Identification of proteins secreted by the fungus Ustilago maydis (DeCandole) Corda (Basidiomicete) grown under in vitro conditions

Andrés Adolfo Estrada-Luna, Alicia Chagolla López, Hilda Eréndira Ramos Aboites, Angelina Guerrero Ambriz, José Ruiz Herrera

Abstract


Introduction: Ustilago maydis is a fungus included in the group of Bacidiomycete that host maize and teocinte plants producing a disease known as common smut or huitlacoche. Today, there are no reports regarding the secretome of this organism grown under in vitro conditions. This information might be useful to characterize the genes involved in several important processes such as those related to nutrition, pathogenicity and fungus diferentiation. The main objective of this study were to identify the proteins differentially expressed and secreted into the culture media by both the sporidia and mycelium forms grown in two conditions of pH and analyzed through the mapping in SDS-PAGE geles.

Methods: We initially generated the two morphological forms (sporidia and mycelium) of Ustilago maydis (strain FB2 a2b2) through its culture in minimal media with adjusted pH of 7 and 3, respectively, and the growth kinetics was determined. The secreted proteins to the culture medium were concentrated in a Sep-Pak Plus C18 and eluted with a solution of acetonitrile (60 Identificación de las proteínas secretadas por el hongo Ustilago maydis (DeCandole) Corda (Basidiomiceto) cultivado en in vitro Revista Electrónica Nova Scientia, Nº 4 Vol. 2 (2), 2010. ISSN 2007 – 0705, pp: 104 – 130 - 107 - %) + trifluoroacetic acid (0.1 %) followed by a partial liophilization and precipitation with trichloroacetic acid-acetone solution. After this step, the protein samples were subjected to a polyacrilamide (SDS-PAGE) electophoresis and stained with Coomassie blue for mapping. The protein bands obtained were cut from the gels and digested with tripsin. Then, the mix of peptide samples were injected in a mass spectrometer for analysis and the obtained MS/MS spectra were then analyzed with Masslynx 4.0 before being subjected to MASCOT (Matrix Science) to perform non-redundant searches on the National Center for Biotechnology Information data base.

Results: After 30 h of inoculation, the growth kinetics of U. maydis cultivated at pH 7 was consistently higher compared to pH 3 (O.D. at 600 nm= 1.35 and 0.85, respectively). The process of dimorphism from sporidia to mycelia at pH 3 began 8 hours after inoculation. Thirty hours after inoculation, we observed that 100 % of cells had differentiated into mycelia. At this time, the supernatant was collected and processed as previously described, obtaining 8 reproducible bands of proteins from the minimal medium at pH 7 and 2 from the minimal medium of pH 3. All proteins were analyzed, however, only 5 from media at pH 7 were identified and their theoretical mass ranged between 31 to 68 kDa showing calculated isoelectrical points between 4.72 y 10.13. Four out of the five proteins were identified as secretion proteins of U. maydis. Three of the five proteins have an unknown function, one of them is related to the Spherulin 4 Precursor, and the remaining one seems to be a GPI (glycosyl phophotidylinositol ) related to Glucanase.

Discussion or Conclusion: Our results are the first set of data describing secreted proteins to the media under conditions in vitro by the sporidial form of U. maydis. With this research we established the bases to study the secretome of the fungus.


Keywords


proteomics; secretome; huitlacoche; Ustilago maydis

References


Agrios, G.N. (2005). Plant Pathology. Fifth Edition. Ed. Elsevier Academic Press. USA. 922 p.

Banuett, F. (1992). Ustilago maydis, the delightful blight. Trends in Genetics 8: 174-180.

Banuett, F. (1995). Genetics of Ustilago maydis, a fungal pathogen that induces tumors in maize. Annual Reviews Genetics 29: 179-208.

Banuett, F. and I. Herkowitz. (1989). Different a alleles of Ustilago maydis are necessary for maintenance of filamentous growth but not for meiosis. Proceedings of the National Academy of Science 86: 5878-5882.

Banuett, F. and I. Herkowitz. (1994). Morphological transitions in the life cycle of Ustilago maydis and their genetic control by the a and b loci. Exp. Mycol. 18: 247-266.

Böhmer, M.; T. Colby; C. Böhmer; A. Bräutigam; J. Schmidt, and M. Bölker. (2007). Proteomic analysis of dimorphic transition in the phytopathogenic fungus Ustilago maydis. Proteomics, 7: 675–685.

Bölke, M. (2001). Ustilago maydis – a valuable model system for the study of fungal dimorphism and virulence. Microbiology 147: 1395-1401.

