STT3A is required for recognition of pathogen-derived sphingolipids in Arabidopsis

STT3A is required for recognition of pathogen-derived sphingolipids in Arabidopsis | 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 STT3A is required for recognition of pathogen-derived sphingolipids in Arabidopsis Seowon Choi , View ORCID Profile Motoki Shimizu , View ORCID Profile Akira Abe , View ORCID Profile Nobuaki Ishihama , Yuko Ishikawa , View ORCID Profile Daigo Takemoto , View ORCID Profile Ken Shirasu , View ORCID Profile Yoshitaka Takano , View ORCID Profile Ryohei Terauchi , View ORCID Profile Hiroaki Kato doi: https://doi.org/10.1101/2025.05.18.654772 Seowon Choi 1 Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University , Kyoto, 606-8502, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Motoki Shimizu 2 Iwate Biotechnology Research Center , Kitakami, 024-0003, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Motoki Shimizu Akira Abe 2 Iwate Biotechnology Research Center , Kitakami, 024-0003, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Akira Abe Nobuaki Ishihama 3 RIKEN Center for Sustainable Resource Science , Yokohama, 230-0045, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nobuaki Ishihama Yuko Ishikawa 1 Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University , Kyoto, 606-8502, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Daigo Takemoto 4 Graduate School of Bioagricultural Sciences, Nagoya University , Nagoya, 464-8610, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Daigo Takemoto Ken Shirasu 3 RIKEN Center for Sustainable Resource Science , Yokohama, 230-0045, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ken Shirasu Yoshitaka Takano 1 Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University , Kyoto, 606-8502, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yoshitaka Takano Ryohei Terauchi 1 Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University , Kyoto, 606-8502, Japan 2 Iwate Biotechnology Research Center , Kitakami, 024-0003, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ryohei Terauchi Hiroaki Kato 1 Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University , Kyoto, 606-8502, Japan 5 PRESTO, Japan Science and Technology Agency , Kawaguchi, 332-0012, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hiroaki Kato For correspondence: kato.hiroaki.6a{at}kyoto-u.ac.jp Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Plants recognize pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors, leading to the activation of pattern-triggered immunity in response to pathogen attack. P hytophthora infestans cer amide D (Pi-Cer D) is a sphingolipid from the oomycete pathogen P. infestans . Pi-Cer D is cleaved by the plant extracellular ceramidase NEUTRAL CERAMIDASE 2 (NCER2), and the resulting 9-methyl-branched sphingoid base is recognized by the plant receptor RESISTANT TO DFPM-INHIBITION OF ABSCISIC ACID SIGNALING 2 (RDA2) at the plasma membrane to transduce a defense signal. However, additional components are likely involved in sphingolipid recognition, which remain to be identified. Here, we employed a screen based on Lumi-Map technology to look for Arabidopsis ( Arabidopsis thaliana ) mutants with altered defense responses to Pi-Cer D. We identified three mutants showing diminished responses to Pi-Cer D and elf18, each carrying mutations in STAUROSPORIN AND TEMPERATURE SENSITIVE 3-LIKE A ( STT3A ), which encodes an oligosaccharyltransferase. The stt3a mutants exhibited higher susceptibility to the pathogen Colletotrichum higginsianum than the wild type and displayed alterations in NCER2 protein modifications. These findings suggest that STT3A contributes to plant immunity via post-translational modification of proteins including NCER2. INTRODUCTION Plants have evolved complex immune systems that detect and respond to pathogens in the environment ( Dodds et al., 2024 ). Plant immune responses begin with the recognition of pathogen-associated molecular patterns (PAMPs); these conserved molecules are found in a wide variety of microbes, including bacteria, fungi, and viruses ( Nejat and Mantri, 2017 ; Segonzac and Zipfel, 2011 ). These PAMPs are recognized by pattern recognition receptors (PRRs), which are typically localized at the plasma membrane and activate pattern-triggered immunity (PTI) ( Bigeard et al., 2015 ). PTI is a critical component of the plant immune system that acts as the first layer of defense, preventing pathogen entry and colonization of plant tissues ( Jones and Dangl, 2006 ). PTI initiates with the perception of a ligand by a PRR, followed by the association of the PRR with co-receptors such as BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) ( Chinchilla et al., 2009 ; Miya et al., 2007 ; Ngou et al., 2022 ). Despite extensive research into the interactions between PAMPs and PRRs, much about PTI signaling pathways remains unknown. Some PAMPs are sphingolipids; members of this class of lipids consist of a long-chain sphingoid base backbone linked to a fatty acid through an amide bond at the 2-amino group and to a polar head group at the C-1 position via an ester bond ( Heung et al., 2006 ). The 9-methyl-branched sphingoid base is absent from plants but present in fungi and oomycetes and is thus recognized by plants as a non-self molecular pattern to activate defense responses and deter pathogen attack ( Koga et al., 1998 ). Cerebrosides, a group of glycosphingolipids, also contain a 9-methyl-branched sphingoid base as part of their epitope structure and have been identified as elicitor molecules from the rice blast pathogen Pyricularia oryzae ( Koga et al., 1998 ; Umemura et al., 2000 ). Sphingolipids are important for membrane structure and as signaling molecules in both microbes and plants. Although sphingolipids in pathogenic fungi are known to play important roles in the initiation and development of infections afflicting humans, the metabolism and function of sphingolipids from plant pathogenic fungi remain to be fully explored ( Zhu et al., 2023 ). Phytophthora infestans ceramide D (Pi-Cer D) is a PAMP sphingolipid purified from the oomycete Phytophthora infestans that promotes resistance in plants ( Monjil et al., 2024 ). In Arabidopsis ( Arabidopsis thaliana ), Pi-Cer D is cleaved by the apoplastic ceramidase NEUTRAL CERAMIDASE 2 (NCER2), yielding a 9-methyl-branched sphingoid base that is recognized by the plasma-membrane-localized lectin receptor-like kinase RESISTANT TO DFPM-INHIBITION OF ABSCISIC ACID SIGNALING 2 (RDA2) ( Kato et al., 2022 ). However, further investigation is necessary to identify and characterize other factors involved in this recognition, including additional receptors, downstream signaling components, and the regulatory networks that modulate sphingolipid-triggered immune responses. Here, we used Lumi-Map ( Kato et al., 2020 ) to identify genes involved in sphingolipid recognition. We identified STAUROSPORIN AND TEMPERATURE SENSITIVE 3-LIKE A ( STT3A ) as an important component of Pi-Cer D recognition-mediated defense signaling and established its function in the sphingolipid response and pathogen resistance in Arabidopsis. RESULTS Mutants showing low responses to Pi-Cer D were identified The expression of the Arabidopsis defense gene WRKY33 is induced