Citation
Soltani, J. (2009, January 14). Host genes involved in Agrobacterium-mediated transformation. Retrieved from https://hdl.handle.net/1887/13400
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/13400
Note: To cite this publication please use the final published version (if applicable).
Methodology for large-scale transformation of Saccharomyces cerevisiae strain collections by
Agrobacterium tumefaciens
A minor part of this chapter is incorporated in: Hooykaas, P.J., Dulk-Ras, A., Bundock, P., Soltani, J., van Attikum, H. and van Heusden, G.P.H. (2006) Agrobacterium Protocols: Yeast (Saccharomyces cerevisiae). Vol 2, pp. 465-473, Eds Wang, K., Humana Press, Totowa, USA.
Abstract
Agrobacterium tumefaciens is a plant pathogen that genetically transforms plant cells.
Under laboratory conditions it can also transform cells from many genera of different non-plant organisms including the yeast Saccharomyces cerevisiae. During the last few years collections of S. cerevisiae strains have been developed with systematic deletion or with systematic GFP- and TAP-tagging of all coding sequences. The availability of such collections allows large scale studies on the mechanisms of Agrobacterium-mediated transformation (AMT) of eukaryotic cells. However, current protocols for AMT are applicable for the transformation of only a limited number of strains in a single experiment. Here, we describe the development of a qualitative AMT protocol for the large scale transformation of a collection of ~4800 different S.
cerevisiae mutant strains.
Introduction
Agrobacterium tumefaciens is a gram-negative soil-born plant pathogen that genetically transforms plant cells by transferring an oncogenic segment of its Ti- plasmid, called the T-DNA, to plant cells at wounded sites (Chilton et al., 1977). The Ti-plasmid encodes a set of virulence (vir) proteins that can mobilize the T-DNA to eukaryotic host cells, function to protect the T-DNA during its journey to the host cell nucleus and may assist in integration of the T-DNA into the host genome (For review, see: Citovsky et al., 2006; Gelvin, 2003; Hooykaas and Schilperoort, 1992; McCullen and Binns, 2006; Tzfira et al., 2004; Zhu et al., 2000). Under laboratory conditions A.
tumefaciens is able to genetically transform cells from many genera of different non- plant organisms (for review see: Soltani et al., 2008), including the yeast Saccharomyces cerevisiae (Bundock et al., 1995; Piers et al., 1996). The observation that Agrobacterium can transform the yeast S. cerevisiae enables the use of the many experimental tools available for this model organism for the elucidation of the molecular mechanism of Agrobacterium-mediated transformation (AMT) of eukaryotic cells.
During the last few years, a number of S. cerevisiae strain collections have been constructed. Deletion mutants have been made for all ~6000 open reading frames.
This resulted in collections of ~4800 viable haploid and homozygous diploid deletion
strains, and of 5916 heterozygous diploid deletion strains (Giaever et al., 2002).
Moreover, other yeast strain collections like collections of TAP- and GFP-tagged strains (Gavin et al., 2002; Ho et al., 2002; Huh et al., 2003) and of strains with inducible promoters are available to answer different scientific questions (Suter et al., 2006). Those collections have successfully been used in many novel screens for different purposes (reviewed by Scherens and Goffeau, 2004). Examples of such screens include plating assays to test for the sensitivity of those collection of mutants to different chemicals and their response to different growth conditions (Aouida et al., 2004; Chang et al., 2002; Huang et al., 2005; Serrano et al., 2004; Zewail et al., 2003) and to study the behavior of Ty- transposons (Griffith et al. 2003). Also for elucidation of the molecular mechanisms involved in AMT such yeast strain collections may be of great importance.
