Pantoea ananatis -triggered systemic resistance requires root sensing through the LORE receptor kinase in Arabidopsis

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View ORCID Profile Romain Schellenberger , Célia Borrego , View ORCID Profile Sandra Villaume , View ORCID Profile Jean-François Guise , View ORCID Profile Qassim Esmaeel , View ORCID Profile Marie-Christine Groleau , View ORCID Profile Maude Cloutier , View ORCID Profile Charles Gauthier , View ORCID Profile Sandrine Dhondt-Cordelier , View ORCID Profile Florence Mazeyrat-Gourbeyre , View ORCID Profile Fabienne Baillieul , View ORCID Profile Stefanie Ranf , View ORCID Profile Eric Déziel , View ORCID Profile Aziz Aziz , View ORCID Profile Stéphan Dorey doi: https://doi.org/10.1101/2025.11.18.688997 Simon Duchateau 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Simon Duchateau For correspondence: simon.duchateau.urca{at}gmail.com aziz.aziz{at}univ-reims.fr stephan.dorey{at}univ-reims.fr Jérôme Crouzet 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jérôme Crouzet Sylvain Cordelier 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sylvain Cordelier Matthieu Touchard 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Matthieu Touchard Romain Schellenberger 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Romain Schellenberger Célia Borrego 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sandra Villaume 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sandra Villaume Jean-François Guise 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jean-François Guise Qassim Esmaeel 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Qassim Esmaeel Marie-Christine Groleau 2 INRS-Centre Armand-Frappier Santé Biotechnologie , Laval, Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Marie-Christine Groleau Maude Cloutier 2 INRS-Centre Armand-Frappier Santé Biotechnologie , Laval, Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maude Cloutier Charles Gauthier 2 INRS-Centre Armand-Frappier Santé Biotechnologie , Laval, Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Charles Gauthier Sandrine Dhondt-Cordelier 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sandrine Dhondt-Cordelier Florence Mazeyrat-Gourbeyre 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Florence Mazeyrat-Gourbeyre Fabienne Baillieul 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Fabienne Baillieul Stefanie Ranf 3 Department of Biology, Faculty of Science and Medicine, University of Fribourg , Fribourg, 1700, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stefanie Ranf Eric Déziel 2 INRS-Centre Armand-Frappier Santé Biotechnologie , Laval, Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Eric Déziel Aziz Aziz 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aziz Aziz For correspondence: simon.duchateau.urca{at}gmail.com aziz.aziz{at}univ-reims.fr stephan.dorey{at}univ-reims.fr Stéphan Dorey 1 University of Reims Champagne-Ardenne , RIBP-USC INRAE 1488, 51100 Reims, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stéphan Dorey For correspondence: simon.duchateau.urca{at}gmail.com aziz.aziz{at}univ-reims.fr stephan.dorey{at}univ-reims.fr Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Systemic immunity induced by beneficial bacteria helps plants to cope with subsequent pathogen attacks. However, how these bacteria are sensed by plants and how this sensing results in a systemic response is not well understood. Recently, the Brassicaceae LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) receptor kinase was identified as the direct sensor of bacterial medium-chain-length 3-hydroxy fatty acids (mc-3-OH-FAs) and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), which activate systemic Brassicaceae immunity. Here, we show that the bacterium Pantoea ananatis BRT175, which produces HAAs, can colonize Arabidopsis thaliana roots independently of the production of these compounds. P. ananatis triggers induced systemic resistance (ISR) to the necrotrophic pathogen Botrytis cinerea , but not to the hemibiotrophic pathogen Pseudomonas syringae pv. tomato . Importantly, this ISR can be mimicked by HAAs or 3-hydroxy-decanoic acid (3-OH-C 10 ). Both P. ananatis -triggered and 3-OH-C 10 -triggered ISR against B. cinerea are mediated by LORE sensing, involve salicylic acid, jasmonic acid and ethylene signaling pathways, and activate the expression of similar defense genes in infected leaves. Thus, 3-OH-C 10 and HAAs are perceived by a pattern recognition receptor (PRR) in the root and activate a local immune response in roots as well as systemic immunity in leaves. Our study demonstrates that these lipidic microbe-associated molecular patterns (MAMPs), produced by beneficial microorganisms, are necessary and sufficient to trigger rhizobacteria-driven ISR. It further highlights the role of these MAMPs in differentially activating systemic resistance against biotrophic and necrotrophic pathogens. Introduction Plants face numerous pathogenic threats in their natural environment. Throughout their evolutionary history, they have developed a defense system to limit infections and diseases ( Finkel et al . 2017 ; Yu et al . 2019 ). Mutualistic bacteria in the rhizosphere can trigger systemic resistance to pathogens ( Pieterse et al . 2014 ; Vlot et al . 2021 ). The term ‘induced systemic resistance (ISR)’ refers to the resistance expressed in plant tissues distant from those initially in contact with the pathogen ( De Kesel et al . 2021 ). Most beneficial bacteria inducing ISR are plant growth-promoting rhizobacteria, often belonging to genera such as Bacillus, Pseudomonas, Enterobacter, Klebsiella, Azospirillum, Paenibacillus , and Pantoea spp. ( Bhat et al . 2022 ; Zhu et al . 2022 ). In plants, activation of an immune response first relies on the perception of specific patterns, including Microbe-Associated Molecular Patterns (MAMPs), that are usually sensed by cell-surface pattern recognition receptors (PRRs), including receptor-like kinases (RLKs) or receptor-like kinase proteins (RLPs) ( Boutrot and Zipfel, 2017 ; Ngou et al . 2022a ; Zhang et al . 2023 ; Dodds et al . 2024 ). This MAMP-triggered immunity restricts pathogen proliferation, but also excessive colonization by beneficial microorganisms ( Zamioudis and Pieterse, 2012 ; Wan et al . 2019 ; Ngou et al . 2022b ). Although MAMPs from pathogenic as well as beneficial bacteria, like flagellin/flg22 or elf18, are recognized at the root level ( Millet et al . 2010 ; Ranf et al . 2011 ; Wyrsch et al . 2015 ; Stringlis et al . 2018 ; Zhou et al . 2020 ), the interplay between MAMPs perception in roots and ISR remains poorly understood. Moreover, the involvement of specific PRRs in triggering ISR against pathogens with different lifestyles is not well documented. Following perception of microbes, ISR activation is regulated by hormonal signaling balance ( Vlot et al . 