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

Humidity-driven ABA depletion determines plant-pathogen competition for leaf water | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Humidity-driven ABA depletion determines plant-pathogen competition for leaf water View ORCID Profile Shigetaka Yasuda , Akihisa Shinozawa , Yuanjie Weng , Arullthevan Rajendram , Taishi Hirase , Haruka Ishizaki , Ryuji Suzuki , Shioriko Ueda , Rahul Sk , Yumiko Takebayashi , Izumi Yotsui , Masatsugu Toyota , View ORCID Profile Masanori Okamoto , View ORCID Profile Yusuke Saijo doi: https://doi.org/10.1101/2025.01.04.631318 Shigetaka Yasuda 1 Graduate School of Science and Technology, Nara Institute of Science and Technology , Ikoma, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shigetaka Yasuda For correspondence: shige-yasuda{at}bs.naist.jp saijo{at}bs.naist.jp Akihisa Shinozawa 2 Department of Bioscience, Tokyo University of Agriculture , Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yuanjie Weng 3 Center for Sustainable Resource Science , RIKEN, Yokohama, Japan 4 United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology , Fuchu, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Arullthevan Rajendram 1 Graduate School of Science and Technology, Nara Institute of Science and Technology , Ikoma, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Taishi Hirase 1 Graduate School of Science and Technology, Nara Institute of Science and Technology , Ikoma, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Haruka Ishizaki 1 Graduate School of Science and Technology, Nara Institute of Science and Technology , Ikoma, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ryuji Suzuki 5 Department of Biochemistry and Molecular Biology, Saitama University , Saitama, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shioriko Ueda 1 Graduate School of Science and Technology, Nara Institute of Science and Technology , Ikoma, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rahul Sk 6 The NODAI Genome Research Center (NGRC), Tokyo University of Agriculture , Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yumiko Takebayashi 3 Center for Sustainable Resource Science , RIKEN, Yokohama, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Izumi Yotsui 2 Department of Bioscience, Tokyo University of Agriculture , Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Masatsugu Toyota 5 Department of Biochemistry and Molecular Biology, Saitama University , Saitama, Japan 7 Suntory Rising Stars Encouragement Program in Life Sciences (SunRiSE), Suntory Foundation for Life Sciences , Kyoto, Japan 8 College of Plant Science and Technology, Huazhong Agricultural University , Wuhan, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Masanori Okamoto 3 Center for Sustainable Resource Science , RIKEN, Yokohama, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Masanori Okamoto Yusuke Saijo 1 Graduate School of Science and Technology, Nara Institute of Science and Technology , Ikoma, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yusuke Saijo For correspondence: shige-yasuda{at}bs.naist.jp saijo{at}bs.naist.jp Abstract Full Text Info/History Metrics Supplementary material Preview PDF 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 Ca 2+ levels via the Ca 2+ 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, particularly AvrPtoB, facilitating water-soaking. These findings provide insights into the mechanisms underlying the competition between plants and bacteria for leaf water under elevated humidity. Introduction Bacterial phytopathogens cause foliar diseases that threaten plant survival and crop yields by proliferating within the leaf apoplast 1 . 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 – 4 . Following ligand perception, PRRs form active complexes with co-receptors to trigger intracellular signaling cascades. This involves rapid cytosolic Ca 2+ influx, a burst of reactive oxygen spices (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 – 4 . To overcome PTI, bacterial pathogens deliver effector proteins into host tissues and cells via type III secretion (T3S) system 1 . The bacterial phytopathogen Pseudomonas syringae pv. tomato ( Pst ) DC3000 has 36 T3S effectors 5 . Recent studies have shown that Pst DC3000 utilizes specific effectors to manipulate the leaf apoplast environment, establishing conditions favorable for infection 6 . High humidity promotes foliar diseases caused by bacterial pathogens through multiple mechanisms. Initially, impaired stomatal closure facilitates bacterial invasion into leaves 7 . Once inside, bacterial pathogens induce apoplast hydration under high humidity, a critical process for disease development known as water-soaking 8 . To induce water-soaking, Pst DC3000 employs the highly conserved 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 water 11 . 