Resumen

ESTRADA-LUNA, Andrés Adolfo et al. Identification of proteins secreted by the fungus Ustilago maydis (DeCandole) Corda (Basidiomicete) grown under in vitro conditions. Nova scientia [online]. 2010, vol.2, n.4, pp.104-130. ISSN 2007-0705.

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 %) + 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.

Palabras llave : proteomics; secretome; huitlacoche; Ustilago maydis.