2021 ). Roles of salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) pathways, three of the key phytohormones involved in plant immunity, are still under debate, depending on the triggering microorganism, plant species and pathogens ( Bürger and Chory, 2019 ). Some beneficial strains of Pantoea are well-known for associating with plants ( Coutinho and Venter, 2009 ) and for activating ISR ( Walterson and Stavrinides, 2015 ; Duchateau et al . 2024 ). For instance, Pantoea agglomerans PTA-AF2 triggers ISR in grapevine against the fungus Botrytis cinerea ( Trotel-Aziz et al . 2008 ; Verhagen et al . 2011 ), while Pantoea ananatis PS27 promotes growth and ISR against Xanthomonas axonopodis pv. vesicatoria in pepper ( Kang et al . 2007 ). Strain BRT175 of P. ananatis is an epiphytic isolate from strawberries that produces effective antibiotics against the fire blight pathogen Erwinia amylovora ( Smith et al . 2013 ). This strain synthesizes 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) as constitutive bricks of ananatoside glycolipids, a group of amphiphilic biosurfactants secreted by the bacterium ( Smith et al . 2016 ; Gauthier et al . 2019 ). HAAs are synthesized by dimerization of ( R )-3-hydroxyalkanoyl-CoA, derived from medium-chain 3-hydroxy fatty acids (mc-3-OH-FAs), through the RhlA enzyme ( Abdel-Mawgoud et al . 2014 ). HAAs and mc-3-OH-FAs are perceived by the Brassicaceae S-domain receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) ( Kutschera et al . 2019 ; Schellenberger et al . 2021 ). Sensing of these ligands by LORE triggers a canonical immune signature, leading to enhanced resistance against Pseudomonas syringae pv. tomato DC3000 ( Pst ) in Arabidopsis thaliana (thereafter Arabidopsis) leaves ( Kutschera et al . 2019 ; Schellenberger et al . 2021 ). Interestingly, plants lacking LORE orthologs, like tomato, a Solanaceae , can gain mc-3-OH-FA immune sensing upon expression of At LORE ( Eschrig et al . 2024 ). Here, we report that colonization of Arabidopsis roots by P. ananatis results in ISR against the necrotrophic pathogen B. cinerea . Although HAA synthesis is not a relevant trait for root colonization by P. ananatis , we demonstrate that HAAs and especially mc-3-OH-FAs mimic Pantoea -induced resistance. We show that P. ananatis -triggered and mc-3-OH-FA-triggered ISRs are mediated by the PRR LORE. Both ISRs require SA, JA and ET pathways and activate similar defense mechanisms in response to B. cinerea . Our data highlight the direct involvement of the PRR LORE and mc-3-OH-FA MAMP sensing in rhizobacteria-triggered ISR. Results P. ananatis colonizes Arabidopsis roots independently of HAA synthesis We first assessed the ability of P. ananatis strain BRT175 to colonize Arabidopsis roots. Root inoculation with P. ananatis does not impede plant development nor provoke visible symptoms ( Fig. 1A ). Using a GFP-tagged P. ananatis BRT175 strain, we observed the colonization of the root system by the bacterium at seven days post-inoculation (dpi) ( Fig. 1B ). To investigate the role of HAAs in root colonization, we generated a GFP-tagged version of an P. ananatis rhlA- mutant, which is unable to synthesize HAAs ( Smith et al . 2016 ). This tagged-mutant strain was also found in association with roots of Arabidopsis ( Fig. 1B ). By enumerating total bacteria at several time points (4 to 14 dpi), we found that both the WT and the mutant strains persist in the root system over the time course at stable titers between 10 5 and 10 6 CFU g FW -1 ( Fig. 1C ), showing that synthesis of HAAs does not impact colonization efficiency. Bacteria were also re-isolated from surface-sterilized roots, indicating their ability to colonize inner root tissues ( Fig. 1C ). No significant differences were observed between the WT and the rhlA- mutant of P. ananatis in this endophytic colonization. We did not retrieve bacteria from leaves of root-inoculated plants even after several days, indicating that P. ananatis colonization is restricted to the rhizosphere. Download figure Open in new tab Fig. 1: P. ananatis BRT175 colonizes Arabidopsis roots, and its association with plant tissues induces rhlA expression. (A) Pictures of mock-treated and root-bacterized A. thaliana Col-0 WT rosettes. Five-week-old plants were root-treated with 10 mM MgSO 4 (Mock) or P. ananatis BRT175 WT (10 8 CFU g of soil -1 ). Photos were taken two weeks after treatment. (B) Microscopic images of A. thaliana Col-0 WT roots colonized by GFP-tagged P. ananatis BRT175 WT or rhlA- mutant. Five-week-old plants were root-treated with a suspension of GFP-tagged P. ananatis BRT175 WT or rhlA- mutant (10 8 CFU g of soil -1 ). Roots were unearthed from potting soil at 7 dpi and directly observed with a fluorescence microscope. (C) Enumeration of P. ananatis BRT175 WT and rhlA- mutant in unsterilized (total bacteria) and surface-sterilized roots (endophytes) of A. thaliana Col-0 WT. Five-week-old plants were root-treated with a suspension of P. ananatis BRT175 WT or rhlA- mutant (10 8 CFU g of soil -1 ). Bacteria were re-isolated from roots at 4, 7, 11, and 14 dpi on LBA medium supplemented with rifampicin (50 mg L -1 ) for unsterilized roots and only at 7 dpi for surface-sterilized roots. Data are represented as individual values with means (black line) (n = 3). No significant differences were observed between both bacteria genotypes based on a non-parametric Mann-Whitney test (p ≤ 0.05). (D) Luminescence of P. ananatis PrhlA-lux mutant in contact with Arabidopsis plantlets grown in liquid MS medium. To evaluate the effect of living plant tissue on rhlA promoter activity, the mutant bacteria was also inoculated in MS medium containing sterile toothpicks (ST). P. ananatis BRT175 WT was used as a negative control. Data are mean ± SD (n = 6). Experiments were conducted twice with similar results. Using a P. ananatis strain containing a transcriptional P rhlA - lux (promoter of rhlA fused with luxCDABE ) reporter to measure the expression from the rhlA promoter, we found that expression was strongly up-regulated when bacteria were exposed to living plant roots compared to bacteria exposed to sterile toothpicks, used as a non-living control matrix, over a 6-h time course ( Fig. 1D ). These data suggest that exposure to living plant tissues significantly enhances HAAs biosynthesis in P. ananatis . Altogether, these observations indicate that while this strain can colonize the root system of Arabidopsis, HAA synthesis does not play an important role in this process, even though HAA production is induced by the presence of the plant. P. ananatis triggers an ISR against B. cinerea but not Pst in Arabidopsis We then investigated