Humidity-driven ABA depletion determines plant-pathogen competition for leaf water

Yasuda, Shigetaka; Shinozawa, Akihisa; Weng, Yuanjie; Rajendram, Arullthevan; Hirase, Taishi; Ishizaki, Haruka; Suzuki, Ryuji; Ueda, Shioriko; Sk, Rahul; Takebayashi, Yumiko; Yotsui, Izumi; Toyota, Masatsugu; Okamoto, Masanori; Saijo, Yusuke

doi:10.1038/s41467-025-67469-y

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Published: 19 December 2025

Humidity-driven ABA depletion determines plant-pathogen competition for leaf water

Nature Communications volume 17, Article number: 787 (2026) Cite this article

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Abstract

Bacterial phytopathogens, such as Pseudomonas syringae pv. tomato (Pst) DC3000, induce water-soaked lesions in the leaf apoplast under high humidity, facilitating infection. However, it remains largely unclear how plants regulate their resistance to restrict bacterial infection in response to humidity. Here, we demonstrate that abscisic acid (ABA)-catabolizing ABA 8’-hydroxylase, encoded by CYP707A3, plays a critical role in this resistance in Arabidopsis thaliana. Elevated humidity induces CYP707A3 expression, which is essential for reducing ABA levels and promoting stomatal opening, thereby limiting bacterial water-soaking and infection following leaf invasion. High humidity also increases cytosolic Ca²⁺ levels via the Ca²⁺ channels CNGC2 and CNGC4, with partial involvement from CNGC9, activating the calmodulin-binding transcription activator CAMTA3 to drive CYP707A3 induction. However, Pst DC3000 counteracts this defense response using type III secretion effectors, including AvrPtoB, facilitating water-soaking. These findings provide insights into the mechanisms underlying the competition between plants and bacteria for leaf water under elevated humidity.

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Introduction

Bacterial phytopathogens cause foliar diseases that threaten plant survival and crop yields by proliferating within the leaf apoplast¹. An important layer of defense against bacterial infection is provided by cell surface receptor-like kinases/proteins (RLKs/RLPs), collectively known as pattern recognition receptors (PRRs). PRRs detect microbe-associated molecular patterns (MAMPs) and endogenous damage-associated molecular patterns (DAMPs) to activate pattern-triggered immunity (PTI)^2,3,4. Following ligand perception, PRRs form active complexes with co-receptors to trigger intracellular signaling cascades. This involves rapid cytosolic Ca²⁺ influx, a burst of reactive oxygen species (ROS), activation of mitogen-activated protein kinase (MAPK) cascades, production of defense-related phytohormones such as salicylic acid (SA), stomatal closure, transcriptional reprogramming, and production of antibacterial compounds^2,3,4. To overcome PTI, bacterial pathogens deliver effector proteins into host tissues and cells via the type III secretion (T3S) system¹. The bacterial phytopathogen Pseudomonas syringae pv. tomato (Pst) DC3000 has 36 T3S effectors⁵. Recent studies have shown that Pst DC3000 utilizes specific T3S effectors to manipulate the leaf apoplast environment, establishing conditions favorable for infection⁶.

High humidity promotes foliar diseases caused by bacterial pathogens through multiple mechanisms. Initially, impaired stomatal closure facilitates bacterial invasion into leaves⁷. Once inside, bacterial pathogens induce apoplast hydration under high humidity, a critical process for disease development known as water-soaking⁸. To induce water-soaking, Pst DC3000 employs the highly conserved T3S effectors HopM1 and AvrE, which manipulate the host plant’s abscisic acid (ABA) pathways, a phytohormone critical for water retention^9,10. Plants typically increase ABA levels under drought conditions to promote stomatal closure and conserve water¹¹. However, under high humidity, when plants naturally reduce ABA levels, Pst DC3000 stimulates ABA biosynthesis and signaling in the host through HopM1 and AvrE, thereby enhancing water-soaking and promoting disease progression^9,10. Similarly, Xanthomonas bacteria exploit host ABA pathways through their T3S effectors to facilitate leaf infection in rice, wheat, and Arabidopsis thaliana (hereafter Arabidopsis)^12,13,14. Despite recent advances in the identification and characterization of bacterial effectors involved in water-soaking, our understanding of the plant resistance mechanisms that counteract bacterial water acquisition remains limited^15,16.

In nature, high humidity often accompanies prolonged rainfall, inducing a wide range of physiological effects in plants, such as defects in cuticle formation, stomatal opening, changes in water uptake and transpiration, and leaf petiole movement (hyponasty)^7,17,18,19. In Arabidopsis, high humidity rapidly induces transcriptional and post-transcriptional changes in leaves, modulating phytohormone pathways^17,19. ABA levels are tightly regulated through the coordination of its biosynthesis and catabolism²⁰. Under high humidity, ABA levels decrease via the action of CYP707A1 and CYP707A3, which encode ABA 8’-hydroxylases, thereby facilitating stomatal opening¹⁷. Concurrently, the production and signaling of defense-promoting salicylic acid (SA) are suppressed, partly due to impaired ubiquitination of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1, an SA receptor and key transcriptional co-activator of SA-responsive genes, reducing its binding to target gene promoters²¹. Additionally, high humidity stimulates the biosynthesis of the gaseous phytohormone ethylene, which promotes leaf hyponasty¹⁹. Despite these extensive changes in phytohormone production and signaling, the mechanisms by which the modulation of host phytohormone pathways under high humidity impacts plant–pathogen interactions remain poorly understood.

In this study, we demonstrate that the high humidity-induced CYP707A3 plays a pivotal role in restricting bacterial water-soaking by promoting stomatal opening in Arabidopsis leaves. While this work was in preparation, Hussain et al. reported that elevated humidity increases cytosolic Ca²⁺ concentrations in a CYCLIC NUCLEOTIDE-GATED CHANNEL 2 (CNGC2)- and CNGC4-dependent manner, thereby inducing CYP707A3 expression through CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3)²². In parallel, our investigation independently identified this signaling module as a key determinant of humidity-triggered CYP707A3 induction. Moreover, we demonstrate that CNGC9 also contributes to this response, and that CAMTA3 is required for resistance to bacterial water-soaking. However, during infection, Pst DC3000 counteracts this signaling pathway and suppresses high humidity-induced transcriptional changes through its T3S effectors, including AvrPtoB. These findings provide valuable insights into the mechanisms regulating plant–pathogen competition for leaf water under high humidity.

Results

CYP707A3 is required for resistance to bacterial water-soaking under high humidity

Upon exposure to high humidity, CYP707A1 and CYP707A3, but not CYP707A2 or CYP707A4, were transiently induced in wild-type (WT) leaves within 0.5 h (Fig. 1a). To investigate the role of CYP707A1 and CYP707A3 in bacterial resistance, we measured ABA levels and assessed bacterial water-soaking in the corresponding mutants (Supplementary Fig. 1a, b). In WT plants, high humidity triggered a marked reduction in ABA levels within 2 h (Fig. 1b). This ABA decline was completely abolished in the cyp707a1 cyp707a3 double mutant, which instead exhibited constitutively high ABA accumulation (Fig. 1b), indicating that these genes are required for humidity-induced ABA catabolism.

We next assessed water-soaking in cyp707a mutants (Supplementary Fig. 1a, b) using Pst ΔhrcC, a strain lacking a functional T3S system. Resistance to water-soaking was evaluated by monitoring leaf water retention following bacterial infiltration under high humidity. At 1 day post-infiltration (dpi), pronounced water-soaking was observed in cyp707a3 and cyp707a1 cyp707a3, but not in WT, cyp707a1, or cyp707a2 (Fig. 1c). The phenotype was more severe in cyp707a1 cyp707a3 than in cyp707a3, suggesting a predominant role for CYP707A3 with a partial contribution from CYP707A1 in water-soaking resistance. Accordingly, we used cyp707a1 cyp707a3 for most subsequent analyses. Under moderate humidity, however, even cyp707a1 cyp707a3 displayed no detectable water-soaking (Fig. 1d). Consistently, Pst ΔhrcC growth was significantly higher in cyp707a1 cyp707a3 than in WT under high humidity, but was indistinguishable under moderate humidity (Fig. 1e). These results demonstrate that CYP707A3 is crucial for restricting water-soaking during Pst DC3000 infection, thereby limiting bacterial proliferation specifically under high humidity.