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 effectors to facilitate leaf infection in rice, wheat, and Arabidopsis 12 – 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 – 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 catabolism 20 . Under high humidity, ABA levels decrease via the action of CYP707A1 and CYP707A3 , which encode ABA 8’-hydroxylases, thereby facilitating stomatal opening 17 . Concurrently, the production and signaling of defense-promoting salicylic acid (SA) are suppressed, partly due to impaired ubiquitination of NPR1, an SA receptor and key transcriptional co-activator of SA-responsive genes, reducing its binding to target gene promoters 21 . Additionally, high humidity stimulates the biosynthesis of the gaseous phytohormone ethylene, which promotes leaf hyponasty 19 . 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 CYP707A 3 plays a pivotal role in restricting bacterial water-soaking by promoting stomatal opening in leaves. High humidity elevates cytosolic Ca 2+ concentrations via the Ca 2+ channels CYCLIC NUCLEOTIDE-GATED CHANNEL 2 (CNGC2), CNGC4, and CNGC9, triggering CYP707A3 induction through calmodulin-binding transcription activator 3 (CAMTA3). However, during infection, Pst DC3000 counteracts this signaling module and suppresses high humidity-induced transcriptional changes through its T3S effectors, with AvrPtoB playing a predominant role. These findings provide valuable insights into the mechanisms regulating plant-pathogen competition for leaf water under high humidity. Result 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. 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. Download figure Open in new tab Fig. 1: CYP707A3 is required for resistance to bacterial water-soaking under high humidity. a, Expression levels of CYP707A genes in humidity-treated leaves. Wild-type (WT) plants were exposed to moderate humidity (MH) or high humidity (HH) for the indicated time. Bars represent means ( n = 3). b , ABA levels in humidity-treated leaves. The indicated plants were exposed to MH or HH for 2 h. Bars represent means ( n = 3). c , d , Water-soaking in Pst Δ hrcC -inoculated leaves. The indicated plants were infiltrated with Pst Δ hrcC (OD 600 = 0.02) and maintained under MH or HH for 1 day. The number of water-soaked leaves was pooled from two independent experiments. e , Bacterial growth in Pst Δ hrcC -inoculated leaves. The indicated plants were infiltrated with Pst Δ hrcC (OD 600 = 0.02) and maintained under MH or HH for 3 days. Bars represent means ( n = 6). Different letters indicate statistically significant differences ( P < 0.05; two-way ANOVA followed by Tukey’s test). dpi, days post-infiltration. Experiments in ( a ) and ( e ) were repeated twice with similar results. 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-inoculation (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 Δ hrcC 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 reports 17 , 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 opening 22 , 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 cyp707a 3 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 Δ hrcC 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. Download figure Open in new tab Fig. 2: CYP707A3 -mediated water-soaking resistance depends on stomatal opening. a , b , Representative images of stomata ( a ) and stomatal aperture ( b ) in humidity-treated leaves. The indicated plants were exposed to moderate humidity (MH) or high humidity (HH) for 1 h. Bars represent the median ( n = 149-264). c , Water-soaking in Pst Δ hrcC -inoculated leaves. The indicated plants were infiltrated with Pst Δ hrcC (OD 600 = 0.02) and maintained under HH for 1 day. The number of water-soaked leaves was pooled from two independent experiments. d , Bacterial growth in Pst Δ hrcC -inoculated leaves. The indicated plants were infiltrated with Pst Δ hrcC (OD 600 = 0.02) and maintained under HH for 3 days. Bars represent means ( n = 6). Different letters indicate statistically significant differences ( P < 0.05; two-way ANOVA followed by Tukey’s test in b ; one-way ANOVA followed by Tukey’s test in d ). WT, wild-type; dpi, days post-infiltration. Experiments in ( b ) and ( d ) were repeated twice with similar results. 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 , 23 , 24 . However, both SA and ABA are required for stomatal closure during early defense responses to bacterial phytopathogens 25 . To assess whether SA influences CYP707A3 -mediated water-soaking resistance, we introduced the sid2-2 mutation, which disrupts ICS1-dependent SA biosynthesis 26 , 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 Δ hrcC 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 aao3 mutant, which is deficient in ABA biosynthesis 27 , 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. S3h), as reported previously 24 . 