions, which\nunderscored the importance of examining the mechanistic basis underlying the differences\nbetween pavement cells and guard cells, including the recommendation to perform single-cell\nsequencing analysis to address this question.\nTo address these points, we expanded our analyses in the following ways:\nFirst, we tested an additional fungal pathogen, Colletotrichum higginsianum (C. higginsianum),\nusing our reporter line pICS1::NLS-3×VENUS CENH3-mRFP (Fig. 4A). At 48 hours post-\ninoculation (hpi), strong NLS-3×VENUS fluorescence was detected in C. higginsianum-\npenetrated epidermal cells containing visible primary hyphae. Fluorescence was also observed in\nsurrounding cells, forming a distinct patchy induction pattern with the highest nuclear signal\nintensity at the center, gradually decreasing toward the periphery. Importantly, ICS1\ntranscriptional activation was not detected in guard cells, consistent with our observations in\npowdery mildew infection. To further investigate the transcriptional differences between cell types\n2\nduring fungal infection, we re-analyzed single-cell transcriptome data generated by Tang et al.\n(2023). Although the dataset covered multiple cell types, we specifically focused on guard cells\nand pavement cells at C. higginsianum infection sites. Our analysis revealed that pavement cells\nand guard cells exhibit distinct gene induction profiles (Fig. 4B). GO term analysis indicated that\nthese two cell types undergo different biological processes during pathogen attack (Fig. 4C).\nNotably, the chorismate biosynthesis pathway was specifically induced in infected pavement cells\nbut not in guard cells. In Arabidopsis, pathogen-induced salicylic acid (SA) synthesis depends on\nthe conversion of chorismate to isochorismate via ICS1. These findings suggest that distinct\ntranscriptional programs in pavement cells versus guard cells lead to divergent pathogen-response\npathways.\nTo extend our study beyond fungal pathogens, we also examined Arabidopsis plants expressing\npICS1::NLS-3×mVENUS and pICS1::ICS1-mSCARLET during infections with various bacterial\nPseudomonas syringae pv. tomato (Pst) DC3000 strains. Similar to our findings with fungal\npathogens, Pst infection triggered robust ICS1 transcription and translation in pavement cells but\nnot in guard cells (Fig. 4D-E, Supplemental Fig. 9). This indicates that the ICS1 induction pattern\nwe observed is conserved across both fungal and bacterial infections. Several independent groups\nhave recently reported cell-type-specific responses to Pst at single-nucleus resolution (Nobri et al.,\n2025; Delannoy et al., 2023; Zhu et al., 2023). Among these, Delannoy et al. provided a\ncomprehensive analysis of cell specialization and coordination during Pst challenge. We\nreanalyzed the datasets generated by Delannoy et al. and compared the induced GO terms between\npavement and guard cells (Fig. 4F). Consistent with our findings, SA signaling was activated only\nin pavement cells, not in guard cells. Moreover, pathogen-induced SA biosynthetic genes,\nincluding ICS1 and PBS3, were strongly upregulated in pavement cells but absent in guard cells\n(Fig. 4G).\nTogether, these new experimental results and our reanalyses of publicly available single-cell\ntranscriptomic datasets reveal a conserved and fundamental distinction between pavement cells\nand guard cells in their transcriptional responses to pathogen attack. These findings directly\naddress the key concerns raised by the Reviewers and further strengthen the conclusions of our\nmanuscript. The corresponding methodological and result descriptions have been incorporated into\nthe revised version as indicated.\nSpecific comments\n3. Line 1: Title: The title is too general (“pathogenic fungi”) as only interactions with powdery\nmildew fungi were looked at.\nResponse: In this revised manuscript, we have extended our analysis to additional pathogen types\nand have updated the title to “Cell-type-specific immune programs orchestrate spatial defense in\nthe Arabidopsis leaf epidermis”.\n3\n4. Line 96: The taxonomy of the barley powdery mildew pathogen has been recently revised, and\nthe fungus renamed “Blumeria hordei” (https://pmc.ncbi.nlm.nih.gov/articles/PMC9157761/).\nResponse: We thank the reviewer for this updated information. The pathogen name has been\nrevised to “Blumeria hordei (B. hordei)” throughout the manuscript.\n5. Lines 130-133: I do not think the conclusion here is complete/correct. An alternative\ninterpretation is that the adapted powdery mildew pathogen is capable of suppressing ICS1\ninduction/cell death in neighboring cells.\nResponse: We carefully re-examined the interactions between the adapted powdery\nmildew\npathogen E. cichoracearum and Arabidopsis. Following E. cichoracearum infection at 26 hpi, we\nobserved the patchy induction pattern of NLS-3×mVENUS in the ICS1 reporter line, similar to\nthat observed with B. hordei infection. The revised manuscript has been updated accordingly.\n“At 26 hpi, induction of both nuclear and cytoplasmic NLS-3×mVENUS signals was evident in E.\ncichoracearum-penetrated cells and their neighboring cells, resembling the ICS1 induction\npattern observed during B. hordei infection (Supplemental Fig. 1B-D). These findings indicate that\nICS1 expression is triggered in response to infection by both adapted and non-adapted powdery\nmildew fungi.” (lines 132-137).\n6. Lines 153-155: I do not think the conclusion here is complete./correct. There is no direct\nevidence that provided that cell death amplifies SA biosynthesis.\nResponse: We thank the reviewer for the insightful comments. The observation of a patchy NLS-\n3×mVENUS induction pattern in the ICS1 reporter line upon a compatible E. cichoracearum\ninfection suggests that cell death is not necessarily required to amplify SA biosynthesis. The\nrevised manuscript has been updated accordingly.