Dokument

Summary:
The plant pathogenic fungus Ustilago maydis secretes effector proteins during the biotrophic interaction with its host plant Zea mays, with the aim being the suppression of the plant immune response virulence. All effector proteins virulence characterized to date were shown to be conventionally secreted proteins. However, over the last decade it could be established that proteins without a signal peptide can also be targeted to the outside of the cell in an ER/Golgi independent manner. A proteomic analysis on the apoplastic fluid of U. maydis infected maize plants identified 65 fungal proteins which did not contain a signal peptide. One of these proteins is the Acyl-CoA binding protein (ACBP) of U. maydis, Acb1, which will be investigated in this thesis. Over the years many studies have been dedicated to the functional analysis of Acyl-CoA binding proteins, in which ACBPs were shown to have many distinct intracellular functions, such as control over the intracellular Acyl-CoA pool size, roles in vesicular trafficking and their relevance for fatty acid biosynthesis. Further, in addition to their metabolic roles, ACBPs were shown to be involved in gene regulation, and also signaling by receptor binding, as was shown for the ACBP of the amoeba Dictyostelium discoideum (AcbA). In this organism it was shown that AcbA is secreted under nutrient limitation and extracellularly processed into the Spore Differentiation Factor 2 (SDF-2), which induces terminal spore differentiation. In this thesis it could be shown that the Acb1 protein of U. maydis is unconventionally secreted, subsequently processed into a SDF-2 like peptide and that this peptide is capable of inducing the sporulation of D. discoideum cells. The study of the protein’s extracellular function in U. maydis, as a full length secreted protein or in its processed form as the SDF-2 like peptide was hampered by the important intracellular functions of Acb1. Deletion of the acb1 gene resulted in strong growth defects and an accumulation of suppressor mutations, which were able to restore growth in a mutation dependent manner. Ultimately it could be shown that a virulence related function of the SDF-2 like peptide in both the solopathogenic strain SG200, as well as the compatible wild type strains FB1 and FB2, has to be considered unlikely. A potential extracellular role of the full length secreted protein has to date been neither confirmed nor ruled out.

Bibliographie / References

1. Chao, H., M. Zhou, A. McIntosh, F. Schroeder & A.B. Kier, (2003) ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT. Journal of lipid research 44: 72-83.
2. Kerkhoff, C., M. Beuck, J. Threige-Rasmussen, F. Spener, J. Knudsen & G. Schmitz, (1997) Acyl-CoA binding protein (ACBP) regulates acyl-CoA:cholesterol acyltransferase (ACAT) in human mononuclear phagocytes. Biochimica et biophysica acta 1346: 163-172.
3. Harris, F.T., S.M. Rahman, M. Hassanein, J. Qian, M.D. Hoeksema, H. Chen, R. Eisenberg, P. Chaurand, R.M. Caprioli, M. Shiota & P.P. Massion, (2014) Acylcoenzyme A-binding protein regulates Beta-oxidation required for growth and survival of non-small cell lung cancer. Cancer prevention research 7: 748-757.
4. Brachmann, A., J. Konig, C. Julius & M. Feldbrugge, (2004) A reverse genetic approach for generating gene replacement mutants in Ustilago maydis. Molecular genetics and genomics : MGG 272: 216-226.
5. Broomfield, P.L. & J.A. Hargreaves, (1992) A single amino-acid change in the ironsulphur protein subunit of succinate dehydrogenase confers resistance to carboxin in Ustilago maydis. Current genetics 22: 117-121.
6. Gillissen, B., J. Bergemann, C. Sandmann, B. Schroeer, M. Bolker & R. Kahmann, (1992) A two-component regulatory system for self/non-self recognition in Ustilago maydis. Cell 68: 647-657.
7. Bruns, C., J.M. McCaffery, A.J. Curwin, J.M. Duran & V. Malhotra, (2011) Biogenesis of a novel compartment for autophagosome-mediated unconventional protein secretion. The Journal of cell biology 195: 979-992.
8. Bell, A.A. & M.H. Wheeler, (1986) Biosynthesis and Functions of Fungal Melanins. Annual review of phytopathology 24: 411-451 24 411-451.