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 flagellin 21 . 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 Δ hrcC infiltration under moderate and high humidity. After 24 h acclimation to the respective humidity conditions, we measured FRK1 , NHL10 , and PHI-1 expression at 1 h post-inoculation (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 bak1-5 bkk1 cerk1 triple mutant (hereafter bbc ), which disrupts PRR activation mediated by the co-receptors BAK1, BKK1, and CERK1 28 (Supplementary Fig. 4b, c). The cyp707a1 cyp707a3 bbc quintuple mutant exhibited pronounced water-soaking following Pst Δ hrcC 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 Δ hrcC 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 up-regulated and 2054 down-regulated genes were identified as differentially expressed (DEGs: fold change > 2, FDR < 0.05) under high humidity compared with moderate humidity ( Fig. 3a and Supplementary Table 1). The DEGs identified at 0.5 h and 5 h showed limited overlap (up-regulated: 43.5% of 0.5 h and 30.5% of 5 h; down-regulated: 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 ). Download figure Open in new tab Fig. 3: CAMTA binding motifs are overrepresented in early-phase high humidity-induced genes. a-c , Transcriptome profiling of humidity-treated leaves by RNA-seq. Wild-type plants were exposed to moderate humidity (MH) or high humidity (HH) for the indicated time. a , Venn diagrams showing differentially expressed genes (DEGs) (fold change > 2; FDR < 0.05) at 0.5 h and 5 h after treatment. b , Principal component analysis (PCA) of transcriptomic data. c , K-means clustering of the 1000 most variable genes. d , Top 10 enriched cis -regulatory elements among high humidity-induced genes identified in the clusters shown in ( c ). K-means clustering of the 1000 most variable genes yielded 10 distinct clusters ( Fig. 3c and Supplementary Table 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 over-represented biological processes in these clusters, including ‘hypoxia response’ (Clusters 1 and 2), ‘jasmonate (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. Download figure Open in new tab Fig. 4: CAMTA3 drives CYP707A3 expression to promote resistance to bacterial water-soaking under high humidity. a , Schematic diagram of the promoter regions of CYP707A3 and CYP707A1 , highlighting the CGCG box. b , GUS expression driven by the CYP707A3 promoter, with or without CGCG boxes, in humidity-treated leaves. The indicated constructs were transiently expressed in NahG leaves via Agrobacterium infiltration. Infiltrated plants were maintained under moderate humidity (MH) for 24 h, then exposed to MH or high humidity (HH) for 0.5 h. Bars represent means ( n = 3). c , d , Expression levels of CYP707A3 and CYP707A1 in humidity-treated leaves of cngc ( c ) and camta3 ( d ) mutants. The indicated plants were exposed to MH or HH for 0.5 h. Bars represent means ( n = 3). e , ABA levels in humidity-treated leaves. The indicated plants were exposed to MH or HH for 2 h. Bars represent means ( n = 5). f , Stomatal aperture in humidity-treated leaves. The indicated plants were exposed to MH or HH for 1 h. Bars represent the median ( n = 220-247). g , Water-soaking in Pst Δ hrcC -inoculated leaves. The indicated plants were infiltrated with Pst Δ hrcC (OD 600 = 0.02) and maintained under HH for 1 day. The number of water-soaked leaves was pooled from two independent experiments. h , i , Bacterial growth in Pst Δ hrcC -inoculated leaves. The indicated plants were infiltrated with Pst Δ hrcC (OD 600 = 0.02) and maintained under HH ( h ) or MH ( i ) for 3 days. Bars represent means ( n = 6). Different letters indicate statistically significant differences ( P < 0.05; two-way ANOVA followed by Tukey’s test in b-f ; one-way ANOVA followed by Tukey’s test in h and i ). WT, wild-type; CAMTA3-A855V, a partial loss-of-function variant; dpi, days post-infiltration. Experiments in ( b - d ), ( f ), ( h ), and ( i ) were repeated twice with similar results. 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 Δ hrcC 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 Ca 2+ signaling to induce CYP707A3 expression CAMTA transcription factors are activated upon binding to Ca 2+ -CaM 29 . Given their putative role in high humidity-induced transcription, we hypothesized that elevated humidity triggers an increase in cytosolic Ca 2+ levels. Using 35S::GCaMP3 plants expressing the Ca 2+ biosensor GCaMP3, we detected a rapid rise in cytosolic Ca 2+ in leaf cells within 10 min of high humidity exposure (Supplementary Fig. 7a, b and Supplementary Movie 1). To test whether cytosolic Ca 2+ influx contributes to high humidity-induced CYP707A3 and CYP707A1 expression, we applied the Ca 2+ channel inhibitors LaCl 3 and GdCl 3 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 Ca 2+ elevation precedes CYP707A3 induction (Supplementary Fig. 7b, d). These findings indicate that humidity elevation triggers cytosolic Ca 2+ 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 Ca 2+ influx in diverse physiological processes 30 . To assess their role in high humidity-triggered Ca 2+ 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 CYP70A3 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 Ca 2+ influx required for high humidity-induced CYP707A3 expression, with partial contribution from CNGC9. Collectively, these findings suggest that CNGC2/4-mediated cytosolic Ca 2+ 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 31 – 34 . 