During the last decade protocols for AMT of S. cerevisiae have been developed (Bundock et al., 1995; Piers et al., 1996). The method introduced by our group makes use of the binary vector system (Hoekema et al., 1983) and is applicable for the transformation of many other fungi and yeasts as well (reviewed by Michielse et al., 2005; Soltani et al., 2008). Mostly, the vectors used are derivatives of pBin19 (Bevan, 1984). Since non-homologous T-DNA integrates at a random position in the yeast and fungal host genomes it can be used for insertional mutagenesis and gene tagging (Bundock et al., 1996; Bundock et al., 2002; de Groot et al., 1998; Michielse et al., 2005). The T-DNA can also be used efficiently for targeted mutagenesis in yeast as homologous T-DNA integrates preferably by homologous recombination (Bundock et al., 1995; 1999; van Attikum et al., 2003).
A method has been described for AMT of yeast strain collections to investigate the host genes involved in the transformation process (Roberts et al. 2003). In this method yeast colonies were replica plated directly to a co-cultivation plate with Agrobacterium. Using this methodology 100,000 yeast transposon insertion mutants were screened that led to the discovery of the inhibitory role of purine biosynthesis in AMT of yeast (Roberts et al., 2003). Genes necessary for AMT of yeast, however, were not discovered in that work. As a non-integrative T-DNA was used, genes involved in the integration process were not identified. In collections of Arabidopsis thaliana mutants many so called rat (resistant to Agrobacterium-mediated
transformation) genes were discovered that play a role in transformation (Crane and Gelvin, 2007; Ditt et al., 2001, 2005, 2006; Veena et al., 2003; Zhu et al., 2003). In the searches done so far, only collections of random mutants were analyzed. We now undertook to systematically analyze the collection of all viable diploid yeast homozygous deletion mutants for AMT. Because the S. cerevisiae strain collections are maintained in 96-well microtiter plates we developed a novel AMT protocol for the transformation of the collection of ~4800 S. cerevisiae deletion strains in 96-well microtiter plates.
Materials and Methods
Yeast strains and media
The collection of S. cerevisiae homozygous diploid deletion strains in BY4743 (MATa/MATα his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0/ura3Δ0)(Giaever et al., 2002) and the isogenic BY4743 parental strain were obtained from InVitrogen (Groningen, the Netherlands) and Euroscarf (Frankfurt, Germany), respectively. Yeast cells were grown at 30°C in liquid YPD medium (Sherman et al., 1986) and deletion mutant strains were grown at 30°C in liquid YPD medium containing G418 (150 μg /ml).
Bacterial strains, plasmids and media
Two derivatives of Agrobacterium tumefaciens strain LBA1100, one carrying the binary vector pRAL7100 containing a T-DNA that integrates into the yeast genome by homologous recombination and the other one carrying binary vector pRAL7101 containing a T-DNA with the yeast 2µ replicator (Bundock et al., 1995) were used in Agrobacterium-mediated transformation screens. The T-DNA of pRAL7100 contains the URA3 gene flanked by PDA1 sequences (Figure 1A) that allows integration at the PDA1 locus via homologous recombination. The T-DNA of pRAL7101 contains the URA3 gene and the yeast 2µ origin of replication (Figure 1B). After AMT this T- DNA generates a plasmid capable of replication in yeast by circularization of the T- DNA. In order to study the random integration of T-DNA in the yeast genome, A.
tumefaciens strain LBA1100 containing non-homologous integrative plasmid pRAL7102 was used. The T-DNA of this plasmid contains URA3 but lacks homology with the yeast genome and also carries no yeast origin of replication (Figure1C).
Agrobacterium strains were grown and maintained as described (Hooykaas et al., 2006).
Figure 1. Binary vectorsused in Agrobacterium-mediated transformation of Saccharomyces cerevisiae in this study (Bundock et al., 1995; 1996) and the mechanisms by which the generated T-DNAs act.