\n7. Lines 198-202: I am surprised that the authors employed the NahG transgene for these\nexperiments. Why didn´t the authors make use of the sid2 mutant? Was the expression of the\nNahG gene checked in the progeny of the crosses? NahG gene expression comes with the\nundesired accumulation of catechol.\nResponse: Following the reviewer’s suggestions, we introduced the ICS1 reporter transgene,\npICS1::NLS-3×mVENUS, into the sid2-1 mutant background. The new data show that, following\nB. hordei infection at 24 hpi, ICS1 reporter expression was induced similarly to that observed in\nthe WT background (Supplemental Fig. 6A-B).\nIn Arabidopsis, the ICS pathway is responsible for approximately 90% of SA production under\nstress conditions (Garcion et al. 2008, https://doi.org/10.1104/pp.108.119420). Therefore, the\nabsence of ICS1 does not completely abolish SA production. By contrast, overexpression of NahG,\nwhich converts SA to catechol, prevents SA accumulation and the establishment of SAR in\nArabidopsis (Gaffney et al. 1993. DOI: 10.1126/science.261.5122.754). For this reason, NahG was\nincluded in our experiments. The presence of NahG in the experimental materials was confirmed\n4\nby PCR-based genotyping (Supplemental Fig. 7B). The revised manuscript has been updated with\nthe following text:\n“To further explore this regulatory network, we introduced the pICS1::NLS-3×mVENUS reporter\ninto the ics1 mutant (sid2-1) and generated dual pICS1::NLS-3×mVENUS pEDS5::NLS-\nmSCARLET reporters in the SA-deficient NahG background through genetic crossing. Notably,\nfollowing B. hordei infection, the reporters displayed comparable induction patterns in WT, sid2-\n1, and NahG backgrounds (Supplementary Fig. 6-7). These results suggest that B. hordei-induced\nactivation of ICS1 and EDS5 occurs independently of SA or its precursor chorismite accumulation.”\n(lines 208-214)\n8. Line 260: It is actually sirofluor, not aniline blue, which stains callose\n(https://pubmed.ncbi.nlm.nih.gov/38739552/).\nResponse: We thank the reviewer for the clarification and have updated the information\naccordingly in the revised manuscript (line 324).\n9. Line 293: Heading is too general – only powdery mildew fungi were tested.\nResponse: For further clarification, in addition to two adapted powdery mildew species (E.\ncichoracearum and E. cruciferum), we also examined the distinct hemibiotrophic fungal pathogen\nC. higginsianum in our pathogenicity assays conducted on pavement and guard cells. In the\nmanuscript, we state:\n“We further tested GC susceptibility to C. higginsianum. In pavement cells, infection proceeded\nthrough appressoria, forming primary hyphae by 3 dpi (Fig. 6D, Supplemental Video 7), which\nsubsequently developed into thin secondary hyphae by 4 dpi (Supplementary Fig. 13). In contrast,\nC. higginsianum infection in GCs was limited to a short initial hypha, which failed to progress\n(Fig. 6D, Supplemental Video 7).” (lines 404-409)\n10. Line 294: “showed” should read “shown”.\nResponse: It is now corrected in the revised manuscript.\n11. Lines 359-360: Isn´t it surprising that guard cells, despite the activation of fewer immune\npathways (as far as one can tell from the present study), have a better immune status? The\nauthors gloss over this conundrum, which should be mentioned/discussed.\nResponse: We appreciate the reviewer’s insightful comments. In addition to expanding the\nDiscussion on the immune status of guard cells, we conducted several experiments to further\nelucidate the molecular mechanisms underlying their enhanced immune status compared with\npavement cells. Our results show that, upon both adapted and non-adapted powdery mildew\ninfection, guard cells activate a rapid and amplified immune response characterized by a\npronounced Ca2+ influx and ROS burst, which is distinct from the response observed in pavement\ncells (Supplementary Fig. 11). We also found that powdery mildew-induced cell death occurs\n5\nindependently of SA accumulation and signaling (Supplementary Fig. 12). In the revised\nmanuscript, we state:\n“Plant defenses against fungal and bacterial pathogens share core signaling molecules, yet their\nspatiotemporal dynamics vary with the host, pathogen lifestyle, and specific molecular\ninteractions. Upon perception of either bacterial or fungal challenges, rapid Ca²⁺ influxes and an\naccompanying ROS burst arise in GCs and pavement cells, serving as early localized signals that\ninitiate downstream immune responses” (lines 495-499)\n“Guard cell death has been observed in several non-adapted rust fungus-plant interactions and\nconsequently stops the fungal infection through the stomata. Similar observations have been\nreported in interactions between wheat leaf rust fungus and non-host Arabidopsis, where fungal\npenetration through stomata triggers transient stomatal closure followed by GC-specific\nhypersensitive cell death that prevents colonization. It is known that that GCs sense fungal chitin\noligosaccharide (GlcNAc)8 (CTOS) through its receptor CERK1 to induce calcium influx and ROS\naccumulation in GCs, which leads to activate Ca²⁺- and anion channel SLAC1-dependent stomatal\nclosure, whereas fungal conversion of CTOS to chitosan oligosaccharides (CSOS) suppresses this\nresponse and instead induces Ca²⁺-dependent, CERK1-independent guard cell death, thereby\nrendering GCs functionally incompatible with host-adapted pathogens. These studies are\nconsistent with our study revealing that GCs engage a rapid and amplified immune signaling mode\nthat is distinct from that of pavement cells. (lines 505-516)\nIn this study, we demonstrate that GC death can be triggered not only by direct infection with non-\nadapted powdery mildew but also by adapted powdery mildews. Key signaling molecules, calcium,\nROS, and SA, coordinate the onset and progression of plant cell death in response to pathogen\ninfection. Using transgenic line expressing pAtML1::NahG-mSCARLET to deplete SA\naccumulation specifically in epidermal cells, we show that B. hordei-induced cell death occurs\nindependently of SA accumulation (Supplemental Fig. 12C–E). We propose that GCs exhibit a\nmore robust calcium influx (Fig. 5D–E, Supplemental Fig. 11C-E) and pronounced ROS bursts\n(Fig. 5F–G, Supplemental Fig. 11A, F-H) compared with pavement cells, enabling them to execute\nhypersensitive cell death in response to pathogen infection without requiring SA signaling.” (lines\n517-525).