CYP707A3-mediated water-soaking resistance depends on stomatal opening

Pst DC3000 induces stomatal closure following leaf invasion to facilitate water-soaking^9,10. Consistent with previous reports¹⁷, loss of CYP707A3 and CYP707A1 impaired high-humidity-induced stomatal opening (Fig. 2a, b). To test whether CYP707A3-mediated bacterial resistance is linked to stomatal regulation, we introduced the ost2-3D mutation, an active allele of the plasma membrane H⁺-ATPase AHA1 that drives constitutive stomatal opening²³, into the cyp707a1 cyp707a3 background (Supplementary Fig. 2a, b). As expected, the cyp707a1 cyp707a3 ost2-3D triple mutant exhibited enhanced stomatal opening compared with cyp707a1 cyp707a3 under both high and moderate humidity (Fig. 2a, b). Correspondingly, water-soaking was almost completely abolished in cyp707a1 cyp707a3 ost2-3D following Pst ΔhrcC infiltration, similar to ost2-3D (Fig. 2c). This suppression was accompanied by markedly reduced Pst ΔhrcC growth in both cyp707a1 cyp707a3 ost2-3D and ost2-3D (Fig. 2d). These findings indicate that CYP707A3 confers resistance to water-soaking, at least in part, by promoting stomatal opening under high humidity.

ABA depletion enhances bacterial resistance by restricting water-soaking independently of SA under high humidity

SA antagonizes many ABA-dependent processes and has been reported to suppress water-soaking under light conditions^16,24,25. However, both SA and ABA are required for stomatal closure during early defense responses to bacterial phytopathogens²⁶. To assess whether SA influences CYP707A3-mediated water-soaking resistance, we introduced the sa induction deficient 2-2 (sid2-2) mutation, which disrupts ISOCHORISMATE SYNTHASE 1 (ICS1)-dependent SA biosynthesis²⁷, into the cyp707a1 cyp707a3 background (Supplementary Fig. 3a, b). The cyp707a1 cyp707a3 sid2-2 triple mutant, but not sid2-2, exhibited extensive water-soaking following Pst ΔhrcC infiltration (Supplementary Fig. 3c), indicating that ICS1-dependent SA biosynthesis is dispensable for CYP707A3-mediated water-soaking resistance.

We next tested whether ABA depletion alone is sufficient to confer bacterial resistance under high humidity in an SA-independent manner. To this end, we examined water-soaking and bacterial growth following Pst DC3000 infiltration in the abscisic aldehyde oxidase 3 (aao3) mutant, which is deficient in ABA biosynthesis²⁸, with or without the sid2-2 mutation (Supplementary Fig. 3d, e). Consistent with previous studies on ABA dependence^9,10, Pst DC3000-induced water-soaking was markedly reduced in aao3, irrespective of sid2-2 mutation (Supplementary Fig. 3f). This reduction correlated with decreased Pst DC3000 growth at 3 dpi in both aao3 and aao3 sid2, although the effect was less pronounced in the latter (Supplementary Fig. 3g). Under moderate humidity, however, suppression of Pst DC3000 growth by the aao3 mutation was observed only in the presence of functional SID2 (Supplementary Fig. 3h), as reported previously²⁵. Collectively, these results demonstrate that ABA depletion confers resistance to Pst DC3000 under high humidity by restricting water-soaking development, independently of the SA-ABA antagonism observed under moderate humidity.

CYP707A3/A1-mediated ABA depletion and PTI contribute additively to bacterial resistance

High humidity differentially affects PTI responses triggered by flg22, a MAMP derived from bacterial flagellin²¹. To test whether high humidity-triggered ABA depletion via CYP707A3 and CYP707A1 contributes to this modulation, we compared PTI marker gene expression in WT and cyp707a1 cyp707a3 following Pst ΔhrcC infiltration under moderate and high humidity. After 24 h acclimation to the respective humidity conditions, we measured FLG22-INDUCED RECEPTOR-LIKE KINASE 1 (FRK1), NON RACE-SPECIFIC DISEASE RESISTANCE 1/HAIRPIN-INDUCED GENE 1-LIKE 10 (NHL10), and PHOSPHATE-INDUCED 1 (PHI-1) expression at 1 h post-infiltration (hpi). Under high humidity, FRK1 induction was enhanced, NHL10 induction was unchanged, and PHI-1 induction was abolished (Supplementary Fig. 4a). These patterns were similar in WT and cyp707a1 cyp707a3, suggesting that CYP707A3/A1-mediated ABA depletion has limited impact on the humidity-dependent PTI gene regulation.

We next examined the relationship between CYP707A3-mediated water-soaking resistance and PTI by crossing cyp707a1 cyp707a3 with the brassinosteroid insensitive 1-associated receptor kinase 1-5 (bak1-5) bak1-like 1 (bkk1) chitin elicitor receptor kinase 1 (cerk1) triple mutant (hereafter bbc), which disrupts PRR activation mediated by the co-receptors BAK1, BKK1, and CERK1²⁹ (Supplementary Fig. 4b, c). The cyp707a1 cyp707a3 bbc quintuple mutant exhibited pronounced water-soaking following Pst ΔhrcC infiltration, similar to cyp707a1 cyp707a3 (Supplementary Fig. 4d). By contrast, bbc showed WT-like or slightly enhanced resistance to water-soaking (Supplementary Fig. 4d), indicating that humidity-triggered ABA depletion restricts water-soaking independently of BAK1/BKK1/CERK1-dependent PTI branches. Notably, Pst ΔhrcC growth was significantly higher in cyp707a1 cyp707a3 bbc compared to either cyp707a1 cyp707a3 or bbc (Supplementary Fig. 4e), indicating additive effects. Together, these results indicate that CYP707A3/A1-mediated ABA depletion and BAK1/BKK1/CERK1-dependent PTI pathways contribute additively to Pst DC3000 resistance under high humidity.

Global transcriptome analysis reveals CAMTA-binding motif enrichment in early-phase high humidity-induced genes

To capture transcriptional reprogramming triggered by high humidity, we performed RNA-sequencing (RNA-seq) on WT leaves exposed to moderate or high humidity for 0.5 and 5 h. In total, 2394 upregulated and 2054 downregulated genes were identified as differentially expressed genes (DEGs; fold change > 2 and FDR < 0.05) under high humidity compared with moderate humidity (Fig. 3a and Supplementary Data 1). The DEGs identified at 0.5 h and 5 h showed limited overlap (upregulated, 43.5% of 0.5 h and 30.5% of 5 h; downregulated, 30% of 0.5 h and 6.7% of 5 h) (Fig. 3a), suggesting that distinct regulatory mechanisms are involved during early and late stages of the response. This distinction was further supported by principal component analysis (PCA), which revealed clear separation of transcriptomes according to both humidity conditions and exposure durations (Fig. 3b).

**Fig. 3: CAMTA-binding motifs are overrepresented in early-phase high humidity-induced genes.**

K-means clustering of the 1000 most variable genes yielded 10 distinct clusters (Fig. 3c and Supplementary Data 2). High humidity-induced genes were distributed across four clusters, with CYP707A1 and CYP707A3 notably present in Cluster 6 (Fig. 3c). Clusters 2 and 6 exhibited rapid transcriptional upregulation within 0.5 h of treatment, Cluster 9 peaked at 0.5 h and gradually declined by 5 h while remaining elevated under high humidity relative to moderate humidity, and Cluster 1 was primarily induced at 5 h (Fig. 3c). Gene ontology analysis highlighted overrepresented biological processes in these clusters, including “hypoxia response” (Clusters 1 and 2), “jasmonic acid (JA) response” (Cluster 6), and “cell wall biogenesis” (Cluster 9) (Supplementary Fig. 5). Notably, Cis-regulatory motif analysis revealed significant enrichment of CAMTA-binding motifs, particularly in Clusters 2 and 6 (Fig. 3d), implicating CAMTA transcription factors as potential key regulators of early transcriptional responses to high humidity.