by diverse PAMPs, including the peptide flagellin 22 (flg22) and the sphingolipid Pi-Cer D ( Denoux et al. 2008 ; Kato et al. 2022 ; Zheng et al. 2006 ). Therefore, we used an Arabidopsis reporter line harboring the firefly luciferase gene ( LUC ) under the control of the WRKY33 promoter (p WRKY33-LUC ) ( Kato et al., 2022 ). We mutagenized the p WRKY33-LUC reporter line (the wild type, WT) with ethyl methane sulfonate and screened 10,000 M 2 seedlings in a previous study ( Kato et al., 2022 ). To select mutants for further analysis, we first confirmed the bioluminescence phenotype in response to Pi-Cer D treatment using M 3 lines and subsequently classified them based on their responses to other elicitors. Among the mutants, L-08, L-13, and L-51 showed low bioluminescence responses to Pi-Cer D treatment: L-08 and L-51 exhibited minimal responses to Pi-Cer D, whereas L-13 showed a response that was approximately half that observed in WT at its peak ( Fig. 1A ). We performed reverse-transcription quantitative PCR (RT-qPCR) to evaluate WRKY33 expression levels in WT and mutant seedlings at 0 and 1 h of treatment with Pi-Cer D. The relative transcript accumulation of WRKY33 was lower in all three mutants compared to WT specifically following Pi-Cer D treatment ( Fig. 1B ). Download figure Open in new tab Figure 1. Classification of mutants with altered responses to Pi-Cer D treatment. (A) Bioluminescence patterns of the three identified mutants under Pi-Cer D treatment (means ± SE). Seven-day-old wild type (p WRKY33-LUC reporter line; WT) and L-08, L-13, and L-51 mutant seedlings were treated with 0.17 µM Pi-Cer D. The bioluminescence of each seedling was monitored at the indicated time points starting at the onset of treatment using a continuous bioluminescence monitoring system. (B) Relative WRKY33 expression levels in response to Pi-Cer D treatment in WT and the three mutants, as determined by RT-qPCR. WRKY33 expression levels were measured in 7-day-old seedlings treated with 0.17 µM Pi-Cer D. (C) Bioluminescence responses of WT and the three mutants following treatment with one of four elicitors (means ± SE). Seven-day-old seedlings were treated with 0.17 µM Pi-Cer D, 0.1 µM flg22, 0.1 µM elf18, or 20 µg/ml chitin, and the bioluminescence of each seedling was monitored. The bioluminescence level in WT was set to 100%, to which the bioluminescence levels in the mutants were normalized. To classify the mutants showing low bioluminescence responses to Pi-Cer D treatment, we investigated the responses of these mutants to three additional PAMPs: flg22, elf18, and chitin ( Fig. 1C ). Flg22, a peptide derived from bacterial flagellin, elicits defense responses and is recognized by the receptor FLS2 ( Chinchilla et al., 2006 ). Elf18 is the N-terminal fragment of the bacterial elongation factor Tu recognized by the EF-TU RECEPTOR (EFR) ( Zipfel et al., 2006 ). Chitin is a component of the fungal cell wall that is recognized by the receptor CERK1 ( Miya et al. 2007 ). All three mutants, L-08, L-13, and L-51, exhibited diminished induction of bioluminescence in response to treatment with Pi-Cer D or elf18 compared to WT, whereas their responses to flg22 and chitin were similar to those of WT ( Fig. 1C ). Therefore, we focused on these three mutants for further analysis. We also examined the responses of the three mutants to the 9-methyl-branched sphingoid base (4E,8E)-9-methyl-4,8-sphingadienine (9Me-Spd), nlp24, 3-hydroxydecanoic acid (3-OH-FA), and cello-oligosaccharides (COS) (Supplementary Fig. S1). Nlp24 is a peptide derived from necrosis-and ethylene-inducing peptide 1-like proteins ( Albert et al., 2015 ). 3-OH-FA is perceived by the lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) ( Kutschera et al., 2019 ). COS are small carbohydrate molecules derived from the partial hydrolysis of cellulose that can elicit a broad-spectrum immune response against several pathogens ( Chen et al., 2021 ; Kongala and Kondreddy, 2023 ). The mutants showed lower induction of bioluminescence in response to treatment with 9Me-Spd or COS compared to WT, whereas they had normal responses to treatment with nlp24 or 3-OH-FA (Supplementary Fig. S1). The similar responses to various elicitors exhibited by the three mutants suggest that the causative genes of the three mutants are likely involved in the same pathway related to plant responses against Pi-Cer D, 9Me-Spd, elf18, and COS. Mutation of STT3A results in the low response to Pi-Cer D To identify the causal mutations of L-08, L-13, and L-51, we crossed each mutant line to the p WRKY33-LUC reporter line (the parental line of the mutants) to produce F 1 progeny, which were self-pollinated to obtain F 2 seeds. We then tested the F 2 segregating population derived from each mutant using Lumi-Map ( Kato et al. 2020 ) to select F 2 seedlings with the mutant phenotype of a reduced bioluminescence response to Pi-Cer D treatment. Following Pi-Cer D treatment, we chose 30 F 2 individuals showing low bioluminescence responses for genomic DNA extraction. For each F 2 population, we obtained equal amounts of leaf material from each individual showing a mutant phenotype, mixed the materials together, and extracted DNA from this mixture. We sequenced the DNA samples from each mutant line to detect single nucleotide polymorphisms (SNPs) relative to the p WRKY33-LUC reporter line, whose genome was also sequenced. When we plotted the SNP-index as a function of SNP position, we identified a single genomic region with SNP-index values close to 1 for all three mutants. Importantly, the three mutants displayed one large SNP-index peak at the identical position (6 Mb) on chromosome 5 ( Fig. 2 ). Download figure Open in new tab Figure 2. MutMap analysis of the L-08, L-13, and L-51 mutants. The highest SNP-index peak within a genomic region indicates the location of the causative mutation. An SNP-index peak around the 6-Mb region on chromosome 5 was observed in all three mutants: L-08, L-13, and L-51. Blue dots represent individual mutations in each mutant. The red line represents average SNP-index values across a 2-Mb sliding window with 10-kb increments. The green and yellow lines indicate the 95% and 99% confidence limits, respectively, of SNP-index values under the null hypothesis of an SNP-index = 0.5 assuming no linkage between the SNP and the causal mutation. Based on the p WRKY33-LUC reporter line reference genome, the region with the highest SNP-index value in the L-08, L-13, and L-51 mutants contains multiple candidate genes, with the gene STT3A harboring mutations in all three mutants (Supplementary Table S1). STT3A encodes an oligosaccharyltransferase that plays a pivotal role in the glycosylation of diverse proteins ( Cheng et al., 2022 ). The three mutants harbored different types of mutations: splicing junctions in L-08 and L-13, and an amino acid substitution in L-51 ( Fig. 3A and Supplementary Table S1). Genetic complementation of the three mutants with a wild-type genomic STT3A fragment led to the recovery of a normal bioluminescence response following Pi-Cer D treatment ( Fig. 3B ). These results demonstrate that the poor induction of the p WRKY33-LUC reporter to Pi-Cer D treatment in the L-08 ( stt3a-3 ), L-13 ( stt3a-4 ), and L-51 ( stt3a-5 ) mutants is caused by mutations