\n12. Line 366 ff: It would have been valuable if the authors, besides the broad-spectrum calcium\nchannel blocker lanthanum chloride, also used more selective calcium channel blockers that\ninhibit members of individual calcium channel families.\nResponse: We thank the reviewer for highlighting the importance of examining more selective\ncalcium channel blockers. In the revised manuscript, we tested a panel of Ca2+ signaling\nantagonists commonly used in plant research (reviewed by De Vriese et al. 2018.\ndoi:10.3390/ijms19051506). Interestingly, our results show that the inhibitors of P-type Ca2+-\nATPase specifically blocked the patchy induction pattern of pICS1::NLS-3×mVENUS following\nB. hordei infection. These findings are highly informative, not only confirming the crucial role of\nCa2+ signalling in the induction of ICS1 expression, but also providing novel insights into how\n6\ncoordinated intracellular and apoplastic Ca2+ homeostasis may contribute to immune-related Ca2+\nsignaling in plants. In the revised manuscript, we state:\n“We next examined the effects of additional Ca2+ signaling antagonists commonly used in plants.\nInterestingly, treatments with erythrosin B or eosin Y, antagonists of P-type Ca2+-ATPase that\nmediate Ca2+ efflux, similarly suppressed the patchy induction pattern of pICS1::NLS-3×mVENUS\nat 24 hpi following B. hordei infection (Supplemental Fig. 4). These results indicate that\nperturbation of Ca2+ fluxes disrupt pathogen-triggered ICS1 expression, underscoring the\nessential role of Ca2+ homeostasis and signaling in this process.” (lines 168-174).\nIn the Discussion section, we state:\n“Intriguingly, among the various Ca²⁺ signaling antagonists commonly used in both animal and\nplant systems, only eosin Y and erythrosin B, two potent inhibitors of ACA-P type Ca²⁺-ATPases,\n2B\nspecifically suppress the infection-induced patchy expression patterns of ICS1 and EDS5\n(Supplemental Fig. 4). Members of the autoinhibited Ca²⁺-ATPase (ACA) family belong to the P-\ntype Ca²⁺-ATPases, a class of active transporters that utilize ATP to pump Ca²⁺ out of the cytosol\ninto intracellular compartments or the apoplast, thereby restoring basal cytosolic Ca²⁺ levels after\ntransient Ca²⁺ elevations. This resetting activity is essential for maintaining Ca²⁺ homeostasis and\nshaping the amplitude, duration, and spatial distribution of Ca²⁺ oscillations. In Arabidopsis, ACA\nfamily members mediate active Ca²⁺ efflux into the apoplast, ER, and vacuole, thereby sustaining\ncalcium gradients across subcellular compartments. Several recent studies have shown that\ngenetic disruption of these pathways leads to altered immune and stress responses. Thus, ACA-\ntype Ca²⁺-ATPases act as key “reset” components of the calcium signaling network, maintaining\nhomeostasis while enabling previse control over the signaling dynamics after exposure to specific\nstimuli. Our findings further suggest that ACA activity may contribute to infection-triggered SA\nbiosynthesis and signaling through intercellular or cell to cell communication, although this\nmechanism warrants further genetic and molecular investigation.” (lines 440-456)\n13. The Discussion omits important points. How do the authors rationalize their findings in the\ncontext of a general SA-responsiveness of guard cells\n(e.g., https://pmc.ncbi.nlm.nih.gov/articles/PMC6130018/, https://pmc.ncbi.nlm.nih.gov/article\ns/PMC6512940/, and many other publications)? Further, findings of guard cells in the context of\nimmunity against filamentous pathogens are not properly integrated into the Discussion\n(e.g., https://www.pnas.org/doi/10.1073/pnas.1922319117 and https://link.springer.com/cont\nent/pdf/10.1186/s12870-024-05426-5.pdf). It is also known that subsidiary cells of the stomatal\ncomplex can exhibit aberrant immunity to powdery mildew fungi. In barley mlo mutants, which\nare otherwise highly resistant against the barley powdery mildew pathogen, subsidiary cells\nremain susceptible\n(see https://www.apsnet.org/publications/phytopathology/backissues/Documents/1977Articles\n/Phyto67n05_678.PDF). The discrepancy between powdery mildew-resistant guard cells and\npowdery mildew-susceptible subsidiary cells would be worthwhile to discuss. A special role of\nsubsidiary cells is also highlighted in the following\nwork: https://www.biorxiv.org/content/10.1101/2022.07.15.499463v1.\nbased immunity. We have carefully reviewed all of the literature mentioned by the reviewer and\nincorporated these studies into a more comprehensive discussion in the Discussion section. In the\nrevised manuscript, we also present our rationales within the context of the general SA-\nresponsiveness of guard cells.\n“Numerous studies investigating ‘stomatal‐based immunity’ across diverse plant species have\nhighlighted stomatal closure as the first line of defense against bacterial and filamentous\npathogens. Key signaling molecules, including abscisic acid (ABA), ROS, Ca2+, salicylic acid (SA)\nand jasmonic acid (JA), play crucial role in regulating the stomatal aperture in response to\npathogen challenge or abiotic stress. With respect to SA involvement in regulating stomatal\nimmunity, the existing literature primarily examines SA function acting at the tissue or whole-\norgan level in modulating GC behavior, rather than exploring the intrinsic capacity of GS\nthemselves to synthesize or respond to SA”. (lines 470-477)\n“Together, these findings reveal for the first time that SA biosynthesis and signaling are not\ncomponents of GC-autonomous immunity, immune responses intrinsic to individual GCs and\ndistinct from their role in regulating stomatal movement. This cell-type-specific immune program\nin GCs versus pavement cells represents a conserved pattern across infections by fungal (C.