High humidity-induced CYP707A3 expression requires CAMTA-binding motifs in its promoter

The promoter region of CYP707A3 contains two tandem CAMTA-binding motifs (CGCG-box), whereas the CYP707A1 promoter lacks these elements (Fig. 4a). To assess the contribution of these motifs to high humidity-induced CYP707A3 expression, we transiently expressed a GUS reporter driven by either a native CYP707A3 promoter (harboring both CGCG boxes) or a modified promoter lacking these motifs (ΔCGCG) in NahG plants (Supplementary Fig. 6a), which are depleted of SA to facilitate Agrobacterium-mediated gene delivery. GUS expression driven by the native promoter was significantly induced 0.5 h after high humidity exposure, whereas the ΔCGCG promoter failed to confer this induction (Fig. 4b). In these plants, high humidity induction of endogenous CYP707A3 expression remained unaffected (Fig. 4b). These results indicate that CAMTA-binding motifs mediate, at least in part, the high humidity responsiveness of the CYP707A3 promoter.

**Fig. 4: CAMTA3 drives *CYP707A3* expression to promote resistance to bacterial water-soaking under high humidity.**

To evaluate the physiological relevance of this regulation, we generated cyp707a3 transgenic plants expressing CYP707A3 fused to a C-terminal 3×FLAG tag (CYP707A3-FLAG), driven by either the native or ΔCGCG promoter (Supplementary Fig. 6b, c). Consistent with the GUS reporter results, high humidity-induced CYP707A3-FLAG expression was markedly reduced in the ΔCGCG promoter line compared with the native promoter line (Supplementary Fig. 6d). Following Pst ΔhrcC infiltration, the impaired water-soaking resistance of cyp707a3 was fully restored by CYP707A3-FLAG expressed under the native promoter, but only partially restored under the ΔCGCG promoter (Supplementary Fig. 6e). Together, these findings indicate that CAMTA-binding motifs in the CYP707A3 promoter are critical for its high humidity-induced expression and for conferring resistance to bacterial water-soaking.

High humidity triggers intracellular Ca²⁺ signaling to induce CYP707A3 expression

CAMTA transcription factors are activated upon binding to Ca²⁺-calmodulin (CaM)³⁰. Given their putative role in high humidity-induced transcription, we hypothesized that elevated humidity triggers an increase in cytosolic Ca²⁺ levels. Using 35S::GCaMP3 plants expressing the Ca²⁺ biosensor GCaMP3, we detected a rapid rise in cytosolic Ca²⁺ in leaf cells within 10 min of high humidity exposure (Supplementary Fig. 7a, b and Supplementary Movie 1). To test whether cytosolic Ca²⁺ influx contributes to high humidity-induced CYP707A3 and CYP707A1 expression, we applied the Ca²⁺ channel inhibitors LaCl₃ and GdCl₃ prior to humidity treatment. Both inhibitors significantly suppressed CYP707A3 induction but did not suppress CYP707A1 (Supplementary Fig. 7c), indicating a specific requirement for Ca²⁺ signaling in CYP707A3 induction. Temporal expression profiling further revealed that high humidity-triggered cytosolic Ca²⁺ elevation precedes CYP707A3 induction (Supplementary Fig. 7b, d). These findings indicate that humidity elevation triggers cytosolic Ca²⁺ influx in leaves, which in turn mediates CYP707A3 expression.

CNGC2/4/9 play a critical role in high humidity-induced CYP707A3 expression

CNGCs, comprising 20 members in Arabidopsis, mediate cytosolic Ca²⁺ influx in diverse physiological processes³¹. To assess their role in high humidity-triggered Ca²⁺ signaling, we examined CYP707A3 induction in loss-of-function mutants of individual CNGCs. An initial screen in 2-week-old plants revealed that high humidity-induced CYP707A3 expression was significantly reduced in cngc2, cngc4, and cngc9 (Supplementary Fig. 8a, b). Subsequent analysis in 5-week-old plants showed that high humidity induction of both CYP707A3 and CYP707A1 was impaired in cngc2 and cngc4, whereas only CYP707A3 induction was reduced in cngc9 (Fig. 4c). These results suggest that CNGC2 and CNGC4 act cooperatively to mediate Ca²⁺ influx required for high humidity-induced CYP707A3 expression, with partial contribution from CNGC9. Collectively, these findings suggest that CNGC2/4-mediated cytosolic Ca²⁺ elevation, together with partial support from CNGC9, is crucial for CYP707A3 and CYP707A1 induction in response to high humidity.

CAMTA3 drives CYP707A3 induction, ABA depletion, and resistance to bacterial water-soaking under high humidity

Among the six CAMTA transcription factors in Arabidopsis, CAMTA3 is a central regulator of responses to diverse stimuli^32,33,34,35. To assess its role in high humidity-induced CYP707A3 expression, we used an inactive CAMTA3-A855V variant, which carries A855V substitution in the IQ domain that disrupts CAMTA3 function without triggering SA defense activation³⁶. Introduction of CAMTA3 or CAMTA3-A855V under the control of a native CAMTA3 promoter in the camta2 camta3 mutant background did not alter plant growth or basal SA levels (Supplementary Fig. 9a–c). CAMTA3 expression restored WT-like responses to high humidity, including CYP707A3 induction, ABA depletion, and stomatal opening (Fig. 4d–f). In contrast, these responses were markedly impaired in CAMTA3-A855V-expressing plants (Fig. 4d–f), indicating that CAMTA3 activity is required for their induction. Consistent with the absence of CGCG motifs in the CYP707A1 promoter, high humidity-induced CYP707A1 expression was unaffected in either CAMTA3- or CAMTA3-A855V-expressing plants (Fig. 4d), underscoring distinct regulatory mechanisms for CYP707A3 and CYP707A1 expression. Moreover, the levels of SA and jasmonoyl-isoleucine (JA-Ile), the major bioactive form of JA, remained unchanged across plant genotypes and humidity treatments (Supplementary Fig. 9c). Together, these findings indicate that CAMTA3 promotes ABA depletion and stomatal opening under high humidity by inducing CYP707A3, without altering SA or JA accumulation.

We next tested whether CAMTA3 activity is required to suppress bacterial water-soaking and proliferation under high humidity. Plants expressing CAMTA3-A855V, but not CAMTA3, exhibited extensive water-soaking and permitted increased growth of Pst Δhrc under high humidity (Fig. 4g, h), whereas bacterial growth was unaffected under moderate humidity (Fig. 4i). These findings demonstrate that CAMTA3 activity is required for resistance to bacterial water-soaking and infection specifically under high humidity. This is consistent with a model in which elevated humidity triggers Ca²⁺ influx through CNGC2/4/9, activating CAMTA3 to drive CYP707A3-mediated resistance.

Pst DC3000 suppresses high humidity-induced CYP707A3 expression via type III secretion effectors

Pst DC3000 enhances host ABA responses by manipulating ABA biosynthesis, signaling, and translocation to promote water-soaking under high humidity^9,10. We tested whether Pst DC3000 also targets high humidity-triggered ABA depletion via CYP707A3 and CYP707A1. WT leaves were inoculated with Pst DC3000, Pst ΔhrcC, and Pst Δ28E (lacking 28 T3S effectors)³⁷. At 8 hpi under moderate humidity, plants were shifted to moderate or high humidity for 0.5 h before RT-qPCR analysis (Fig. 5a). At this point, bacterial populations were comparable between Pst DC3000 and Pst ΔhrcC (Fig. 5b), minimizing potential effects of differential bacterial growth. Under these conditions, CYP707A1 was not induced by high humidity, even in the absence of bacterial inoculation (Fig. 5C), likely due to desensitization from water infiltration. By contrast, CYP707A3 was significantly induced by high humidity in mock-inoculated leaves and in those inoculated with Pst ΔhrcC or Pst Δ28E, but this induction was markedly suppressed by Pst DC3000 (Fig. 5c). CAMTA3 protein accumulation was unaffected by inoculation with the WT or mutant bacteria (Supplementary Fig. 10), suggesting that suppression is not due to CAMTA3 depletion. Consistent with previous reports^9,10, Pst DC3000, but not Pst ΔhrcC or Pst Δ28E, also induced NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), encoding a key ABA biosynthesis enzyme, under these conditions (Fig. 5c). Together, these results indicate that Pst DC3000 employs T3S effectors to suppress CYP707A3 induction while promoting NCED3 expression, thereby maintaining elevated ABA levels under high humidity to facilitate infection.

