Department of Plant Sciences, Tel Aviv University
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Salomon, D., Sessa, G. Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast. J. Vis. Exp. (37), e1865, doi:10.3791/1865 (2010).
I. Designing a Yeast Expression System for Type III Effectors
Calibrating a yeast system appropriate for expression of the type III effector(s) of interest is an important task and may require some trial and error. Factors of major relevance that should be considered and optimized when designing such a system are: 1) the promoter driving expression of the effector(s), 2) the copy number of the effector gene, 3) the epitope tag used to verify protein expression, and 4) the yeast strain.
Because bacterial type III effectors may be toxic to yeast cells, an inducible promoter should be used to control their expression. The GAL1 promoter is commonly used for this purpose and its activity is regulated by the carbon source present in the growth medium: it is repressed by glucose and induced by galactose. However, this promoter was reported as slightly leaky under repressing conditions resulting in undesired toxicity. If a similar problem is encountered, it is advisable to minimize the copy number of the effector gene as described below, or to use alternative inducible promoters, such as the MET3 or CUP1 promoters1. Here we describe procedures for expressing effector proteins from a galactose-inducible promoter.
2) Gene copy-number
An additional parameter affecting the level of protein expression is the number of copies of the effector gene present in the cell. High expression levels are achieved when the effector gene is carried by a 2-micron plasmid (40-60 copies per cell). Intermediate expression is obtained by using a centromere-containing plasmid (1-3 copies per cell), whereas low expression is achieved when the effector gene is integrated into the yeast genome by homologous recombination. By combining a centromere-containing vector and the GAL1 promoter we have successfully expressed about 50 effectors of the plant pathogens Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato at levels detectable by immunoblot analysis and with negligible leakage under repressing conditions (Salomon and Sessa, unpublished).
3) Epitope tag
If antibodies against the effector of interest are not available, an epitope tag is fused to the effector protein to monitor its expression by immunoblot. Commonly used tags are Myc, Hemagglutinin (HA) or Flag, to which reliable commercial antibodies are available. We suggest using only one copy of the tag to minimize undesired deleterious effects on structure and activity of the effector protein.
4) Yeast strain
A fundamental requirement for the yeast strain to be used for expression is auxotrophy to the selectable marker of the expression vector. It is important to note that different yeast strains may display different sensitivities to expression of certain effectors. For example, we observed that the BY4741 and W303 strains have different sensitivities to several Xanthomonas campestris pv. vesicatoria effectors (Salomon and Sessa, unpublished). Therefore, it is preferable to test the effector(s) of interest in different yeast strains.
II. Preparation of Yeast Media
For growth of yeast strains that do not contain any vector, use YPD (10 g/L yeast extract, 20 g/L peptone and 2% [w/v] glucose) as the growth medium. For solid media, add 2% [w/v] agar to the solution (if the solid medium is very soft, add 0.05% [v/v] from a 5 N NaOH stock solution to the medium before autoclaving). We recommend preparing the medium without glucose in 90% of the final volume and autoclaving it. Shortly before use, fill the volume to 100% with a filter-sterilized 20% [w/v] glucose solution (keep the 20% glucose solution at 4°C to prevent contamination).
For growth of yeast strains containing a vector that provides prototrophy to a selectable marker (leucine, uracil, histidine or tryptophan), use synthetic drop-out medium without leucine, uracil, histidine and tryptophan (6.7 g/L yeast nitrogen base without amino acids, 1.4 g/L yeast synthetic drop-out medium supplement) and containing 2% [w/v] glucose, or 2% [w/v] galactose and 1% [w/v] raffinose. For solid media, add 2% [w/v] agar to the solution. We recommend preparing the medium in 90% of the final volume and autoclaving it. Shortly before use, fill the volume to 100% with a filter-sterilized 20% glucose solution, or a filter-sterilized 20% galactose + 10% raffinose solution, depending on the desired carbon source (keep the 20% glucose and the 20% galactose + 10% raffinose solutions at 4°C to prevent contamination). Depending on the selectable marker of the expression vector, add the other amino acids or uracil before use (10 ml/L from a 1 g/100 ml leucine stock, 10 ml/L from a 200 mg/100 ml uracil stock, 2 ml/L from a 1 g/100 ml histidine stock, or 2 ml/L from a 1 g/100 ml tryptophan stock). When preparing leucine and uracil stocks, sterilize them by autoclaving and keep at room temperature. When preparing histidine and tryptophan stocks, sterilize them by filtering, wrap the bottle with aluminum foil to protect from light, and keep at 4°C. In the procedures described below, synthetic drop-out medium supplemented with leucine, uracil, histidine and tryptophan is designated as synthetic complete medium.