in STT3A . Furthermore, we obtained the Arabidopsis line stt3a-2 , with a T-DNA insertion in STT3A ( Fig. 3A , Supplementary Fig. S2). This mutant showed compromised induction of WRKY33 expression after treatment with Pi-Cer D, as determined by RT-qPCR ( Fig. 3C ). These results suggest that STT3A is involved in defense signaling mediated by Pi-Cer D in Arabidopsis. Download figure Open in new tab Figure 3. STT3A is involved in the recognition of Pi-Cer D in plants. (A) STT3A gene structure and the locations of the causative mutations in the three mutants, stt3a-3 (L-08), stt3a-4 (L-13), and stt3a-5 (L-51). Black rectangles indicate exons; lines represent introns. The locations of causative mutations are represented by white triangles, with their mutation types indicated. (B) Genetic complementation of the three mutants with a wild-type copy of the STT3A gene. The wild-type STT3A gene was introduced into each mutant background. The p WRKY33-LUC reporter line (WT), the L-08, L-13, L-51 mutants, and complementation lines for each mutant were treated with 0.17 µM Pi-Cer D (means ± SE); bioluminescence was monitored using a continuous bioluminescence monitoring system. (C) Relative WRKY33 expression levels in Col-0 and the T-DNA insertion lines stt3a-2 and rda2-3 , as determined by RT-qPCR. Seven-day-old seedlings were used for the analysis. The seedlings were sampled at 0, 1, and 3 h of treatment with 0.17 µM Pi-Cer D. STT3A is required for defense signaling in Arabidopsis following sphingolipid recognition To determine the role of STT3A in defense signaling, we evaluated the expression of defense-related genes and the activation of Mitogen-Activated Protein Kinase (MAPK) signaling following Pi-Cer D treatment of WT, stt3a-3 (L-08), stt3a-4 (L-13), and stt3a-5 (L-51) seedlings. PENETRATION 2 ( PEN2 ) encodes a glycosyl hydrolase that localizes to peroxisomes and acts as a component of an inducible preinvasion resistance mechanism ( Lipka et al., 2005 ). CYTOCHROME P450 FAMILY 81 SUBFAMILY F2 ( CYP81F2 ) is involved in glucosinolate metabolism, and its loss-of-function mutants show impaired resistance to fungal attack ( Bednarek et al., 2009 ; Hunziker et al., 2020 ). ETHYLENE RESPONSIVE FACTOR 6 ( ERF6 ) encodes a central regulator of stress-induced inhibition of plant growth that is involved in the response to reactive oxygen species ( Li et al., 2025 ; Sewelam et al., 2013 ). RT-qPCR analysis detected lower expression of these three marker genes in the three stt3a mutant lines compared to WT following treatment with Pi-Cer D for 1 h or 3 h ( Fig. 4A ). The stt3a-2 T-DNA insertion line also showed compromised induction of PEN2 , CYP81F2 , and ERF6 expression in response to the same treatment (Supplementary Fig. S3A). Download figure Open in new tab Figure 4. STT3A is required for the defense response following the recognition of Pi-Cer D. (A) Relative expression levels of the defense-related genes PEN2 , CYP81F2 , and ERF6 as determined by RT-qPCR (means ± SE). Seven-day-old seedlings of the p WRKY33-LUC reporter line (WT), stt3a-3 (L-08), stt3a-4 (L-13), and stt3a-5 (L-51) were treated with 0.17 µM Pi-Cer D. The seedlings were sampled at 0, 1, and 3 h after treatment ( n = 3). (B) MAPK phosphorylation assay in WT and the three stt3a mutants. Seven-day-old seedlings were sampled at 0, 15, or 30 min of treatment with 0.17 µM Pi-Cer D. Phosphorylated MAPKs were visualized with anti-phospho-p44/p42 MAPK antibody. Equal protein loading was determined by staining the membrane with Coomassie brilliant blue (CBB). We also investigated MAPK activation in WT and the three mutants following the treatment of seedlings with Pi-Cer D ( Fig. 4B ). WT seedlings exhibited clear MAPK activation at 30 min after the onset of treatment. By contrast, all mutants exhibited diminished MAPK activation. Similarly, the stt3a-2 T-DNA insertion line showed lower MAPK activation compared to Col-0 (Supplementary Fig. S3B). As a negative control, the rda2-3 mutant exhibited no induction of marker gene expression and no MAPK activation. We also observed lower expression of the marker genes and lower MAPK activation when we used 9Me-Spd as the elicitor instead of Pi-Cer D (Supplementary Fig. S4, Supplementary Fig. S5). STT3A contributes to resistance against the fungal pathogen Colletotrichum higginsianum The lower expression of defense-related genes and the diminished activation of MAPK in the stt3a mutants suggested that STT3A is required for plant defense against pathogens. To test this hypothesis, we inoculated WT, stt3a-3 (L-08) stt3a-5 (L-51), and pad3-1 (susceptible control; Glazebrook and Ausubel, 1994 ), which lacks PHYTOALEXIN DEFICIENT 3 function, with the pathogenic fungus Colletotrichum higginsianum . The stt3a-3 and stt3a-5 mutants were more susceptible to C. higginsianum than WT, as evidenced by the size of lesions developing on leaves ( Fig. 5 ). Furthermore, the stt3a mutants were more susceptible to C. higginsianum than rda2-5 ( Fig. 5 ). These results suggest that STT3A affects the defense of Arabidopsis plants against fungal infection. Download figure Open in new tab Figure 5. The stt3a mutants show enhanced susceptibility to C. higginsianum . Six-week-old plants of the p WRKY33-LUC reporter line (WT), rda2-5 , stt3a-3 , stt3a-5 , and pad3-1 were inoculated with a C. higginsianum spore suspension containing 1.5 × 10 5 conidiospores/ml. Top, photograph showing typical lesions on the leaves of WT and the mutants following inoculation with C. higginsianum . Bottom, boxplots showing lesion area in WT and the stt3a mutants rda2-5 , and pad3-1 . Lesion size was measured 4 days after inoculation. Box bounds represent the lesion area, center line represents the median, and whiskers indicate the range of the maximum or minimum data. Different lowercase letters represent statistically significant differences ( p < 0.05), as revealed by Tukey’s test. The results were replicated in three separate experiments. STT3A affects the molecular size of NCER2 STT3A is involved in the post-translational modification of proteins ( Koiwa et al. 2003 ). We determined that the reduction in the bioluminescence response in the stt3a mutants was more pronounced in response to Pi-Cer D vs. 9Me-Spd treatment ( Fig. 1C and Supplementary Fig. S1), suggesting that STT3A exerts a greater influence on NCER2 than RDA2. The neutral ceramidase of fruit fly ( Drosophila melanogaster ) undergoes glycosylation, a common modification involved in protein secretion and stability (Yoshimura et al., 2022). Therefore, we focused on the abundance and molecular size of NCER2 in the WT and stt3a mutants. NCER2 comprises two distinct structural units; when the N-terminal neutral/alkaline non-lysosomal ceramidase domain is cleaved from the C-terminal portion of the protein, the two resulting fragments form a complex ( Kato et al., 2022 ). We performed immunoblotting with an antibody that recognizes the C-terminus of NCER2 (anti-NCER2) in WT, the stt3a mutants ( stt3a-3 and stt3a-5 ), and a ncer2 mutant ( ncer2-2 ; Kato et al., 2022 ), using total protein extracts and apoplast wash fluid, which should be enriched in secreted proteins ( Fig. 6 ). In WT, we detected a single band (of ∼50 kDa) corresponding to the expected molecular weight of the C-terminus of NCER2, whereas