**Fig. 5: *Pst* DC3000 blocks high humidity-driven plant transcriptome reprogramming via T3S effectors.**

Pst DC3000 blocks high humidity-driven plant transcriptome reprogramming via type III secretion effectors

To further investigate how Pst DC3000 modulates plant responses to high humidity, we performed RNA-seq analysis on leaves inoculated with Pst DC3000, Pst ΔhrcC, and Pst Δ28E for 8 h, followed by a 0.5 h of exposure to moderate or high humidity. PCA revealed a clear separation of leaf transcriptomes based on humidity conditions in non-inoculated leaves and those inoculated with Pst ΔhrcC or Pst Δ28E (Fig. 5d). In contrast, this separation was absent in leaves inoculated with Pst DC3000 (Fig. 5d), indicating that Pst DC3000 broadly disrupts transcriptomic responses to high humidity via T3S effectors. K-means clustering of the 2000 most variable genes resulted in 10 distinct clusters (Fig. 5e and Supplementary Data 3). High humidity-induced genes were enriched in three clusters, with Clusters 3 and 8 containing CYP707A1 and CYP707A3, respectively (Fig. 5e). Pst DC3000 strongly suppressed high humidity-induced upregulation in Clusters 3 and 8, and partially in Cluster 4 (Fig. 5e). Notably, Cluster 3 genes were upregulated following Pst DC3000 inoculation under moderate humidity, irrespective of subsequent humidity conditions (Fig. 5e). These findings indicate that Pst DC3000, through T3S effectors, not only suppresses CAMTA3-regulated genes but also broadly alters early transcriptional networks responsive to high humidity during infection.

AvrPtoB contributes to suppression of CYP707A3-mediated water-soaking resistance

To identify the Pst DC3000 T3S effector(s) responsible for suppressing CYP707A3 induction under high humidity, we analyzed multiple effector-deletion mutants of Pst DC3000³⁷. High humidity-induced CYP707A3 expression was significantly suppressed in WT leaves at 8 hpi with Pst Δ14E (lacking 14 T3S effectors) and Pst Δ20E (lacking 20 T3S effectors), similar to Pst DC3000 (Fig. 6a), at a time when T3S-dependent bacterial growth was not evident (Fig. 5b). In contrast, Pst Δ28E failed to suppress CYP707A3 induction, indicating that one or more of the remaining eight effectors (HopK1, HopY1, HopB1, HopAF1, AvrPtoB, AvrPto, HopE1, and HopA1) are required for this suppression (Fig. 6a).

**Fig. 6: T3S effector AvrPtoB suppresses *CYP707A3*-mediated water-soaking resistance.**

Given that in-planta expression of AvrPtoB has been associated with increased ABA levels through induction of NCED3³⁸, we tested whether AvrPtoB contributes to CYP707A3 suppression. To this end, we generated transgenic plants expressing either AvrPtoB or its partially redundant counterpart, AvrPto³⁹, under a DEX-inducible promoter (DEX::AvrPtoB and DEX::AvrPto; Fig. 6b, c and Supplementary Fig. 11a). Following DEX treatment, high humidity-induced CYP707A3 expression was specifically suppressed in DEX::AvrPtoB, but not in DEX::AvrPto, despite lower protein accumulation of AvrPtoB compared to AvrPto (Fig. 6b, c and Supplementary Fig. 11b). An E3 ubiquitin ligase-deficient variant, AvrPtoB-F479A⁴⁰, retained the ability to suppress CYP707A3 induction (Fig. 6d and Supplementary Fig. 11a), suggesting that this activity is independent of its E3 ligase function. In contrast, high humidity induction of CYP707A1 was specifically suppressed in AvrPto-expressing leaves (Supplementary Fig. 11c–e). Collectively, these findings suggest that Pst DC3000 selectively targets CYP707A3 and CYP707A1 with distinct T3S effectors, AvrPtoB and AvrPto, respectively, under high humidity.

Consistent with the predominant role for CYP707A3 in water-soaking resistance (Fig. 1c), high humidity-triggered ABA depletion and stomatal opening were also compromised in DEX::AvrPtoB following DEX treatment (Fig. 6e, f). Importantly, DEX-induced AvrPtoB expression substantially restored water-soaking in leaves infiltrated with Pst DC3000 ΔhrcC (Fig. 6g). Together, these results indicate that AvrPtoB counteracts CYP707A3-mediated water-soaking resistance under high humidity, although its contribution may involve indirect effects or additional factors.

Pst DC3000 employs AvrPtoB to suppress CYP707A3 expression and promote water-soaking under high humidity

We next evaluated the physiological relevance of AvrPtoB and AvrPto in promoting ABA-dependent bacterial infection under high humidity using Pst DC3000 mutant strain lacking both AvrPtoB and AvrPto (Pst ΔavrPtoB ΔavrPto). Plants were shifted from moderate to high humidity at 2 hpi, following conditions conventionally used in previous studies^8,9,10 (Fig. 7a and Supplementary Fig. 12a). RT-qPCR analysis revealed that CYP707A3 expression was suppressed from 6 to 24 hpi with Pst DC3000, despite continuous high humidity from 2 hpi (Supplementary Fig. 12b). This suppression was absent with Pst Δ28E, whereas with Pst ΔavrPtoB ΔavrPto it was impaired at 6 hpi but restored at 12–24 hpi (Supplementary Fig. 12b), indicating that AvrPtoB and/or AvrPto mediate early suppression of CYP707A3, while other effectors contribute to later suppression. Consistently, ABA levels increased at 6 hpi with Pst DC3000, but not with Pst ΔavrPtoB ΔavrPto or Pst Δ28E (Supplementary Fig. 12c). This initial increase was followed by further accumulation at 12 and 24 hpi with Pst DC3000 and partially with Pst ΔavrPtoB ΔavrPto, but not with Pst Δ28E. Together, these findings indicate that AvrPtoB- and/or AvrPto-mediated suppression of CYP707A3 is essential for the initial phase of ABA accumulation under high humidity, which in turn facilitates subsequent water-soaking and bacterial proliferation.

To dissect individual contributions of these effectors, we generated Pst DC3000 mutant strains lacking either AvrPtoB (Pst ΔavrPtoB) or AvrPto (Pst ΔavrPto) (Fig. 7b and Supplementary Fig. 13a, b). The two effectors did not affect each other’s expression during infection (Fig. 7b). Consistent with DEX induction (Fig. 6b, c), high humidity-induced CYP707A3 expression was suppressed by Pst DC3000 and Pst ΔavrPto, but permitted with Pst ΔavrPtoB and Pst ΔavrPtoB ΔavrPto (Fig. 7b). By contrast, enhanced CYP707A1 expression relative to Pst DC3000 was observed only with Pst ΔavrPtoB ΔavrPto (Fig. 7b), suggesting redundancy in CYP707A1 suppression. For NCED3 induction, Pst ΔavrPto resembled Pst DC3000, whereas Pst ΔavrPtoB and Pst ΔavrPtoB ΔavrPto failed to induce it (Fig. 7b), indicating a predominant role of AvrPtoB in both CYP707A3 suppression and NCED3 induction. Correspondingly, ABA accumulation at 6 hpi was enhanced with Pst DC3000 and Pst ΔavrPto, but not with Pst ΔavrPtoB or Pst ΔavrPtoB ΔavrPto (Fig. 7c). Together, these results indicate that AvrPtoB drives ABA accumulation under high humidity, acting through CYP707A3 suppression and NCED3 induction.