the ability of P. ananatis to trigger ISR to the necrotrophic fungus B. cinerea and the hemibiotrophic bacterium Pst in soil-grown Arabidopsis. We inoculated roots with P. ananatis by soil drenching two weeks prior to leaf infection with B. cinerea or Pst. B. cinerea- induced necrosis symptoms were significantly reduced in leaves of root-bacterized plants as compared to leaves of mock-treated plants ( Fig. 2A and 2B ). In contrast, root treatment with P. ananatis did not reduce the titer of Pst nor associated disease symptoms in leaves ( Fig. 2C and 2D ). Download figure Open in new tab Fig. 2: P. ananatis BRT175 induces a systemic resistance against B. cinerea but not against Pst in Arabidopsis. (A) Representative symptoms on Arabidopsis leaves caused by B. cinerea at 96 hpi. Five-week-old plants were root-treated with 10mM MgSO 4 (Mock) or P. ananatis BRT175 WT (10 8 CFU g of soil -1 ). Leaves were infected two weeks later with a 5 μL drop of B. cinerea spores (10 6 spores·mL -1 ). (B) Necrotic symptoms provoked by B. cinerea . Lesions were measured at 96 hpi with ImageJ (n is indicated above each boxplot). (C) Representative symptoms on Arabidopsis leaves caused by Pst at 120 hpi. A. thaliana Col-0 WT were root-treated as previously described. Leaves were infected with Pst by spraying pathogens at 10 9 CFU mL -1 . (D) Enumeration of Colony-Forming Units (CFU) of Pst -infected leaves at 120 hpi. Data are represented individually (dots) with means (black line) (n = 6 different plants, for each, 6 foliar disks from 3 leaves are sampled and pooled). Asterisks indicate significant differences (p ≤ 0.05) based on a non-parametric Mann-Whitney test. Experiments were conducted three times with similar results. ISR triggered by P. ananatis against B. cinerea requires mc-3-OH-FA/HAA sensing by the PRR LORE We then asked whether HAAs play a role in the ISR triggered by the beneficial bacterium. The P. ananatis rhlA- mutant strain induced less resistance against B. cinerea than the WT bacteria ( Fig. 3A ). Interestingly, HAAs alone induced a similar resistance phenotype as WT bacteria ( Fig. 3A ). Addition of HAAs to P. ananatis rhlA- during the inoculation process restored the ISR phenotype to the level observed with the WT strain ( Fig. 3A ). Since HAAs are sensed by the PRR LORE ( Schellenberger et al . 2021 ), we tested the ability of P. ananatis WT to induce resistance in lore -5 mutant plants and in p LORE:LORE complemented lore -5 plants ( Ranf et al . 2015 ). As shown in figure 3B , the P. ananatis- triggered ISR was lost in lore -5 plants but restored in p LORE:LORE complemented lore -5 plants. Additionally, we observed that both the rhlA- mutant strain and HAA were not able to trigger ISR in lore -5 plants (Supplemental Fig. 1). Download figure Open in new tab Fig. 3: MAMP perception by the PRR LORE drives P. ananatis BRT175-triggered immunity in Arabidopsis against B. cinerea . (A) Necrotic symptoms provoked by B. cinerea in A. thaliana Col-0 WT. Plants were root-treated with 10mM MgSO 4 (Mock), P. ananatis BRT175 WT or P. ananatis BRT175 rhlA- (both bacteria at 10 8 CFU g of soil -1 ) or HAAs (directly supplied in bacterial suspension or in 10 mM MgSO 4 at a final concentration of 500 μM). Leaves were infected two weeks later with a 5 μL drop of B. cinerea spores (10 6 spores mL -1 ). Lesions were measured at 96 hpi with ImageJ. (B) Necrotic symptoms provoked by B. cinerea in A. thaliana Col-0 WT, lore -5 and p LORE:LORE , plants, root-treated and infected as previously described. Lesions were measured at 96 hpi with ImageJ. (C) Necrotic symptoms provoked by B. cinerea in A. thaliana Col-0 WT, lore -5 and p LORE:LORE , plants. Root were treated with MetOH 0.1 % (Mock) or with 3-OH-C 10 (in 10 mM MgSO 4 at a final concentration of 50 μM). Leaves were infected as previously described. Lesions were measured at 96 hpi with ImageJ. Different letters and asterisks indicate significant differences (p ≤ 0.05) based on a non-parametric Mann-Whitney test (n is indicated above each boxplot). All experiments were conducted three times with similar results. We also investigated ISR activation against B. cinerea induced by 3-hydroxydecanoic acid [3-OH-C10:0] (thereafter 3-OH-C 10 ), the strongest LORE agonist among the mc-3-OH-FAs group ( Kutschera et al . 2019 ; Schellenberger et al . 2021 ). 3-OH-C 10 triggers ISR against B. cinerea ( Fig. 3C ). This MAMP-mediated resistance was abolished in the lore -5 mutant but restored in p LORE:LORE complemented lore -5 plants ( Fig. 4A ). Like P. ananatis , 3-OH-C 10 was unable to trigger ISR against Pst (Supplemental Fig. 2). Interestingly, 3-OH-C 10 did not trigger resistance against B. cinerea when locally applied to leaves (Supplemental Fig. 3). Download figure Open in new tab Fig. 4: 3-OH-C 10 and HAAs activate root defense mechanisms in Arabidopsis through perception by the LORE receptor. (A) Callose deposits stained with aniline blue observed on treated roots of A. thaliana Col-0 WT or lore -5 plants. Roots were challenged with EtOH 0.1 % (Mock), 3-OH-C 10 (10 μM) or HAAs (10 μM) and stained 24 hours later. (B) Phosphorylation of MAPK 6 in A. thaliana Col-0 WT and lore -5 plants. Roots were treated for 10 minutes with MetOH 0.1 % (Mock), 3-OH-C 10 (1 μM) or HAAs (10 μM). Actin was used as a loading control. Full pictures are provided in Supplemental figure 4. Experiments were conducted twice with similar results. We then investigated local defense responses in roots following challenge with 3-OH-C 10 and HAAs as marker of local perception. As an event of late plant defense response, we assayed callose deposition ( Millet et al . 2010 ) ( Fig. 4A ). In WT plants, we observed callose deposits in plant roots treated with 3-OH-C 10 or HAAs, but not in mock-treated roots or lore -5 mutant plants. We also evaluated MAPK phosphorylation as a marker of early immune signaling ( Kutschera et al . 2019 ) ( Fig. 4B ). Both 3-OH-C 10 and HAAs triggered phosphorylation of MPK6 in WT, but not in lore -5 plants. These results demonstrate that HAAs and 3-OH-C 10 are perceived by the PRR LORE at the root level, inducing a local immune response and activating ISR against B. cinerea in leaves. P. ananatis BRT175 (both WT and rhlA- mutants) also induce phosphorylation of MAPKs. Interestingly, we also observed MAPK activation by P. ananatis BRT175 in lore -5 mutant plants (Supplemental Fig. 4). P. ananatis and 3-OH-C 10 -triggered ISR require similar signaling pathways and trigger similar defense mechanisms We aimed to decipher the contribution of hormone signal transduction pathways in P. ananatis -driven ISR. P. ananatis -triggered ISR against B. cinerea was compromised in sid2 -1 and npr1 -1 mutant plants ( Fig. 5A ), which are impaired in SA accumulation ( Wildermuth et al . 