Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. EVs from pathogenic bacteria play functions in host immune modulation, elimination of host defenses, and acquisition of nutrients from the host. Here, we observed EV production of the bacterial speck disease causal agent, Pseudomonas syringae pv. tomato (Pto) DC3000, as outer membrane vesicle release. Mass spectrometry identified 369 proteins enriched in Pto DC3000 EVs. The EV samples contained known immunomodulatory proteins and could induce plant immune responses mediated by bacterial flagellin. Having identified two biomarkers for EV detection, we provide evidence for Pto DC3000 releasing EVs during plant infection. Bioinformatic analysis of the EV-enriched proteins suggests a role for EVs in antibiotic defense and iron acquisition. Thus, our data provide insights into the strategies this pathogen may use to develop in a plant environment. IMPORTANCE The release of extracellular vesicles (EVs) into the environment is ubiquitous among bacteria. Vesiculation has been recognized as an important mechanism of bacterial pathogenesis and human disease but is poorly understood in phytopathogenic bacteria. Our research addresses the role of bacterial EVs in plant infection. In this work, we show that the causal agent of bacterial speck disease, Pseudomonas syringae pv. tomato, produces EVs during plant infection. Our data suggest that EVs may help the bacteria to adapt to environments, e.g., when iron could be limiting such as the plant apoplast, laying the foundation for studying the factors that phytopathogenic bacteria use to thrive in the plant environment.

Bavarian Center for Biomolecular Mass Spectrometry Technical University of Munich Gregor Mendel Strasse Freising United Kingdom

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Prague Czechia

Faculty of Science University of South Bohemia in České Budějovice České Budějovice Czechia

John Innes Centre Norwich Research Park Norwich United Kingdom

LMU Munich Biocenter Ludwig Maximilian University of Munich Munich Germany

The Sainsbury Laboratory Norwich Research Park Norwich United Kingdom

University of East Anglia Norwich Research Park Norwich United Kingdom

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Büttner D, Bonas U. 2010. Regulation and secretion of Xanthomonas virulence factors. FEMS Microbiol Rev 34:107–133. doi:10.1111/j.1574-6976.2009.00192.x PubMed DOI

Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Dow M, Verdier V, Beer SV, Machado MA, Toth I, Salmond G, Foster GD. 2012. Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13:614–629. doi:10.1111/j.1364-3703.2012.00804.x PubMed DOI PMC

Wilson M, Campbell HL, Ji P, Jones JB, Cuppels DA. 2002. Biological control of bacterial speck of tomato under field conditions at several locations in North America. Phytopathology 92:1284–1292. doi:10.1094/PHYTO.2002.92.12.1284 PubMed DOI

Melotto M, Underwood W, Koczan J, Nomura K, He SY. 2006. Plant stomata function in innate immunity against bacterial invasion. Cell 126:969–980. doi:10.1016/j.cell.2006.06.054 PubMed DOI

Xin XF, He SY. 2013. Pseudomonas syringae pv. tomato DC3000: a model pathogen for probing disease susceptibility and hormone signaling in plants. Annu Rev Phytopathol 51:473–498. doi:10.1146/annurev-phyto-082712-102321 PubMed DOI

Xin XF, Kvitko B, He SY. 2018. Pseudomonas syringae: what it takes to be a pathogen. Nat Rev Microbiol 16:316–328. doi:10.1038/nrmicro.2018.17 PubMed DOI PMC

Yuan M, Jiang Z, Bi G, Nomura K, Liu M, Wang Y, Cai B, Zhou JM, He SY, Xin XF. 2021. Pattern-recognition receptors are required for NLR-mediated plant immunity. Nature 592:105–109. doi:10.1038/s41586-021-03316-6 PubMed DOI PMC

Couto D, Zipfel C. 2016. Regulation of pattern recognition receptor signalling in plants. Nat Rev Immunol 16:537–552. doi:10.1038/nri.2016.77 PubMed DOI

Dodds PN, Rathjen JP. 2010. Plant immunity: towards an integrated view of plant-pathogen interactions. Nat Rev Genet 11:539–548. doi:10.1038/nrg2812 PubMed DOI

Kvitko BH, Park DH, Velásquez AC, Wei C-F, Russell AB, Martin GB, Schneider DJ, Collmer A. 2009. Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes reveal functional overlap among effectors. PLoS Pathog 5:e1000388. doi:10.1371/journal.ppat.1000388 PubMed DOI PMC