We next assessed the impact of AvrPtoB on bacterial water-soaking and infection. Under high humidity, Pst DC3000 induced extensive water-soaking, which was strongly reduced in leaves infiltrated with Pst ΔavrPtoB or Pst ΔavrPtoB ΔavrPto, while Pst ΔavrPto retained partial activity (Supplementary Fig. 13c, d). These results indicate a predominant role for AvrPtoB, with a moderate contribution from AvrPto. Importantly, delayed suppression of CYP707A3 by Pst ΔavrPtoB ΔavrPto at 12–24 hpi (Supplementary Fig. 12b) was insufficient to promote water-soaking, highlighting the importance of early ABA depletion for this virulence activity. Moreover, Pst ΔavrPtoB induced smaller water-soaked area compared to Pst DC3000 even in cyp707a1 cyp707a3 leaves (Fig, 7d, e). This reduction correlated with milder disease symptoms and lower bacterial growth in both WT and cyp707a1 cyp707a3 plants (Fig. 7f, g). These findings indicate that AvrPtoB also targets an additional, CYP707A1/CYP707A3-independent layer of water-soaking resistance, which may align with the additive roles of BAK1/BKK1/CERK1-mediated PTI and ABA catabolism-mediated defenses in restricting bacterial growth under high humidity (Supplementary Fig. 4d, e).

Discussion

Terrestrial plants are constantly exposed to fluctuations in air humidity and have evolved elaborate adaptation mechanisms^{17,19,41,42,43}. High humidity, often following prolonged rainfall, substantially exacerbates plant diseases caused by fungal and bacterial pathogens^8,44. It is therefore plausible that plants pre-condition or activate defense responses in anticipation of these threats when sensing increased humidity. In parallel with our study, the involvement of a humidity-induced CNGC2/4-CAMTA3 module in activating CYP707A3 expression was recently reported²², yet its physiological relevance in plant-microbe interactions remained unresolved. Here, we demonstrate that the CNGC2/4/9-CAMTA3-CYP707A3 signaling module contributes to bacterial resistance by limiting water-soaking under high humidity (Fig. 7h). Our findings suggest that elevated humidity is perceived as a danger signal to enhance defense readiness. This aligns with earlier studies that high humidity induces ROS accumulation in Arabidopsis leaf apoplast, a hallmark of defense responses during pathogen challenge^19,21. Additionally, high humidity increases cuticle permeability, which has been associated with enhanced resistance to the fungal pathogen Botrytis cinerea^18,45,46. Rainfall and submergence have also been linked to strengthened disease resistance in plants^35,47. These findings suggest that plants possess the mechanisms linking the sensing of increased water or humidity levels to the activation or reinforcement of defense responses. Our findings reveal a mechanism linking humidity perception to the activation of defenses that prevent bacterial access to leaf water.

Antagonistic interactions between ABA and SA signaling have been well documented^24,25,48,49, yet their modulation under varying environmental conditions remain under-explored. In Arabidopsis, CYP707A3 overexpression enhances transpiration rates⁵⁰. ABA application suppresses systemic acquired resistance (SAR) inducers acting both upstream and downstream of SA accumulation²⁴. Enhanced SA production under continuous light suppresses Pst DC3000-induced water-soaking without interfering with NCED3 induction¹⁶. We show that CYP707A3-mediated ABA depletion confers water-soaking resistance independently of SA under high humidity, whereas ABA deficiency enhances bacterial resistance in an SA-dependent manner under moderate humidity. These observations underscore a humidity-dependent differentiation in bacterial virulence strategies and the role of ABA depletion in bacterial resistance. The distinct regulation of SA-ABA antagonism aligns with the decrease in SA production and effectiveness under high humidity²¹. Notably, however, genes associated with SA defense, such as SARD1, CBP60g, and BCS1^51,52,53, are induced under high humidity even in the absence of pathogen challenge (Cluster 3 in Fig. 5e and Supplementary Data 3), suggesting potential coupling between humidity sensing and SA-mediated resistance, which may be further potentiated by PRR signaling (Supplementary Fig. 4e).

To our knowledge, this study provides the first evidence that phytopathogens promote infection by suppressing high humidity responses in host plants through effector proteins. Our RNA-seq analyses (Fig. 5d, e) demonstrate that Pst DC3000 employs multiple T3S effectors to inhibit humidity-triggered transcriptomic changes, thereby circumventing CYP707A3-mediated water-soaking resistance (Fig. 7h). Interestingly, Pst DC3000 T3S effectors also induce a subset of humidity-inducible genes, including CYP707A1, regardless of humidity conditions (Fig. 5e). The functional significance of their induction represents an open question for future studies. CYP707A1 is predominantly induced in guard cells under high humidity¹⁷, and Pst DC3000 elevates ABA levels in guard cells¹⁰. Whether bacterial CYP707A1 induction occurs specifically in guard cells or in other tissues remains to be determined. Suppression of CYP707A3 induction alone is sufficient to permit ABA-mediated stomatal closure, facilitating water-soaking under high humidity, as indicated by the impaired water-soaking resistance in cyp707a3 mutants (Fig. 1c). Recent findings indicate that post-invasion stomatal reopening, rather than the initial closure to restrict bacterial entry, is critical for resisting bacterial water-soaking and infection⁵⁴, emphasizing the role of CYP707A-mediated ABA depletion as a core defense barrier limiting bacterial access to apoplastic water.

We identify AvrPtoB as a T3S effector suppressing this defense. Genetic analyses show that among the 36 T3S effectors, AvrPtoB has a role in blocking high humidity-induced CYP707A3 expression (Fig. 7h). Earlier studies reported that AvrPtoB can target CYP707A3 and its paralogs for proteasomal degradation when these proteins are transiently expressed in Nicotiana benthamiana⁵⁵. By suppressing ABA catabolism and simultaneously promoting ABA biosynthesis via NCED3 induction^38,55, AvrPtoB manipulates both arms of host ABA balance, facilitating stomatal closure and maintaining elevated ABA levels under high humidity. These effects on CYP707A3 and NCED3 expression may arise indirectly as downstream consequences of AvrPtoB activity. How AvrPtoB, known to act on membrane-localized immune receptors and transcription cofactors^{56,57,58,59,60,61}, contributes to the modulation of CYP707A3 and NCED3 expression remains to be elucidated.

Our work identifies CAMTA3 transcription regulator and CNGC2/4/9 Ca²⁺ channels as key regulators of humidity-induced CYP707A3 expression, consistent with recent findings²². While the oomycete effector AVRblb2 has been shown to suppress CNGC activity⁶², the potential targeting of CNGCs by Pst DC3000 T3S effectors remains unexplored. Notably, the presence of a dominant-negative CAMTA3-A855V allele significantly impairs resistance to bacterial and fungal pathogens, SAR, as well as early transcriptional responses shared by PTI, effector-triggered immunity, and abiotic stress responses^63,64,65. This underscores the critical role of CAMTA3 in promoting and/or priming defense activation under high humidity. The disruption of CAMTA1/2/3 is also linked to nucleotide-binding leucine-rich repeat receptor activation leading to SA defense activation and autoimmunity^36,66, suggesting that CAMTAs might be targeted by pathogen effectors to counteract humidity-induced defenses. Future studies on AvrPtoB and other T3S effectors, including HopAM1, HopM1, and AvrE, which promote ABA responses^9,43,67, and their interactions with the CNGC2/4/9-CAMTA3-CYP707A3 signaling module and another water-soaking defense(s) will deepen our understanding of the interplay between humidity-triggered defenses and effector-mediated host manipulation. This work advances our knowledge of how plants and pathogens optimize immunity and virulence strategies under high humidity, driven by their competition for water as a decisive factor.

Methods

Plant materials and growth conditions

Arabidopsis accession Columbia-0 (Col-0) was used as the wild-type (WT) in this study. The cyp707a1 cyp707a3 ost2-3D, cyp707a1 cyp707a3 sid2-2, aao3 sid2-2, bak1-5 bkk1 cerk1 (bbc), and cyp707a1 cyp707a3 bbc mutants were generated by genetic crossing. The genotypes of Arabidopsis mutants and transgenic lines were verified by PCR, Sanger sequencing, or chemical selection. Plant materials and genotyping primers used are listed in Supplementary Data 4, 6, respectively.