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 activation 35 . 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 Δ hrc 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 Δ hrcC , and Pst Δ28E (lacking 28 T3S effectors) 36 . 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 Δ hrcC ( 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 Δ hrcC 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 Δ hrcC or Pst Δ28E, also induced 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. Download figure Open in new tab Fig. 5: Pst DC3000 blocks high humidity-driven plant transcriptome reprogramming via T3S effectors. a , Schematic overview of the experimental design for analyzing high humidity-responsive gene expression in Pst DC3000-inoculated leaves. b , Bacterial titers in wild-type (WT) leaves infiltrated with Pst DC3000 or Pst Δ hrcC (OD 600 = 0.02) at 8 hours post-infiltration (hpi) under moderate humidity (MH). Bars represent means ( n = 6). c , Expression levels of CYP707A3 , CYP707A1 , and NCED3 in Pst DC3000-inoculated leaves following humidity treatment. WT plants were infiltrated with the indicated Pst DC3000 strains (OD 600 = 0.02), maintained under MH for 8 h, and then exposed to MH or high humidity (HH) for 0.5 h. Bars represent means ( n = 3). d , e , Transcriptome profiling of Pst DC3000-inoculated leaves following humidity treatment as described in ( a ) and ( c ). d , Principal component analysis (PCA) of transcriptomic data. e , K-means-clustering of the 2000 most variable genes. Different letters indicate statistically significant differences ( P < 0.05; Welch’s t -test in b ; two-way ANOVA followed by Tukey’s test in c ). Experiments in ( b ) and ( c ) were repeated twice with similar results. 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 Δ hrcC , 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 Δ hrcC 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 Table 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 up-regulation 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 bacterial effector(s) responsible for suppressing CYP707A3 induction under high humidity, we analyzed multiple effector-deletion mutants of Pst DC3000 36 . 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 ). Download figure Open in new tab Fig. 6: T3S effector AvrPtoB suppresses CYP707A3 -mediated water-soaking resistance. a , Expression levels of CYP707A3 in Pst DC3000-inoculated leaves following humidity treatment. Wild-type (WT) plants were infiltrated with the indicated Pst DC3000 strains (OD 600 = 0.02), maintained under moderate humidity (MH) for 8 h, and then exposed to MH or high humidity (HH) for 0.5 h. Bars represent means ( n = 3). The box highlights the effectors retained in Pst Δ20E but absent in Pst Δ28E. b - d , Expression levels of CYP707A3 in humidity-treated leaves with or without induction of AvrPtoB ( b ), AvrPto ( c ), or AvrPtoB-F479A ( d ). The indicated plants were infiltrated with 0.1% ethanol (−DEX) or 10 μM DEX (+DEX), maintained under moderate humidity (MH) for 24 h, then exposed to MH or HH for 0.5 h. Bars represent means ( n = 3). e , ABA levels in humidity-treated leaves with or without AvrPtoB induction. The indicated plants were treated with DEX as described in ( b-d ), and then exposed to MH or HH for 2 h. Bars represent means ( n = 3). f , Stomatal aperture in humidity-treated leaves with or without AvrPtoB induction. The indicated were treated with DEX as described in ( b-d ), and then exposed to MH or HH for 1 h. Bars represent the median ( n = 253-367). g , Water-soaking in Pst Δ hrcC -inoculated leaves with or without AvrPtoB induction. The indicated plants were infiltrated with Pst Δ hrcC (OD 600 = 0.02) supplemented with −DEX or +DEX and maintained under HH for 1 day. The number of water-soaked leaves was pooled from two independent experiments. Different letters indicate statistically significant differences ( P < 0.05; two-way ANOVA followed by Tukey’s test). AvrPtoB-F479A, E3 ligase deficient variant; ND, not detected. Experiments in ( a-d ) and ( f ) were repeated twice with similar results. Given that in-planta expression of AvrPtoB has been associated with increased ABA levels through induction of NCED3 37 , we tested whether AvrPtoB contributes to CYP707A3 suppression. To this end, we generated transgenic plants expressing either AvrPtoB or its partially redundant counterpart, AvrPto 38 , 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-F479A 39 , 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 bacteria selectively target 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 ). The results indicate that AvrPtoB plays a critical role in counteracting CYP707A3 -mediated water-soaking resistance under high humidity. 