Abstract
Sphingolipids play an important role in signal transduction pathways that regulate physiological functions and stress responses in eukaryotes. In plants, recent evidence suggests that their metabolic precursors, the long-chain bases (LCBs) act as bioactive molecules in the immune response. Interestingly, the virulence of two unrelated necrotrophic fungi, Fusarium verticillioides and Alternaria alternata, which are pathogens of maize and tomato plants, respectively, depends on the production of sphinganine-analog mycotoxins (SAMs). These metabolites inhibit de novo synthesis of sphingolipids in their hosts causing accumulation of LCBs, which are key regulators of programmed cell death. Therefore, to gain more insight into the role of sphingolipids in plant immunity against SAM-producing necrotrophic fungi, we disrupted sphingolipid metabolism in Nicotiana benthamiana through virus-induced gene silencing (VIGS) of the serine palmitoyltransfersase (SPT). This enzyme catalyzes the first reaction in LCB synthesis. VIGS of SPT profoundly affected N. benthamiana development as well as LCB composition of sphingolipids. While total levels of phytosphingosine decreased, sphinganine and sphingosine levels increased in SPT-silenced plants, compared with control plants. Plant immunity was also affected as silenced plants accumulated salicylic acid (SA), constitutively expressed the SA-inducible NbPR-1 gene and showed increased susceptibility to the necrotroph A. alternata f. sp. lycopersici. In contrast, expression of NbPR-2 and NbPR-3 genes was delayed in silenced plants upon fungal infection. Our results strongly suggest that LCBs modulate the SA-dependent responses and provide a working model of the potential role of SAMs from necrotrophic fungi to disrupt the plant host response to foster colonization.
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Abbreviations
- SPT:
-
Serine palmitoyltransferase
- LCB:
-
Long-chain base
- d18:0:
-
Sphinganine
- d18:1(Δ4):
-
Sphingosine
- t18:0:
-
Phytosphingosine
- SAMs:
-
Sphinganine-analog mycotoxins
- FB1:
-
Fumonisin B1
- JA:
-
Jasmonic acid
- PCD:
-
Programmed cell death
- VIGS:
-
Virus-induced gene silencing
- PR:
-
Pathogenesis-related
- SA:
-
Salicylic acid
References
Abbas HK, Tanaka T, Duke SO, Porter JK, Wray EM, Hodges L, Sessions A, Wang E, Merril AH, Riley RT (1994) Fumonisin- and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases. Plant Physiol 106:1085–1093
Akamatsu H, Itoh Y, Kodama M, Otani H, Kohmoto K (1997) AAL-toxin-deficient mutants of Alternaria alternata tomato pathotype by restriction enzyme-mediated integration. Phytopathology 87:967–972
An C, Mou Z (2011) Salicylic acid and its function in plant immunity. J Int Plant Biol 53:412–428
Asai T, Stone JM, Heard JE, Kovtun Y, Yorgey P, Sheen J, Ausubel FM (2000) Fumonisin B1-induced cell death in Arabidopsis protoplasts requires jasmonate-, ethylene-, and salicylate-dependent signaling pathways. Plant Cell 12:1823–1835
Aubert A, Marion J, Boulogne C, Bourge M, Abreu S, Bellec Y, Faure JD, Satiat-Jeunemaitre B (2011) Sphingolipids involvement in plant endomembrane differentiation: the BY2 case. Plant J 65:958–971
Berkey R, Bendigeri D, Xiao S (2012) Sphingolipids and plant defense/disease: the “death” connection and beyond. Front Plant Sci 3:68
Borner GH, Sherrier DJ, Weimar T, Michaelson LV, Hawkins ND, Macaskill A, Napier JA, Beale MH, Lilley KS, Dupree P (2005) Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts. Plant Physiol 137:104–116
Brandwagt BF, Kneppers TJA, Van der Weerden GM, Nijkamp HJJ, Hille J (2001) Most AAL toxin-sensitive Nicotiana species are resistant to the tomato fungal pathogen Alternaria alternata f. sp. lycopersici. Mol Plant Microbe Interact 14:460–470