Surface-sterilized seeds were sown on half-strength Murashige and Skoog (MS) medium [2.3 g/L Murashige and Skoog Plant Salt Mixture (FUJIFILM Wako Pure Chemical, Cat# 392-00591), 1% (w/v) sucrose, 0.5 g/L MES, 1% (w/v) agar (FUJIFILM Wako Pure Chemical, Cat# 016-11875), pH 5.6–5.8 adjusted by KOH] followed by stratification for 1–4 days in the dark at 4°C. Plates were placed vertically in a growth chamber [22°C, 16 h light/8 h dark (long-day) or 8 h light/16 h dark (short-day), illuminated with daylight white fluorescent lamps (2 lamps/side)]. Seedlings were then transplanted into a soli mixture [50% (v/v) Super Mix A (Sakata Seed), 50% (v/v) Vermiculite G20 (Nittai)] and cultivated in either a growth chamber [22°C, 60% RH, 8 h light/16 h dark, illuminated with daylight white fluorescent lamps (2 lamps/side)] or a growth room [22°C, 30-70% RH, 16 h light/8 h dark, illuminated with daylight white fluorescent lamps (4 lamps/shelf)]. Plants were irrigated with either tap water or a modified Hoagland solution [1.67 mM KNO₃, 0.67 mM MgSO₄, 5.21 μM KH₂PO₄, 28.3 μM KCl, 15.5 μM H₃BO₄, 3.07 μM MnCl₂, 0.28 μM ZnSO₄, 0.11 μM CuSO₄, 13.3 μg/L 80% molybdic acid (FUJIFILM Wako Pure Chemical, Cat# 134-03332), 1.67 mM Ca(NO₃)₂, 46.3 μM Fe(III)-EDTA (Dojindo, Cat# 345-01245)].

For general cultivation, 2-week-old seedlings were transplanted and grown under long-day conditions with irrigation using the modified Hoagland solution. For experiments using 5-week-old plants, 2-week-old seedlings were transplanted and grown under short-day conditions with irrigation using the modified Hoagland solution. For experiments using 2-week-old plants, 4-day-old seedlings were transplanted and grown under long-day conditions with irrigation using tap water.

Plasmid construction

For the generation of Arabidopsis transgenic plants, the promoter region (−2776 bp to −1 bp relative to the start codon) and the genomic complementation sequence (−2776 to +1930 bp relative to the start codon) of CYP707A3 were amplified from Arabidopsis WT genomic DNA. The full-length coding sequences (excluding the stop codon) of AvrPtoB and AvrPto were amplified from Pst DC3000 genomic DNA. A deletion of the CGCG boxes in the CYP707A3 promoter (−2220 bp to −2150 bp relative to the start codon) and the AvrPtoB-F479A substitution were introduced by overlap extension PCR. PCR amplifications were performed using PrimeSTAR GXL DNA Polymerase (Takara Bio, Cat# R050A). Amplified products were cloned into pENTR/D-TOPO using either the pENTR/D-TOPO Cloning Kit (Thermo Scientific, Cat# K240020SP) or the In-Fusion Snap Assembly Master Mix (Takara Bio, Cat# 638948). The resulting entry clones were verified by Sanger sequencing with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Cat# 4337455) and recombined with destination vectors using Gateway LR Clonase II Enzyme mix (Invitrogen, Cat# 11791020). The following destination vectors were used: pGWB533⁶⁸ for GUS reporter constructs, pGWB501 (C-3×FLAG) for CYP707A3 complementation constructs, and pTA70001-DEST (Addgene, Cat# 71745) for DEX-inducible constructs.

The pGWB501 (C-3×FLAG) vector was generated by cloning the 3×FLAG tag coding sequence from pAMPAT-GW-3×FLAG⁶⁹ into the AfeI site of pGWB501⁶⁸ using In-Fusion Snap Assembly Master Mix. For constructs encoding C-terminally 3×FLAG-tagged AvrPtoB, AvrPto, and AvrPtoB-F479A, entry clones were recombined with pAMPAT-GW-3×FLAG, and the resulting coding sequences were subsequently cloned into pENTR/D-TOPO.

For the generation of Pst DC3000 mutant strains, the flanking 1 kb regions upstream and downstream of either avrPtoB or avrPto were amplified from Pst DC3000 genomic DNA by PCR using PrimeSTAR GXL DNA Polymerase. Amplified fragments were assembled and cloned into EcoRI- and XbaI-digested pK18mobsacB using In-Fusion Snap Assembly Master Mix. The resulting constructs were verified by Sanger sequencing with the BigDye Terminator v3.1 Cycle Sequencing Kit. The pK18mobsacB vector was obtained from the National BioResource Project (NIG, Japan): E. coli.

All cloning procedures were performed according to the manufacturer’s instructions. Plasmids generated in this study and the primers for cloning are listed in Supplementary Data 5, 6, respectively.

Generation of Arabidopsis transgenic plants

Expression vectors were introduced into Agrobacterium tumefaciens strain GV3101::pMP90 by electroporation. Transgenic plants were generated by transforming WT or cyp707a3 mutant plants using the floral dip method⁷⁰. For the CYP707A3 complementation experiments, T2 segregating lines were analyzed, whereas T3 or T4 homozygous lines were selected and used for all other experiments. The transgenic plants used in this study are listed in Supplementary Data 4.

Generation of Pst DC3000 mutant strains

Pst DC3000 mutant strains lacking either avrPtoB or avrPto were generated by introducing the pK18mobsacB-based constructs into Pst DC3000, as previously described⁷¹. Deletions of avrPtoB and avrPto were verified by PCR using EmeraldAmp MAX PCR Master Mix (Takara Bio, Cat# RR320A) and Sanger sequencing with the BigDye Terminator v3.1 Cycle Sequencing Kit. The primers used for PCR and Sanger sequencing are listed in Supplementary Data 6.

Chemicals

The following stock solutions were prepared and stored at −30°C: LaCl₃ (FUJIFILM Wako Pure Chemical, Cat# 123-04222), 1 M in water; GdCl₃ (FUJIFILM Wako Pure Chemical, Cat# 078-02661), 1 M in water; Acetosyringone (FUJIFILM Wako Pure Chemical, Cat# 320-29611), 100 mM in DMSO; Dexamethasone (DEX) (FUJIFILM Wako Pure Chemical, Cat# 047-18863), 10 mM in ethanol.

Humidity treatment

High humidity treatment was performed by adding approximately 200 mL of modified Hoagland solution or tap water (for 2-week-old plants grown under long-day conditions) to a plastic container (Daiso, Cat# 4550480269726), spraying tap water onto the container walls, placing the plants inside, sealing the container with plastic wrap, and immediately returning it to the growth conditions. Moderate humidity treatment was performed by adding approximately 500 mL of modified Hoagland solution or tap water (for 2-week-old plants grown under long-day conditions) to a plastic tray (As One, Cat# 1-4618-01), placing the plants inside, and immediately returning it to the growth conditions.

For inhibitor treatments and Pst DC3000 inoculation experiments, the indicated solutions were infiltrated into three leaves per 5-week-old WT plant using a needleless syringe (Terumo, Cat# SS-01T). Excess solution on the leaf surfaces was gently removed using tissue paper (Elleair, Cat# DSI20703128). The infiltrated plants were maintained under their growth conditions for 24 h (for inhibitor treatments) or 8 h (for Pst DC3000 inoculation), followed by humidity treatment. During Pst DC3000 inoculation experiments, lights were kept on continuously from inoculation until sampling.

Agrobacterium-mediated transient expression

Agrobacterium-mediated transient expression was performed as previously described⁷² with minor modifications. Agrobacterium tumefaciens strain GV3101::pMP90 carrying the relevant expression vectors was cultured overnight at 28°C with shaking (180 rpm) in 2×YT liquid medium [1% (w/v) yeast extract, 1.6% (w/v) tryptone, 0.5% (w/v) NaCl] supplemented with 50 μg/mL gentamicin and 100 μg/mL spectinomycin. Bacterial cells were collected by centrifugation at 2000 × g for 10 min at 25°C, washed once with sterile water, and resuspended in sterile water to an OD₆₀₀ of 0.05. Acetosyringone (100 mM stock solution) was added to the suspension to a final concentration of 100 μM.