9. Laemmli, U.K., (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
10. Ausubel, F.M., R. Brenz, R.E. Kongston, D.D. Moore, J.G. Seidmann, J.A. Smith & K. Strukl, (1987) Current protocols in molecular microbiology. John Wiley & Sons, Inc, USA.
11. Kretschmer, M., J. Klose & J.W. Kronstad, (2012) Defects in mitochondrial and peroxisomal beta-oxidation influence virulence in the maize pathogen Ustilago maydis. Eukaryotic cell 11: 1055-1066.
12. Gaigg, B., T.B. Neergaard, R. Schneiter, J.K. Hansen, N.J. Faergeman, N.A. Jensen, J.R. Andersen, J. Friis, R. Sandhoff, H.D. Schroder & J. Knudsen, (2001) Depletion of acyl-coenzyme A-binding protein affects sphingolipid synthesis and causes vesicle accumulation and membrane defects in Saccharomyces cerevisiae. Molecular biology of the cell 12: 1147-1160.
13. Fyrst, H., J. Knudsen, M.A. Schott, B.H. Lubin & F.A. Kuypers, (1995) Detection of acylCoA-binding protein in human red blood cells and investigation of its role in membrane phospholipid renewal. The Biochemical journal 306 part 3: 793-799.
14. Banuett, F. & I. Herskowitz, (1996) Discrete developmental stages during teliospore formation in the corn smut fungus, Ustilago maydis. Development 122: 2965- 2976.
15. Callow, J.A., (1975) Endopolyploidy in maize smut neoplasms induced by the maize smut fungus, Ustilago maydis. New Phytology 75: 253-257.
16. Burton, M., T.M. Rose, N.J. Faergeman & J. Knudsen, (2005) Evolution of the acyl-CoA binding protein (ACBP). The Biochemical journal 392: 299-307.
17. Cabral, M., C. Anjard, W.F. Loomis & A. Kuspa, (2006) Genetic evidence that the acyl coenzyme A binding protein AcbA and the serine protease/ABC transporter TagA function together in Dictyostelium discoideum cell differentiation. Eukaryotic cell 5: 2024-2032.
18. Bölker, M., S. Genin, C. Lehmler & R. Kahmann, (1995) Genetic regulation of mating and dimorphism in Ustilago maydis. Canadian Journal of Botany 73(S1): 320- 325, 10.1139/b95-262.
19. Besman, M.J., K. Yanagibashi, T.D. Lee, M. Kawamura, P.F. Hall & J.E. Shively, (1989) Identification of des-(Gly-Ile)-endozepine as an effector of corticotropindependent adrenal steroidogenesis: stimulation of cholesterol delivery is mediated by the peripheral benzodiazepine receptor. Proceedings of the National Academy of Sciences of the United States of America 86: 4897-4901.
20. Brachmann, A., G. Weinzierl, J. Kamper & R. Kahmann, (2001) Identification of genes in the bW/bE regulatory cascade in Ustilago maydis. Molecular microbiology 42: 1047-1063.
21. Bendtsen, J.D., H. Nielsen, G. von Heijne & S. Brunak, (2004) Improved prediction of signal peptides: SignalP 3.0. Journal of molecular biology 340: 783-795.
22. Kamper, J., R. Kahmann, M. Bolker, L.J. Ma, T. Brefort, B.J. Saville, F. Banuett, J.W. Kronstad, S.E. Gold, O. Muller, M.H. Perlin, H.A. Wosten, R. de Vries, J. RuizHerrera, C.G. Reynaga-Pena, K. Snetselaar, M. McCann, J. Perez-Martin, M. Feldbrugge, C.W. Basse, G. Steinberg, J.I. Ibeas, W. Holloman, P. Guzman, M. Farman, J.E. Stajich, R. Sentandreu, J.M. Gonzalez-Prieto, J.C. Kennell, L. Molina, J. Schirawski, A. Mendoza-Mendoza, D. Greilinger, K. Munch, N. Rossel, M. Scherer, M. Vranes, O. Ladendorf, V. Vincon, U. Fuchs, B. Sandrock, S. Meng, E.C. Ho, M.J. Cahill, K.J. Boyce, J. Klose, S.J. Klosterman, H.J. Deelstra, L. Ortiz-Castellanos, W. Li, P. Sanchez-Alonso, P.H. Schreier, I. Hauser-Hahn, M. Vaupel, E. Koopmann, G. Friedrich, H. Voss, T. Schluter, J. Margolis, D. Platt, C. Swimmer, A. Gnirke, F. Chen, V. Vysotskaia, G. Mannhaupt, U. Guldener, M. Munsterkotter, D. Haase, M. Oesterheld, H.W. Mewes, E.W. Mauceli, D. DeCaprio, C.M. Wade, J. Butler, S. Young, D.B. Jaffe, S. Calvo, C. Nusbaum, J. Galagan & B.W. Birren, (2006) Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature 444: 97-101.