in ncer2-2 , no band was observed. By contrast, we observed a change in the mobility of NCER2 in stt3a-3 and stt3a-5 , indicating a difference in the molecular mass of NCER2. These results suggest that NCER2 is regulated by STT3A-mediated protein modification. Download figure Open in new tab Figure 6. Modification of NCER2 is influenced by STT3A . Five-week-old plants were used for immunoblotting (IB) of total proteins and apoplast wash fluid (AWF) in the p WRKY33-LUC reporter line (WT) and the stt3a -3, stt3a-5 , and necr2-2 mutants. NCER2 was detected with a specific antibody against NCER2. Equal protein loading was determined by staining the membrane with CBB. The results were replicated in three separate experiments. DISCUSSION Our Lumi-Map analysis revealed that STT3A is required for defense responses in Arabidopsis following the recognition of sphingolipids ( Figs. 1 – 3 ). STT3A is involved in protein N-glycosylation, which supports proper protein folding and function. For example, STT3A-mediated N-glycosylation is important for the function of cellulose biosynthesis enzymes ( Kang et al., 2008 ). In Arabidopsis stt3a mutants, defects in N-glycosylation result in insufficient glycosylation of β-glucosidase, an enzyme responsible for converting conjugated forms of the phytohormones abscisic acid and auxin into their active forms ( Jiao et al., 2020a ). STT3A also participates in temperature sensitivity, salinity tolerance, and responses to abiotic stress through increased stomatal density ( Jiao et al., 2020b ; Liu et al., 2018 ; Rips et al., 2014 ; Zhang et al., 2009 ). Although all three mutants analyzed harbor mutations in STT3A , their phenotypes in reporter assays and their defense responses differed ( Fig. 1 , Fig. 4 , and Supplementary Fig. S4). The stt3a-3 and stt3a-5 mutants showed the weakest defense responses, whereas stt3a-4 exhibited intermediate defense responses compared to WT ( Figs. 1 and 4 ). Since stt3a-3 and stt3a-4 are predicted to affect splicing, STT3A transcripts in these two mutants might be different from those of WT. STT3A in stt3a-5 harbors a G616D mutation that is likely located in the catalytic domain or the dolichol-binding lipid-exposed groove, both of which are essential for oligosaccharyltransferase activity, thus potentially impairing N-glycosylation efficiency ( Niu et al., 2020 ; Wild et al., 2018 ). Therefore, the mutation in stt3a-5 might inhibit the enzymatic activity of STT3A or affect its stability. NCER2 is a ceramidase located in the plant apoplast that converts microbial-derived ceramides into sphingoid bases ( Kato et al., 2022 ). We observed a band shift for C-terminus of NCER2 in apoplast fluid collected from the stt3a mutants ( Fig. 6 ), suggesting that NCER2 is post-translationally modified by STT3A . Our results suggest that NCER2 is a target of STT3A-mediated N-glycosylation. From the perspective of plant immunity, STT3A is known to maintain the stability and function of plasma membrane–localized PRRs including EFR and leucine-rich repeat receptor-like kinases (LRR-RLKs) via their N-glycosylation ( Häweker et al., 2010 ; Saijo et al., 2009 ). In this study, WRKY33 transcription in the three stt3a mutants was also insensitive to treatment with elf18 ( Fig. 1 ), confirming a role for STT3A in EFR regulation ( Häweker et al., 2010 ; Saijo et al., 2009 ). Moreover, in experiments with various elicitors, the stt3a mutants showed less pronounced responses to 9Me-Spd and COS (Supplementary Fig. S1). RDA2 recognizes 9Me-Spd and triggers PTI ( Kato et al., 2022 ). Therefore, RDA2 may also require modification by STT3A for its full function. Since we were not able to detect RDA2 in immunoblots, we did not examine its potential modifications but hope to explore this question in the future. Cellulose-derived COS, along with xylooligosaccharides (XOS), are recognized by leucine-rich repeat-malectin receptor kinases such as IMPAIRED GLYCAN PERCEPTION 1 (IGP1, also reported as CELLOOLIGOMER-RECEPTOR KINASE 1 [CORK1]), IGP3, and IGP4 ( Fernández-Calvo et al., 2024 ; Pring et al., 2023 ; Tseng et al., 2022 ); these receptors may also be targets of modification by STT3A. The stt3a mutants were less resistant to the fungal pathogen C. higginsianum than WT ( Fig. 5 ). C. higginsianum contains sphingolipids including 9Me-Spd; their recognition by RDA2 might be important for plant defense against this pathogen. The lower expression of PEN2 and CYP81F2 in the stt3a mutants following sphingolipid treatment ( Fig. 4 and Supplementary Fig. S4) may result in the compromised production and accumulation of antimicrobial compounds, leading to the lower resistance against filamentous fungi ( Fig. 5 ). Notably, the stt3a mutants were more susceptible to C. higginsianum than the rda2-5 mutant ( Fig. 5 ). This difference suggests that in addition to signaling mediated by NCER2 and RDA2, multiple pathways for the recognition of PAMPs and damage-associated molecular patterns are affected by mutations in STT3A ( Fig. 1 , Supplementary Fig. S1). In this study, we demonstrated that STT3A is involved in the recognition of sphingolipids and affects the molecular mass of NCER2. In addition, stt3a mutants showed a reduced response to 9-methyl-branched sphingoid base, suggesting that STT3A might also regulate RDA2 or its downstream signaling factors. Comparative glycoproteomics between WT and stt3a plants should help reveal the importance of N-glycosylation in PTI signaling. MATERIALS AND METHODS Plant materials and growth conditions All Arabidopsis ( Arabidopsis thaliana ) lines used in this study were in the Col-0 background. Seeds of the stt3a-2 (SALK_058814) and rda2-3 (SALK_143489C) mutants were obtained from the Arabidopsis Biological Resource Center (ABRC). The rda2-5 and ncer2-2 mutants were described previously ( Kato et al., 2022 ). All seeds were surface sterilized using a sodium hypochlorite solution and incubated at 4°C in the dark for 4 days prior to seed sowing. Seeds were plated on Murashige and Skoog (MS) ( Murashige and Skoog, 1962 ) liquid medium containing 0.5% (w/v) sucrose, B5 vitamin solution, and 2 mM MES (pH 5.7) and incubated at 23°C in the light (24-h photoperiod, 57 μmol m -2 s -1 ). For seed propagation, Arabidopsis plants were grown on MS agar medium (MS salt, 1.5% sucrose, B5 vitamin solution, 2 mM MES [pH 5.7], and 0.8% agar) for 10 to 14 days, transferred to soil, and growth under continuous light at 23°C (57 μmol m -2 s -1 ). Soil-grown Arabidopsis plants were grown under controlled conditions (23°C, 10-h photoperiod, 57 μmol m -2 s -1 ) Genotyping T-DNA insertion lines Genomic DNA was extracted from seedlings in sucrose lysis buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 300 mM sucrose. PCR products were obtained with KOD One PCR Master Mix (TOYOBO) and separated by electrophoresis on 1% (w/v) agarose gels. Left primer (LP) and border primer (BP) were used as forward primers, and right primer (RP) was used as the reverse primer (Supplementary Table S2) to identify lines homozygous for the respective T-DNA insertion. The PCR conditions were 98°C for denaturation, 55°C for annealing, and 68°C for extension. Elicitors and chemicals Pi-Cer D was purified from Phytophthora infestans as described by Monjil et al. (2024) . Other elicitors and