The bacterial suspensions were infiltrated into three leaves per 5-week-old NahG plant using a needleless syringe. Excess solution on the leaf surfaces was gently removed using tissue paper. The infiltrated plants were maintained under their growth conditions for 24 h before humidity treatment.

Pst DC3000 inoculation assays

The following Pst DC3000 strains were used in this study: WT, ΔhrcC⁷³, Δ28E (CUCPB5585)³⁷, Δ14E (CUCPB5459)⁷⁴, Δ20E (CUCPB5520), ΔavrPtoB ΔavrPto³⁹, ΔavrPtoB, and ΔavrPto. Strains were cultured overnight at 28°C with shaking (180 rpm) in King’s B liquid medium [2% (w/v) Bacto Proteose Peptone No. 3 (Gibco, Cat# 211693), 0.15% (w/v) K₂HPO4, 1% (v/v) glycerol, 5 mM MgSO₄] supplemented with 50 μg/mL rifampicin and additional antibiotics as appropriate. Bacterial cells were collected by centrifugation at 2000 × g for 10 min at 25°C, washed once with sterile water, and resuspended in sterile water to the OD₆₀₀ values indicated in figure legends.

Bacterial suspensions were infiltrated into three leaves per plant using a needleless syringe. Excess solution on the leaf surfaces was gently removed using tissue paper. Infiltrated plants were maintained under their respective growth conditions for the indicated time. To ensure high humidity (~95% RH), infiltrated plants were covered with a plastic container as described in the humidity treatment.

For water-soaking assays, the abaxial sides of infiltrated leaves were photographed at 1 day post-infiltration (dpi). The number of leaves exhibiting water-soaking was recorded, or the water-soaked area relative to total leaf area was quantified using ImageJ software (https://imagej.net/ij/). For bacterial growth assays, three inoculated leaves were collected from three different plants at the indicated dpi. Leaves were homogenized in 1 mL sterile water, and 10 uL aliquots of serial dilutions (10⁻¹ to 10⁻⁸) were spotted onto NYGA medium [0.5% (w/v) Bacto Peptone (Gibco, Cat# 211677), 0.3% (w/v) yeast extract, 2% (v/v) glycerol. 1.5% agar] supplemented with 50 μg/mL rifampicin. Colony-forming units (CFUs) were determined after 2 days of incubation at 28°C.

RT-qPCR

Total RNA was extracted from three leaves or a single shoot segment using Sepasol-RNA I Super G (Nacalai Tesque, Cat# 09379-55), followed by DNase treatment and reverse transcription (RT) with the PrimeScript RT reagent Kit with the gDNA Eraser (Takara Bio, Cat# RR047B), according to the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Cat# 4368702) on a Thermal Cycler Dice Real Time System III (Takara Bio, Cat# TP950). Gene expression levels were calculated using the ΔΔCt method, with normalization to ACTIN2 or gap-1 as internal reference genes. Primers used for qPCR are listed in Supplementary Data 6.

Immunoblotting

Frozen plant tissue (three leaves per sample) was ground into a fine powder and resuspended in protein extraction buffer [62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 5% (v/v) 2-mercaptoethanol, 0.02% bromophenol blue] supplemented with 1 mM EDTA (pH 8.0) and protease inhibitor cocktail (Roche, Cat# 11873580001). The buffer was added at a ratio of 3 μL per mg of tissue weight. Extracts were incubated at 95°C for 5 min and subsequently centrifuged at 20,000 × g for 10 min at 25°C, and the supernatants were collected.

Proteins were separated on a 10% polyacrylamide gel and transferred to a PVDF membrane (Millipore, Cat# IPVH00010). The membrane was blocked 2% (w/v) skim milk (Nacalai Tesque, Cat# 31149-75) in TBS-T [25 mM Tris, 192 mM glycine, 0.1% (v/v) Tween-20] for 1 h at 25°C with agitation, rinsed once with deionized water, and once with TBS-T. The blocked membrane was incubated with the primary antibody solution overnight at 4°C with agitation, rinsed five times with deionized water, and washed once in TBS-T for 5 min at 25°C with agitation. Subsequently, the membrane was incubated with the secondary antibody solution for 1 h at 25°C with agitation, rinsed five times with deionized water, and washed once with TBS-T for 5 min at 25°C with agitation.

Chemiluminescent detection was performed using Chemi-Lumi One L (Nacalai Tesque, Cat# 07880) and a FUSION FX imaging system (Vilber). The following antibodies were used for immunoblotting: anti-FLAG (Sigma-Aldrich, Cat# F1804; 1:5000 dilution), anti-GFP (MBL, Cat# 598; 1:5000 dilution), anti-mouse IgG HRP-linked (Cell Signaling Technology, Cat# 7076; 1:5000 dilution), and anti-rabbit IgG HRP-linked (Cell Signaling Technology, Cat# 7074; 1:5000 dilution). All antibodies were diluted in TBS-T.

Phytohormone quantification

SA, ABA, and JA-Ile were extracted and purified from freeze-dried samples as previously described⁷⁵. Frozen samples were lyophilized for 12 h using a freeze dryer (TAITEC-OnLine; VD-250R) and pulverized with a bead-based crusher (Shake Master, Bio Medical Science; BMS-A20TP). The powder was extracted twice with 500 µL and 1.0 mL of acetonitrile/water (80:20 v/v). The extraction buffer for the first step was supplemented with internal standards, including d₆-SA, d₆-ABA, and ¹³C₆-JA-Ile for the quantification of SA, ABA, and JA-Ile, respectively. During each extraction step, samples were homogenized at room temperature (18–22°C) for 5 min. The samples were then centrifuged at 5900 × g for 5 min at 25°C, and the supernatants from both extraction steps were combined into a 2 mL tube. Aliquots (1.1–1.2 mL) of the combined extracts were evaporated to dryness using a vacuum centrifuge (Thermo Fisher Scientific; SPD121P). The dried samples were homogenized at room temperature for 5 min and stored at −20°C for at least 30 min. After removal from the freezer, the samples were completely thawed, homogenized again for 5 min at room temperature, and centrifuged at 20,600 × g for 5 min at 4°C. The resulting supernatants (50–100 µL) were filtered through a 0.22 µm filter (Corning) and centrifuged at 800 × g for 3 min at 4°C. Finally, aliquots (50–80 µL) were transferred into HPLC vials (polypropylene, 12 × 32 mm screw-neck vial; Waters). Plant hormones were analyzed by an LC-MS/MS system (ACQUITY UPLC I-Class PLUS FTN/Xevo TQ-S micro; Waters) equipped with a short ODS column (TSKgel ODS-120H 2.0 mm I.D. x 5 cm, 1.9 µm; TOSHO) or a long ODS column (TSKgel ODS-120H 2.0 mm I.D. x 15 cm, 1.9 µm; TOSHO). A binary solvent system composed of 0.1% acetic acid (v/v) ACN (A) and 0.1% acetic acid (v/v) H₂O (B) was used. SA, ABA, and JA-Ile were measured using the short ODS column, and separation was performed at a flow rate of 0.2 mL/min with the following linear gradient of solvent B: 0–2 min (1–5%), 2–5 min (5–15%), 5–5 min (15–40%), 25.1–27 min (100%), and 27.1–30 min (1%). The retention times for each compound were as follows: d₆-SA (7.39), SA (7.46), d₆-ABA (12.59), ABA (12.67), ¹³C₆-JA-Ile (21.64), and JA-Ile (21.94). The MS/MS analysis was performed under the following conditions: capillary (kV) = 1.00, source temperature (°C) = 150, desolvation temperature (°C) = 500, cone gas flow (L/h) = 50, desolvation gas flow (L/h) = 1000. Ion mode was negative. The MS/MS transitions (m/z) and collision energies (V) for each compound were as follows: d₆-SA (142.1/98.0, 18 V), SA (137.0/93.4, 18 V), d₆-ABA (269.1/159.1, 8 V), ABA (263.1/153.1, 8 V), ¹³C₆-JA-Ile (328.2/136.1, 20 V), and JA-Ile (322.2/130.1, 20 V). Data analysis were performed using MassLynx 4.2 (Waters).