23. Aubry, L. & R. Firtel, (1999) Integration of signaling networks that regulate Dictyostelium differentiation. Annual review of cell and developmental biology 15: 469-517.
24. Guidotti, A., C.M. Forchetti, M.G. Corda, D. Konkel, C.D. Bennett & E. Costa, (1983) Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors. Proceedings of the National Academy of Sciences of the United States of America 80: 3531-3535.
25. Anselmi, C., M. Grininger, P. Gipson & J.D. Faraldo-Gomez, (2010) Mechanism of substrate shuttling by the acyl-carrier protein within the fatty acid megasynthase. Journal of the American Chemical Society 132: 12357-12364.
26. Djamei, A., K. Schipper, F. Rabe, A. Ghosh, V. Vincon, J. Kahnt, S. Osorio, T. Tohge, A.R. Fernie, I. Feussner, K. Feussner, P. Meinicke, Y.D. Stierhof, H. Schwarz, B. Macek, M. Mann & R. Kahmann, (2011) Metabolic priming by a secreted fungal effector. Nature 478: 395-398.
27. Anjard, C., S. Pinaud, R.R. Kay & C.D. Reymond, (1992) Overexpression of Dd PK2 protein kinase causes rapid development and affects the intracellular cAMP pathway of Dictyostelium discoideum. Development 115: 785-790.
28. Anjard, C. & W.F. Loomis, (2005) Peptide signaling during terminal differentiation of Dictyostelium. Proceedings of the National Academy of Sciences of the United States of America 102: 7607-7611.
29. Anjard, C., W.T. Chang, J. Gross & W. Nellen, (1998a) Production and activity of spore differentiation factors (SDFs) in Dictyostelium. Development 125: 4067-4075.
30. Apoga, D., J. Barnard, H.G. Craighead & H.C. Hoch, (2004) Quantification of substratum contact required for initiation of Colletotrichum graminicola appressoria. Fungal genetics and biology : FG & B 41: 1-12.
31. Anjard, C., C.J. Zeng, W.F. Loomis & W. Nellen, (1998b) Signal transduction pathways leading to spore differentiation in Dictyostelium discoideum. Developmental biology 193: 146-155.
32. Bölker, M., M. Urban & R. Kahmann, (1992) The a mating type locus of U. maydis specifies cell signaling components. Cell 68: 441-450.
33. Knudsen, J., S. Mandrup, J.T. Rasmussen, P.H. Andreasen, F. Poulsen & K. Kristiansen, (1993) The function of acyl-CoA-binding protein (ACBP)/diazepam binding inhibitor (DBI). Molecular and cellular biochemistry 123: 129-138.
34. Kinseth, M.A., C. Anjard, D. Fuller, G. Guizzunti, W.F. Loomis & V. Malhotra, (2007) The Golgi-associated protein GRASP is required for unconventional protein secretion during development. Cell 130: 524-534.
35. Brefort, T., G. Doehlemann, A. Mendoza-Mendoza, S. Reissmann, A. Djamei & R. Kahmann, (2009) Ustilago maydis as a Pathogen. Annual review of phytopathology 47: 423-445.
36. Knudsen, J., N.J. Faergeman, H. Skott, R. Hummel, C. Borsting, T.M. Rose, J.S. Andersen, P. Hojrup, P. Roepstorff & K. Kristiansen, (1994) Yeast acyl-CoAbinding protein: acyl-CoA-binding affinity and effect on intracellular acyl-CoA pool size. The Biochemical journal 302 part 2: 479-485.

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