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 Δ avrPtoB Δ avrPto ). Plants were shifted from moderate to high humidity at 2 hpi, following conditions conventionally used in previous studies 8 – 10 ( Fig. 7a and Supplementary Fig. S12a). 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. S12b). This suppression was absent with Pst Δ 28E, whereas with Pst Δ avrPtoB Δ avrPto it was impaired at 6 hpi but restored at 12-24 hpi (Supplementary Fig. S12b), 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 Δ avrPtoB Δ avrPto or Pst Δ 28E (Supplementary Fig S12c). This initial increase was followed by further accumulation at 12 and 24 hpi with Pst DC3000 and partially with Pst Δ avrPtoB Δ avrPto , but not with Pst Δ 28E. 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 is critical for subsequent water-soaking and bacterial proliferation. Download figure Open in new tab Fig. 7: Pst DC3000 employs AvrPtoB to suppress CYP707A3 expression and promote water-soaking under high humidity. a , Schematic overview of the experimental design for analyzing gene expression, ABA accumulation, water-soaking, and bacterial growth in Pst DC3000-inoculated leaves. b , Expression levels of CYP707A3 , CYP707A1 , and NCED3 in Pst DC3000-inoculated leaves at 6 hours post-infiltration (hpi) under HH. Wild-type (WT) plants were infiltrated with the indicated Pst DC3000 strains (OD 600 = 0.2), maintained under moderate humidity (MH) for 2 h, and then transferred to HH for 4 h. Bars represent means ( n = 3). c , ABA levels in Pst DC3000-inoculated leaves. WT plants were inoculated with the indicated Pst DC3000 strains as described in ( b ). Bars represent means ( n = 6). d , e , Representative images ( d ) and quantification ( e ) of water-soaking in Pst DC3000-inoculated leaves. WT and cyp707a1 cyp707a3 mutant plants were infiltrated with the indicated Pst DC3000 strains (OD 600 = 0.2), maintained under MH for 2 h, and then transferred to HH for 20-22 h. Bars represent the median ( n = 36; pooled from two independent experiments). f , g , Disease symptoms ( f ) and bacterial growth ( g ) in Pst DC3000-inoculated leaves. WT and cyp707a1 cyp707a3 mutant plants were infiltrated with the indicated Pst DC3000 strains (OD 600 = 0.0002), maintained under MH for 2 h, and then transferred to HH for 3 days. Bars represent means ( n = 6). Different letters indicate statistically significant differences ( P < 0.05; one-way ANOVA followed by Tukey’s test in b and c ; two-way ANOVA followed by Tukey’s test in e and g ). dpi, days post-infiltration. Experiments in ( b ), ( f ), and ( g ) were repeated twice with similar results. g , Working model of CYP707A3 -mediated water-soaking resistance and its suppression by Pst DC3000 via T3S effector AvrPtoB under high humidity. To dissect individual contributions of these effectors, we generated Pst DC3000 mutant strains lacking either AvrPtoB ( Pst Δ avrPtoB ) or AvrPto ( Pst Δ avrPto ) ( Fig. 7b and Supplementary Fig. S13a, 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 Δ avrPto , but permitted with Pst Δ avrPtoB and Pst Δ avrPtoB Δ avrPto ( Fig. 7b ). By contrast, enhanced CYP707A1 expression relative to Pst DC3000 was observed only with Pst Δ avrPtoB Δ avrPto ( Fig. 7b ), suggesting redundancy in CYP707A1 suppression. For NCED3 induction, Pst Δ avrPto resembled Pst DC3000, whereas Pst Δ avrPtoB and Pst Δ avrPtoB Δ avrPto 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 Δ avrPto , but not with Pst Δ avrPtoB or Pst Δ avrPtoB Δ avrPto ( Fig. 7c ). Together, these results indicate that AvrPtoB is the primary determinant of ABA accumulation under high humidity, acting through both 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 Δ avrPtoB Δ avrPto 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 Δ avrPtoB 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 , 40 – 42 . High humidity, often following prolonged rainfall, substantially exacerbates plant diseases caused by fungal and bacterial pathogens 8 , 43 . It is therefore plausible that plants pre-condition or activate defense responses in anticipation of these threats when sensing increased humidity. In this study, we demonstrate that the CNGC2/4/9-CAMTA3-CYP707A3 module is involved in response to elevated humidity, conferring bacterial resistance ( Fig. 7h ). Our findings suggest that high humidity acts 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 , 44 , 45 . Rainfall and submergence have also been linked to strengthened disease resistance in plants 34 , 46 . 