Abstract
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 cellular zinc 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).
Keywords: Plant defensin, Abiotic stress, Zinc tolerance, Yeast
Materials and Reagents
Equipment
Procedure
Note: For each yeast clone to be analyzed. On day -1
On day 0 Prepare Petri dish for drop assay.
Representative data
Figure 1. In vitro functional characterisation of amino-acid substitutions impacting zinc tolerance capability in AhPDF1.4 and AtPDF1.4. Serial dilutions of the S. cerevisiae BY4741 strain expressing the pYX212 empty vector (EV) or pYX212 harbouring AhPDF1.4, AtPDF1.4 or its mutated versions of were analysed. Replacement of either A28 to G (A28G) or S54 to R (S54R) or both (A28G- S54R) were generated. The various yeast transformants were spotted on medium supplemented with 1.4 µM (control), or 27.5 mM ZnSO4, as indicated above the panels. Each spot was made with 10 µl of a yeast culture diluted at the OD600nm mentioned below the drops. Pictures were taken at day 2 for control and day 11 for the Zn treatments; they are representative of the three experiments, which have been performed with three independent yeast transformants. Reproduced from (Shahzad et al., 2013; Supplementary Figure 4).
Notes
The BY4741 S. cerevisiae strain has auxotrophic markers, in particular for the production of the amino acid histidine (his3∆1). Free histidine is a potent metal chelator and could, in particular, chelate zinc if this metal is present in the culture medium. The histidine (his3∆1) auxotrophy must thus be complemented by the expression of the corresponding HIS gene (here expressed from the pFL38 plasmid) and not by the addition of free histidine, which would bias the zinc tolerance test.
Recipes
Acknowledgments
This work was supported by the University of Montpellier (LM), the French Ministère de l’Enseignement Supérieur et de la Recherche (PB), the Pakistani Higher Education Commission (ZS) and the Centre National de la Recherche Scientifique (FG).
References
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