Bölker, M.; C.W. Basse, and J. Schirawski. (2008). Ustilago maydis secondary metabolism—From genetics to biochemistry. Fungal Genetics and Biology 45: S88-S93.

Bradford, M.M. (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.

Brown, D. and G.L. Waneck. (1992). Glycosyl-phosphatidylinositol-anchored membrane proteins. J Am Soc Nephrol 3: 895-906.

Guevara-Olvera, L.; C. Calvo-Méndez, and J. Ruíz-Herrera. (1993). The role of polyamine metabolism in dimorphism of Yarrowia lipolytica. J. Gen. Microbiol. 139; 485-493.

Haskins, R.H. and J.A. Thorn. (1951). Biochemistry of the ustilaginales. II Antibiotic activity of ustilagic acid. Canadian Journal of Botany 18C: 585-592.

Holliday, R. (1974). Ustilago maydis. In: King RC (ed) Handbook of Genetics. Vol 1. New York: Plenum Press, pp 575–595.

Holliday, R. (2004). Early studies on recombination and DNA repair in Ustilago maydis. DNA Repair (Amst.). 3: 671-682.

Immer, F.R. and J.J. Christensen. (1928). Determination of losses due to smut infections in selfed lines of corn. Phytopathology 18: 599-602.

Immer, F.R. and J.J. Christensen. (1931). Further studies on reaction of corn to smut on yield. Phytopathology 21: 661-674.

Kahmann, R. and J. Kämper. (2004). Ustilago maydis: how its biology relates to pathogenic development. New Phytologist 164: 31-42.

Kämper, J.; R. Kahmann; M. Bölker; L.-J. Ma; T. Brefort; B.J. Saville; F. Banuett; J.W. Kronstad; S.E. Gold; O. Müller; M.H. Perlin; H.A.B. Wösten; R. de Vries; J. Ruíz-Herrera; C.G. Reynaga-Peña; K. Snetselaar; M. McCann; J. Pérez-Martín; M. Feldbrügge; C.W. Basse; G. Steinberg; J.I. Ibeas; W. Holloman; P. Guzmán; M. Farman; J.E. Stajich; R. Sentandreu; J.M. González-Prieto; J.C. Kennell; L. Molina; J. Schirawski; A. Mendoza-Mendoza; D. Greilinger; K. Munich; N. Rössel; M. Scherer; M. Vranes; O. Ladendorf; V. Vincon; U. Fuchs; B. Sandrock; S. Meng; E.C.H. Ho; M.J. Cahill; K.J. Boyce; J. Klose; S.J. Klosterman; H.J. Deelstra; L. Ortiz-Castellanos; W. Li; P. Sánchez-Alonso; P.H. Schreier; I. Häuser-Hahn; M. Vaupel; E. Koopmann; G. Friedrich; H. Voss; T. Schlüter; J. Margolis; D. Platt; C. Swimmer; A. Gnirke; F. Chen; V. Vysotskaia; G. Mannhaupt; U. Güldener; M. Münsterkötter; 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, and B.W. Birren. (2006). Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature 44: 97-101.

Klosterman, S.J. (2007). Genetics of Morphogenesis and Pathogenic Development of Ustilago maydis. Advances in Genetics 57: 1-24.

Klosterman, S.J.; M.H. Perlin; M. García-Pedrejas; S.F. Covert, and S.E. Gold. (2007). Genetics of Morphogenesis and Pathogenic Development of Ustilago maydis. Advances in Genetics 57: 1-47.

Kurtz, W.G. and L.E. Ericson. (1962). Microbial production of amino acids. II. The influence of carbon and nitrogen sources and metal ions on growth of Ustilago maydis (DC.) Cda. and on on lysine and threonine production. Biotechnol. Bioeng. 4: 37-52.

Martínez-Espinoza, A.D.; M.D. García-Pedrejas, and S.E. Gold. (2002). The Ustilaginales as Plant pests and Model Systems. Fungal Genetics and Biology 35: 1-20.

Martínez-Espinoza, A.D.; C. León; G. Elizarraraz, and J. Ruiz-Herrera. (1997). Monomorphic nonpathogenic mutants of Ustilago maydis. Phytopathology 87:259–265.

Merrian-Webster On Line. http://www.merriam-webster.com/dictionary/spherulin.

Mueller, O.; R. Kahman; G. Aguilar; B. Trejo-Aguilar, and A. Wu. (2008). The secretome of the maize pathogen Ustilago maydis. Fungal Genetics and Biology 45: S63-S70.

Müller, O.; P.H. Schreier, and J.F. Uhrig. (2008). Identification and characterization of secreted and patogénesis-related proteins in Ustilago maydis. Mol. Genet. Genomics 279: 27-39.