2001 ) and signaling ( Wu et al . 2012 ), respectively. ISR was also compromised in the jar1 -1 mutant ( Fig. 5A ), deficient in JA-isoleucine synthesis ( Staswick et al . 2002 ) and in the ein2 -1 mutant ( Fig. 5A ), deficient in ET signaling transduction ( Alonso et al . 1999 ). Altogether, these observations indicate that SA, JA and ET phytohormones contribute to P. ananatis -triggered ISR against the necrotrophic fungus. We then investigated whether these hormone signaling pathways are also involved in ISR activation after 3-OH-C 10 sensing. Our results show that the ISR was indeed compromised in sid2 -1, npr1 -1, jar1 -1 and ein2 -1 mutant plants compared to WT upon pre-treatment of roots with 3-OH-C 10 ( Fig. 5B ). To further compare P. ananatis and 3-OH-C 10 -triggered ISR, we quantified expression of defenses genes AtPR1 and AtPR4 in infected leaves of root-treated Arabidopsis. AtPR1 is well known as a SA-dependent defense gene marker ( Lebel et al . 1998 ; Vlot et al . 2009 ), whereas AtPR4 expression is dependent on ET ( Lawton et al . 1994 ; Broekaert et al . 2006 ). We observed that transcript levels of both AtPR1 and AtPR4 were significantly higher at 24 hpi in plants root-treated with P. ananatis ( Fig. 5C ) or 3-OH-C 10 ( Fig. 5D ) in comparison with mock-treated plants. Both of these observations show that P. ananatis - and 3-OH-C 10 -triggered ISR involve similar hormone signaling pathways and potentiate similar defense mechanisms at the leaf level against B. cinerea . Download figure Open in new tab Fig. 5: P. ananatis BRT175 and 3-OH-C 10 -triggered ISRs require identical signaling pathways and activates similar defense mechanisms against B. cinerea . Necrotic symptoms provoked by B. cinerea in A. thaliana Col-0 WT, sid2- 1, npr1- 1, jar1- 1 and ein2 -1 mutants, root-treated either with (A) 10mM MgSO 4 (Mock) or P. ananatis BRT175 WT (10 8 CFU g of soil -1 ), or with (B) MetOH 0.1 % (Mock) or 3-OH-C 10 (in 10 mM MgSO 4 at a final concentration of 50 μM). Leaves were infected two weeks later with a 5 μL drop of B. cinerea spores (10 6 spores mL -1 ). Lesions were measured at 96 hpi with ImageJ. Different letters indicate significant differences (p ≤ 0.05) based on a non-parametric Mann-Whitney test (n is indicated above each boxplot). Experiments were conducted three times with similar results. Quantification of AtPR1 and AtPR4 expression in A. thaliana Col-0 WT leaves infected with B. cinerea . Plants were root-treated either with (C) 10mM MgSO4 (Mock) or P. ananatis BRT175 WT (10 8 CFU g of soil-1), or with (D) MetOH 0.1 % (Mock) or 3-OH-C 10 (in 10 mM MgSO4 at a final concentration of 50 μM). Leaves were infected with a spray of B. cinerea spores (10 5 spores mL -1 ). Samples were collected at 0 and 24 hpi. For each timepoint and for each condition, 3 plants were samples (one sample consisting of 3 leaves pooled). Transcripts were quantified by qRT-PCR in triplicate for each samples. Results are expressed as fold change, calculated with the ΔΔCq method using AtACT7 and AtUBQ10 as housekeeping genes and mock-treated plants sampled at 0 hpi as the reference condition. Data are pooled from two independent biological replicates (6 different samples per condition in total, each quantified in triplicate). Asterisks indicate significant differences (p ≤ 0.05) based on a non-parametric Mann-Whitney test (n is indicated above each boxplot). Discussion In this work, we first demonstrated that strain BRT175 of P. ananatis can colonize the root system of Arabidopsis as an endophyte. Many Pantoea bacteria can positively interact with plants ( Walterson and Stavrinides et al . 2015 ; Duchateau et al . 2024 ). Some P. ananatis strains are characterized by their ability to colonize plant tissues ( Kang et al . 2007 ; Gasser et al . 2012 ). We found that the lack of HAA biosynthesis does not affect the capacity of P. ananatis BRT175 to colonize the root system. Besides being released by some bacteria, HAAs are precursors for the synthesis of biosurfactants like rhamnolipids and ananatosides ( Smith et al . 2016 ; Gauthier et al . 2019 ). Like their glycolipidic counterparts, HAAs play a direct role in swarming motility or adhesion to surfaces ( Déziel et al . 2003 ; Smith et al . 2016 ). The role of microbial amphiphilic metabolites in plant colonization is dependent on the nature of the molecules. For instance, the cyclic lipopeptide massetolide A contributes to tomato colonization by Pseudomonas fluorescens ( Tran et al . 2007 ). On the other hand, the lipopeptide poaeamide, synthesized by Pseudomonas poae , is a restrictive factor in this process ( Zachow et al . 2015 ). Plant colonization by bacteria involves multiple factors. For example, the presence of pili/flagella on P. ananatis ( Weller-Stuart et al . 2017 ) could participate in this process. Despite not playing a role in Arabidopsis root colonization, we provide evidence that HAA synthesis is induced when P. ananatis is exposed to Arabidopsis tissues, via activation of RhlA expression from the rhlA promoter. A positive effect of plant tissues on bacterial metabolite synthesis was also described for surfactin in Bacillus spp. ( Nihorimbere et al . 2009 ; Debois et al . 2015 ; Hoff et al . 2021 ), syringopeptin ( Wang et al . 2006 ) and amphisin ( Koch et al . 2002 ) in Pseudomonas spp. Molecules such as small organic compounds, found in root exudates, could be involved in this process ( Raaijmakers et al . 2010 ; Hoff et al . 2021 ). Through its interaction with roots, P. ananatis activates a protection of Arabidopsis leaves against B. cinerea . While several strains of P. ananatis exhibit antagonistic features against plant pathogens ( Torres et al . 2005 ; Gasser et al . 2012 ; Aman and Rai 2016 ), we found only one example of a systemic resistance inducer ( Kang et al . 2007 ). As P. ananatis was not detected in leaf tissues, we postulate that this protection is solely driven by the induction of ISR upon sensing the bacteria or MAMPs they release in root tissues. We hence aimed to decipher the molecular actors involved in this ISR. As precursors of glycolipid biosynthesis, free HAAs are generally found in minimal concentrations in bacterial culture supernatants ( Dubeau et al . 2009 ; Tiso et al . 2017 ; Germer et al . 2020 ). When synthesized by plant-associated bacteria, HAAs are thus most likely in contact with plant cells and membrane receptors. A recent study characterized the perception of HAAs by the PRR LORE, inducing Arabidopsis local immunity to pathogenic bacteria ( Schellenberger et al . 