chemicals were acquired or purchased from commercial sources: flg22, elf18, and nlp24 (Life Technologies, Tokyo, Japan); chitin (C9752, Sigma); (4E,8E)-9-methyl-4,8-sphingadienine (9Me-Spd, NS440901; Nagara Science); 3-hydroxy decanoic acid (3-OH-FA, 24613; Cayman). Cello-oligosaccharides (COS) were prepared via hydrolysis of cotton linters or microcrystalline cellulose (Avicel PH 101, Sigma-Aldrich, Burlington, MA, USA). Measurement of bioluminescence responses Surface-sterilized seeds were incubated at 4°C in the dark for 4 days and sown in the wells of 96-well microplates (Luminunc TM Plates White F96; Thermo Fisher Scientific) containing 150 µl liquid MS medium supplemented with 50 µM D-luciferin, potassium salt (Biosynth). Seed germination was conducted under continuous illumination (23°C, 57 μmol m -2 s -1 ). Following a 7-day incubation period, the seedlings were treated with elicitors (100-fold dilution), and the 96-well plates were sealed with a plate seal (100-THER-PLT, Excel Scientific) instead of a plastic cover. Bioluminescence from each well was measured automatically and immediately after elicitor addition using a bioluminescence monitoring system (model CL96-4; Churitsu Electric Corp.) with a robotic plate conveyor (model CI-08L; Churitsu Electric Corp.). Bioluminescence data were analyzed using the software provided with the instrument (SL00-01; Churitsu Electric Corp.). Generation of F 2 progeny and whole-genome sequencing F 1 progeny were generated by crossing the p WRKY33-LUC reporter line (W33-1B, the parental line of the mutants) ( Kato et al., 2022 ) with each of the mutants identified in the screen. All F 1 plants were subsequently self-pollinated to obtain F 2 seeds. The bioluminescence responses of the F 2 seedlings were tested using Pi-Cer D treatment as described above. F 2 seedlings with the mutant phenotype were selected and their genomic DNA extracted. The same quantity of genomic DNA from 30 F 2 seedlings with the mutant phenotype was combined to obtain a bulk (pooled) DNA sample for MutMap analysis. For whole-genome sequencing, genomic DNA samples were extracted from young (3-week-old) leaves using a DNeasy Plant Mini Kit (Qiagen). Sequencing libraries were prepared for the L-08, L-13, and L-51 mutant lines using an Illumina TruSeq DNA LT Sample Prep Kit (Illumina). All libraries were sequenced as paired-end 150-bp reads. The libraries were sequenced on a HiSeq High-Output system. The whole-genome sequencing data have been deposited in DDBJ BioProject under accession number PRJDB20249. MutMap analysis The p WRKY33-LUC reporter line reference sequence was constructed by replacing nucleotides in Col-0 with those of the p WRKY33 - LUC reporter line W33-1B ( Kato et al. 2022 ). Bulked segregant analysis, as implemented in MutMap ( Abe et al., 2012 ), was performed using the MutMap pipeline ( https://github.com/YuSugihara/MutMap ) ( Sugihara et al., 2022 ). The Arabidopsis Col-0 reference genome was downloaded from ftp://ftp.ensemblgenomes.org/pub/release-36/plants/fasta/Arabidopsis_thaliana/dna/Arabidopsis_thaliana.TAIR10.dna.toplevel.fa . Whole-genome sequencing of the three mutants identified 773 ± 334 (mean ± s.d.; range 398– 1,038) SNPs relative to the p WRKY33-LUC reporter parental line W33-1B. Plasmid construction and plant transformation To complement the stt3a mutants ( stt3a-3 , stt3a-4 , stt3a-5 ), a 10.7-kb genomic fragment containing the STT3A genomic coding region and its native promoter was amplified and inserted into pBIB-KAN at the SalI and SacI restriction sites using an In-Fusion HD Cloning Kit (Takara). The primer sequences used for plasmid construction are listed in Supplementary Table S2. The plasmids were introduced into Agrobacterium ( Agrobacterium tumefaciens ) strain GV3101::pMP90 by electroporation and used to transform Arabidopsis plants via the floral dip method ( Clough and Bent, 1998 ). T 1 seeds were collected from these T 0 plants, and T 1 seedlings were grown on MS medium containing kanamycin (50 μg/ml) and carbenicillin (150 μg/ml) to select transgene-positive T 1 plants. T 2 seeds were then collected from the positive T 1 plants and sown on MS medium containing kanamycin; T 2 lines showing a 3:1 segregation ratio were thought to contain a single T-DNA. T 3 plants homozygous for the transgene were obtained and used for subsequent analysis. MAPK activation assay Seedlings germinated from surface-sterilized seeds were grown in 96-well microplates in MS liquid medium for 7 days. Samples were obtained from sphingolipid-treated seedlings at 15 and 30 min of treatment (0.17 µM Pi-Cer D, 0.5 µM 9Me-Spd) using the same method used for screening. All samples were immediately frozen in liquid nitrogen. Proteins were extracted from the samples in extraction buffer (50 mM HEPES, pH 7.4, 5 mM EDTA, 0.5 mM EGTA, 50 mM β-glycerophosphate, 10 mM Na 3 VO 4 , 10 mM NaF, 2 mM DTT). MAPK activation was monitored by immunoblot analysis using an antibody that recognizes the dual phosphorylation of the activation loop of MAPK (pTEpY). Phospho-p44/42 MAPK (Erk1/2; Thr-202/Tyr-204, Cell Signaling Technology), and rabbit monoclonal antibodies were used to detect phosphorylated MAPK according to the manufacturer’s protocol (#9101, Cell Signaling Technology). Immunoblotting detection solution (ECL Prime Western Blotting Detection Reagents, Amersham) was used for signal detection. The PVDF membrane was stained with Coomassie Brilliant Blue (PageBlue Protein Staining Solution, Thermo Fisher Scientific) to verify equal protein loading. RNA extraction and RT-qPCR Seven-day-old seedlings were treated with sphingolipids, and samples were collected at the specified time points before being frozen in liquid nitrogen. Total RNA was extracted from the samples using an RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. Following RNA extraction, first-strand cDNA was synthesized using Takara PrimeScript RT Master Mix (Takara). Quantitative PCR was conducted using rTaq DNA polymerase (TAP201, TOYOBO) and EvaGreen Dye (31000, Biotium, Inc.) on a CFX Connect Real-Time System (BIO-RAD) with the primers listed in Supplementary Table S2. Relative gene expression levels were calculated using the ΔΔCt method, with ubiquitin-conjugating enzyme 21 ( UBC21 , At5g25760) ( Czechowski et al., 2005 ) serving as a reference for normalization. Pathogen inoculation assay The inoculation assay was performed as previously reported ( Singkaravanit-Ogawa et al., 2021 ) with some modifications. Colletotrichum higginsianum strain MAFF_305635 was cultured on potato dextrose agar (BD Biosciences, Franklin Lakes, NJ) at 24°C in the dark. For plant inoculation, four drops (5 µl per drop) of a spore suspension containing 1.5 × 10 5 conidiospores/ml were placed onto one leaf of a 5-to 6-week-old Arabidopsis plant without punching. The plants were covered with a transparent lid to maintain high humidity conditions and placed in a growth cabinet at 23°C under a 10-h light/14-h dark photoperiod until the day of observation (4 dpi). The inoculated leaves were excised and photographed after 4 days for analysis. The lesion size (area) was quantified using ImageJ ( https://imagej.net/ij/ ). The statistical significance of differences between means was determined using Tukey’s test. In the figures, different lowercase