An Assay to Test the Capacity of Arabidopsis Plant Defensin Type1 Protein to Induce Cellular Zinc (Zn) Tolerance in Yeast
Résumé
Heterologous expression of genes in budding yeast Saccharomyces cerevisiae (S.cerevisiae) is especially suitable to functionally study the corresponding encoded protein at the cellular level (Bonneaud et al., 1991). This is mainly because many strains defective in specific activities are available and could be complemented by homologous genes existing across the eukaryotic kingdom (http://www.yeastgenome.org/). However, the protocol we describe here is not a complementation but a “gain-of-function” assay. It is based on a drop-test assay that we have set up to assess the zinc cellular tolerance conferred by the expression of heterologous genes in the wild-type S. cerevisiae. Different dilutions of a yeast culture expressing the heterologous gene of interest are grown on a range of zinc-enriched plates, and are then compared to the control yeast expressing the empty vector. Working with different concentrations of both yeast and zinc are essential to succeed in describing zinc tolerance phenotype upon yeast transformation (Mirouze et al., 2006). This test has also proven to be valuable to differentiate among related members of gene families as exemplified for Arabidopsis Plant Defensin type1 (Shahzad et al., 2013).