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 23 , 24 , 47 , 48 , yet their modulation under varying environmental conditions remain under-explored. In Arabidopsis, CYP707A3 overexpression enhances transpiration rates 49 . ABA application suppresses systemic acquired resistance (SAR) inducers acting both upstream and downstream of SA accumulation 23 . Enhanced SA production under continuous light suppresses Pst DC3000-induced water-soaking without interfering with NCED3 induction 16 . 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 humidity 21 . Notably, however, genes associated with SA defense, such as SARD1 , CBP60g , and BCS1 50 – 52 , are induced under high humidity even in the absence of pathogen challenge (Cluster 3 in Fig 5e and Supplementary Table 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 humidity 17 and Pst DC3000 elevates ABA levels in guard cells 10 . 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 infection 53 , emphasizing the role of CYP707A-mediated ABA depletion as a core defense barrier limiting bacterial access to apoplastic water. We identify AvrPtoB as a major T3S effector suppressing this defense. Genetic analyses show that among the 36 T3S effectors, AvrPtoB has a prominent role in blocking high humidity-induced CYP707A3 expression ( Fig. 7h ). Earlier studies reported that AvrPtoB can target CYP707A3 and paralogs for proteasomal degradation when these proteins are transiently expressed in Nicotiana benthamiana 54 . By suppressing ABA catabolism and simultaneously promoting ABA biosynthesis via NCED3 induction 37 , 54 , AvrPtoB manipulates both arms of host ABA balance, facilitating stomatal closure and maintaining elevated ABA under high humidity. How AvrPtoB E3 ubiquitin ligase activity, known to act on membrane-localized immune receptors and transcription cofactors 55 – 60 , contributes to suppression of CYP707A3 and induction of NCED3 remains to be determined. Our work identifies CAMTA3 transcription regulator and CNGC2/4/9 Ca 2+ channels as key regulators of humidity-induced CYP707A3 expression, consistent with recent findings 61 . While the oomycete effector AVRblb2 has been shown to suppress CNGC activity 62 , the potential targeting of CNGCs by Pst DC3000 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 – 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-biding leucine-rich repeat receptor activation leading to SA defense activation and autoimmunity 35 , 66 , suggesting that CAMTAs might be targeted by pathogen effectors to counteract humidity-induced defenses. Future studies on AvrPtoB and other effectors, including HopAM1, HopM1, and AvrE, which promote ABA responses 9 , 42 , 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 Tables 4 and 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 KNO 3 , 0.67 mM MgSO 4 , 5.21 μM KH 2 PO 4 , 28.3 μM KCl, 15.5 μM H 3 BO 4 , 3.07 μM MnCl 2 , 0.28 μM ZnSO 4 , 0.11 μM CuSO 4 , 13.3 μg/L 80% molybdic acid (FUJIFILM Wako Pure Chemical, Cat# 134-03332), 1.67 mM Ca(NO 3 ) 2 , 46.3 μM Fe(III)-EDTA (Dojindo, Cat# 345-01245)]. For general cultivation, 2-week-old seedings 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: pGWB533 68 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×FLAG 69 into the AfeI site of pGWB501 68 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. 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 Tables 5 and 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 method 70 . 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 Table 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 described 71 . 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 Table 6. Chemicals The following stock solutions were prepared and stored at −30°C: LaCl 3 (FUJIFILM Wako Pure Chemical, Cat# 123-04222), 1 M in water; GdCl 3 (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 described 72 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 2,000 g for 10 min at 25°C, washed once with sterile water, and resuspended in sterile water to an OD 600 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 approximately 48 h before humidity treatment. Pst DC3000 inoculation assays The following Pst DC3000 strain were used in this study: WT, Δ hrcC 73 , Δ28E 36 , Δ avrPtoB Δ avrPto 38 , Δ avrPtoB , and Δ av r Pto . 