Solid-media plates can be stored for up to two months at 4°C. Before use, dry the plates for 20 min in a sterile laminar flow hood at room temperature.
III. Yeast Transformation
IV. Preparing a Yeast Protein Extract to Verify Effector Expression by Immunoblot
V. Spotting Assay to Detect Effector-induced Growth Inhibition Phenotypes
VI. Representative Results
A representative yeast spotting assay and detection of growth inhibition phenotypes induced by expression of type III effectors are shown in Fig. 1. In this experiment, the type III effectors AvrPto, HopAA1-1 and AvrE1 of the Gram-negative phytopathogenic bacterium Pseudomonas syringae pv. tomato (Pst) were expressed from the centromere-containing plasmid pGML10 in the yeast strain BY4741, and tested for their ability to inhibit yeast growth. Individual yeast cultures expressing Pst type III effectors under the control of the galactose inducible GAL1 promoter or containing an empty vector were serially diluted and plated onto repressing (glucose) or inducing (galactose) media (Fig. 1). On repressing medium, yeast strains carrying plasmids for the expression of AvrPto and HopAA1-1 exhibited similar growth as the control strain containing an empty vector. However on the same medium, yeast carrying AvrE1 displayed a slightly reduced growth, probably related to some degree of leakage of the GAL1 promoter and to the high cytotoxic effect of AvrE1. In inducing conditions, expression of the AvrE1 effector caused a drastic growth inhibition phenotype reflected by the lack of colonies in any dilution. As previously observed by Munkvold et al.3, expression of HopAA1-1 also resulted in severe inhibition of growth, while AvrPto did not show any effect.
Figure 1. Yeast growth inhibition caused by expression of the Pseudomonas syringae pv. tomato (Pst) type III effectors AvrE1 and HopAA1-1. Yeast strains (BY4741) containing the plasmid pGML10, either empty or carrying GAL1-driven cassettes for galactose-inducible expression of AvrPto, HopAA1-1 or AvrE1, were grown overnight in synthetic complete medium supplemented with glucose (2%) as a carbon source, and lacking leucine. Cultures were washed, normalized to OD600=1.0 and serial 10-fold dilutions were spotted onto synthetic complete solid media lacking leucine and containing glucose (2%), or galactose (2%) and raffinose (1%). Photographs were taken after 2 and 3 days of growth at 30°C for yeast growing in glucose and galactose media, respectively.
In this presentation, we illustrated how to use the budding yeast Saccharomyces cerevisiae as a heterologous system for the expression of type III bacterial effector proteins and how to identify effector-induced growth inhibition phenotypes. Importantly, these phenotypes can be utilized in genetic screens to identify suppressors of the negative impact of effectors on yeast growth. Suppressors may represent either direct targets of the effector studied or proteins that participate in cellular processes affected by the effector. Numerous type III effectors from animal and plant bacterial pathogens have been expressed in yeast to date, and growth inhibition phenotypes caused by several of them were used to identify processes or pathways that they target1,4. In several studies, effector toxicity in yeast was also successfully exploited to identify novel bacterial effectors5. Moreover, effector-induced growth inhibition phenotypes may be exploited for drug discovery in screens of chemical libraries for compounds that suppress the toxicity of effectors in yeast6.
This work was supported by the Israel Science Foundation.
|Yeast extract||Difco Laboratories||212750|
|Sodium hydroxide (NaOH)||Sigma-Aldrich||S8045|
|Yeast nitrogen base w/o amino acids||Difco Laboratories||291940|
|Yeast synthetic drop-out medium supplement||Sigma-Aldrich||Y2001|
|D-galactose||Sigma-Aldrich||G0750||>99%; <0.1% glucose|
|DNA, single stranded, from salmon testes||Sigma-Aldrich||D7656|
|Dimethyl sulfoxide (DMSO)||Sigma-Aldrich||D5879||Desiccate|
|Hydrochloric acid (HCl)||Sigma-Aldrich||H1758|
|Polyethylene glycol (PEG) 3350||Sigma-Aldrich||P4338|
|Lithium acetate (LiAc)||Sigma-Aldrich||L4958|
|Tris (base)||JT Baker||4109-02|
|Ethylenediamine-tetraacetic acid (EDTA)||Sigma-Aldrich||E5134|
|Dodecyl sulfate sodium salt (SDS)||Merck & Co., Inc.||8.22050.1000|
|Centrifuge tubes (15 ml)||Corning||430052||Sterile|
|Spectrophotometer cuvette (10x4x45 mm)||Sarstedt Ltd||67.742|
|pH indicator strip, pH 6.5-10.0||Merck & Co., Inc.||1.09543.0001|