Methods for Studying the Polyamines in Ustilago maydis.

Polyamines are small aliphatic molecules with two or more amino groups. They are present in almost all living organisms and regulate fundamental cellular processes through interactions with the DNA, RNA, and proteins. Although the mechanisms by which these micromolecules regulate cellular processes are not fully understood, the intracellular concentration of polyamines is crucial for the development of organisms. In fungi, the importance of polyamines lies in the cellular processes they regulate, since they are involved in cellular growth and differentiation and even in the virulence and pathogenesis of pathogenic fungi. Ustilago maydis is a basidiomycete, dimorphic fungus, and phytopathogen with a biotrophic lifestyle and synthesizes only two of the three most common polyamines. These features and advantages make this fungus a eukaryotic model for studying polyamines. The study of polyamines requires a suitable model that facilitates in vitro and in vivo experimentation and adequate methodologies for the appropriate quantification and determination of specific polyamines. This chapter presents the detailed protocols for the quantification of polyamines by liquid and gas chromatography, as well as the techniques to quantify the activity of enzymes involved in the biosynthesis of polyamines, and some relevant aspects to work with polyamine-auxotrophic strains of U. maydis as well. Collectively, the methods described here contribute to the study of polyamines in Ustilaginomycetes and even in evolutionarily distant fungi.

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