Breslow DK, Collins SR, Bodenmiller B, Aebersold R, Simons K, Shevchenko A, Ejsing CS, Weissman JS (2010) Orm family proteins mediate sphingolipid homeostasis. Nature 463:1048–1055
Brodersen P, Petersen M, Pike HM, Olszak B, Skov S, Odum N, Jorgensen LB, Brown RE, Mundy J (2002) Knockout of Arabidopsis accelerated cell death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defense. Genes Dev 16:490–502
Brodersen P, Malinovsky FG, Hématy K, Newman MA, Mundy J (2005) The role of salicylic acid in the induction of cell death in Arabidopsis acd11. Plant Physiol 138:1037–1045
Buré C, Cacas JL, Wang F, Gaudin K, Domergue F, Mongrand S, Schmitter JM (2011) Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry. Rapid Commun Mass Spectrom 25:3131–3145
Catinot J, Buchala A, Abou-Monsour E, Métraux JP (2008) Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana. FEBS Lett 582:473–478
Chen M, Han G, Dietrich CR, Dunn TM, Cahoon EB (2006) The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase. Plant Cell 18:3576–3593
Chen M, Markham JE, Dietrich CR, Jaworski JG, Cahoon EB (2008) Sphingolipid long-chain base hydroxylation is important for growth and regulation of sphingolipid content and composition in Arabidopsis. Plant Cell 20:1862–1878
Chen M, Cahoon EB, Saucedo-García M, Plasencia J, Gavilanes-Ruíz M (2009) Plant sphingolipids: structure, synthesis and function. In: Wada H, Murata N, Govindjee (eds) Lipids in photosynthesis: essential and regulatory functions. Springer, Dordrecht, pp 77–115
Chen M, Markham JE, Cahoon EB (2012) Sphingolipid Δ8 unsaturation is important for glucosylceramide biosynthesis and low-temperature performance in Arabidopsis. Plant J 69:769–781
Coursol S, Fan LM, Le Stunff H, Spiegel S, Gilroy S, Assmann SM (2003) Sphingolipid signalling in Arabidopsis guard cells involves heterotrimeric G proteins. Nature 423:651–654
Dana M, Pintor-Toro J, Cubero B (2006) Transgenic tobacco plants overexpressing chitinases of fungal origin show enhanced resistance to biotic and abiotic stress agents. Plant Physiol 142:722–730
de la Torre-Hernández ME, Rivas-San Vicente M, Greaves-Fernández N, Cruz-Ortega R, Plasencia J (2010) Fumonisin B1 induces nuclease activation and salicylic acid accumulation through long-chain sphingoid base build-up in germinating maize. Physiol Mol Plant Pathol 74:337–345
Denoux C, Galletti R, Mammarella N, Gopalan S, Werck D, De Lorenzo G, Ferrari S, Ausubel FM, Dewdney J (2008) Activation of defense response pathways by OGs and Flg22 elicitors in Arabidopsis seedlings. Mol Plant 1:423–445
Desjardins AE, Plattner RD, Nelsen TC, Leslie JF (1995) Genetic analysis of fumonisin production and virulence of Gibberella fujikuroi mating population A (Fusarium monilliforme) on maize (Zea mays) seedlings. Appl Environ Microbiol 61:79–86
Dietrich CR, Han G, Chen M, Howard R, Dunn TM, Cahoon EB (2008) Loss-of-function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability. Plant J 54:284–298
Dutilleul C, Benhassaine-Kesri G, Demandre C, Rézé N, Launay A, Pelletier S, Renou JP, Zachowski A, Baudouin E, Guillas I (2012) Phytosphingosine-phosphate is a signal for AtMPK6 activation and Arabidopsis response to chilling. New Phytol 194:181–191
Egusa M, Ozawa R, Takabayashi J, Otani H, Kodama M (2009) The jasmonate signaling pathway in tomato regulates susceptibility to a toxin-dependent necrotrophic pathogen. Planta 229:965–976
El Oirdi M, El Rahman TA, Rigano L, El Hadrami A, Rodriguez MC, Daayf F, Vojnov A, Bouarab K (2011) Botrytis cinerea manipulates the antagonistic effects between immune pathways to promote disease development in tomato. Plant Cell 23:2405–2421
Gable K, Slife H, Bacikova D, Monaghan E, Dunn TM (2000) Tsc3p is an 80-amino acid protein associated with serine palmitoyltransferase and required for optimal enzyme activity. J Biol Chem 275:7597–7603
