title: A role for beta-sitosterol to stigmasterol conversion in plant-pathogen interactions.
abstract: Upon inoculation with pathogenic microbes, plants induce an array of metabolic changes that potentially contribute to induced resistance or even enhance susceptibility. When analysing leaf lipid composition during the Arabidopsis thaliana-Pseudomonas syringae interaction, we found that accumulation of the phytosterol stigmasterol is a significant plant metabolic process that occurs upon bacterial leaf infection. Stigmasterol is synthesized from beta-sitosterol by the cytochrome P450 CYP710A1 via C22 desaturation. Arabidopsis cyp710A1 mutant lines impaired in pathogen-inducible expression of the C22 desaturase and concomitant stigmasterol accumulation are more resistant to both avirulent and virulent P. syringae strains than wild-type plants, and exogenous application of stigmasterol attenuates this resistance phenotype. These data indicate that induced sterol desaturation in wild-type plants favours pathogen multiplication and plant susceptibility. Stigmasterol formation is triggered through perception of pathogen-associated molecular patterns such as flagellin and lipopolysaccharides, and through production of reactive oxygen species, but does not depend on the salicylic acid, jasmonic acid or ethylene defence pathways. Isolated microsomal and plasma membrane preparations exhibited a similar increase in the stigmasterol/beta-sitosterol ratio as whole-leaf extracts after leaf inoculation with P. syringae, indicating that the stigmasterol produced is incorporated into plant membranes. The increased contents of stigmasterol in leaves after pathogen attack do not influence salicylic acid-mediated defence signalling but attenuate pathogen-induced expression of the defence regulator flavin-dependent monooxygenase 1. P. syringae thus promotes plant disease susceptibility through stimulation of sterol C22 desaturation in leaves, which increases the stigmasterol to beta-sitosterol ratio in plant membranes.
authors.affiliation: Julius-von-Sachs-Institute of Biological Sciences, University of Würzburg, Julius-von-Sachs Platz 3, D-97082 Würzburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Griebel
authors.affiliation: Department of Biology, Plant Biology Section, University of Fribourg, Route Albert Gockel 3, CH-1700 Fribourg, Switzerland.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Zeier
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D020171:Botrytis / Q000472:pathogenicity; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D054884:Host-Pathogen Interactions; D010935:Plant Diseases / Q000382:microbiology; D010937:Plant Growth Regulators / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism / Q000382:microbiology; D044224:Pseudomonas syringae / Q000472:pathogenicity; D012855:Sitosterols / Q000378:metabolism*; D013265:Stigmasterol / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D010937:Plant Growth Regulators; D012855:Sitosterols; C025473:gamma-sitosterol; D003577:Cytochrome P-450 Enzyme System; D013265:Stigmasterol; C527746:CYP710A1 protein, Arabidopsis
references.citation: Alonso, J.M., Stepanova, A.N., Leisse, T.J. et al. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science, 301, 653-657.
references.citation: Alvarez, M.E., Pennell, R.I., Meijer, P.-J., Ishikawa, A., Dixon, R.A. and Lamb, C. (1998) Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell, 92, 773-784.
references.citation: Arnqvist, L., Persson, M., Jonsson, L., Dutta, P.C. and Sitbon, F. (2008) Overexpression of CYP710A1 and CYP710A4 in transgenic Arabidopsis plants increases the level of stigmasterol at the expense of sitosterol. Planta, 227, 309-317.
references.citation: Attaran, E., Rostás, M. and Zeier, J. (2008) Pseudomonas syringae elicits emission of the terpenoid (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene in Arabidopsis leaves via jasmonate signaling and expression of the terpene synthase TPS4. Mol. Plant Microbe Interact. 21, 1482-1497.
references.citation: Attaran, E., Zeier, T.E., Griebel, T. and Zeier, J. (2009) Methyl salicylate production and jasmonate signaling are not essential for systemic acquired resistance in Arabidopsis. Plant Cell, 21, 954-971.
references.citation: Bartsch, M., Gobbato, E., Bednarek, P., Debey, S., Schultze, J.L., Bautor, J. and Parker, J.E. (2006) Salicylic acid-independent ENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monoxygenase FMO1 and the nudix hydrolase NUDT7. Plant Cell, 18, 1038-1051.
references.citation: Benveniste, P. (2004) Biosynthesis and accumulation of sterols. Annu. Rev. Plant Biol. 55, 429-457.
references.citation: Bhat, R.A. and Panstruga, R. (2005) Lipid rafts in plants. Planta, 223, 5-19.
references.citation: Bhat, R.A., Miklis, M., Schmelzer, E., Schulze-Lefert, P. and Panstruga, R. (2005) Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain. Proc. Natl Acad. Sci. USA, 102, 3135-3140.