\nhigginsianum) and bacterial (P. syringae) pathogens (Fig. 4, Supplemental Fig. 9). Single-cell\nRNA-seq data further corroborate this distinction, demonstrating that GCs and pavement cells\npossess unique transcriptional signature under both fungal and bacterial pathogen challenge (Fig.\n4, Supplemental Fig. 10). Collectively, these observations suggest that although GCs can perceive\npathogen attack, their intrinsic and cell-autonomous immune responses are selective and\nmechanistically distinct from those of pavement cells.” (lines 484-494)\nAdditionally, we have also discussed the discrepancy between powdery mildew-resistant guard\ncells and powdery mildew-susceptible subsidiary cells in the revised discussion section.\n“In monocots, each stoma comprises GCs accompanied by specialized subsidiary cells that\nsupport GC function. In barley mlo mutants, epidermal pavement cells exhibit strong resistance to\nbarley powdery mildew, yet the subsidiary cells remain comparatively susceptible. Moreover,\nepidermal cells adjacent to invaded subsidiary cells also exhibit elevated susceptibility. These\nobservations indicate that distinct epidermal cell types in barley differ in their vulnerability to\npowdery mildew and that successfully colonized subsidiary cells can influence neighboring cells,\nrendering them more susceptible to pathogen invasion. Unlike the powdery mildew fungi,\nZymoseptoria tritici, the causal agent of Septoria tritici blotch in wheat, cannot directly penetrate\nthe cuticle and instead enters the plant through stomata. Wheat cultivars with different types of\nresistance have been shown to block invasion by avirulent Z. tritici isolates during stomatal\npenetration. Using both wheat-Z. tritici and wheat-barley powdery mildew (B. hordei)\npathosystems, Valente et al. (2022) demonstrated that subsidiary cells in the wheat epidermis were\nthe first to respond to Z. tritici infection by forming cell wall papillae enriched with callose at\nfungus contact sites. Similarly, B. hordei infection triggers papilla formation and callose\ndeposition in subsidiary cells, although the callose deposition patterns differ between two different\nfungal pathogens. Notably, no significant callose deposition is detectable in GCs. Furthermore,\nnone of these studies assessed the immune response or immune status of the GCs themselves. Using\nrevealing a marked deficiency in callose deposition in GCs upon powdery mildew infection (Fig.\n5A-B, Fig. 6A-C, Supplemental Fig. 13). Our systemic and comparative analyses of immune\nresponses in GCs versus pavement cells will therefore be instrumental for advancing our\nunderstanding of epidermal cell-type-specific immunity. Nonetheless, stomatal architecture and\nmechanics vary substantially among plant species, a potential divergence in cell-type-specific\nimmune mechanisms warrants further investigation. ” (lines 526-549)\nReviewer #2 (Remarks to the Author)\nTitle: Deciphering Guard Cell-Autonomous Immunity Against Pathogenic Fungi (NCOMMS-25-\n57522)\n1. In this study, authors used transgenic immune reporter systems and live imaging to analyze\ncell-type specific immune responses in Arabidopsis leaves infected with different non-adapted\nand adapted pathogenic fungi. Study showed that while neighboring epidermal cells activated\nsalicylic acid signaling and callose deposition, guard cells showed distinct immunity\ncharacterized by strong calcium influx, reactive oxygen species bursts, and hypersensitive cell\ndeath. Although the findings of the study are indeed novel and will advance our understanding\nof the autonomous and unique defense mechanisms occurring in guard cells, the actual\nmechanism of GC-mediated defense response is not clear even though the authors showed Ca\nsignaling and ROS are involved. In addition, the authors need to address the following issues\nregarding some technical aspects and the claims made.\nResponse: We thank the reviewer for their positive assessment of the novelty of our work and for\ntheir constructive comments. We agree that our study reveals previously unrecognized intrinsic,\nguard cell-autonomous immune features, and we appreciate the reviewer’s request for additional\nclarification regarding the mechanisms underlying guard cell-mediated defense. Our primary\nobjective in this study was to map cell-type-specific immune outputs at high spatial and temporal\nresolution, rather than to fully delineate the signaling cascade involving Ca2+, ROS, and\ndownstream components in guard cells. We concur that the mechanistic link between these\nsignaling events and the observed hypersensitive responses warrants further exploration. To\naddress this concern, we have performed several key experiments and analyses to elucidate\npotential mechanisms underlying guard cell-mediated defense. We briefly highlight these findings\nas follows:\nFirst, our new imaging data demonstrate that guard cells do not activate SA biosynthesis or\ncanonical SA signaling, even when they are directly infected. Guard cells adjacent to penetration\nsites show no induction of pICS1::NLS-3×mVENUS, pEDS5::NLS-mSCARLET, or SA-\nresponsive PR-genes, whereas neighboring pavement cells strongly activate these pathways (Fig.\n3A-D; Supplementary Fig. 8). This establishes that SA is not an intrinsic component of guard cell-\nautonomous immunity.