letters indicate significant differences ( p < 0.05). Production of the anti-NCER2 antibody A peptide from the C-terminus of NCER2 (ADVPPKSTFRR) with an N-terminal cysteine was chemically synthesized (Scrum). The antigen peptide was conjugated to a keyhole limpet hemocyanin carrier via SS-linkage through the N-terminal cysteine, and polyclonal antisera were raised in rabbits (Scrum). Anti-NCER2 antibodies were purified from the antisera by affinity chromatography using antigen-immobilized beads (HiTrap NHS-activated HP; GE Healthcare). Extraction of apoplast wash fluid and detection of NCER2 Apoplast wash fluid (AWF) was extracted from the samples as described by Gentzel et al. (2019) , with minor modifications. Extraction buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl) was introduced into leaves via syringe infiltration. The leaves were blotted dry with a paper towel, wrapped around a 1-ml pipette tip using Parafilm, and placed in a 15-ml conical tube. The tube was subjected to centrifugation at 1,000 × g for 3 min at room temperature using a swing rotor. AWF proteins were precipitated by acetone precipitation and resuspended in extraction buffer containing 1% SDS. The protein content of the AWF was quantified using the Bradford method, and a 10 µg equivalent of AWF proteins was separated by electrophoresis on a 12% (w/v) SDS acrylamide gel. Immunoblot analysis was performed with anti-NCER2 antibodies (1:2,000 dilution) and HRP-conjugated secondary antibodies (1:2,000 dilution). Accession numbers The accession numbers of the Arabidopsis genes mentioned in this article are as follows: STT3A (At5g19690), RDA2 (At1g11330), NCER2 (At2g38010), and WRKY33 (At2g38470). FUNDING Japan Science and Technology Agency (JST)-PRESTO, JPMJPR22D2 (H.K.) Japan Society for the Promotion of Science KAKENHI grant 23K20042, 24H00010 (R.T.) Japan Society for the Promotion of Science KAKENHI grant 25H00431 (Y.T.) AUTHOR CONTRIBUTIONS Conceptualization: SC, RT, HK Methodology: MS, AA, RT, HK Investigation: SC, NI, YI Visualization: SC, MS, AA Funding acquisition: YT, RT, HK Project administration: DT, YT, RT, HK Supervision: DT, KS, YT, RT, HK Writing – original draft: SC Writing – review & editing: SC, DT, YT, KS, RT, HK COMPETING INTERESTS The authors declare no conflicts of interest. Supplementary Information Download figure Open in new tab Supplementary Fig. S1. Bioluminescence responses of the three mutants to treatment with four elicitors. Seven-day-old wild type (p WRKY33-LUC reporter line; WT), L-08, L-13, and L-51 seedlings were treated with 0.5 µM (4E,8E)-9-methyl-4,8-sphingadienine (9Me-Spd), 0.1 µM nlp24, 1 µM 3-hydroxy fatty acid (3-OH-FA), or 20 µg/ml cello-oligosaccharides (COS). The bioluminescence level of the WT was set to 100%, to which the bioluminescence levels in the mutants were normalized. Download figure Open in new tab Supplementary Fig. S2. Confirmation of the T-DNA insertion in the stt3a-2 and rda2-3 mutant lines. Gel electrophoresis of the PCR products confirmed the genotypes of the T-DNA insertion lines and Col-0. LP and RP indicate the left primer and right primer of the respective gene, respectively; BP indicates the T-DNA border primer. Download figure Open in new tab Supplementary Fig. S3. Immune response analysis in the stt3a T-DNA insertion mutant treated with Pi-Cer D. (A) Relative expression levels of the defense-related genes PEN2 , CYP81F2 , and ERF6 as determined by RT-qPCR. Seven-day-old Col-0, stt3a-2 , and rda2-3 seedlings were treated with 0.17 µM Pi-Cer D, and sampled at 0, 1, or 3 h after treatment ( n = 3). (B) MAPK phosphorylation assay in Col-0, stt3a-2 , and rda2-3. Seven-day-old seedlings were sampled at 0, 15, or 30 min of treatment with 0.17 µM Pi-Cer D. Phosphorylated MAPKs were visualized with anti-phospho-p44/p42 MAPK antibody. Equal protein loading was determined by staining the membrane with Coomassie brilliant blue (CBB). Download figure Open in new tab Supplementary Fig. S4. Immune response analysis in the stt3a-3, stt3a-4, and stt3a-5 mutants treated with 9Me-Spd. (A) Relative expression levels of the defense-related genes PEN2 , CYP81F2 , and ERF6 as determined by RT-qPCR (means ± SE). Seven-day-old seedlings of the p WRKY33-LUC reporter line (WT), stt3a-3 (L-08), stt3a-4 (L-13), and stt3a-5 (L-51) were treated with 0.5 µM 9Me-Spd, and sampled at 0, 1, and 3 h after treatment ( n = 3). (B) MAPK phosphorylation assay in WT and each stt3a mutant. Seven-day-old seedlings were sampled at 0, 15, and 30 min of treatment with 0.5 µM 9Me-Spd. Phosphorylated MAPKs were visualized with anti-phospho-p44/p42 MAPK antibody. Equal protein loading was determined by staining the membrane with CBB. Download figure Open in new tab Supplementary Fig. S5. Immune response analysis in the stt3a T-DNA insertion mutant treated with 9Me-Spd. (A) Relative expression levels of the defense-related genes PEN2 , CYP81F2 , and ERF6 as determined by RT-qPCR. Seven-day-old seedlings of Col-0, stt3a-2 , and rda2-3 were treated with 0.5 µM 9Me-Spd, and sampled at 0, 1, and 3 h after treatment ( n = 3). (B) MAPK phosphorylation assay in Col-0, stt3a-2 , and rda2-3. Seven-day-old seedlings were sampled at 0, 15, and 30 min of treatment with 0.5 µM 9Me-Spd. Equal protein loading was determined by staining the membrane with CBB. View this table: View inline View popup Download powerpoint Supplementary Table S1. Candidate genes and polymorphisms identified in the high SNP-index intervals on Arabidopsis chromosome 5. View this table: View inline View popup Download powerpoint Supplementary Table S2. Primers used in this study. ACKNOWLEDGEMENTS We thank Y. Nakagawa (Kyoto University) for technical support. We also thank Prof. Kazuhito Kawakita and Makoto Ojika (Nagoya University, Japan) for valuable suggestions and for Pi-Cer D purification. Some of the computational analysis was performed on the National Institute of Genetics (NIG) supercomputer at the Research Organization of Information and Systems (ROIS), NIG, Tokyo, Japan. Funder Information Declared Japan Society for the Promotion of Science, https://ror.org/00hhkn466 , 23K20042 , 24H00010 , 25H00431 Japan Science and Technology Agency, https://ror.org/00097mb19 , JPMJPR22D2 REFERENCES ↵ Abe , A. , Kosugi , S. , Yoshida , K. , Natsume , S. , Takagi , H. , Kanzaki , H. , Matsumura , H. , Yoshida , K. , Mitsuoka , C. , Tamiru , M. , Innan , H. , Cano , L. , Kamoun , S. , and Terauchi , R . 2012 . Genome sequencing reveals agronomically important loci in rice using MutMap . Nat. Biotechnol . 30 : 174 – 178 . OpenUrl CrossRef PubMed ↵ Albert , I. , Böhm , H. , Albert , M. , Feiler , C.E. , Imkampe , J. , Wallmeroth , N. , Brancato , C. , Raaymakers , T.M. , Oome , S. , Zhang , H. , Krol , E. , Grefen , C. , Gust , A.A. , Chai , J. , Hedrich , R. , Ackerveken , G.V.D. , and Nürnberger , T . 2015 . An RLP23–SOBIR1–BAK1 complex mediates NLP-triggered immunity . Nat. Plants 1 : 15140 . OpenUrl CrossRef PubMed ↵ Bednarek , P. , Pislewska-Bednarek , M. , Svatoš , A. , Schneider , B. , Doubský , J. , Mansurova , M. , Humphry , M. , Consonni , C. , Panstruga , R. , Sanchez-Vallet , A. , Molina , A. , and Schulze-Lefert , P. 2009 . A glucosinolate metabolism pathway in living plant cells mediates broad-spectrum antifungal defense . Science 323 : 101 – 106 . OpenUrl Abstract / FREE Full Text ↵ Bigeard , J. , Colcombet , J. , and Hirt , H . 