Gan Y, Zhang L, Zhang Z, Dong S, Li J, Wang Y, Zheng X (2009) The LCB2 subunit of the sphingolipid biosynthesis enzyme serine palmitoyltransferase can function as an attenuator of the hypersensitive response and Bax-induced cell death. New Phytol 181:127–146
Gechev TS, Gadjev IZ, Hille J (2004) An extensive microarray analysis of AAL-toxin-induced cell death in Arabidopsis thaliana brings new insights into the complexity of programmed cell death in plants. Cell Mol Life Sci 61:1185–1197
Gilchrist DG, Grogan RG (1976) Production and nature of a host-specific toxin from Alternaria alternata f. sp. lycopersici. Phytopathology 66:165–171
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43:205–227
Glenn AE, Zitomer NC, Zimeri AM, Williams LD, Riley RT, Proctor RH (2008) Transformation-mediated complementation of a FUM gene cluster deletion in Fusarium verticillioides restores both fumonisin production and pathogenicity on maize seedlings. Mol Plant Microbe Interact 21:87–97
Greenberg JT, Silverman P, Liang H (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses from disease resistance in the Arabidopsis mutant acd5. Genetics 156:341–350
Jach G, Gornhardt B, Mundy J, Logemann J, Pinsdorf E, Leah R, Schell J, Maas C (1995) Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J 8:97–109
Koga J, Yamauchi T, Shimura M, Ogawa N, Oshima K, Umemura K, Kikuchi M, Ogasawara N (1998) Cerebrosides A and C, sphingolipid elicitors of hypersensitive cell death and phytoalexin accumulation in rice plants. J Biol Chem 273:31985–31988
Kommedahl T, Windels CE (1981) Root-, stalk-, and ear-infecting Fusarium species on corn in the USA. In: Nelson PE, Toussoun TA, Cook RJ (eds) Fusarium: diseases, biology, and taxonomy. The Pennsylvania State University Press, University Park, pp 94–103
Koornneef A, León-Reyes A, Ritsema T, Verhage A, Den Otter FC, Van Loon LC, Pieterse CMJ (2008) Kinetics of salicylate-mediated suppression of jasmonate signaling reveal a role for redox modulation. Plant Physiol 147:1358–1368
Lachaud C, Da Silva D, Cotelle V, Thuleau P, Xiong TC, Jauneau A, Brière C, Graziana A, Bellec Y, Faure JD, Ranjeva R, Mazars C (2010) Nuclear calcium controls the apoptotic-like cell death induced by D-erythro-sphinganine in tobacco cells. Cell Calcium 47:92–100
Lachaud C, Da Silva D, Amelot N, Béziat C, Brière C, Cotelle V, Graziana A, Grat S, Mazars C, Thuleau P (2011) Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H2O2 production. Mol Plant 4:310–318
Lamprecht SC, Marasas WFO, Alberts JF, Cawood ME, Gelderblom WCA, Shephard GS, Thiel PG, Calitz FJ (1994) Phytotoxicity of fumonisins and TA-toxin to corn and tomato. Phytopathology 84:383–391
Lawrence CB, Josten MHAJ, Tuzun S (1996) Differential induction of pathogenesis-related proteins in tomato by Alternaria solani and the association of basic chitinase isozyme with resistance. Physiol Mol Plant Pathol 48:361–377
Leon-Reyes A, Spoel SH, De Lange ES, Abe H, Kobayashi M, Tsuda S, Millenaar FF, Welschen RAM, Ritsema T, Pieterse CMJ (2009) Ethylene modulates the role of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 in cross talk between salicylate and jasmonate signaling. Plant Physiol 149:1797–1809
Leon-Reyes A, Du Y, Koornneef A, Proietti S, Körbes AP, Memelink J, Pieterse CMJ, Ritsema T (2010) Ethylene signaling renders the jasmonate response of Arabidopsis insensitive to future suppression by salicylic acid. Mol Plant Microbe Int 23:187–197
Liang H, Yao N, Song JT, Luo S, Lu H, Greenberg JT (2003) Ceramides modulate programmed cell death in plants. Genes Dev 17:2636–2641
Lieberherr D, Thao NP, Nakashima A, Umemura K, Kawasaki T, Shimamoto K (2005) A sphingolipid elicitor-inducible mitogen-activated protein kinase is regulated by the small GTPase OsRac1 and heterotrimeric G-protein in rice. Plant Physiol 138:1644–1652