\nboth reporter assays and for single-cell RNA-seq. These data reveal that guard cells and pavement\ncells exhibit distinct pathogen-induced transcriptional signatures (Fig. 4; Supplementary Fig. 9-\n10). The transcriptomic profiles further support a mechanistic divergence in immune programming\nbetween the two epidermal cell types: pavement cells show strong engagement, and enrichment\nfor SA biosynthesis and SA-responsive pathways, whereas guard cells are largely impaired in\nactivating these responses.\nThird, additional pharmacological perturbations using a panel of selective Ca²⁺ channel inhibitors\nfurther strengthen the causal link between Ca²⁺ signaling and infection-triggered SA biosynthesis.\nOur results show that dynamic Ca²⁺ influx, rather than elevated intracellular Ca²⁺ concentration\nper se, is required for pathogen-induced SA biosynthesis (Supplementary Fig. 4).\nFourth, our new data show that guard cells mount a faster and more amplified Ca²⁺ influx and ROS\nburst upon pathogen perception than pavement cells, and that these early signaling events precede\nSA biosynthesis in pavement cells (Fig. 5D-G; Supplementary Fig. 11). Notably, similarly robust\nROS bursts occur during infection by the adapted powdery mildew E. cichoracearum, indicating\nthat guard cells elicit stronger and more rapid immune responses than pavement cells upon\npathogen detection.\nFifth, we provide new genetic evidence that hypersensitive cell death in guard cells occurs\nindependently of SA accumulation. Using an epidermis-specific SA-depleted line\n(pAtML1::NahG-mSCARLET), we show that B. hordei-induced guard cell death is unaffected by\nthe loss of SA (Supplementary Fig. 12C-E). These findings suggest that infection-triggered guard\ncell death is likely driven by Ca²⁺-dependent ROS signaling rather than activation of the SA\npathway.\nFinally, we substantially revised the Discussion to clarify how guard cells deploy a distinct,\nintrinsic, cell-autonomous immune program that is mechanistically separate from both pavement-\ncell immunity and the classical stomatal aperture-based immune responses.\nTogether, these new experiments clearly establish a clear mechanistic framework for guard cell-\nautonomous immunity, centered on strong Ca²⁺ influx, amplified ROS production, and SA-\nindependent cell death, thereby fully addressing the reviewer’s concern.\n2• How many transgenic lines exhibited similar expression patterns for pICS1::ICS1-mSCARLET\nand pEDS5::NLS-mSCARLET? Since these lines have not been reported before, please present\ndata from at least 2–3 independent lines showing consistent expression patterns in response to\nfungal pathogens. These data can be included as supplementary material.\nResponse: We generated at least 5 independent transgenic lines for both pICS1::ICS1-mSCARLET\nand pEDS5::NLS-mSCARLET constructs, all of which exhibited similar expression patterns. In the\nrevised manuscript, we have included an additional independent line for each reporter (See\nSupplemental Fig. 2B, Supplemental Fig. 3A).\n3• This study used several mutants (e.g., eds5, sard1, cbp60g, camta123, etc.) related to\ndifferent defense-related pathways, which were crossed with reporter lines. Although the primer\n10\nsequences used for genotyping are provided in the supplementary material, no data confirming\nthe genotyping of these lines for homozygosity and knockout status of the target genes are\nincluded. Please provide these data as supplementary material.\nResponse: As suggested by the reviewer, we have included the genotyping results confirming the\nhomozygosity and knockout status of the target genes for these lines in the revised manuscript\n(please see Supplemental Fig. 5A-B, Supplemental Fig. 6B, Supplemental Fig. 7B).\n4• Although the study analyzed different fungi, terms such as 'pathogen attack,' 'pathogen-\ninduced SA,' or 'pathogen-induced' such as in lines 53 and 57, are used throughout the\nmanuscript when reporting observations in response to fungal infection. Please be specific to\nfungal pathogens, as responses to other pathogens (bacterial or viral) were not tested and may\ndiffer.\nResponse: In this revised manuscript, we have extended our study to include additional fungal\n(Colletotrichum higginsianum) and bacterial (Pseudomonas syringae) pathogens. In addition, we\nhave revised the terminology throughout the text to specify the type of pathogen accordingly.\n5• How would you explain a faint cyan color in the neighboring cells for adapted fungus\n(supplemental Fig. 1A)?\nResponse: The ICS1 reporter transgene pICS1::NLS-3×mVENUS exhibits basal expression under\nnormal conditions, as shown in Fig. 1A-B. In the revised manuscript, we carefully examined the\ninteraction between the adapted powdery mildew pathogen E. cichoracearum and Arabidopsis.\nFollowing E. cichoracearum infection at 26 hpi, we observed a patchy induction pattern of NLS-\n3×mVENUS in the ICS1 reporter line, resembling the pattern observed during B. hordei infection.\nThe revised manuscript has been updated to reflect these findings.\n6• In Fig. 2D, the pICS1-driven reporter construct shows low activity; therefore, the claim in lines\n187–188 that NLS-3×mVENUS signals are completely absent in the cbp60g sard1 mutants does\nnot appear to be accurate. Additionally, there is no evident saturation of pICS1::NLS-3×mVENUS\nsignals in the camta1/2/3 mutant upon B.g. hordei infection. In fact, the signal appears weaker\nin B.g. hordei-infected tissue (Fig. 2D) compared to the non-infected control (Fig. 2C). Why is\nsignal quantification provided for the reporter in non-infected tissues in Supplemental Fig. 4B\n(based on Fig. 2C), but not for the infected tissues shown in Supplemental Fig. 4 (based on Fig.\n2D)?\nResponse: We meant the patchy induction pattern of NLS-3×mVENUS signals was absent in the\ncbp60g sard1-2 mutant background. To avoid any confusion, we have revised our statement.\n“Upon B. hordei infection at 24 hpi, NLS-3×mVENUS signals were strongly induced at the\ninfection sites in WT plants but remained at basal levels in the cbp60g sard1-2 mutants.” (lines\n196-198)\nmeasurements for the infected tissues (see Supplemental Fig. 5D).