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) K 2 HPO4, 1% (v/v) glycerol, 5 mM MgSO 4 ] supplemented with 50 μg/mL rifampicin and additional antibiotics as appropriate. Bacterial cells were collected by centrifugation at 2,000 g for 10 min at 25°C, washed once with sterile water, and resuspended in sterile water to the OD 600 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 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 Image J software ( https://imagej.net/ij/ ). For 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 (Nacalai Tesque, Cat# 15838-45), 2% (v/v) glycerol. 1.5% agar (Nacalai Tesque, Cat# 01162-15)] 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, 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, Cat# TP950). Gene expression levels were calculated using the ΔΔCt method, with normalization to ACTIN2 as an internal reference. Primers used for qPCR are listed in Supplementary Table 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 overnight at 4°C with the primary antibody solution under agitation, rinsed five times with deionized water, and washed once in TBS-T for 5 min at 25°C. 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. 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 described 74 . For hormone measurement, isotope-labeled internal standards were added at the initial extraction procedure. Plant hormones were analyzed by a 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) H 2 O (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%), 5–25 min (15%–40%), 25.1–27 min (100%), and 27.1–30 min (1%). The retention times for each compound were as follows: d 6 -SA (7.39), SA (7.46), d 6 -ABA (12.59), ABA (12.67), 13 C 6 -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: d 6 -SA (142.1/98.0, 18V), SA (137.0/93.4, 18V), d 6 -ABA (269.1/159.1, 8V), ABA (263.1/153.1, 8V), 13 C 6 -JA-Ile (328.2/136.1, 20V), and JA-Ile (322.2/130.1, 20V). 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 10× objective lens. Stomatal aperture was quantified using Image J software. Real-time cytosolic Ca 2+ imaging Real-time cytosolic Ca 2+ imaging of whole plants expressing GCaMP3 was performed as described previously 75 , 76 . 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 instruction. 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 instruction. PCR for library amplification was performed for 7 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 differs 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 8 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 reads 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, options are the same as above analysis. The obtained read counts were processed and analyzed using iDEP 2.01 ( https://bioinformatics.sdstate.edu/idep/ ) 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 Table 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. Data availability The raw RNA sequencing data have been submitted in the DDJB database under accession numbers DRR545835 to DRR545870. Author 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. Competing interests The authors declare no competing interests. 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.). Footnotes We added two co-authors for their contributions to the revision experiments; revised the manuscript throughout to improve readability; revised the Figures and Supplementary Figures to address the reviewers' comments; and updated the Methods, Acknowledgments, Author Contributions, References, and Supplementary Tables 5 and 6. References ↵ Xin , X. F. , Kvitko , B. & He , S. Y . Pseudomonas syringae: what it takes to be a pathogen . Nat. Rev. Microbiol . 16 , 316 – 328 ( 2018 ). 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Share Humidity-driven ABA depletion determines plant-pathogen competition for leaf water Shigetaka Yasuda , Akihisa Shinozawa , Yuanjie Weng , Arullthevan Rajendram , Taishi Hirase , Haruka Ishizaki , Ryuji Suzuki , Shioriko Ueda , Rahul Sk , Yumiko Takebayashi , Izumi Yotsui , Masatsugu Toyota , Masanori Okamoto , Yusuke Saijo bioRxiv 2025.01.04.631318; doi: https://doi.org/10.1101/2025.01.04.631318 Share This Article: Copy Citation Tools Humidity-driven ABA depletion determines plant-pathogen competition for leaf water Shigetaka Yasuda , Akihisa Shinozawa , Yuanjie Weng , Arullthevan Rajendram , Taishi Hirase , Haruka Ishizaki , Ryuji Suzuki , Shioriko Ueda , Rahul Sk , Yumiko Takebayashi , Izumi Yotsui , Masatsugu Toyota , Masanori Okamoto , Yusuke Saijo bioRxiv 2025.01.04.631318; doi: https://doi.org/10.1101/2025.01.04.631318 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7622) Biochemistry (17650) Bioengineering (13871) Bioinformatics (41881) Biophysics (21424) Cancer Biology (18566) Cell Biology (25461) Clinical Trials (138) Developmental Biology (13365) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22476) Immunology (17713) Microbiology (40331) Molecular Biology (17148) Neuroscience (88473) Paleontology (666) Pathology (2827) Pharmacology and Toxicology (4816) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)