2021 ). Using a P. ananatis mutant unable to synthesize HAAs, we demonstrated here that these lipids play a significant role in P. ananatis -mediated ISR against the necrotrophic fungus B. cinerea . In line with this observation, the external supply of HAAs to the rhlA- mutant strain restores the protection against B. cinerea . Concomitantly, we found that this ISR is fully dependent on bacterial perception involving the PRR LORE, as ISR was abolished in the lore -5 mutant plants, but restored in the complemented line. The partial protection observed in WT plants challenged with P. ananatis rhlA- could be explained by the perception of mc-3-OH-FAs by LORE ( Kutschera et al . 2019 ). These molecules are also present in rhlA- bacteria, as a vital part of their primary lipid metabolism, and are potentially released by bacteria ( Schellenberger et al . 2021 ). The role of MAMPs in bacterial-triggered ISR through PRR perception is not fully understood. Flagellin, for instance, is not a central actor of some bacterial-mediated ISR, although this peptide can trigger ISR when used as a purified compound ( Meziane et al . 2005 ) and is also known for activating local root defenses ( Millet et al . 2010 ; Zhou et al . 2020 ; Colaianni et al . 2021 ). Recently, the MAMP chitin was shown to drive a CERK1-dependent activation ISR to Pst in several plants, including Arabidopsis and lettuce ( Makechemu et al . 2025 ). In line with our previous observations, we demonstrated that the MAMPs involved in P. ananatis -triggered ISR, HAAs and particularly mc-3-OH-FAs (here used in the form of 3-OH-C 10 ), activate ISR against B. cinerea through sensing by the PRR LORE. We also showed that 3-OH-C 10 and HAAs trigger MAPK phosphorylation and callose deposition in WT, but not in lore -5 Arabidopsis roots. Activation of LORE-dependent defense in roots by 3-OH-C 10 has already been mentioned as 3-OH-C 10 elicits defense responses in the elongation zone, but not in the differentiation zone of this organ ( Zhou et al . 2020 ). P. ananatis BRT175 is supposed to present other MAMPs, like flg22 for instance ( De Maayer and Cowan, 2016 ), which could also be sensed by plant cells. This could explain our observation that P. ananatis activate MAPK phosphorylation in roots of both WT and lore -5 mutant plants. Taken together, our results clearly demonstrate that LORE sensing of 3-OH-C 10 /HAAs is necessary and sufficient to trigger ISR to the necrotrophic fungus. P. ananatis triggers ISR against the necrotrophic fungus B. cinerea but not against the hemibiotrophic bacterium Pst . Similarly, 3-OH-C 10 , albeit activating an ISR to the necrotrophic fungus, is unable to trigger ISR against Pst . The opposite effects on necrotrophic and hemibiotrophic pathogens may imply differential early or late signaling pathways post-perception. Interestingly, we found that P. ananatis and 3-OH-C 10 mediated-ISR to B. cinerea involve SA, JA and ET pathways. Current models of signaling pathways in Arabidopsis immunity usually depict JA and ET as the most important phytohormones in ISR against necrotrophic pathogens while SA appears mostly involved in resistance against (hemi)biotrophic pathogens ( Pieterse et al . 2014 ; Vlot et al . 2021 ). There is, however, some evidence that synergies of SA and JA pathways may occur in ISR, principally depending on the quantities and the timing of hormone production ( Mur et al . 2006 ; Pieterse et al . 2012 ; Spoel and Dong, 2024 ). Moreover, our results are consistent with other studies showing that beneficial microorganisms, either bacteria ( Nguyen et al . 2022 ) or fungi ( Salas-Marina et al . 2011 ), can activate Arabidopsis ISR to necrotrophic pathogens via these two hormonal pathways. Regarding MAMPs, flg22 also triggers the accumulation of both phytohormones ( Tsuda et al . 2008 ; Chang et al . 2017 ) and enhances the expression of genes involved in the SA and JA pathways ( Denoux et al . 2008 ). P. ananatis - and 3-OH-C 10 -triggered ISR are both characterized by a potentiation of AtPR1 and AtPR4 expression in Arabidopsis leaves upon infection with B. cinerea . Activation of AtPR1 is an outcome of an interplay between simultaneous low activations of the SA and the JA pathways ( Mur et al . 2006 ; Loake and Grant, 2007 ). On the other hand, AtPR4 expression depends on the JA and ET pathways ( Lorenzo et al . 2003 ; Van Loon et al . 2006 ). Altogether, our data further emphasize the essential role of 3-OH-C 10 /HAAs in P. ananatis -triggered ISR, as we observed shared mechanisms between the MAMP-triggered and the bacteria-triggered defense response. Based on previous studies ( Kutschera et al . 2019 ; Schellenberger et al . 2021 ), we were also able to prove that 3-OH-C 10 binding to LORE triggers different types of induced resistance depending on its site of perception. We found here that root perception of 3-OH-C 10 activates ISR against the necrotrophic fungus B. cinerea but not against the hemibiotrophic bacteria Pst . Furthermore, leaf perception of this molecule does not trigger resistance against B. cinerea , while interestingly, previous studies conducted by Schellenberger et al . (2021) and Kutschera et al . (2019) showed an activation of local resistance against Pst in leaves through perception by the PRR LORE. Overexpression of this PRR in tomato leaves was also shown to enhance resistance against Pst ( Eschrig et al . 2024 ). One explanation for this organ-dependent dichotomy may lie in the downstream signaling activated by LORE. To date, LORE is known to activate RIPK, involved in RBOHD activation ( Wang et al . 2023 ), and the RLCKs PBL34, 35 and 36 ( Luo et al . 2020 ). Further investigations into the direct and indirect targets of LORE could potentially reveal organ-related specificities in the signal transduction of mc-3-OH-FA sensing. Recently, LORE was also described as the key component governing perception of the pathogenic bacteria Ralstonia solanacearum and controlling xylem immunity in roots ( Wang et al . 2023 ). In conclusion, we demonstrate here that, when applied to Arabidopsis roots, P. ananatis BRT175 activates an ISR to the necrotroph B. cinerea but not to the hemibiotroph Pst in leaves. This ISR is solely driven by the perception of mc-3-OH-FAs/HAAs by the PRR LORE and involves SA, JA and ET signaling pathways ( Fig. 6 ). As LORE sensing of 3-OH-mc-FAs/HAAs is activating a local resistance to Pst ( Schellenberger et al . 2021 ) but not to B. cinerea (this study) in leaves, our work also highlights a differential effect on local/systemic resistance against necrotrophic/biotrophic pathogens following perception of a lipid MAMP by a PRR receptor. Download figure Open in new tab Fig. 6: P. ananatis BRT175 triggers systemic immunity in Arabidopsis against B. cinerea through 3-OH-C 10 and HAAs interaction with the PRR LORE. P. ananatis BRT175 synthetizes HAAs and 3-OH-C 10 . Root perception of these MAMPs by LORE activates root defense mechanisms. The defense signal reaches leaves, where it activates a defense response against B. cinerea . While triggering an ISR against a necrotrophic fungus, neither P. ananatis nor 3-OH-C 10 mediated a systemic resistance against Pst . Material and Methods Plant material and growth conditions Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used in all experiments. In addition to the wild-type (WT) genotype, the lore -5, sid2- 1, jar1- 1, npr1- 1 and ein2 -1 mutants of the Col-0 ecotype ( Schellenberger et al . 2021 ; Nguyen et al . 2022 ) and the pLORE:LORE complemented lore -5 line CLF12 ( Ranf et al . 2015 ) were used in this study. All plants were grown in 50 g of potting soil (Terreau Motte, Sorexto) within a growth chamber (20°C, 12 h light/12 h dark, 150 μmol m -2 s -1 , 60 % relative humidity). For the quantification of the rhlA promoter activity, plantlets were floating-cultivated in Murashige-Skoog (MS) medium. Seeds were first surface-sterilized as previously described ( Millet et al . 2010 ), and disposed in 24-well plates, in MS medium with vitamins (Duchefa Biochemie) at pH 5.7 supplemented with sucrose (0.5 %) and MES monohydrate (0.05 %). Plates were sealed with Parafilm and incubated in a growth chamber under the same conditions. The medium was renewed after 8 days of incubation. For protection assays in hydroponics, plantlets were grown in Araponic systems. Seeds were placed in a seed-holder on a small plug of agar (0.6 %). The hydroponic fertilizer (Flora Series, Terra Aquatica) was added to the tray and changed every week. Araponic systems were placed in growth chamber (20°C, 12 h light/12 h dark, 150 μmol m -2 s -1 , 60 % relative humidity). Microbial material and growth conditions Both the wild-type (WT) Pantoea ananatis BRT175, isolated from a strawberry plant ( Smith et al . 2013 ), and the isogenic rhlA- mutant ( Smith et al . 2016 ), were used in this study. Bacteria were grown overnight in Lysogeny Broth (LB) medium at 28°C with shaking at 180 rpm. P. ananatis BRT175 is rifampicin-resistant, while P. ananatis BRT175 rhlA- is also kanamycin-resistant. When required, antibiotics were added to the liquid culture media at 50 mg L -1 . For plant treatment, bacteria were collected by centrifugation (20 min, 3200 x g , 4°C) and suspended in sterile 10 mM MgSO 4 . Concentration of the bacterial suspension was adjusted to 10 9 CFU mL -1 . Botrytis cinerea Bc 630 was used as a necrotrophic pathogen ( Nguyen et al . 2022 ). Conidia of B. cinerea were first germinated in Potato Dextrose Broth (PDB, 24 g L -1 ) for one week at 20°C under shaking (80 rpm). The mycelium from germinated conidia was then ground and spread on PDB Agar medium, followed by a two-week incubation at 20°C. Conidia were then harvested by scraping the mycelium with 4 mL of PDB. For plant infection, conidia were counted using a Malassez cell and the concentration was adjusted to 10 6 conidia mL -1 . Prior to infection, conidia were pre-germinated at 20°C under shaking (80 rpm) for two hours prior to the infection. Pseudomonas syringae pv. tomato DC3000 ( Pst ) was used as a hemibiotrophic pathogen ( Schellenberger et al . 2021 ; Nguyen et al . 2022 ). Pst was grown overnight in King’s B (KB) medium supplemented with rifampicin (50 mg L -1 ) at 28°C and 180 rpm. For infections, bacteria were collected by centrifugation (20 min, 3,200 x g , 4°C) and suspended in sterile 10 mM MgSO 4 (10 mM). The concentration of the bacterial suspension was adjusted to 10 9 CFU mL -1 . Sterile Silwett-L77 was added to the bacterial suspension (0.02 %) as a wetting agent. Molecules Purified 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs) as C 10 -C 10 congeners ( Schellenberger et al . 2021 ) were used in this study. 3-Hydroxydecanoic acid (3-OH-C 10 ) was purchased from Ark Pharm. All molecules were conserved at 100 mM in methanol (MetOH) or in ethanol (EtOH) at −20°C. GFP-tagging of P. ananatis BRT175 and its mutant P. ananatis BRT175 (WT and rhlA- ) were tagged with GFP to assess their ability to colonize root systems using epifluorescence microscopy. Competent cells were obtained from fresh cultures of both strains. Once the cultures reached an OD 600 of 0.6, they were chilled on ice for 20 minutes. Pellets were collected by centrifugation (15 min, 16000 x g , 4°C) and washed twice with 20 mL of sterile water at 4°C. Pellets were then suspended in 4.5 mL of 10 % glycerol at 4°C and centrifuged again (10 min, 7500 x g , 4°C). Four milliliters of supernatant were discarded, and competent cells were suspended in the remaining 500 μL. Plasmid pIN301, carrying GFP and a chloramphenicol-resistance gene ( Rabhi et al . 2018 ), was used to tag P. ananatis BRT175. 50 nanograms of plasmid were added to 100 μL of competent cells. The entire mixture was transferred to an electroporation cuvette (Gene Pulser/MicroPulser Electroporation Cuvettes, 0.2 cm gap, Bio-Rad). Electroporation was performed with an Eppendorf electroporator at 2.5 kV, 25 μF, and 200 Ω. Cells were immediately recovered after electroporation with 900 μL of LB medium supplemented with sterile dextrose (20 g L -1 ) and incubated for 2 h at 28°C with agitation at 180 rpm. Recovered cells were centrifuged (5 min, 3200 x g , 20°C) after incubation. 400 μl of supernatant were removed, and pellets were suspended in the remaining medium. 200 μl of suspended bacteria were spread on LB agar supplemented with chloramphenicol (50 mg L -1 ). After two days of incubation at 28°C, successful transformation was verified through direct observation of bacteria with an epifluorescence microscope (Olympus BH2) with UV light source (BH2-RFL-T3; Olympus). Root and leaf colonization assays Five-week-old A. thaliana Col-0 WT plants were root-treated with different suspensions of P. ananatis BRT175 (WT or rhlA- mutant). Five milliliters of bacterial suspensions were inoculated directly into the soil, near the basis of the stem, to reach a final concentration of 10 8 CFU g -1 of soil. Negative control plants were mock-treated with 5 mL of sterile 10 mM MgSO 4 in a similar manner. Treated plants were placed in a growth chamber for a maximum of 14 days. Colonization was evaluated in roots and leaves at 4, 7, 11 and 14 days post-inoculation. At each time point and for each condition, six plants were collected. Whole plants were gently extracted from soil and roots were separated from the stem and leaves. The residual soil on the root system was removed with a gentle wash in water before sampling. To assess the endophytic capacity of both P. ananatis BRT175 strains (WT and mutant rhlA- ), three out of the six roots were surface-sterilized, at each time point and for each condition. The sterilization process involved a general wash of roots with sterile MgSO 4 , followed by transfer to a diluted commercial bleach solution (0.5 % of active chlorine with 0.01 % Tween added) for 5 min. Sterilization ended with three 30 s washes of bleached roots with sterile MgSO 4 . All samples were ground in sterile mortars with 1 mL of sterile MgSO 4 and diluted ten times. For each dilution, 10 μL were deposited twice on LB agar medium, supplemented with 50 mg L -1 rifampicin for selection of both phenotypes of P. ananatis BRT175. CFUs were counted after 2 days of incubation at 28°C. Results were expressed as a number of CFU per gram of root fresh weight. As negative controls, three root samples were collected from plants mock-treated with sterile MgSO 4 . Samples were ground using the same method and deposited on the same medium to assess the absence of rifampicin-resistant bacteria in Arabidopsis tissues. To control the efficiency of the sterilization process, 10 μL from the last wash with sterile 10 mM MgSO 4 were deposited twice on the same medium. Microscopic observations of A. thaliana colonized roots Following the same methodology described for colonization assays, GFP-tagged P. ananatis BRT175 were directly observed in the roots of Arabidopsis. Five-weeks old A. thaliana Col-0 WT plants were root-treated with a suspension of tagged-bacteria, and root systems were observed at 7 dpi. Roots were gently washed with water to remove remaining soil particles and were immediately observed using an epifluorescence microscope (Olympus BH2) with a UV light source (BH2-RFL-T3; Olympus). Roots were first observed under white light, and then under UV light. Both pictures were merged using the ImageJ software. Reporter gene construction The activity from the rhlA promoter of P. ananatis BRT175 was evaluated by a fusion with the reporter genes luxCDABE . The rhlA promoter was amplified from genomic DNA of P. ananatis BRT175 (extracted using the Easy Pure Genomic DNA Kit, Trans) using the PaBRT175prhlA primers (sequences are listed in Supplemental Table I). PCR was conducted with the Q5® High Fidelity polymerase (New England Biolab) in a T100 Thermal Cycler (Bio-Rad). The PCR product was recovered from agarose using a FavorPrep Gel/PCR Purification Kit (Favorgen). The suicide vector pIJ514, containing a promoterless luxCDABE operon and a chloramphenicol-resistance gene within a Tn 7 transposon ( Bessaiah et al . 2019 ), was linearized by digestion with EcoRI and SacI restriction enzymes (Thermo Scientific). The PCR product and linearized plasmid were assembled using the pEasy-Uni Seamless Cloning and Assembly Kit (Transgen) according to the manufacturer’s recommendations. The assembly product was introduced into competent E. coli SM-10 by heat shock. The construction was confirmed by PCR, using the same primers. After verification, the plasmid was transferred to competent P. ananatis BRT175 alongside the pSTNSK helper plasmid (Crépin et al . 2012) using electroporation. Chloramphenicol-resistant colonies of P. ananatis BRT175 were first selected at 30°C and then incubated at 37°C to lose the thermosensitive helper plasmid. Light emission of positive clones was verified using a Cytation multimode plate reader (BioTek). rhlA promoter activity assay Evaluation of promoter activity was indirectly measured by monitoring the light emission of P. ananatis BRT175 PrhlA-lux cultivated alongside floating-cultivated A. thaliana plantlets. After 10 days of incubation, mutant bacteria were directly inoculated into MS medium at a final concentration of 10 8 CFU mL -1 . To compare the impact of living plant tissues on promoter activity, mutant bacteria were also inoculated in MS medium containing sterile toothpicks. Plant-bacteria co-culture medium was sampled (100 μL) at 2 and 6 h post-inoculation. Samples were placed in white 96-wells plate (OptiPlate, Perkin Elmer). Light emission and optical density at 600 nm were then measured directly using a CM SPARK (TECAN). Plantlets inoculated with P. ananatis BRT175 WT were used as negative control. For each condition, 6 wells (each containing approximately 15 plantlets) were inoculated. Data are expressed as the ratio between light emission and optical density. Bioprotection assays against B. cinerea and Pst For the protection assays, plants were root-treated with bacterial suspensions as described for the colonization assays. However, these assays incorporated different genotypes of A. thaliana Col-0 (WT, lore -5, pLORE:LORE, sid2 -1, npr1 -1, jar1 -1 and ein2 -1). In addition to both P. ananatis BRT175 strains (WT or mutant rhlA- ), plants were also root-treated with 3-OH-C 10 or HAAs suspension, either alone or mixed with bacteria. 3-OH-C 10 and HAAs were directly diluted (at 50 and 500 μM, respectively) in sterile 10 mM MgSO 4 or in bacterial suspension for HAAs. After 2 weeks of incubation in a growth chamber, leaves of control and treated plants were infected as previously described ( Nguyen et al . 2022 ). One day prior to infection, plants of similar conditions were placed in a plastic box containing 300 mL of tap water. Boxes were sealed with micropore adhesive tape and replaced in growth chamber for 24 hours. For B. cinerea , leaves were infected by placing a 5 μL drop of conidial suspension on the adaxial face of medium foliar-stage leaves. Infected plants were then replaced in sealed boxes. Infection was evaluated after 4 days of incubation in a growth chamber, by measuring the necrosis area provoked by B. cinerea using the ImageJ software. For each condition, approximately 15 to 30 leaves were infected on 6 different plants. For the infection with Pst , leaves were uniformly sprayed with bacterial suspension. After 5 days of incubation in sealed boxes, 3 leaves were sampled for bacterial quantification. On each plant, 6 leaf discs (5 mm diameter) were excised from the 3 infected leaves and pooled together for grinding in sterile 10 mM MgSO 4 . Samples were then ten-fold diluted. Ten μL of the dilutions were spread on KB medium supplemented with rifampicin (50 mg L -1 ). CFUs were counted after 2 days of incubation at 28°C. Results are expressed as CFU per gram of fresh weight. For protection assays conducted in hydroponic devices, 4-week-old plants were transferred to 10 mL vials containing hydroponic solution. Treatments were carried out by adding appropriate molecules (HAAs or 3-OH-C 10 ) directly into the hydroponic solution, at respective final concentrations of 1 μM and 10 μM. 0.1 % EtOH was used as a negative control. After two days of incubation, leaves were infected with a drop of B. cinerea conidia suspension (10 μL at 10 5 conidia mL -1 ). Necrosis areas were measured as described above. For the infection with Pst , leaves were uniformly sprayed with bacterial suspension (10 7 CFU mL -1 ). Sterile Silwet L-77 was added to the bacterial suspension (0.02%) as a wetting agent. Three leaves were sampled and pooled per plant after 3 days of incubation in a growth chamber. CFUs were isolated and counted as described above. For local protection assays, 5-week-old plants were leaf-treated by the spraying of a molecular suspension of 3-OH-C 10 at 10 μM. EtOH 0.1 % was used as a negative control. After two days of incubation in a growth chamber, leaves were infected with a drop of B. cinerea conidia suspension (10 μL at 10 5 conidia mL -1 ). Necrosis areas were measured as previously described. MAPK phosphorylation assays MAPK phosphorylation state was measured in 2-week-old A. thaliana grown in hydroponic medium. The day prior to elicitation, twelve plantlets were incubated overnight in split Petri dishes to specifically treat the root systems. 3-OH-C 10 (1 μM) or HAAs (10 μM) were added to root tips. MetOH 0.1 % was used as a negative control. Roots were then sampled and immediately placed in liquid nitrogen to preserve the phosphorylation state. The frozen roots were then ground. Protein extraction buffer (0.35 M Tris-HCl pH 6.8 containing 30 % (v/v) glycerol, 10 % (v/v) SDS, 0.6 M dithiothreitol, and 0.012 % (w/v) bromophenol blue) was then added (1 μL per 1 mg of root powder). Samples were then heated (10 min at 95°C) and centrifuged (10 min at 16000 x g ). Supernatants were loaded into a polyacrylamide gel (SDS-PA gels 12 %). Proteins were separated by electrophoresis (15 min at 90 V, followed by 90 min at 150 V). Gel was then transferred to iBlot2 gel transfer system (Invitrogen) for protein migration on a PVDF membrane (7 min at 25 V). The membrane was then blocked using saturation buffer (3 % low-fat dry milk in Tris Buffered Saline (TBS)–Tween 20 (0.05 %)) for 30 min. Following 2 washes using washing buffer (0.5 % low-fat dry milk in TBS–Tween 20), the membrane was incubated overnight at 4°C with primary antibodies (Anti-phospho-p44/42 MAP kinase, Cell Signaling Technologies, diluted 1:2000 in washing buffer). The membrane was then washed 3 times and incubated for 1 h at room temperature with secondary antibodies (HRP-conjugated anti-rabbit, Bio-Rad, diluted 1:3000 in washing buffer). SuperSignal® West Femto (Thermo Fisher Scientific) was used as a revealing reagent. Luminescence was detected using Odyssey® Fc Dual-Mode Imaging System (LI-COR). Actin was revealed to normalize protein loading. The membrane was incubated for 30 min in 0.25 M NaOH solution, then blocked with saturation buffer. The membrane was incubated for 1 h at room temperature with primary antibodies (Plant monoclonal anti-actin, CusAb, 1:1000 in washing buffer), washed 3 times and incubated for 1 h at room temperature with secondary antibodies (Anti-mouse IgG HRP-conjugated, Cell Signaling, 1:3000 in washing buffer). The membrane was revealed as described above. Callose deposition assays Callose deposits were observed in 2-week-old A. thaliana plantlets grown in hydroponics. 3-OH-C 10 or HAAs were directly added into the culture medium at a final concentration of 10 μM. EtOH 0.1% was used as a negative control. After 24 h of treatment, root systems were sampled and placed in a discoloration solution (ethanol: acetic acid [3:1/v:v]) for 2 h. Roots were rehydrated in EtOH 70 % for 2 h, then in EtOH 50 % for 2 h, and finally in MilliQ water overnight. Subsequently, roots were incubated for 1 h at 37°C in NaOH 10 %, and in K 2 HPO 4 150 mM for 30 min, following several washes with MilliQ water. Roots were finally incubated in staining solution (K 2 HPO 4 150 mM and Aniline Blue 0.01 %) for 3 h in the dark. Stained roots were observed under an epifluorescent microscope (Olympus BH2) equipped with a UV light source (BH2-RFL-T3; Olympus). Quantification of defense gene expression RNA extractions were performed on leaf samples of infected A. thaliana Col-0 WT. Soil-grown plants were root-treated with P. ananatis BRT175 WT or 3-OH-C 10 and infected with B. cinerea as described above. Leaves were sampled at 0 and 24 hpi. For each time point and condition, 3 different plants were sampled (3 leaves were collected on each plant and pooled to make a unique sample per plant). Samples were freeze-dried in liquid nitrogen and ground to fine powder. Total RNA was extracted from 50 mg of ground tissue using QIAzol Lysis Reagent (Qiagen) according to the manufacturer’s instructions. RNA samples were reverse-transcribed using the Verso cDNA synthesis kit (Thermo Scientific) according to the manufacturer’s recommendations. To quantify gene expression, qPCR reactions were performed with the qPCRBIO SyGreen Blue Mix Lo-ROX (Eurobio Scientific, PCR Biosystem) on cDNA samples (1:10 diluted in RNAse-free water). Primer pairs were used at a final concentration of 0.3 or 0.1 μM, depending on the targeted gene. Sequences are listed in Supplemental Table I. PCR reactions were performed in white 384-well plate (Sorenson) with the CFX Opus 384 Real-Time PCR System (Bio-Rad) using the following procedure: first denaturation (15 min, 95°C), 40 cycles of denaturation (5 s, 95°C) and annealing/extension (30 s, 65°C), melting curve between 60°C and 95°C. Reactions were conducted in triplicate for each sample. Wells presenting a melting temperature difference of more than 0.5°C were excluded from the analysis (based on the melting curve of a PCR reaction performed on a pool of randomly selected samples). Expression of defense genes was normalized with the mean Cq of AtACT7 and AtUBQ10 , validated as housekeeping genes with the GeNorm algorithm. Expression was then calculated as fold change with the ΔΔCq method based on control samples (Mock-treated A. thaliana at 0 hpi). Statistical analysis Statistical analyses were conducted using GraphPad Prism v10.6.1. Statistical differences of means were tested using Mann-Whitney tests. Differences were considered statistically significant at p ≤ 0.05. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment This work was supported by grants from the GLYCOBAC project, co-funded by the Région Grand Est and the ABIES Doctoral school. The authors also would like to gratefully acknowledge the MOBDOC program at the University of Reims Champagne-Ardenne and ABIES doctoral school for funding international mobility. Financial support from MESRI (Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation) is also gratefully acknowledged. The authors also wish to thank C.M. Dozois (INRS-Centre Armand-Frappier Santé Biotechnologie, Laval, Québec, Canada) for providing plasmids pIJ514 and pSTNSK. Funder Information Declared AgroParisTech, https://ror.org/02kbmgc12 Conseil régional du Grand Est Ministère de l’Enseignement Supérieur et de la Recherche, https://ror.org/03sjk9a61 Université de Reims Champagne-Ardenne, https://ror.org/03hypw319 References 1. ↵ A. M. Abdel-Mawgoud , F. Lépine , E. 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