\n7• Line 217-188, the authors mention examining the reporter gene expression in infected GCs.\nHow often do they find this? Some numbers or frequency will help.\nResponse: We observed at least 10 independent sites where guard cells were directly infected by\nB. hordei. None of these infection events triggered ICS1 expression. The corresponding data have\nbeen included in the revised figure legend of Fig. 3D.\n8• Lines 267–268: There appears to be callose deposition in one of the guard cells (GCs) of the\nkidney-shaped structure in Fig. 4B and C. Although it seems slightly away from the penetration\nsite, it still appears to be part of the GC. A similar pattern of callose deposition is observed in the\nGCs in Supplemental Fig. 7B for the adapted fungal pathogen. Therefore, it is unclear from these\nimages whether the callose was deposited by the GCs or by neighboring pavement cells, as\ncallose is always deposited in the apoplast (outside the cell membrane).\nResponse: We appreciate the reviewer’s careful attention to the callose deposition patterns. In our\nstudy, callose was visualized using aniline blue staining in a basic solution (pH 9.5). Because\naniline-blue-stained callose produces strong autofluorescence, the resulting fluorescence signal\ncan extend into neighboring cells, potentially giving the impression of signal within guard cells.\nThe apparent signal inside portions of the guard cell is therefore most likely due to the fluorescence\nsaturation caused by the intense aniline blue staining.\nIn Fig. 4B&C (now revised as Fig. 5B&C) and Supplemental Fig. 7B (revised Supplemental Fig.\n13B), the callose signals originate from the surrounding pavement cells rather than from the guard\ncells. In both Fig. 5B&C and Supplemental Fig. 13B, the discontinuous pattern of callose\ndeposition, including the distinct gap corresponding to the intercellular space between two\npavement cells, clearly indicates that the deposition occurs in pavement cells adjacent to the\ninfected guard cell. Supplemental Videos 2 and 3 support this conclusion by providing three-\ndimensional visualization of the signal distribution.\nAdditionally, we note that callose deposition is absent at the penetration sites of guard cells\nthemselves, both for the non-adapted B. hordei (Fig. 5B & C) and the adapted E. cruciferarum\n(Supplemental Fig. 13B), further indicating that guard cells do not deposit callose in these\ninstances.\n9• The GC-mediated defense against adapted pathogen is intriguing but the mechanism is not\nshown. For non-adapted pathogen, the authors show Ca signaling ROS play a role in cell\ndefense. Is it the same for adapted pathogens? This can be easily tested. This observation also\nwarrants more discussion. Why an adapted pathogen not able to suppress defense response in\nGCs? Does GCs lack components needed for fungal cell differentiation and development?\nResponse: We thank the reviewer for their insightful comments. In response to the suggestion,\nwe\nconducted additional experiments to examine whether Ca2+ influx and ROS accumulation also\n12\noccur during infection by the host-adapted powdery mildew. These new data (Supplemental Fig.\n11C-H) now allow a direct comparison of guard cell responses to both non-adapted and adapted\npathogens.\nOur results show that infection by the adapted powdery mildew E. cichoracearum triggers\npronounced Ca2+ elevations and strong ROS accumulation in guard cells, surpassing the responses\nobserved in adjacent pavement cells (Supplemental Fig. 11C-F). As now described in the revised\ntext:\n“Notably, infection by compatible E. cichoracearum induces higher Ca²⁺ elevation and ROS\naccumulation in GCs adjacent to the infected pavement cell than in the surrounding pavement\ncells at 22-26 hpi (Supplemental Fig. 11C-F). These results suggest that GCs respond more\nvigorously to pathogen challenges than pavement cells, demonstrated by their more rapid and\npronounced Ca²⁺ elevation as well as heightened intracellular ROS accumulation.” (Lines 359-\n364).\nThus, guard cells mount enhanced early Ca²⁺- and ROS-associated responses to both adapted and\nnon-adapted pathogens, and their signaling amplitude remains high even during compatible\ninteractions.\nIn response to the reviewer’s second point regarding potential mechanisms underlying GC-\nmediated defense against adapted pathogens, we refer to our detailed reply to Comment 1.\nAlthough guard cells are impaired in SA biosynthesis and SA signaling, our new results show that\nthey exhibit a faster and more amplified Ca²⁺ influx and ROS burst than pavement cells upon\nperception of the non-adapted B. hordei infection (Supplemental Fig. 11C-E). Moreover, stronger\nROS accumulation in guard cells relative to pavement cells is also observed in regions adjacent to\ninfection sites of the adapted powdery mildew E. cichoracearum (Supplemental Fig. 11F-H). We\nfurther demonstrate that B. hordei-induced cell death accompanied by ROS accumulation occurs\ndespite the absence of SA accumulation (Supplemental Fig. 12A-E).\nTaken together, these findings support our proposal that early immune activation, characterized by\nCa²⁺ influx and ROS burst, is robustly executed in guard cells, potentially contributing to their\nincompatibility even during otherwise compatible interactions. Nonetheless, we cannot exclude\nthe possibility that guard cells may simply lack components required for fungal differentiation and\ndevelopment, as the reviewer suggested. We now discuss potential mechanisms underlying guard\ncell innate immunity more in great depth in the revised Discussion section.\n10• Discussion section is very poorly written. It is just a summary of results and repetitive rather\nthan discussion with reference to previous work. There are numerous studies regarding the role\nof GCs in plant defense. Surprisingly, only three additional references are added in the\ndiscussion! In addition, the authors could hypothesize or come up with a model regarding how\nthe GC-mediated defense response is triggered? For example, is the defense response triggered\ndirectly by PAMPs or internal signal from neighboring cells?