Rowell, J.D. (1955-a). Functional role of compatibility factors and in vitro test for sexual compatibility with haploid lines of Ustilago zeae. Phytopathology 45: 370-374.

Rowell, J.D. (1955-b). Segregation of sex factors in a diploid line of Ustilago zeae induce by  radiation. Science 1231: 304-306.

Ruíz-Herrera, J. (2008). Ustilago maydis: ascenso de un hongo mexicano de la gastronomía local al mundo científico. Nova Scientia 1: 118-135.

Ruíz-Herrera, J.; C.G. León; L. Guevara-Olvera, and A. Cárabez-Trejo. (1995). Yeast-mycelial dimorphism of haploid and diploid strais of Ustilago maydis. Microbiology 141: 695-703.

Ruíz-Herrera, J. and A.D. Martínez-Espinoza. (1998). The fungus Ustilago maydis, from the aztec cuisine to the research laboratory. International Microbiology 1: 149-158.

Ruíz-Herrera, J.; L. Ortiz-Castellanos; A.I. Martínez; C. León-Ramírez, and R. Sentandreu. (2008). Analysis of the proteins involved in the structure and síntesis of the cell wall of Ustilago maydis. Fungal Genetics and Biology, 45: S71–S76.

Schägger, H. and G. von Jagow. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analytical Biochemistry, 166: 368.379.

Shevchenko, A.; M. Wilm; O. Vorm, and M. Mann. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal.Chem. 68: 850–858.

Soll, D.R. (1985). Candida albicans. In: Fungal Dimorphism with Emphasis on Fungi Pathogenic for Humans. Szaniszlo PJ. Ed. Plenum Press. New York, USA.

Stevens, D.A.; H.B. Levine, and D.R. Ten Eyck. (1975). Sensibilidad dérmica a diferentes dosis de esferulina y coccidioidina. Boletín de la Oficina Sanitaria Panamericana – Julio: 48-53.

Tjalsma, A.; J. Bolhuis; D.H. Jongbloed; S. Bron, and J.M. van Dijl. (2000) Signal peptide-dependent protein transport in Bacillus subtilis: A genome-based survey of the secretome. Microbiol. Mol. Biol. Rev., 64: 515–547.

Teesrtstra, W.R.; H.J. Deelstra; M. Vranes; R. Lohlmann; R. Kahmann; J. Kamper, and H.A. Wolsten. (2006). Repellents have functionally replaced hydrophobins in mediating attachment to a hydrophobic surface and in formation of hydrophobic aerial hyphae in Ustilago maydis. Microbiology, 152: 3607-3612.

Villanueva, C. (1997). “Huitlacoche” (Ustilago maydis) as a food in Mexico. Micología Neotropical Aplicada 10:73-81.

Villanueva Verduzco, C.; E. Sánchez Ramírez, and E. Villanueva Sánchez. 2007. El huitlacoche y su cultivo. Ed. Mundi-Prensa México, S.A. de C.V. México, D.F. 96 p.

Wösten , H.A.B. 2001. Hydrophobins: Multipurpose Proteins. Annu. Rev. Microbiol., 55: 625-646.

Yonghyun, K.; M.P. Nandakumar, and M.R. Marten. 2007. Proteomics of filamentous fungi. TRENDS in Biotechnology, 25: 395-400.




DOI: https://doi.org/10.21640/ns.v2i4.213

Refbacks

  • There are currently no refbacks.


Copyright (c) 2015 Nova Scientia

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

Scope

Nova Scientia is a multidisciplinary, electronic publication that publishes twice a year in the months of May and November; it is published by the Universidad De La Salle Bajío and aims to distribute unpublished and original papers from the different scientific disciplines written by national and international researchers and academics. It does not publish reviews, bibliographical revisions, or professional applications.

Nova Scientia, year 12, issue 24, May – October 2020, is a biannual journal printed by the Universidad De La Salle Bajío, with its address: Av. Universidad 602, Col. Lomas del Campestre, C. P. 37150, León, Gto. México. Phone: (52) 477 214 3900, http://novascientia.delasalle.edu.mx/. Chief editor: Ph.D. Ramiro Rico Martínez. ISSN 2007 - 0705. Copyright for exclusive use No. 04-2008-092518225500/102, Diffusion rights via computer net 04 - 2008 – 121011584800-203 both granted by the Instituto Nacional del Derecho de Autor.

Editor responsible for updating this issue: Direction of Research Department of the Universidad De La Salle Bajío, last updated on May 15th, 2020.