2015 . Signaling mechanisms in pattern-triggered immunity (PTI) . Mol. Plant 8 : 521 – 539 . OpenUrl CrossRef PubMed ↵ Chen , P. , Shrotri , A. , and Fukuoka , A . 2021 . Synthesis of cello-oligosaccharides by depolymerization of cellulose: A review . Appl. Catal. A Gen . 621 : 118177 . OpenUrl CrossRef ↵ Cheng , J. , Xia , L. , Hao , X. , Gan , F. , Bai , Y. , Zhang , C. , Mao , Y. , Zhu , Y. , Pu , Q. , Park , D.W. , Tavolari , S. , Mei , J. , Chen , Y. , Deng , S. , and Liu , L . 2022 . Targeting STT3A produces an anti-tumor effect in lung adenocarcinoma by blocking the MAPK and PI3K/AKT signaling pathway . Transl. Lung Cancer Res . 11 : 1089 – 1107 . OpenUrl PubMed ↵ Chinchilla , D. , Bauer , Z. , Regenass , M. , Boller , T. , and Felix , G . 2006 . The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception . Plant Cell 18 : 465 – 476 . OpenUrl Abstract / FREE Full Text ↵ Chinchilla , D. , Shan , L. , He , P. , de Vries , S. , and Kemmerling , B. 2009 . One for all: the receptor-associated kinase BAK1 . Trends Plant Sci . 14 : 535 – 541 . OpenUrl CrossRef PubMed Web of Science ↵ Clough , S.J. , and Bent , A.F . 1998 . Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana . Plant J . 16 : 735 – 743 . OpenUrl CrossRef PubMed Web of Science ↵ Czechowski , T. , Stitt , M. , Altmann , T. , Udvardi , M.K. , and Scheible , W.R . 2005 . Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis . Plant Physiol . 139 : 5 – 17 . OpenUrl Abstract / FREE Full Text ↵ Denoux , C. , Galletti , R. , Mammarella , N. , Gopalan , S. , Werck , D. , De Lorenzo , G. , Ferrari , S. , Ausubel , F.M. , and Dewdney , J. 2008 . Activation of defense response pathways by OGs and Flg22 elicitors in Arabidopsis seedlings . Mol. Plant 1 : 423 – 445 . OpenUrl CrossRef PubMed Web of Science ↵ Dodds , P.N. , Chen , J. , and Outram , M.A . 2024 . Pathogen perception and signaling in plant immunity . Plant Cell 36 : 1465 – 1481 . OpenUrl CrossRef PubMed ↵ Fernández-Calvo , P. , López , G. , Martín-Dacal , M. , Aitouguinane , M. , Carrasco-López , C. , González-Bodí , S. , Bacete , L. , Mélida , H. , Sánchez-Vallet , A. , and Molina , A . 2024 . Leucine rich repeat-malectin receptor kinases IGP1/CORK1, IGP3 and IGP4 are required for arabidopsis immune responses triggered by β-1, 4-D-Xylo-oligosaccharides from plant cell walls . Cell Surf . 11 : 100124. ↵ Gentzel , I. , Giese , L. , Zhao , W. , Alonso , A.P. , and Mackey , D . 2019 . A simple method for measuring apoplast hydration and collecting apoplast contents . Plant Physiol . 179 : 1265 – 1272 . OpenUrl Abstract / FREE Full Text ↵ Glazebrook , J. , and Ausubel , F.M . 1994 . Isolation of phytoalexin-deficient mutants of Arabidopsis thaliana and characterization of their interactions with bacterial pathogens . Proc. Natl. Acad. Sci. U.S.A . 91 : 8955 – 8959 . OpenUrl Abstract / FREE Full Text ↵ Heung , L.J. , Luberto , C. , and Del Poeta , M . 2006 . Role of sphingolipids in microbial pathogenesis . Infect. Immun . 74 : 28 – 39 . OpenUrl FREE Full Text ↵ Häweker , H. , Rips , S. , Koiwa , H. , Salomon , S. , Saijo , Y. , Chinchilla , D. , Robatzek , S. , and von Schaewen , A. 2010 . Pattern recognition receptors require N-glycosylation to mediate plant immunity . J. Biol. Chem . 285 : 4629 – 4636 . OpenUrl Abstract / FREE Full Text ↵ Hunziker , P. , Ghareeb , H. , Wagenknecht , L. , Crocoll , C. , Halkier , B.A. , Lipka , V. , and Schulz , A . 2020 . De novo indol-3-ylmethyl glucosinolate biosynthesis, and not long-distance transport, contributes to defence of Arabidopsis against powdery mildew . Plant Cell Environ . 43 : 1571 – 1583 . OpenUrl CrossRef ↵ Jiao , Q. , Niu , G. , Wang , F. , Dong , J. , Chen , T. , Zhou , C. , and Hong , Z . 2020a . N-glycosylation regulates photosynthetic efficiency of Arabidopsis thaliana . Photosynthetica 58 : 72 – 79 . OpenUrl CrossRef ↵ Jiao , Q. , Chen , T. , Niu , G. , Zhang , H. , Zhou , C. , and Hong , Z . 2020b . N-glycosylation is involved in stomatal development by modulating the release of active abscisic acid and auxin in Arabidopsis . J. Exp. Bot . 71 : 5865 – 5879 . OpenUrl CrossRef PubMed ↵ Jones , J.D. , and Dangl , J.L . 2006 . The plant immune system . Nature 444 : 323 – 329 . OpenUrl CrossRef PubMed Web of Science ↵ Kang , J.S. , Frank , J. , Kang , C.H. , Kajiura , H. , Vikram , M. , Ueda , A. , Kim , S. , Bahk , J.D. , Triplett , B. , Fujiyama , K. and Lee , S.Y. , von Schaewen , A. , and Koiwa , H. 2008 . Salt tolerance of Arabidopsis thaliana requires maturation of N-glycosylated proteins in the Golgi apparatus . Proc. Natl. Acad. Sci. U.S.A . 105 : 5933 – 5938 . OpenUrl Abstract / FREE Full Text ↵ Kato , H. , Onai , K. , Abe , A. , Shimizu , M. , Takagi , H. , Tateda , C. , Utsushi , H. , Singkarabanit-Ogawa , S. , Kitakura , S. , Ono , E. , Zipfel , C. , Takano , Y. , Ishiura , M. , and Terauchi , R . 2020 . Lumi-Map, a real-time luciferase bioluminescence screen of mutants combined with MutMap, reveals Arabidopsis genes involved in PAMP-triggered immunity . Mol. Plant-Microbe Interact . 33 : 1366 – 1380 . OpenUrl CrossRef PubMed ↵ Kato , H. , Nemoto , K. , Shimizu , M. , Abe , A. , Asai , S. , Ishihama , N. , Matsuoka , S. , Daimon , T. , Ojika , M. , Kawakita , K. , Onai , K. , Shirasu , K. , Yoshida , M. , Ishiura , M. , Takemoto , D. , Takano , Y. , and Terauchi , R . 2022 . Recognition of pathogen-derived sphingolipids in Arabidopsis . Science 376 : 857 – 860 . OpenUrl CrossRef PubMed ↵ Koga , J. , Yamauchi , T. , Shimura , M. , Ogawa , N. , Oshima , K. , Umemura , K. , Kikuchi , M. , and Ogasawara , N . 1998 . Cerebrosides A and C, sphingolipid elicitors of hypersensitive cell death and phytoalexin accumulation in rice plants . J. Biol. Chem . 273 : 31985 – 31991 . OpenUrl Abstract / FREE Full Text ↵ Koiwa , H. , Li , F. , McCully , M.G. , Mendoza , I. , Koizumi , N. , Manabe , Y. , Nakagawa , Y. , Zhu , J. , Rus , A. , Pardo , J.M. , Bressan , R.A. , and Hasegawa , P.M . 2003 . The STT3a subunit isoform of the Arabidopsis oligosaccharyltransferase controls adaptive responses to salt/osmotic stress . Plant Cell 15 : 2273 – 2284 . OpenUrl Abstract / FREE Full Text ↵ Kongala , S.I. , and Kondreddy , A . 2023 . A review on plant and pathogen derived carbohydrates, oligosaccharides and their role in plant’s immunity . Carbohydr. Polym . 6 : 100330 . OpenUrl ↵ Kutschera , A. , Dawid , C. , Gisch , N. , Schmid , C. , Raasch , L. , Gerster , T. , Schäffer , M. , Smakowska-Luzan , E. , Belkhadir , Y. , Vlot , A.C. , Chandler , C.E. , Schellenberger , R. , Schwudke , D. , Ernst , R.L. , Dorey , S. , Hückelhoven , R. , Hofmann , T. , and Ranf , S . 2019 . Bacterial medium-chain 3-hydroxy fatty acid metabolites trigger immunity in Arabidopsis plants . Science 364 : 178 – 181 . OpenUrl Abstract / FREE Full Text ↵ Li , T. , Peng , Z. , Kangxi , D. , Inzé , D. , and Dubois , M . 2025 . ETHYLENE RESPONSE FACTOR6, A central regulator of plant growth in response to stress . Plant Cell Environ . 48 : 882 – 892 . OpenUrl CrossRef ↵ Lipka , V. , Dittgen , J. , Bednarek , P. , Bhat , R. , Wiermer , M. , Stein , M. , Landtag , J. , Brandt , W. , Rosahl , S. , Scheel , D. , Llorente , F. , Molina , A. , Parker , J. , Somerville , S. , and Schulze-Lefert , P . 