\nResponse: We thank the reviewer for this important comment. In response, we have substantially\nrewritten, expanded, and reorganized the Discussion to provide a more integrative and mechanistic\n13\ninterpretation of our findings, firmly grounded in the existing literature. The revised Discussion\nnow includes the following improvements:\na) A deeper integration of our live-cell imaging data using immune reporters with prior\nstudies on spatial single-cell immune transcriptomics (lines 415-432; 487-494).\nb) A new conceptual framework explaining how dynamic Ca²⁺ homeostasis regulates SA\nbiosynthesis through intercellular communication (lines 433-456).\nc) An expanded comparison between classical stomatal-based immunity, where stomata\nserve as active immune gates restricting pathogen entry, and our findings on intrinsic, guard\ncell-autonomous immunity (lines 470-494).\nd) Additional discussion of potential mechanisms underlying guard cell immunity,\nincluding Ca²⁺/ROS signaling, CERK1-mediated chitin perception (lines 500-517), and\nCa²⁺/ROS-mediated cell death independent of SA signaling (lines 517-525).\ne) A proposed rationale for the incompatibility of guard cells with adapted pathogens (lines\n517-525).\nf) A comprehensive comparison of stomatal cell-type-specific responses across wheat-rust,\nwheat-Z. tritici, and barley-B. hordei pathosystems, drawing on the studies highlighted by\nReviewer 1 (lines 526-549).\nWe believe these major revisions substantially strengthen the Discussion and fully address the\nreviewer’s concerns.\nMinor Comments\n11• Figure legend 4A. is it really a “cyan color or magenta” representing aniline blue staining?\nResponse: We have doubled checked the figure legend and corrected the description in the\nrevised version of figures.\n12• Also include a paragraph on comparing cell-type specific defense responses (Ca2+ signaling,\nROS accumulation, SA production) between fungal and bacterial pathogens in the discussion\nsection.\nResponse: we have added a paragraph to discuss cell-type specific defense responses in the\nrevised manuscript. Please see lines 470-477 and 484-494.\n“Numerous studies investigating ‘stomatal‐based immunity’ across diverse plant species have\nhighlighted stomatal closure as the first line of defense against bacterial and filamentous\npathogens. Key signaling molecules, including abscisic acid (ABA), ROS, Ca2+, salicylic acid (SA)\nand jasmonic acid (JA), play crucial role in regulating the stomatal aperture in response to\npathogen challenge or abiotic stress. With respect to SA involvement in regulating stomatal\nimmunity, the existing literature primarily examines SA function acting at the tissue or whole-\norgan level in modulating GC behavior, rather than exploring the intrinsic capacity of GS\nthemselves to synthesize or respond to SA”\n14\n“Together, these findings reveal for the first time that SA biosynthesis and signaling are not\ncomponents of GC-autonomous immunity, immune responses intrinsic to individual GCs and\ndistinct from their role in regulating stomatal movement. This cell-type-specific immune program\nin GCs versus pavement cells represents a conserved pattern across infections by fungal (C.\nhigginsianum) and bacterial (P. syringae) pathogens (Fig. 4, Supplemental Fig. 9). Single-cell\nRNA-seq data further corroborate this distinction, demonstrating that GCs and pavement cells\npossess unique transcriptional signature under both fungal and bacterial pathogen challenge (Fig.\n4, Supplemental Fig. 10). Collectively, these observations suggest that although GCs can perceive\npathogen attack, their intrinsic and cell-autonomous immune responses are selective and\nmechanistically distinct from those of pavement cells.”\n13• Line 108: “emerge: instead of “merge”?\nResponse: We have revised the statement in the revised manuscript.\n“By 21 hpi, however, visible induction of nuclear-associated fluorescent signals became apparent\nin “Patient 0” and adjacent L1 cells (Fig. 1A-B).” (lines 115-117)\n14• Line 213: replace GSs (GCs)\nResponse: Corrected.\n15• Line 282: Expand or explain H2DCF-DA. Readers may not be familiar with this.\nResponse: We have provided additional explanation of H2DCF-DA, see the revised manuscript.\n“H2DCF-DA, a cell-permeable fluorescent probe that is deacetylated by intracellular esterases\nand oxidized by ROS, enabling visualization of ROS accumulation in live GCs.” (lines 350-351)\n16• Line 294: replace “non-adapted fugus pathogens” with “non-adapted fungal pathogens”.\nResponse: Corrected.\n17• Throughout the manuscript either use A. thaliana or Arabidopsis (common name with no\nitalics).\nResponse: We have revised it to “Arabidopsis” throughout the revised manuscript.\n18• The title of the manuscript can be a bit more descriptive and interesting to describe the\nactual findings of the manuscript.\nResponse: In this revised manuscript, we have updated the tittle to “Cell-type-specific immune\nprograms orchestrate spatial defense in the Arabidopsis leaf epidermis ”.\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We thank this reviewer for their time and effort to provide constructive comments.\nPlease see our point-by-point responses above.\nResponse to Reviewers’ Comments\nWe sincerely thank the reviewers for the time and effort they devoted to evaluating our\nmanuscript. We greatly appreciate their thoughtful and supportive comments.\nReviewers' comments:\nReviewer #1 (Remarks to the Author):\nThe resubmitted version of the manuscript by Song and co-workers shows impressive\nimprovements. The authors addressed all my concerns and now provide several new\nexperimental data and in silico analyses of single-cell RNA-Seq data. Major improvements cover\nthe following aspects:\n1.) Marker fluorescence intensities were quantified. This approach further strengthens the claims\nof the authors.