Stomatal aperture measurement

Following humidity treatment, detached leaves were immediately immersed in 4% (v/v) formaldehyde and incubated overnight at 25°C. Images of stomata on the abaxial leaf surface were captured using a Leica DMI6000 B microscope equipped with a 40× objective lens. Stomatal aperture (pore width/pore length) was quantified using ImageJ Fiji software (https://imagej.net/software/fiji/downloads).

Real-time cytosolic Ca²⁺ imaging

Real-time cytosolic Ca²⁺ imaging of whole p35S::GCaMP3 plants was conducted as described previously^76,77. Two-week-old plants grown under long-day conditions were used for Ca²⁺ imaging. Fluorescence imaging was initiated immediately following humidity treatment. To reduce unintended stimulation by excitation light, plants were pre-exposed to light of a wavelength proximal to the excitation wavelength for ~20 min before humidity treatment.

Fluorescence signals were captured using an Axio Zoom stereomicroscope (ZEISS) equipped with an ORCA-Flash4.0 V2 digital CMOS camera (Hamamatsu) and a mercury lamp (motorized Intensilight Hg Illuminator, Nikon), with excitation at 470 nm and emission at 535 nm. Images were acquired for ~17 min with a 2 s exposure and a 5 s interval, during which the excitation light was switched off between exposures. Quantification of GCaMP3 fluorescence was performed using ZEN Pro (ZEISS). Fluorescence intensity across the leaf surface was measured, and relative fluorescence values were calculated by normalizing each time point to the initial intensity (set to 1).

RNA-sequencing and data analysis

Total RNA was extracted from three leaves and treated with DNase using the NucleoSpin RNA Plant kit (Macherey-Nagel, Cat# 740949.50) according to the manufacturer’s instructions. RNA quality was assessed using a bioanalyzer (Agilent Technologies).

Each raw read data stored in DDBJ with accession numbers DRR545835 to DRR545846 was generated and analyzed as follows. Each RNA library was prepared from 1000 ng of total RNA using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Cat# E7770) according to the manufacturer’s instructions. PCR for library amplification was performed for seven cycles. The average length of the library was 350 bp, as measured by a bioanalyzer. Concentrations were measured by Kapa Library Quantification Kit (Kapa Biosystems, Cat# KK4824), each sample was diluted to 10 nM, and equal volumes were mixed. Single reads of 75 bp were obtained using a NextSeq 500 system (Illumina). CLC genomics workbench version 21 (Qiagen) was used to trim raw reads and map to the TAIR10 Arabidopsis genome (https://www.arabidopsis.org/). In trimming, the following parameters were changed (quality limit = 0.001, number of 5′ terminal nucleotides =15, number of 3′ terminal nucleotides = 5, and minimum number of nucleotides in reads = 25) and other options were default values. In mapping, all options were default values.

The generation and analysis of each raw read data stored in DDBJ with accession numbers DRR545847 to DRR545870 differ from the above analysis in three points. (1) Each library was prepared from 500 ng total RNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Cat# 7760). PCR for library amplification was performed for eight cycles. Each sample was diluted to 4 nM, and equal volumes were mixed. (2) Single reads of 100 bp were obtained using a NextSeq 1000 system (Illumina). (3) CLC genomics workbench version 24 (Qiagen) was used for read trimming and mapping. In trimming and mapping, the following parameters were changed (minimum number of nucleotides in reads = 40, and strand setting = Revers). All other methods, numbers, and options are the same as the above analysis.

The obtained read counts were processed and analyzed using iDEP 2.01 (https://bioinformatics.sdstate.edu/idep20/) with default settings. Gene Ontology and cis-regulatory enrichment analyses were performed using ShinyGO 0.81 (https://bioinformatics.sdstate.edu/go/) with default settings. For cis-regulatory enrichment analysis, the AGRIS database was used as the pathway reference.

Accession numbers

The gene sequences analyzed in this study were obtained from The Arabidopsis Information Resource (https://www.arabidopsis.org/) and The Pseudomonas Genome Database (https://www.pseudomonas.com/), under the accession numbers listed in Supplementary Data 7.

Statistical analysis

All data, except RNA-seq, were analyzed using GraphPad Prism 10 software (https://www.graphpad.com/features). No statistical methods were used to predetermine sample size, and no data were excluded from analyses. Experiments were not randomized, and investigators were not blinded to group allocation or outcome assessment. Statistical comparisons were performed using two-tailed Welch’s t-test, or one-way or two-way ANOVA followed by Tukey’s multiple comparison test, as specified in the figure legends.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The raw RNA-sequencing data have been deposited in the DDJB database under accession code PRJDB17982. Source data are provided with this paper.

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Acknowledgements

We thank Chika Tateda, Mie Matsubara, Taiga Ishihara, Natsuki Tsuchida, and Temma Takazawa (Nara Institute of Science and Technology) for technical assistance; Yuri Kanno (RIKEN CSRS) for supporting plant hormone analysis; Akira Mine (Kyoto University) for cyp707a seeds; Atsushi Takemiya (Yamaguchi University) for ost2-3D seeds; Michael F. Thomashow (Michigan State University) for camta seeds; Alan Collmer (Cornell University) for Pst DC3000 strains. We also thank the NAIST Life Science Collaboration Center (LiSCo) for supporting this study. This work was supported in part by JSPS KAKENHI Grant Numbers 21K14829 (S.Y.), 24H00565 (M.T.) and 21H02507 (Y.S.), JST ACT-X Grant Number JPMJAX22BN (S.Y.), ERATO Grant Number JPMJER2403 (M.T.), Cooperative Research Grant of the Genome Research for BioResource, NODAI Genome Research Center, Tokyo University of Agriculture (S.Y.), and Institute for Fermentation, Osaka Grant Number G-2025-2-089 (S.Y.).

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These authors contributed equally: Akihisa Shinozawa, Yuanjie Weng, Arullthevan Rajendram, Taishi Hirase.

Authors and Affiliations

Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Japan
Shigetaka Yasuda, Arullthevan Rajendram, Taishi Hirase, Haruka Ishizaki, Shioriko Ueda & Yusuke Saijo
Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan
Akihisa Shinozawa & Izumi Yotsui
Center for Sustainable Resource Science, RIKEN, Yokohama, Japan
Yuanjie Weng, Yumiko Takebayashi & Masanori Okamoto
Department of Biochemistry and Molecular Biology, Saitama University, Saitama, Japan
Ryuji Suzuki & Masatsugu Toyota
The NODAI Genome Research Center (NGRC), Tokyo University of Agriculture, Tokyo, Japan
Rahul Sk
Suntory Rising Stars Encouragement Program in Life Sciences (SunRiSE), Suntory Foundation for Life Sciences, Kyoto, Japan
Masatsugu Toyota
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
Masatsugu Toyota

Authors

Shigetaka Yasuda
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Akihisa Shinozawa
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Yuanjie Weng
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Arullthevan Rajendram
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Taishi Hirase
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Haruka Ishizaki
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Ryuji Suzuki
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Shioriko Ueda
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Rahul Sk
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Yumiko Takebayashi
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Izumi Yotsui
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Masatsugu Toyota
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Masanori Okamoto
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Yusuke Saijo
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Contributions

S.Y. and Y.S. conceived the study. S.Y. designed the research, conducted most of the experiments, and analyzed the data. A.R., T.H., and S.U. performed part of the RT-qPCR and Pst DC3000 inoculation assay. Y.W., Y.T., and M.O. performed phytohormone quantification. H.I., R.Suzuki, and M.T. performed GCaMP3 imaging. A.S., R.Sk, and I.Y. performed RNA-sequencing. S.Y. and Y.S. wrote the manuscript with input from the other authors.

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Correspondence to Shigetaka Yasuda or Yusuke Saijo.

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Yasuda, S., Shinozawa, A., Weng, Y. et al. Humidity-driven ABA depletion determines plant-pathogen competition for leaf water. Nat Commun 17, 787 (2026). https://doi.org/10.1038/s41467-025-67469-y

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Received: 22 January 2025
Accepted: 01 December 2025
Published: 19 December 2025
Version of record: 21 January 2026
DOI: https://doi.org/10.1038/s41467-025-67469-y