references.citation: Bisgrove, S.R., Simonich, M.T., Smith, N.M., Sattler, A. and Innes, R.W. (1994) A disease resistance gene in Arabidopsis with specificity for two different pathogen avirulence genes. Plant Cell, 6, 927-933.
references.citation: Bleecker, A.B., Estelle, M.A., Somerville, C. and Kende, H. (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science, 241, 1086-1089.
references.citation: Borner, G.H., Sherrier, D.J., Weimar, T., Michaelson, L.V., Hawkins, N.D., Macaskill, A., Napier, J.A., Beale, M.H., Lilley, K.S. and Dupree, P. (2005) Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts. Plant Physiol. 137, 104-116.
references.citation: Bowling, S.A., Clarke, J.D., Liu, Y., Klessig, D.F. and Dong, X. (1997) The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance. Plant Cell, 9, 1573-1584.
references.citation: Cameron, R.K., Dixon, R.A. and Lamb, C.J. (1994) Biologically induced systemic acquired resistance in Arabidopsis thaliana. Plant J. 5, 715-725.
references.citation: Cao, H., Bowling, S.A., Gordon, A.S. and Dong, X.N. (1994) Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance. Plant Cell, 6, 1583-1592.
references.citation: Century, K.S., Holub, E.B. and Staskawicz, B.J. (1995) NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a fungal pathogen. Proc. Natl Acad. Sci. USA, 92, 6597-6601.
references.citation: 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.
references.citation: Dean, J.V. and Delaney, S.P. (2008) Metabolism of salicylic acid in wild-type, ugt74f1 and ugt74f2 glucosyltransferase mutants of Arabidopsis thaliana. Physiol. Plant, 132, 417-425.
references.citation: Delledonne, M., Xia, Y., Dixon, R.A. and Lamb, C. (1998) Nitric oxide functions as a signal in plant disease resistance. Nature, 394, 585-588.
references.citation: Drazkiewicz, M., Skorzynska-Polit, E. and Krupa, Z. (2004) Copper-induced oxidative stress and antioxidant defence in Arabidopsis thaliana. Biometals, 17, 379-387.
references.citation: Fabro, G., Di Rienzo, J.A., Voigt, C.A., Savchenko, T., Dehesh, K., Somerville, S. and Alvarez, M.E. (2008) Genome-wide expression profiling Arabidopsis at the stage of Golovinomyces cichoracearum haustorium formation. Plant Physiol. 146, 1421-1439.
references.citation: Felix, G., Duran, J.D., Volko, S. and Boller, T. (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J. 18, 265-276.
references.citation: Felle, H.H., Herrmann, A., Hückelhoven, R. and Kogel, K.H. (2005) Root-to-shoot signalling: apoplastic alkalinization, a general stress response and defence factor in barley (Hordeum vulgare). Protoplasma, 227, 17-24.
references.citation: Glazebrook, J. (2005) Contrasting mechanisms of defence against biotrophic and necrotrophic pathogens. Annu. Rev. Phytopathol. 43, 205-227.
references.citation: Glazebrook, J., Zook, M., Mert, F., Kagan, I., Rogers, E.E., Crute, I.R., Holub, E.B., Hammerschmidt, R. and Ausubel, F.M. (1997) Phytoalexin-deficient mutants of Arabidopsis reveal that PAD4 encodes a regulatory factor and that four PAD genes contribute to downy mildew resistance. Genetics, 146, 381-392.
references.citation: Gomez-Gomez, L., Felix, G. and Boller, T. (1999) A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana. Plant J. 18, 277-284.
references.citation: Govrin, E.M. and Levine, A. (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea. Curr. Biol. 10, 751-757.
references.citation: Granado, J., Felix, G. and Boller, T. (1995) Perception of fungal sterols in plants (subnanomolar concentrations of ergosterol elicit extracellular alkalinization in tomato cells). Plant Physiol. 107, 485-490.
references.citation: Grandmougin-Ferjani, A., Schuler-Muller, I. and Hartmann, M.A. (1997) Sterol modulation of the plasma membrane H+-ATPase activity from corn roots reconstituted into soybean lipids. Plant Physiol. 113, 163-174.
references.citation: Grant, M. and Lamb, C. (2006) Systemic immunity. Curr. Opin. Plant Biol. 9, 414-420.
references.citation: Griebel, T. and Zeier, J. (2008) Light regulation and daytime dependency of inducible plant defenses in Arabidopsis: phytochrome signaling controls systemic acquired resistance rather than local defense. Plant Physiol. 147, 790-801.
references.citation: Grun, C., Berger, S., Matthes, D. and Mueller, M.J. (2007) Early accumulation of non-enzymatically synthesized oxylipins in Arabidopsis thaliana after infection with Pseudomonas syringae. Funct. Plant Biol. 34, 1-7.