Lynch DV, Dunn TM (2004) An introduction to plant sphingolipids and a review of recent advances in understanding their metabolism and function. New Phytol 161:677–702
Markham JE, Li J, Cahoon EB, Jaworski JG (2006) Separation and identification of major plant sphingolipid classes from leaves. J Biol Chem 281:22684–22694
Markham JE, Molino D, Gissot L, Bellec Y, Hématy K, Marion J, Belcram K, Palauqui JC, Satiat-JeuneMaître S, Faure JD (2011) Sphingolipids containing very-long-chain fatty acids define a secretory pathway for specific polar plasma membrane protein targeting in Arabidopsis. Plant Cell 23:2362–2378
Mase K, Mizuno T, Ishihama N, Fuji T, Mori H, Kodama M, Yoshioka H (2012) Ethylene signaling pathway and MAPK cascades are required for AAL-toxin-induced programmed cell death. Mol Plant Microbe Interact 25:1015–1025
Mesbah LA, van der Weerden GM, Nijkamp HJJ, Hille J (2000) Sensitivity among species of Solanaceae to AAL toxins produced by Alternaria alternata f. sp. lycopersici. Plant Pathol 49:734–741
Meuwly P, Métraux JP (1993) Ortho-anisic acid as internal standard for the simultaneous quantitation of salicylic acid and its putative biosynthetic precursors in cucumber leaves. Anal Biochem 214:500–505
Mongrand S, Morel J, Laroche J, Claverol S, Carde JP, Hartmann MA, Bonneu M, Simon-Plas F, Lessire R, Bessoule JJ (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
Mur LAJ, Kenton P, Atzorn R, Miersch O, Wasternack C (2006) The outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death. Plant Physiol 140:249–262
Ng CK, Carr K, McAinsh MR, Powell B, Hetherington AM (2001) Drought-induced guard cells signal transduction involves sphingosine-1-phosphate. Nature 410:596–599
Pata MO, Hannun YA, Ng CKY (2010) Plant sphingolipids: decoding the enigma of the sphinx. New Phytol 185:611–630
Peer M, Stegmann M, Mueller MJ, Waller F (2010) Pseudomonas syringae infection triggers de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana. FEBS Lett 584:4053–4056
Pieterse CMJ, León-Reyes A, Van der Ent S, Van Wees SCM (2009) Networking by small-molecule hormones in plant immunity. Nat Chem Biol 5:308–316
Pieterse CMJ, Van der Does D, Zamioudis C, Leon-Reyes A, Van Wees SCM (2012) Hormonal modulation of plant immunity. Annu Rev Cell Dev Biol 28:28.1–28.33
Ratcliff F, Martin-Hernandez AM, Baulcombe DC (2001) Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J 25:237–245
Rivas-San Vicente M, Plasencia J (2011) Salicylic acid beyond defence: its role in plant growth and development. J Exp Bot 62:3321–3338
Robert-Seilaniantz A, Grant M, Jones JDG (2011) Hormone crosstalk in plant disease and defense: more than just JASMONATE-SALICYLATE antagonism. Annu Rev Phytopathol 49:26.1–26.27
Sánchez-Rangel D, Plasencia J (2010) The role of sphinganine analog mycotoxins on the virulence of plant pathogenic fungi. Toxin Rev 29:73–86
Saucedo-García M, Guevara-García A, González-Solís A, Cruz-García F, Vázquez-Santana S, Markham JE, Lozano-Rosas MG, Dietrich CR, Ramos-Vega M, Cahoon EB, Gavilanes-Ruíz M (2011) MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis. New Phytol 191:943–957
Shi L, Bielawski J, Mu J, Dong H, Teng C, Zhang J, Yang X, Tomishige N, Hanada K, Hannun YA, Zuo J (2007) Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis. Cell Res 17:1030–1040
Spassieva SD, Markham JE, Hille J (2002) The plant disease resistance gene Asc-1 prevents disruption of sphingolipid metabolism during AAL-toxin-induced programmed cell death. Plant J 32:561–572
Sperling P, Heinz E (2003) Plant sphingolipids: structural diversity, biosynthesis, first genes and functions. Biochim Biophys Acta 1632:1–15
Sperling P, Franke S, Lüthje S, Heinz E (2005) Are glucocerebrosides the predominant sphingolipids in plant plasma membranes? Plant Physiol Biochem 43:1031–1038
Spoel SH, Dong X (2008) Making sense of hormone crosstalk during plant immune responses. Cell Host Microbe 3:348–351
Spoel SH, Johnson JS, Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestyles. Proc Natl Acad Sci USA 104:18842–18847
Stone JM, Heard JE, Asai T, Ausubel FM (2000) Simulation of fungal-mediated cell death by fumonisin B1 and selection of fumonisin B1-resistant (fbr) Arabidopsis mutants. Plant Cell 12:1811–1822
Suharsono U, Fujisawa Y, Kawasaki T, Iwasaki Y, Satoh H, Shimamoto K (2002) The heterotrimeric G protein α subunit acts upstream of the small GTPase Rac in disease resistance of rice. Proc Natl Acad Sci USA 99:13307–13312
Takahashi Y, Berberich T, Kanzaki H, Matsumura H, Saitoh H, Tomonobu K, Terauchi R (2009) Serine palmitoyltransferase, the first step enzyme in sphingolipid biosynthesis, is involved in nonhost resistance. Mol Plant Microbe Int 22:31–38
Teng C, Dong H, Shi L, Deng Y, Mu J, Zhang J, Yang X, Zuo J (2008) Serine palmitoyltransferase, a key enzyme for de novo synthesis of sphingolipids, is essential for male gametophyte development in Arabidopsis. Plant Physiol 146:1322–1332
Vlot AC, Dempsey DMA, Klessig DF (2009) Salicylic acid, a multifaceted hormone to combat disease. Annu Rev Phytopathol 47:177–206
Wang W, Yang X, Tangchaiburana S, Ndeh R, Markham JE, Tsegaye Y, Dunn TM, Wang GL, Bellizi M, Parsons JF, Morrissey D, Bravo JE, Lynch DV, Xiao S (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed cell death associated with defense in Arabidopsis. Plant Cell 20:3163–3179
Watanabe N, Lam E (2011) Arabidopsis metacaspase 2d is a positive mediator of cell death induced during biotic and abiotic stresses. Plant J 66:969–982
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp 315–322
Willemsen V, Frimi J, Grebe M, van der Toorn A, Palme K, Scheres B (2003) Cell polarity and PIN protein positioning in Arabidopsis require STEROL METHYLTRANSFERASE1 function. Plant Cell 15:612–625
Worrall D, Ng CKY, Hetherington AM (2003) Sphingolipids, new players in plant signaling. Trends Plant Sci 8:317–320
Zäuner S, Ternes P, Warnecke D (2010) Biosynthesis of sphingolipids in plants (and some of their functions). Adv Exp Med Biol 688:249–263
Zhang L, Jia C, Liu L, Zhang Z, Li C, Wang Q (2011) The involvement of jasmonates and ethylene in Alternaria alternata f. sp. lycopersici toxin-induced tomato cell death. J Exp Bot 62:5405–5418
Acknowledgments
We thank to Dr. Baulcombe and Sainsbury Laboratory (Norwich, UK) for the TRV vector and Agrobacterium strains used for VIGS, Dr. Liancheng Du (University of Nebraska) for the Alternaria alternata f. sp. lycopersici aa strain, Dra. Helena Porta-Ducoing. for the Alternaria brassiciola strain, and Dr. Arturo Guevara-García (IBT, UNAM) for providing N. benthamiana seeds. The authors gratefully acknowledge the critical comments and support received from Drs. Yvonne Rosenstein-Azoulay and Felipe Cruz-García. We appreciate the technical assistance of Manuela Nájera-Martínez and Diana Sánchez-Rangel, and Laurel Fabila-Ibarra for greenhouse work. This study was funded by Consejo Nacional de Ciencia y Tecnología (Grant No. CONACYT 50503,) and Facultad de Química, UNAM (PAIP 6290-08). Mariana Rivas-San Vicente received a doctoral fellowship (170394) from CONACYT.
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Rivas-San Vicente, M., Larios-Zarate, G. & Plasencia, J. Disruption of sphingolipid biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f. sp. lycopersici . Planta 237, 121–136 (2013). https://doi.org/10.1007/s00425-012-1758-z
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DOI: https://doi.org/10.1007/s00425-012-1758-z