2005 . Pre-and postinvasion defenses both contribute to nonhost resistance in Arabidopsis . Science 310 : 1180 – 1183 . OpenUrl Abstract / FREE Full Text ↵ Liu , C. , Niu , G. , Zhang , H. , Sun , Y. , Sun , S. , Yu , F. , Lu , S. , Yang , Y. , Li , J. , and Hong , Z . 2018 . Trimming of N-glycans by the Golgi-localized α-1, 2-mannosidases, MNS1 and MNS2, is crucial for maintaining RSW2 protein abundance during salt stress in Arabidopsis . Mol. Plant 11 : 678-690. ↵ Miya , A. , Albert , P. , Shinya , T. , Desaki , Y. , Ichimura , K. , Shirasu , K. , Narusaka , Y. , Kawakami , N. , Kaku , H. , and Shibuya , N . 2007 . CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis . Proc. Natl. Acad. Sci. U.S.A . 104 : 19613 – 19618 . OpenUrl Abstract / FREE Full Text ↵ Monjil , M.S. , Kato , H. , Ota , S. , Matsuda , K. , Suzuki , N. , Tenhiro , S. , Tatsumi , A. , Pring , S. , Miura , A. , Camagna , M. , Suzuki , T. , Tanaka , A. , Terauchi , R. , Sato , I. , Chiba , S. , Kawakita , K. , Ojika , M. , and Takemoto , D . 2024 . Two structurally different oomycete lipophilic microbe-associated molecular patterns induce distinctive plant immune responses . Plant Physiol . 196 : 479 – 494 . OpenUrl CrossRef PubMed ↵ Murashige , T. , and Skoog , F . 1962 . A revised medium for rapid growth and bioassays with tobacco tissue cultures . Physiol. Plant . 15 : 473 – 497 . OpenUrl CrossRef ↵ Nejat , N. , and Mantri , N . 2017 . Plant immune system: crosstalk between responses to biotic and abiotic stresses the missing link in understanding plant defence . Curr. Issues Mol. Biol . 23 : 1 – 16 . OpenUrl CrossRef PubMed ↵ Ngou , B.P.M. , Jones , J.D. , and Ding , P . 2022 . Plant immune networks . Trends Plant Sci . 27 : 255 – 273 . OpenUrl CrossRef PubMed ↵ Niu , G. , Shao , Z. , Liu , C. , Chen , T. , Jiao , Q. , and Hong , Z . 2020 . Comparative and evolutionary analyses of the divergence of plant oligosaccharyltransferase STT3 isoforms . FEBS Open Bio 10 : 468 – 483 . OpenUrl CrossRef PubMed ↵ Pring , S. , Kato , H. , Imano , S. , Camagna , M. , Tanaka , A. , Kimoto , H. , Chen , P. , Shrotri , A. , Kobayashi , H. , Fukuoka , A. , Saito , M. , Suzuki , T. , Terauchi , R. , Sato , I. , Chiba , S. , and Takemoto , D . 2023 . Induction of plant disease resistance by mixed oligosaccharide elicitors prepared from plant cell wall and crustacean shells . Physiol. Plant . 175 : 14052 . OpenUrl CrossRef ↵ Rips , S. , Bentley , N. , Jeong , I.S. , Welch , J.L. , von Schaewen , A. , and Koiwa , H. 2014 . Multiple N-glycans cooperate in the subcellular targeting and functioning of Arabidopsis KORRIGAN1 . Plant Cell 26 : 3792 – 3808 . OpenUrl Abstract / FREE Full Text ↵ Saijo , Y. , Tintor , N. , Lu , X. , Rauf , P. , Pajerowska-Mukhtar , K. , Häweker , H. , Dong , X. , Robatzek , S. , and Schulze-Lefert , P. 2009 . Receptor quality control in the endoplasmic reticulum for plant innate immunity . EMBO J . 28 : 3439 – 3449 . OpenUrl Abstract / FREE Full Text ↵ Segonzac , C. , and Zipfel , C . 2011 . Activation of plant pattern-recognition receptors by bacteria . Curr. Opin. Microbiol . 14 : 54 – 61 . OpenUrl CrossRef PubMed ↵ Sewelam , N. , Kazan , K. , Thomas-Hall , S.R. , Kidd , B.N. , Manners , J.M. , and Schenk , P.M . 2013 . Ethylene response factor 6 is a regulator of reactive oxygen species signaling in Arabidopsis . PLoS One 8 : e70289 . OpenUrl CrossRef PubMed ↵ Singkaravanit-Ogawa , S. , Kosaka , A. , Kitakura , S. , Uchida , K. , Nishiuchi , T. , Ono , E. , Fukunaga , S. , and Takano , Y. 2021 . Arabidopsis CURLY LEAF functions in leaf immunity against fungal pathogens by concomitantly repressing SEPALLATA3 and activating ORA59 . Plant J . 108 : 1005 – 1019 . OpenUrl CrossRef PubMed ↵ Sugihara , Y. , Young , L. , Yaegashi , H. , Natsume , S. , Shea , D.J. , Takagi , H. , Booker , H. , Innan , H. , Terauchi , R. , and Abe , A . 2022 . High-performance pipeline for MutMap and QTL-seq . PeerJ 10 : e13170 . OpenUrl CrossRef PubMed ↵ Tseng , Y.H. , Scholz , S.S. , Fliegmann , J. , Krüger , T. , Gandhi , A. , Furch , A.C. , Kniemeyer , O. , Brakhage , A.A. , and Oelmüller , R . 2022 . CORK1, a LRR-malectin receptor kinase, is required for cellooligomer-induced responses in Arabidopsis thaliana . Cells 11 : 2960 . OpenUrl CrossRef ↵ Umemura , K. , Ogawa , N. , Yamauchi , T. , Iwata , M. , Shimura , M. , and Koga , J . 2000 . Cerebroside elicitors found in diverse phytopathogens activate defense responses in rice plants . Plant Cell Physiol . 41 : 676 – 683 . OpenUrl CrossRef PubMed Web of Science ↵ Wild , R. , Kowal , J. , Eyring , J. , Ngwa , E.M. , Aebi , M. , and Locher , K.P . 2018 . Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation . Science 359 : 545 – 550 . OpenUrl Abstract / FREE Full Text Yoshimura , Y. , Okino , N. , Tani , M. , and Ito , M . 2002 . Molecular cloning and characterization of a secretory neutral ceramidase of Drosophila melanogaster . J. Biochem . 132 : 229 – 236 . OpenUrl CrossRef PubMed Web of Science ↵ Zhang , M. , Henquet , M. , Chen , Z. , Zhang , H. , Zhang , Y. , Ren , X. , Van Der Krol , S. , Gonneau , M. , Bosch , D. , and Gong , Z. 2009 . LEW3, encoding a putative α-1, 2-mannosyltransferase (ALG11) in N-linked glycoprotein, plays vital roles in cell-wall biosynthesis and the abiotic stress response in Arabidopsis thaliana . Plant J . 60 : 983 – 999 . OpenUrl CrossRef PubMed Web of Science ↵ Zheng , Z. , Qamar , S.A. , Chen , Z. , and Mengiste , T . 2006 . Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens . Plant J . 48 : 592 – 605 . OpenUrl CrossRef PubMed Web of Science ↵ Zhu , X.M. , Li , L. , Bao , J.D. , Wang , J.Y. , Daskalov , A. , Liu , X.H. , Poeta , M.D. , and Lin , F.C . 2023 . The biological functions of sphingolipids in plant pathogenic fungi . PLoS Pathog . 19 : e1011733 . OpenUrl CrossRef PubMed ↵ Zipfel , C. , Kunze , G. , Chinchilla , D. , Caniard , A. , Jones , J.D. , Boller , T. , and Felix , G . 2006 . Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation . Cell 125 : 749 – 760 . 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Share STT3A is required for recognition of pathogen-derived sphingolipids in Arabidopsis Seowon Choi , Motoki Shimizu , Akira Abe , Nobuaki Ishihama , Yuko Ishikawa , Daigo Takemoto , Ken Shirasu , Yoshitaka Takano , Ryohei Terauchi , Hiroaki Kato bioRxiv 2025.05.18.654772; doi: https://doi.org/10.1101/2025.05.18.654772 Share This Article: Copy Citation Tools STT3A is required for recognition of pathogen-derived sphingolipids in Arabidopsis Seowon Choi , Motoki Shimizu , Akira Abe , Nobuaki Ishihama , Yuko Ishikawa , Daigo Takemoto , Ken Shirasu , Yoshitaka Takano , Ryohei Terauchi , Hiroaki Kato bioRxiv 2025.05.18.654772; doi: https://doi.org/10.1101/2025.05.18.654772 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 (7643) Biochemistry (17717) Bioengineering (13910) Bioinformatics (42016) Biophysics (21477) Cancer Biology (18628) Cell Biology (25536) Clinical Trials (138) Developmental Biology (13392) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22530) Immunology (17755) Microbiology (40437) Molecular Biology (17200) Neuroscience (88704) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4832) Physiology (7657) Plant Biology (15171) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9828) Zoology (2272)