references.citation: Hartmann, M.-A. (1998) Plant sterols and the membrane environment. Trends Plant Sci. 3, 170-175.
references.citation: Hodzic, A., Rappolt, M., Amenitsch, H., Laggner, P. and Pabst, G. (2008) Differential modulation of membrane structure and fluctuations by plant sterols and cholesterol. Biophys. J. 94, 3935-3944.
references.citation: Katagiri, F., Thilmony, R. and He, S.Y. (2002) The Arabidopsis thaliana-Pseudomonas syringae interaction. In The Arabidopsis Book (Somerville, C.R. and Meyerowitz, E.M., eds). Rockville, MD: American Society of Plant Biologists, doi 10.1199-tab.0039.
references.citation: Kloek, A.P., Verbsky, M.L., Sharma, S.B., Schoelz, J.E., Vogel, J., Klessig, D.F. and Kunkel, B.N. (2001) Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms. Plant J. 26, 509-522.
references.citation: Koch, M., Vorwerk, S., Masur, C., Sharifi-Sirchi, G., Olivieri, N. and Schlaich, N.L. (2006) A role for a flavin-containing mono-oxygenase in resistance against microbial pathogens in Arabidopsis. Plant J. 47, 629-639.
references.citation: Kunze, G., Zipfel, C., Robatzek, S., Niehaus, K., Boller, T. and Felix, G. (2004) The N-terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell, 16, 3496-3507.
references.citation: Laloi, M., Perret, A.M., Chatre, L. et al. (2007) Insights into the role of specific lipids in the formation and delivery of lipid microdomains to the plasma membrane of plant cells. Plant Physiol. 143, 461-472.
references.citation: Lamb, C. and Dixon, R.A. (1997) The oxidative burst in plant disease resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 251-275.
references.citation: Maldonado, A.M., Doerner, P., Dixon, R.A., Lamb, C. and Cameron, R.C. (2002) A putative lipid transfer protein involved in systemic resistance signalling in Arabidopsis. Nature, 419, 399-403.
references.citation: Mateo, A., Funck, D., Mühlenbock, P., Kular, B., Mullineaux, P.M. and Karpinski, S. (2006) Controlled levels of salicylic acid are required for optimal photosynthesis and redox homeostasis. J. Exp. Bot. 57, 1795-1807.
references.citation: Meyer, A., Puhler, A. and Niehaus, K. (2001) The lipopolysaccharides of the phytopathogen Xanthomonas campestris pv. campestris induce an oxidative burst reaction in cell cultures of Nicotiana tabacum. Planta, 213, 214-222.
references.citation: Mikes, V., Milat, M.L., Ponchet, M., Panabières, F., Ricci, P. and Blein, J.P. (1998) Elicitins, proteinaceous elicitors of plant defense, are a new class of sterol carrier proteins. Biochem. Biophys. Res. Commun. 245, 133-139.
references.citation: Mishina, T.E. and Zeier, J. (2006) The Arabidopsis flavin-dependent monooxygenase FMO1 is an essential component of biologically induced systemic acquired resistance. Plant Physiol. 141, 1666-1675.
references.citation: Mishina, T.E. and Zeier, J. (2007a) Interactions of Arabidopsis with non-adapted Pseudomonas syringae strains: possible determinants of bacterial non-host resistance. Physiol. Plant, 131, 448-461.
references.citation: Mishina, T.E. and Zeier, J. (2007b) Pathogen-associated molecular pattern recognition rather than development of tissue necrosis contributes to bacterial induction of systemic acquired resistance in Arabidopsis. Plant J. 50, 500-513.
references.citation: Mishina, T.E., Griebel, T., Geuecke, M., Attaran, E. and Zeier, J. (2008) New insights into the molecular events underlying systemic acquired resistance. In Biology of Plant-Microbe Interactions Volume 6 (Lorito, M., Woo, S.L. and Scala, F., eds). St Paul, MN: International Society for Molecular Plant-Microbe Interactions, paper 81.
references.citation: Mongrand, S., Morel, J., Laroche, J., Claverol, S., Carde, J.P., Hartmann, M.A., Bonneu, M., Simon-Plas, F., Lessire, R. and Bessoule, J.J. (2004) Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane. J. Biol. Chem. 279, 36277-36286.
references.citation: Morikawa, T., Mizutani, M., Aoki, N. et al. (2006) Cytochrome P450 CYP710A encodes the sterol C-22 desaturase in Arabidopsis and tomato. Plant Cell, 18, 1008-1022.
references.citation: Morikawa, T., Saga, H., Hashizume, H. and Ohta, D. (2009) CYP710A genes encoding sterol C22-desaturase in Physcomitrella patens as molecular evidence for the evolutionary conservation of a sterol biosynthetic pathway in plants. Planta, 229, 1311-1322.