\n2.) Pre-exisiting single cell RNA-Seq data from infection experiments (conducted by other\ngroups) with C. higginsianum and P. syringae were analyzed. Careful examination of these data\nsets supports the key conclusions of the present manuscript. Using pre-existing published single-\ncell transcriptomic data is a creative approach to address this issue. Supplementing this,\ninfection experiments with ICS1 reporter lines were performed to examine for a putative cell\ntype-specific activation of the SA signaling pathway in these interactions.\n3.) In addition to the NahG transgene, the sid2 mutant was used for crossings with the ICS1\nreporter line. The results of both approaches are consistent. I do appreciate the rationale of the\nauthors to keep the NahG transgene, as this shows a stronger effect.\n4.) A panel of more specific calcium inhibitors has been tested (at single concentrations only,\nthough), which revealed a key role for P-type calcium ATPases in the activation of pathogen-\ninduced ICS1 expression. Maybe a short note could be added to justify the concentrations used.\n5.) The Discussion was largely expanded and improved. It covers many more aspects, placing\nthe findings in a broader context. It now has sufficient breadth and depth.\nIn addition to the key issues, the authors also dealt with the minor points mentioned.\nI have the impression that also the concerns of reviewer #2 were addressed adequately to the\nmost part, as we shared some similar comments, e.g., regarding the lack of more mechanistic\ninsights.\nIn summary, I feel that the current version has improved substantially. The authors have made a\nmajor and serious effort to advance their study. In my opinion, the work has reached the level\nrequired for publication in a journal like Nature Communications. Unraveling cell type\nspecificity in immune responses is a topic of broad scientific relevance. I congratulate the\nauthors on an important advancement in our understanding of plant immunity.\nUnclassified / Non classifié\nReviewer #2 (Remarks to the Author):\nThe authors have substantially improved the manuscript by adding a new dataset that supports\ntheir findings on distinct defense responses in pavement and guard cells, namely salicylic acid\nsignalling and callose deposition in pavement cells and strong Ca²⁺ influx, amplified ROS\nproduction, and SA-independent cell death in guard cells. Furthermore, the conservation of\nthese cell-type–specific responses to both bacterial and fungal infections make the study\nparticularly interesting and novel. Additionally, the authors have successfully addressed the\nissues raised in the previous submission\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The resubmitted version of the manuscript by Song and co-workers shows impressive improvements. The authors addressed all my concerns and now provide several new experimental data and in silico analyses of single-cell RNA-Seq data. Major improvements cover the following aspects:\n1.) Marker fluorescence intensities were quantified. This approach further strengthens the claims of the authors.\n2.) Pre-exisiting single cell RNA-Seq data from infection experiments (conducted by other groups) with C. higginsianum and P. syringae were analyzed. Careful examination of these data sets supports the key conclusions of the present manuscript. Using pre-existing published single-cell transcriptomic data is a creative approach to address this issue. Supplementing this, infection experiments with ICS1 reporter lines were performed to examine for a putative cell type-specific activation of the SA signaling pathway in these interactions.\n3.) In addition to the NahG transgene, the sid2 mutant was used for crossings with the ICS1 reporter line. The results of both approaches are consistent. I do appreciate the rationale of the authors to keep the NahG transgene, as this shows a stronger effect.\n4.) A panel of more specific calcium inhibitors has been tested (at single concentrations only, though), which revealed a key role for P-type calcium ATPases in the activation of pathogen-induced ICS1 expression. Maybe a short note could be added to justify the concentrations used.\n5.) The Discussion was largely expanded and improved. It covers many more aspects, placing the findings in a broader context. It now has sufficient breadth and depth.\nIn addition to the key issues, the authors also dealt with the minor points mentioned.\nI have the impression that also the concerns of reviewer #2 were addressed adequately to the most part, as we shared some similar comments, e.g., regarding the lack of more mechanistic insights.\nIn summary, I feel that the current version has improved substantially. The authors have made a major and serious effort to advance their study. In my opinion, the work has reached the level required for publication in a journal like Nature Communications. Unraveling cell type specificity in immune responses is a topic of broad scientific relevance. I congratulate the authors on an important advancement in our understanding of plant immunity.", "reviewer_2": "The authors have substantially improved the manuscript by adding a new dataset that supports their findings on distinct defense responses in pavement and guard cells, namely salicylic acid signalling and callose deposition in pavement cells and strong Ca²⁺ influx, amplified ROS production, and SA-independent cell death in guard cells. Furthermore, the conservation of these cell-type–specific responses to both bacterial and fungal infections make the study particularly interesting and novel. Additionally, the authors have successfully addressed the issues raised in the previous submission", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter