The budding yeast (Saccharomyces cerevisiae) can serve as a unique experimental system for functional studies of heterologous genes, allowing not only complementation of readily available yeast mutations but also generation of overexpression phenotypes and in some cases also rescue of such phenotypes. Here we summarize the main considerations that have to be taken into account when using the yeast expression system for investigating the function of plant genes participating in cell morphogenesis; outline the strategies of experiment planning, yeast strain selection (or construction), and expression vector choice; and provide detailed protocols for yeast transformation, transformant selection, and phenotype evaluation.
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This work has been supported by the MSM0021620858 project. We thank Marta Čadyová for expert technical assistance and members of the A. Ragnini laboratory (University of Vienna, Austria) for the rapid yeast transformation protocol.
Novick P, Field C, Schekman R (1980) Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21:205–215PubMedCrossRefGoogle Scholar
Žárský V, Cvrčková F, Bischoff F et al (1997) At-GDI1 from Arabidopsis thaliana encodes a rab-specific GDI that complements the sec19 mutation of Saccharomyces cerevisiae. FEBS Lett 403:303–308PubMedCrossRefGoogle Scholar
Hála M, Eliáš M, Žárský V (2005) A specific feature of the angiosperm Rab escort protein (REP) and evolution of the REP/GDI superfamily. J Mol Biol 348:1299–1313PubMedCrossRefGoogle Scholar
Kranz JE, Holm C (1990) Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms. Proc Natl Acad Sci USA 87: 6629–6633PubMedCrossRefGoogle Scholar
Ueda T, Matsuda N, Anai T et al (1996) An Arabidopsis gene isolated by a novel method for detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase. Plant Cell 8:2079–2091PubMedGoogle Scholar
Cherry JM, Hong EL, Amundsen C et al (2012) Saccharomyces genome database: the genomics resource of budding yeast. Nucleic Acids Res 40:D700–D705PubMedCrossRefGoogle Scholar
Mumberg D, Muller R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119–122PubMedCrossRefGoogle Scholar
Albert S, Gitter AD, Lindquist S (2007) A suite of gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast 24:913–919CrossRefGoogle Scholar
Boeke JD, Trueheart J, Natsoulis G et al (1987) 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol 154:164–175PubMedCrossRefGoogle Scholar
Elledge SJ, Muligan JT, Ramer SW et al (1991) Lambda YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations. Proc Natl Acad Sci USA 88: 1731–1735PubMedCrossRefGoogle Scholar
Minet M, Dufour ME, Lacroute F (1992) Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs. Plant J 2:417–422PubMedGoogle Scholar