Yeast strain PJ69-4A propagating plasmid pTS195.1 was successively transformed with different amounts of vector pCL1 using a standard transformation procedure [22]. Transformation efficiencies ranged between 0.01% (0.1 μg DNA per 10⁸ cells) and 0.1% (5 μg DNA per 10⁸ cells, Figure 1). Extrapolation of the data revealed saturation at approximately 0.11%.

As a standard mating protocol we used a method from [23], suitable for yeast two-hybrid library screenings, a further development of the original interaction-mating procedure described in [24]. Mating of two haploid yeast strains of opposite mating type, each harboring one of the respective plasmids, results in the formation of doubly transformed diploid zygotes. A major advantage of this approach is the possibility of using frozen aliquots of yeast cells carrying the cDNA library; hence transformation of the whole library in every single screening experiment becomes unnecessary. Yeast strain YTS1 (MATα) was transformed with plasmid pCL1, propagated overnight in liquid medium and frozen in aliquots. A melted aliquot of YTS1/pCL1 was combined with a freshly raised culture of PJ69-4A/pTS195.1 (MATa) and then subjected to the standard mating procedure. Because the main focus of our experiments was reduction of the total cell number in our assays, we calculated mating efficiencies as the ratio of zygotes versus total cell number (viable and nonviable MATα cells from the frozen culture plus MATa cells). The mating efficiency of the standard method was found to be 2.8% on average. In terms of the usual way of quantifying mating (the ratio of zygotes versus viable MATα cells) this corresponds to a value of 12.2% in accordance with values of around 10% reported in the literature [23,25,26].

To enable short-term quantification of mating efficiencies we set up a model system using green fluorescent protein (GFP) as a reporter. Expression of the yeast-enhanced green fluorescent protein (yEGFP [27]) from plasmid pTS195.1 was controlled by the Gal1 promoter and was therefore dependent on the coexpression of the Gal4 protein from plasmid pCL1. Using the yeast strains YTS1 (MATα) and PJ69-4A (MATa), transformed with pCL1 and pTS195.1, respectively, and the standard mating procedure for producing doubly transformed zygotes, GFP fluorescence was detectable in dumbbell-shaped and in budding three-part zygotes as early as 4 hours after setting up the mating mixture (Figure 2). Both the fluorescence of a mating mixture measured in a fluorescence reader and the number of fluorescent zygotes counted in a hemocytometer were found to be linearly correlated with the actual mating efficiency as determined by growth selection of zygotes. This system was thus suitable for quickly assessing mating efficiencies and was used thereafter to evaluate experimental conditions.

Apart from mating on filters, as in the standard mating procedure [23,28], other approaches have been described to yield optimal mating efficiencies and/or the maximum number of zygotes. These protocols aim at synchronizing the yeast cultures to maximize mating ability and/or enriching zygotes in the suspension by chemically induced cell arrest and zonal centrifugation [29,30]. Additionally, preincubation on specific media before mating has been shown to increase the mating competence of yeast cells [31,32,33,34,35]. We tested the applicability of these procedures to our frozen/thawed yeast cultures, as the use of frozen cells is one major benefit of the interaction-mating procedure with respect to library screening. GFP fluorescence in the resulting cell suspension was measured as a marker for zygote formation and improvements were confirmed by counting cells and propagating aliquots on SD-LU medium to calculated the exact mating efficiency.

Neither enrichment of mating-competent cells by zonal centrifugation nor the use of chemically synchronized cells significantly improved mating efficiencies in our experimental set-up. However, preincubation of the two yeast strains in low pH medium before mating turned out to significantly increase mating efficiency. Preincubation of a mixture of a mid-log-phase culture of PJ69-4A/pTS195.1 and a melted aliquot of YTS1/pCL1 at high cell density (10⁸ cells/ml) in YCM medium (pH 3.5) for 105 minutes, a subsequent washing step and incubation on a filter on agar plates (YCM, pH 4.5) for another 4.5 hours gave an average mating rate of 10%.

An optimal cell ratio of freshly grown MATa versus MATα cells of 1:1 has been reported for most mating protocols [32,36]. Because of freezing and thawing, however, the interaction-mating mixture is a heterogeneous cell suspension composed of viable and nonviable cells. Therefore, nonequivalent quantities of the two strains have been suggested to optimize mating [26]. On the basis of the improved mating protocol described above, we systematically investigated the effects of cell ratio on mating efficiency. Varying amounts of freshly raised PJ69-4A/pTS195.1 cells from 0.1- to 25-fold excess over YTS1/pCL1 cells were tested. The ratio between the yeast strains (MATa:MATα ratio) was calculated as the number of uracil prototrophic cells versus leucine prototrophic cells, thus representing the proportion of viable cells in the assay. Mating efficiency was assessed by the numbers of zygotes versus total cell number (including nonviable cells). Mating efficiencies were found to be strongly dependent on the MATa:MATα ratio, showing an optimum at a ratio of 2.5:1, leading to a maximum mating efficiency of 17% (Figure 3). As a measure of mating competence of the frozen/thawed YTS1/pCL1 cells we calculated the ratio of zygotes versus viable leucine prototrophic cells. For optimal MATa:MATα ratio of 2.5:1, up to 80% of the viable MATα cells have formed zygotes (Figure 3). This almost quantitative mating is of particular importance for the screening of cDNA libraries, where a complete coverage of the clones present in the library is necessary to ensure a nonbiased experimental set-up. Table 1 summarizes the results with respect to the number of cells needed to obtain 5 × 10⁶ double transformants.

The mating conditions found to be optimal for the model system were applied to the screening of a 'real' two-hybrid cDNA library of A. thaliana using a set of 'real' bait proteins. The cDNA library (> 10⁶ independent clones) was transformed into Y187 (MATα), colonies were propagated, pooled and stored at -70°C. The fraction of viable cells in melted aliquots was determined as 39%.