references.citation: \Nawrath, C. and Métraux, J.P. (1999) Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation. Plant Cell, 11, 1393-1404.
references.citation: Ohyama, K., Suzuki, M., Kikuchi, J., Saito, K. and Muranaka, T. (2009) Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis. Proc. Natl Acad. Sci. USA, 106, 725-730.
references.citation: Schaeffer, A., Bronner, R., Benveniste, P. and Schaller, H. (2001) The ratio of campesterol to sitosterol that modulates growth in Arabidopsis is controlled by STEROL METHYLTRANSFERASE 2;1. Plant J. 25, 605-615.
references.citation: Schaller, H. (2003) The role of sterols in plant growth and development. Prog. Lipid Res. 42, 163-175.
references.citation: Schaller, H. (2004) New aspects of sterol biosynthesis in growth and development of higher plants. Plant Physiol. Biochem. 42, 465-476.
references.citation: Schlaich, N.L. (2007) Flavin-containing monooxygenases in plants: looking beyond detox. Trends Plant Sci. 12, 412-418.
references.citation: Schuler, I., Milon, A., Nakatani, Y., Ourisson, G., Albrecht, A.M., Benveniste, P. and Hartman, M.A. (1991) Differential effects of plant sterols on water permeability and on acyl chain ordering of soybean phosphatidylcholine bilayers. Proc. Natl Acad. Sci. USA, 88, 6926-6930.
references.citation: Staswick, P.E., Su, W. and Howell, S.H. (1992) Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant. Proc. Natl Acad. Sci. USA, 89, 6837-6840.
references.citation: Stefanato, F.L., Abou-Mansour, E., Buchala, A., Kretschmer, M., Mosbach, A., Hahn, M., Bochet, C.G., Métraux, J.P. and Schoonbeek, H.-J. (2009) The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana. Plant J. 58, 499-510.
references.citation: Thomma, B.P., Nelissen, I., Eggermont, K. and Broekaert, W.F. (1999) Deficiency in phytoalexin production causes enhanced susceptibility of Arabidopsis thaliana to the fungus Alternaria brassicicola. Plant J. 19, 163-171.
references.citation: de Torres Zabala, M., Bennett, M.H., Truman, W.H. and Grant, M.R. (2009) Antagonism between salicylic and abscisic acid reflects early host-pathogen conflict and moulds plant defence responses. Plant J. 59, 375-386.
references.citation: Torres, M.A., Dangl, J.L. and Jones, J.D. (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc. Natl Acad. Sci. USA, 99, 517-522.
references.citation: Von Malek, B., van der Graaff, E., Schneitz, K. and Keller, B. (2002) The Arabidopsis male-sterile mutant dde2-2 is defective in the ALLENE OXIDE SYNTHASE gene encoding one of the key enzymes of the jasmonic acid biosynthesis pathway. Planta, 216, 187-192.
references.citation: Wildermuth, M.C., Dewdney, J., Wu, G. and Ausubel, F.M. (2001) Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature, 414, 562-565.
references.citation: Yaeno, T., Matsuda, O. and Iba, K. (2004) Role of chloroplast trienoic fatty acids in plant disease defense responses. Plant J. 40, 931-941.
references.citation: Yousef, L.F., Yousef, A.F., Mymryk, J.S., Dick, W.A. and Dick, R.P. (2009) Stigmasterol and cholesterol regulate the expression of elecitin genes in Phytophtora sojae. J. Chem. Ecol. 35, 824-832.
references.citation: Yu, I.-c., Parker, J. and Bent, A.F. (1998) Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant. Proc. Natl Acad. Sci. USA, 95, 7819-7824.
references.citation: Zappel, N.F. and Panstruga, R. (2008) Heterogeneity and lateral compartmentalization of plant plasma membranes. Curr. Opin. Plant Biol. 11, 632-640.
references.citation: Zeier, J. (2005) Age-dependent variations of local and systemic defense responses in Arabidopsis leaves towards an avirulent strain of Pseudomonas syringae. Physiol. Mol. Plant Pathol. 66, 30-39.
references.citation: Zeier, J., Pink, B., Mueller, M.J. and Berger, S. (2004) Light conditions influence specific defence responses in incompatible plant-pathogen interactions: uncoupling systemic resistance from salicylic acid and PR-1 accumulation. Planta, 219, 673-683.
references.citation: Zhou, N., Tootle, T.L. and Glazebrook, J. (1999) Arabidopsis PAD3, a gene required for camalexin biosynthesis, encodes a putative cytochrome P450 monooxygenase. Plant Cell, 11, 2419-2428.
references.citation: Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L. and Gruissem, W. (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol. 136, 2621-2632.