Nobori T, Wang Y, Wu J, Stolze SC, Tsuda Y, Finkemeier I, Nakagami H, Tsuda K. 2020. Multidimensional gene regulatory landscape of a bacterial pathogen in plants. Nat Plants 6:1064. doi:10.1038/s41477-020-0746-8 PubMed DOI

Nobori T, Wang Y, Wu J, Stolze SC, Tsuda Y, Finkemeier I, Nakagami H, Tsuda K. 2019. In planta bacterial multi-omics analysis illuminates regulatory principles underlying plant-pathogen interactions. Plant Biol. doi:10.1101/822932 DOI

Nomura K, Debroy S, Lee YH, Pumplin N, Jones J, He SY. 2006. A bacterial virulence protein suppresses host innate immunity to cause plant disease. Science 313:220–223. doi:10.1126/science.1129523 PubMed DOI

Schwechheimer C, Kuehn MJ. 2015. Outer-Membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat Rev Microbiol 13:605–619. doi:10.1038/nrmicro3525 PubMed DOI PMC

Bielska E, Birch PRJ, Buck AH, Abreu-Goodger C, Innes RW, Jin H, Pfaffl MW, Robatzek S, Regev-Rudzki N, Tisserant C, Wang S, Weiberg A. 2019. Highlights of the mini-symposium on extracellular vesicles in inter-organismal communication, held in Munich, Germany, August 2018. J Extracell Vesicles 8:1590116. doi:10.1080/20013078.2019.1590116 PubMed DOI PMC

Rybak K, Robatzek S. 2019. Functions of extracellular vesicles in immunity and virulence. Plant Physiol 179:1236–1247. doi:10.1104/pp.18.01557 PubMed DOI PMC

Raposo G, Stoorvogel W. 2013. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200:373–383. doi:10.1083/jcb.201211138 PubMed DOI PMC

Roier S, Zingl FG, Cakar F, Durakovic S, Kohl P, Eichmann TO, Klug L, Gadermaier B, Weinzerl K, Prassl R, Lass A, Daum G, Reidl J, Feldman MF, Schild S. 2016. A novel mechanism for the biogenesis of outer membrane vesicles in gram-negative bacteria. Nat Commun 7:10515. doi:10.1038/ncomms10515 PubMed DOI PMC

Pérez-Cruz C, Delgado L, López-Iglesias C, Mercade E. 2015. Outer-inner membrane vesicles naturally secreted by gram-negative pathogenic bacteria. PLoS One 10:e0116896. doi:10.1371/journal.pone.0116896 PubMed DOI PMC

Toyofuku M, Nomura N, Eberl L. 2019. Types and origins of bacterial membrane vesicles. Nat Rev Microbiol 17:13–24. doi:10.1038/s41579-018-0112-2 PubMed DOI

Roszkowiak J, Jajor P, Guła G, Gubernator J, Żak A, Drulis-Kawa Z, Augustyniak D. 2019. Interspecies outer membrane Vesicles (Omvs) modulate the sensitivity of pathogenic bacteria and pathogenic yeasts to Cationic peptides and serum complement. Int J Mol Sci 20:5577. doi:10.3390/ijms20225577 PubMed DOI PMC

Kaparakis-Liaskos M, Ferrero RL. 2015. Immune modulation by bacterial outer membrane vesicles. Nat Rev Immunol 15:375–387. doi:10.1038/nri3837 PubMed DOI

McMillan HM, Kuehn MJ. 2021. The extracellular vesicle generation paradox: a bacterial point of view. EMBO J 40:e108174. doi:10.15252/embj.2021108174 PubMed DOI PMC

McMillan HM, Rogers N, Wadle A, Hsu-Kim H, Wiesner MR, Kuehn MJ, Hendren CO. 2021. Microbial vesicle-mediated communication: convergence to understand interactions within and between domains of life. Environ Sci Process Impacts 23:664–677. doi:10.1039/d1em00022e PubMed DOI

Bahar O, Mordukhovich G, Luu DD, Schwessinger B, Daudi A, Jehle AK, Felix G, Ronald PC. 2016. Bacterial outer membrane vesicles induce plant immune responses. Mol Plant Microbe Interact 29:374–384. doi:10.1094/MPMI-12-15-0270-R PubMed DOI

Feitosa-Junior OR, Stefanello E, Zaini PA, Nascimento R, Pierry PM, Dandekar AM, Lindow SE, da Silva AM. 2019. Proteomic and metabolomic analyses of Xylella fastidiosa OMV-enriched fractions reveal association with virulence factors and signaling molecules of the DSF family. Phytopathology 109:1344–1353. doi:10.1094/PHYTO-03-19-0083-R PubMed DOI

Chowdhury C, Jagannadham MV. 2013. Virulence factors are released in association with outer membrane vesicles of Pseudomonas syringae pv. tomato T1 during normal growth. Biochim Biophys Acta 1834:231–239. doi:10.1016/j.bbapap.2012.09.015 PubMed DOI

Sidhu VK, Vorhölter F-J, Niehaus K, Watt SA. 2008. Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium Xanthomonas campestris pv. campestris. BMC Microbiol 8:87. doi:10.1186/1471-2180-8-87 PubMed DOI PMC

Mitre LK, Teixeira-Silva NS, Rybak K, Magalhães DM, de Souza-Neto RR, Robatzek S, Zipfel C, de Souza AA. 2021. The Arabidopsis immune receptor EFR increases resistance to the bacterial pathogens Xanthomonas and Xylella in transgenic sweet orange. Plant Biotechnol J 19:1294–1296. doi:10.1111/pbi.13629 PubMed DOI PMC

McMillan HM, Zebell SG, Ristaino JB, Dong X, Kuehn MJ. 2021. Protective plant immune responses are elicited by bacterial outer membrane vesicles. Cell Rep 34:108645. doi:10.1016/j.celrep.2020.108645 PubMed DOI PMC

Klimentová J, Stulík J. 2015. Methods of isolation and purification of outer membrane vesicles from gram-negative bacteria. Microbiol Res 170:1–9. doi:10.1016/j.micres.2014.09.006 PubMed DOI

Bachurski D, Schuldner M, Nguyen P-H, Malz A, Reiners KS, Grenzi PC, Babatz F, Schauss AC, Hansen HP, Hallek M, Pogge von Strandmann E. 2019. Extracellular vesicle measurements with nanoparticle tracking analysis - an accuracy and repeatability comparison between Nanosight NS300 and Zetaview. J Extracell Vesicles 8:1596016. doi:10.1080/20013078.2019.1596016 PubMed DOI PMC

Nobori T, Velásquez AC, Wu J, Kvitko BH, Kremer JM, Wang Y, He SY, Tsuda K. 2018. Transcriptome landscape of a bacterial pathogen under plant immunity. Proc Natl Acad Sci U S A 115:E3055–E3064. doi:10.1073/pnas.1800529115 PubMed DOI PMC

Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. 2000. Gene ontology: tool for the unification of biology.The Gene Ontology Consortium. Nat Genet 25:25–29. doi:10.1038/75556 PubMed DOI PMC

The Gene Ontology Consortium . 2019. The gene ontology resource: 20 years and still going strong. Nucleic Acids Res 47:D330–D338. doi:10.1093/nar/gky1055 PubMed DOI PMC

Huang DW, Sherman BT, Lempicki RA. 2009. Systematic and integrative analysis of large gene Lists using DAVID bioinformatics resources. Nat Protoc 4:44–57. doi:10.1038/nprot.2008.211 PubMed DOI

Huang DW, Sherman BT, Lempicki RA. 2009. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene Lists. Nucleic Acids Res 37:1–13. doi:10.1093/nar/gkn923 PubMed DOI PMC

Kulp A, Kuehn MJ. 2010. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Rev Microbiol 64:163–184. doi:10.1146/annurev.micro.091208.073413 PubMed DOI PMC

Kramer J, Özkaya Ö, Kümmerli R. 2020. Bacterial siderophores in community and host interactions. Nat Rev Microbiol 18:152–163. doi:10.1038/s41579-019-0284-4 PubMed DOI PMC

Manabe T, Kato M, Ueno T, Kawasaki K. 2013. Flagella proteins contribute to the production of outer membrane vesicles from Escherichia coli W3110. Biochem Biophys Res Commun 441:151–156. doi:10.1016/j.bbrc.2013.10.022 PubMed DOI

Bredow M, Sementchoukova I, Siegel K, Monaghan J. 2019. Pattern-triggered oxidative burst and Seedling growth inhibition assays in Arabidopsis Thaliana. J Vis Exp. doi:10.3791/59437 PubMed DOI

Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JDG, Felix G, Boller T. 2004. Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428:764–767. doi:10.1038/nature02485 PubMed DOI

Wang Y, Garrido-Oter R, Wu J, Winkelmüller TM, Agler M, Colby T, Nobori T, Kemen E, Tsuda K. 2019. Site-specific cleavage of bacterial Mucd by secreted proteases mediates Antibacterial resistance in Arabidopsis. Nat Commun 10:2853. doi:10.1038/s41467-019-10793-x PubMed DOI PMC

Vinatzer BA, Teitzel GM, Lee MW, Jelenska J, Hotton S, Fairfax K, Jenrette J, Greenberg JT. 2006. The type III effector repertoire of Pseudomonas syringae pv. syringae B728a and its role in survival and disease on host and non-host plants. Mol Microbiol 62:26–44. doi:10.1111/j.1365-2958.2006.05350.x PubMed DOI

Lovelace AH, Smith A, Kvitko BH. 2018. Pattern-triggered immunity alters the transcriptional regulation of virulence-associated genes and induces the sulfur starvation response in Pseudomonas syringae pv. tomato DC3000. Mol Plant Microbe Interact 31:750–765. doi:10.1094/MPMI-01-18-0008-R PubMed DOI

Schechter LM, Vencato M, Jordan KL, Schneider SE, Schneider DJ, Collmer A. 2006. Multiple approaches to a complete inventory of Pseudomonas syringae pv. tomato DC3000 type III secretion system effector proteins. Mol Plant Microbe Interact 19:1180–1192. doi:10.1094/MPMI-19-1180 PubMed DOI

Block A, Alfano JR. 2011. Plant targets for Pseudomonas syringae type III effectors: virulence targets or guarded decoys? Curr Opin Microbiol 14:39–46. doi:10.1016/j.mib.2010.12.011 PubMed DOI PMC

Tran TM, Chng CP, Pu X, Ma Z, Han X, Liu X, Yang L, Huang C, Miao Y. 2022. Potentiation of plant defense by bacterial outer membrane vesicles is mediated by membrane nanodomains. Plant Cell 34:395–417. doi:10.1093/plcell/koab276 PubMed DOI PMC

Rutter BD, Innes RW. 2017. Extracellular vesicles isolated from the leaf apoplast carry stress-response proteins. Plant Physiol 173:728–741. doi:10.1104/pp.16.01253 PubMed DOI PMC

Couto N, Schooling SR, Dutcher JR, Barber J. 2015. Proteome profiles of outer membrane vesicles and extracellular matrix of Pseudomonas aeruginosa biofilms . J. Proteome Res 14:4207–4222. doi:10.1021/acs.jproteome.5b00312 PubMed DOI

Choi DS, Kim DK, Choi SJ, Lee J, Choi JP, Rho S, Park SH, Kim YK, Hwang D, Gho YS. 2011. Proteomic analysis of outer membrane vesicles derived from Pseudomonas aeruginosa . Proteomics 11:3424–3429. doi:10.1002/pmic.201000212 PubMed DOI

Reales-Calderón JA, Corona F, Monteoliva L, Gil C, Martínez JL. 2015. Quantitative proteomics unravels that the post-transcriptional regulator CRC modulates the generation of vesicles and secreted virulence determinants of Pseudomonas aeruginosa. Data Brief 4:450–453. doi:10.1016/j.dib.2015.07.002 PubMed DOI PMC

Toyofuku M, Roschitzki B, Riedel K, Eberl L. 2012. Identification of proteins associated with the Pseudomonas aeruginosa biofilm extracellular matrix. J Proteome Res 11:4906–4915. doi:10.1021/pr300395j PubMed DOI

Tamber SH, Robert EWH. 2004. The outer membranes of pseudomonads, . In Ramos J (ed), Pseudomonas. Springer. doi:10.1007/978-1-4419-9086-0 DOI

Buzas EI. 2022. Opportunities and challenges in studying the extracellular vesicle corona. Nat Cell Biol 24:1322–1325. doi:10.1038/s41556-022-00983-z PubMed DOI

Devos S, Van Putte W, Vitse J, Van Driessche G, Stremersch S, Van Den Broek W, Raemdonck K, Braeckmans K, Stahlberg H, Kudryashev M, Savvides SN, Devreese B. 2017. Membrane vesicle secretion and prophage induction in multidrug-resistant Stenotrophomonas maltophilia in response to ciprofloxacin stress. Environ Microbiol 19:3930–3937. doi:10.1111/1462-2920.13793 PubMed DOI

Orench-Rivera N, Kuehn MJ. 2016. Environmentally controlled bacterial vesicle-mediated export. Cell Microbiol 18:1525–1536. doi:10.1111/cmi.12676 PubMed DOI PMC

Wan W-L, Fröhlich K, Pruitt RN, Nürnberger T, Zhang L. 2019. Plant cell surface immune receptor complex signaling. Curr Opin Plant Biol 50:18–28. doi:10.1016/j.pbi.2019.02.001 PubMed DOI

Bauman SJ, Kuehn MJ. 2006. Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response. Microbes Infect 8:2400–2408. doi:10.1016/j.micinf.2006.05.001 PubMed DOI PMC

Zingl FG, Kohl P, Cakar F, Leitner DR, Mitterer F, Bonnington KE, Rechberger GN, Kuehn MJ, Guan Z, Reidl J, Schild S. 2020. Outer membrane vesiculation facilitates surface exchange and in vivo adaptation of Vibrio cholerae. Cell Host Microbe 27:225–237. doi:10.1016/j.chom.2019.12.002 PubMed DOI PMC

Devos S, Van Oudenhove L, Stremersch S, Van Putte W, De Rycke R, Van Driessche G, Vitse J, Raemdonck K, Devreese B. 2015. The effect of imipenem and diffusible signaling factors on the secretion of outer membrane vesicles and associated Ax21 proteins in Stenotrophomonas maltophilia. Front Microbiol 6:298. doi:10.3389/fmicb.2015.00298 PubMed DOI PMC

Campos ML, de Souza CM, de Oliveira KBS, Dias SC, Franco OL. 2018. The role of antimicrobial peptides in plant immunity. J Exp Bot 69:4997–5011. doi:10.1093/jxb/ery294 PubMed DOI

Vogel CM, Potthoff DB, Schäfer M, Barandun N, Vorholt JA. 2021. Protective role of the Arabidopsis leaf microbiota against a bacterial pathogen. Nat Microbiol 6:1537–1548. doi:10.1038/s41564-021-00997-7 PubMed DOI PMC

Manning AJ, Kuehn MJ. 2011. Contribution of bacterial outer membrane Vesicles to innate bacterial defense. BMC Microbiol 11:258. doi:10.1186/1471-2180-11-258 PubMed DOI PMC

Park J, Kim M, Shin B, Kang M, Yang J, Lee TK, Park W. 2021. A novel decoy strategy for polymyxin resistance in Acinetobacter Baumannii. Elife 10:e66988. doi:10.7554/eLife.66988 PubMed DOI PMC

Aznar A, Dellagi A. 2015. New insights into the role of siderophores as triggers of plant immunity: what can we learn from animals? J Exp Bot 66:3001–3010. doi:10.1093/jxb/erv155 PubMed DOI

Prados-Rosales R, Weinrick BC, Piqué DG, Jacobs WR, Casadevall A, Rodriguez GM. 2014. Role for Mycobacterium tuberculosis membrane vesicles in iron acquisition. J Bacteriol 196:1250–1256. doi:10.1128/JB.01090-13 PubMed DOI PMC

Parys K, Colaianni NR, Lee H-S, Hohmann U, Edelbacher N, Trgovcevic A, Blahovska Z, Lee D, Mechtler A, Muhari-Portik Z, Madalinski M, Schandry N, Rodríguez-Arévalo I, Becker C, Sonnleitner E, Korte A, Bläsi U, Geldner N, Hothorn M, Jones CD, Dangl JL, Belkhadir Y. 2021. Signatures of antagonistic pleiotropy in a bacterial flagellin epitope. Cell Host Microbe 29:620–634. doi:10.1016/j.chom.2021.02.008 PubMed DOI

Kunze G, Zipfel C, Robatzek S, Niehaus K, Boller T, Felix G. 2004. The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell 16:3496–3507. doi:10.1105/tpc.104.026765 PubMed DOI PMC

Zipfel C, Kunze G, Chinchilla D, Caniard A, Jones JDG, Boller T, Felix G. 2006. Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125:749–760. doi:10.1016/j.cell.2006.03.037 PubMed DOI

Jones AM, Wildermuth MC. 2011. The phytopathogen Pseudomonas syringae pv. tomato DC3000 has three high-affinity iron-scavenging systems functional under iron limitation conditions but dispensable for pathogenesis. J Bacteriol 193:2767–2775. doi:10.1128/JB.00069-10 PubMed DOI PMC

Katagiri F, Thilmony R, He SY. 2002. The Arabidopsis thaliana-Pseudomonas Syringae interaction. Arabidopsis Book 1:e0039. doi:10.1199/tab.0039 PubMed DOI PMC

Liang J, Klingl A, Lin Y-Y, Boul E, Thomas-Oates J, Marín M. 2019. A subcompatible rhizobium strain reveals infection duality in Lotus . J Exp Bot 70:1903–1913. doi:10.1093/jxb/erz057 PubMed DOI PMC

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. 2012. Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. doi:10.1038/nmeth.2019 PubMed DOI PMC

Flechsler J, Heimerl T, Pickl C, Rachel R, Stierhof YD, Klingl A. 2020. 2D and 3D immunogold localization on (epoxy) ultrathin sections with and without osmium tetroxide. Microsc Res Tech 83:691–705. doi:10.1002/jemt.23459 PubMed DOI

Krcková Z, Kocourková D, Danek M, Brouzdová J, Pejchar P, Janda M, Pokotylo I, Ott PG, Valentová O, Martinec J. 2018. The Arabidopsis thaliana non-specific phospholipase C2 is involved in the response to Pseudomonas syringae attack. Ann Bot 121:297–310. doi:10.1093/aob/mcx160 PubMed DOI PMC

Béziat C, Kleine-Vehn J, Feraru E. 2017. Histochemical staining of β-glucuronidase and its spatial quantification. Methods Mol Biol 1497:73–80. doi:10.1007/978-1-4939-6469-7_8 PubMed DOI

Andriankaja A, Boisson-Dernier A, Frances L, Sauviac L, Jauneau A, Barker DG, de Carvalho-Niebel F. 2007. AP2-ERF transcription factors mediate Nod factor dependent Mt ENOD11 activation in root hairs via a novel cis-regulatory motif. Plant Cell 19:2866–2885. doi:10.1105/tpc.107.052944 PubMed DOI PMC

Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR. 2005. Genome-Wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17. doi:10.1104/pp.105.063743 PubMed DOI PMC

Marshall OJ. 2004. Perlprimer: Cross-platform, graphical Primer design for standard, Bisulphite and real-time PCR. Bioinformatics 20:2471–2472. doi:10.1093/bioinformatics/bth254 PubMed DOI

Mersmann S, Bourdais G, Rietz S, Robatzek S. 2010. Ethylene signaling regulates accumulation of the FLS2 receptor and is required for the oxidative burst contributing to plant immunity. Plant Physiol 154:391–400. doi:10.1104/pp.110.154567 PubMed DOI PMC

Park AJ, Murphy K, Krieger JR, Brewer D, Taylor P, Habash M, Khursigara CM. 2014. A temporal examination of the planktonic and biofilm proteome of whole cell Pseudomonas aeruginosa PAO1 using quantitative mass spectrometry. Molecular & Cellular Proteomics 13:1095–1105. doi:10.1074/mcp.M113.033985 PubMed DOI PMC

Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi:10.1006/abio.1976.9999 PubMed DOI

Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. 2006. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860. doi:10.1038/nprot.2006.468 PubMed DOI

Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M. 2011. Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10:1794–1805. doi:10.1021/pr101065j PubMed DOI

Tyanova S, Temu T, Cox J. 2016. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11:2301–2319. doi:10.1038/nprot.2016.136 PubMed DOI

Winsor GL, Griffiths EJ, Lo R, Dhillon BK, Shay JA, Brinkman FSL. 2016. Enhanced Annotations and features for comparing thousands of Pseudomonas Genomes in the Pseudomonas genome database. Nucleic Acids Res 44:D646–D653. doi:10.1093/nar/gkv1227 PubMed DOI PMC

Sambrook J, Russell DW. 2001. Molecular cloning: A laboratory manual, . In Cold spring harbor laboratory, 3rd ed. Cold Spring Harbor, N.Y.

Perez-Riverol Y, Csordas A, Bai J, Bernal-Llinares M, Hewapathirana S, Kundu DJ, Inuganti A, Griss J, Mayer G, Eisenacher M, Pérez E, Uszkoreit J, Pfeuffer J, Sachsenberg T, Yilmaz S, Tiwary S, Cox J, Audain E, Walzer M, Jarnuczak AF, Ternent T, Brazma A, Vizcaíno JA. 2019. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res 47:D442–D450. doi:10.1093/nar/gky1106 PubMed DOI PMC