Twenty library screenings have been carried out using 15 proteins from A. thaliana and five proteins from Nicotiana tabacum as baits. The bait proteins included small Ras-like GTPases, kinases and several proteins of unknown function. The efficiency of the optimized mating protocol made it possible to set up a standard screening protocol with the potential to cover the expected bait-dependent variation in mating efficiencies. We used a mating mixture containing 6 × 10⁸ cells in total, composed of 50% bait cells, 20% viable library cells and 30% nonviable cells, corresponding to a MATa:MATα ratio of 2.5:1. Aliquots of the mated cells were propagated onto selective medium to determine mating efficiencies, and 10⁸ cells were plated onto a single 500 cm² agar plate to select for interacting clones promoting Leu/Trp/His prototrophic growth. The protocol allowed us to process up to 20 screenings in parallel with standard laboratory equipment. Mating efficiencies were bait dependent and ranged between 4.8% and 17.3%, indicating a general applicability of the method (Figure 4a). In only one case was our aim of 5 × 10⁶ zygotes (corresponding to a mating efficiency of 5%, see above) not achieved. A direct comparison of the efficiency of the optimized protocol versus the standard protocol was done by repeating screenings 1, 4 and 18 using both protocols in parallel. Mating efficiencies using the optimized protocol were found to be greater by factors of 5, 3 and 13, respectively (Figure 4b).

The mean mating efficiency of about 8% in these 20 experiments is somewhat lower than the optimal mating efficiencies using the model system, but is clearly sufficient to be used in the standardized screening protocol described, obviating the need to adjust conditions to every individual bait protein.

The improved pretreatment and mating conditions prepare the cells optimally for mating, as can be seen by the fact that the experiment with the most efficient bait protein (Figure 4a, screen 18) resulted in almost quantitative mating; more than 85% of the viable library cells have formed zygotes.

Yeast strains PJ69-4A [37] and Y187 [38] were maintained using standard conditions [39]. A MATα strain which is deleted in the URA3 gene was obtained by streaking out an overnight culture of Y187 onto YPAD medium containing 0.05% 5-fluoroorotic acid (TRC, Canada). Several colonies were picked and examined for uracil auxotrophy while maintaining the other markers of yeast strain Y187. The resulting yeast strain with the desired genomic markers was called YTS1 (MATα, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4Δ, met, gal80Δ). Plasmid pCL1 [4] encodes the full-length, wild-type Gal4 protein under control of the ADH promoter and was obtained from Clontech. Vector pTS195.1 provides the yeast-enhanced green fluorescent protein (yEGFP [27], GenBank accession number U73901) under control of the GAL1 promoter and was cloned as follows: yEGFP was amplified by PCR using primers YGFP-1 (5'-GAGAGAAAGCTTGGATCCATGTCTAAAGGTGAAGAAT-TATTC-3') and YGFP-1 reverse (5'-GAGAGACTCGAGAA-TTCTTATTTGTACAATTCATCCATAC-3') and cloned as a HindIII/XhoI fragment into vector pCUG1, a derivative of pRS316 (GenBank U03442) containing an EcoRI/HindIII fragment from pBM272 (GenBank U03497) carrying the GAL1/GAL10 promoter. Plasmid pCUG1 is a gift from Philip James (University of Wisconsin, Madison, USA). The construct pCUG1-yEGFP was cut with KpnI and XbaI and the fragment containing the GAL1/GAL10 promoter and yEGFP was ligated into the same sites of plasmid YEplac195 ([40]; GenBank X75459) to obtain pTS195.1. Yeast full media and selective drop-out media were prepared using standard recipes [39].

Transformation of plasmid or library DNA into yeast was done according to the LiAc transformation method [22,41].

Yeast strain YTS1 transformed with pCL1 was raised in liquid medium lacking leucine (SD-L medium) and was frozen in aliquots as a 25% glycerol stock at -70°C. For mating, a melted aliquot of approximately 7 × 10⁷ cells was pooled with a threefold excess of a freshly raised culture of PJ69-4A propagating pTS195.1. This mixture was distributed onto a YPAD plate (140 mm diameter) and set on 30°C for 4.5 h. Cells were washed off the plate with 1 M sorbitol, sonicated, counted in a hemocytometer (Fuchs-Rosenthal-chamber) and titrated onto the appropriate drop-out media.

GFP fluorescence was investigated by fluorescence microscopy using fluorescein isothiocyanate (FITC) filters (485/510 nm, excitation/emission) and by fluorescence measurements in microtiter plates (Dynatech Microfluor) using a Fluoroscan II fluorescence reader (Labsystems Oy, Helsinki, Finland; excitation 485 nm, half-width 14 nm, transmission > 50%; emission 510 nm, half-width 11 nm, transmission 40%).

This protocol focused on stimulation of a/α-factor expression and optimized cell-cell contact during mating. The two yeast strains were mixed in the desired cell ratios, resuspended in YCM (1% yeast extract, 1% bactopeptone, 2% dextrose) at pH 3.5 and a cell density of 10⁸ cells/ml and shaken for 105 min at 30°C. The cells were then diluted 1:100 in sterile water and cell clumps were resolved by sonification. The cells were then treated as follows.

For screening cDNA libraries, yeast strain Y187 was transformed with the activation domain fused cDNA library from A. thaliana in plasmid pACT2 [42], plated onto SD-L medium and incubated for 2-3 days. Colonies were collected from the plates using 1 M sorbitol, pooled, sonicated and stored in aliquots after addition of 1 vol 50% glycerol. A thawed aliquot was propagated on SD-L medium and cells counted in a hemocytometer to determine the survival rate of leucine prototrophic cells.