Protein extraction methods for the two-dimensional gel electrophoresis analysis of the slow growing fungus Undifilum oxytropis

The fungus Undifilum oxytropis produces the toxin swainsonine and is symbiotic with locoweeds, which are toxic Oxytropis and Astragalus species. The genus Oxytropisincludes perennial legumes that are widespread in many rangeland regions around the world. Consumption of locoweeds causes significant livestock poisoning and severe economic losses. Information about swainsonine synthesis by the U. oxytropis endophyte in locoweeds is limited and the interactions between the fungus and locoweed plant are poorly understood. Since U. oxytropis is a slow growing fungus that does not readily sporulate, its genetic characterization has been limited. An understanding of its proteome can be an important component in its biological characterization. The goal of this study was to develop an efficient protein extraction method for U. oxytropis. To develop an optimized protein extraction protocol for U. oxytropis, five protein extraction methods were evaluated. Of the five procedures assessed, trichloroacetic acid (TCA) in acetone was shown to be the best method for the fungus. The U. oxytropis proteins extracted using the TCA-acetone method were further characterized using two dimensional gel electrophoresis (2-DE) followed by mass spectrometry. The high resolution of the 2-DE reference map provided a useful approach for proteomic analysis of slow growing fungi.   Key words: Endophytes, mass spectrometry, protein extraction method, swainsonine, two-dimensional polyacrylamide gel electrophoresis, Undifilum oxytropis.