The binary vector pBIN19 is the backbone of these plasmids. (A) pRAL7100 allows integration of URA3 at the yeast PDA1 locus via homologous recombination. (B) pRAL7101 generates a plasmid capable of replication in yeast by circularization of the T-DNA. (C) Non-homologous integrative plasmid pRAL7102 allows random integration of T-DNA in the yeast genome. Restriction enzymes: H, HindIII; S, SalI; X, XhoI. LB,left border repeat; RB, right border repeat; MB, mixed border; 2μ ori, origin of replication from the yeast 2μ plasmid. ColE1 ori, origin of replication from ColE1 plasmid, M13 ori, origin of replication from M13 phage; OriV, vegetative origin of replication; NPT, neomycin phosphotransferase gene; Pnos, nopaline synthase promoter; Tnos, nopaline synthase terminator; Ch DNA, chromosomal DNA.
Agrobacterium-mediated transformation
AMT of S. cerevisiae according to the standard protocol was performed as described by Bundock et al., 1995. All materials and media were prepared as described (Hooykaas et al., 2006). The final protocol developed for AMT of S. cerevisiae strain collections grown in microtiter plates developed here (figure 5) is described in Chapter 3.
(A)
(B)
(C)
Results
Basic protocols for AMT of S. cerevisiae (Bundock et al., 1995; Piers et al., 1996) are applicable for the transformation of only a limited number of strains in a single experiment. For transformation of strain collections used for genome wide studies, these protocols need to be adapted. Mutant strain collections are usually maintained in 96-well microtiter plates. Therefore, we wanted to adapt our standard protocol for AMT as developed in our group (Bundock et al., 1995) to allow transformation of yeast strain collections grown in microtiter plates.
For the development of a protocol for AMT of yeast strains grown in microtiter plates, we made use of the BY4743 strain, the parent strain of the collection of systematic gene deletion strains. This strain can grow on the induction medium (IM) used during the co-cultivation of yeast with Agrobacterium, if supplemented with uracil, leucine and histidine (data not shown). Furthermore, Agrobacterium strains containing pRAL7100, pRAL7101 and pRAL7102 were able to transform this strain using the standard transformation protocol (data not shown). For this transformation addition of acetosyringone to the co-cultivation medium was essential. Moreover, either Select agar (Gibco BRL) or Micro agar (Duchefa) can be used (Fig 2). Using the standard protocol, we then investigated in two independent experiments the optimal co-cultivation time of Agrobacterium containing plasmids pRAL7100 or pRAL7101 with S. cerevisiae strain BY4743 on IM plates. As shown in figure 3 the number of transformants rises during the first six to seven days and then drops until the eleventh day of co-cultivation. Hence, for further experiments we chose to co- cultivate Agrobacterium with S. cerevisiae for 6 to 7 days.
In an initial experiment to investigate AMT of BY4743 grown in microtiter plates, different volumes (5, 10, 20, 30, 40, 50 µl) of an overnight culture of OD620=1.2 (approximately 50×106 cell/mL) of BY4743 in YPD were added to the wells of a microtiter plate and YPD medium was added to a final volume of 200 µl. After shaking for 6 hours at 30°C, the microtiter plate was centrifuged and, after washing, the yeast cells were resuspended in 180 µl of Agrobacterium culture of OD620=0.6
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
pRAL7100 pRAL7101 pRAL7102
Agrobacterium strains
Frequency (×10-5)
Select Micro
Figure 2. AMT efficiency of S. cerevisiae strain BY4743 with Agrobacterium containing either pRAL7100, pRAL7101 or pRAL7102 on Select- or Micro-agar. AMT frequency is calculated by dividing the number of transformants to the number of yeast output after 6 days of co-cultivation. The data are the average of 4 experiments. Error bars: standard deviation.
0 0.2 0.4 0.6 0.8 1 1.2
1 2 3 4 5 6 7 8 9 10 11
Days of co-cultivation
Frequency (×10-5)
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
1 2 3 4 5 6 7 8 9 10 11
Days of co-cultivation
Frequency (×10-5)
Figure 3. The effect of co-cultivation time on AMT efficiency. Agrobacterium containing either pRAL7100 (A) or pRAL7101 (B) co-cultivated with the S. cerevisiae strain BY4743. AMT frequency is calculated by dividing the number of transformants to the number of yeast output. The data are averages from 2 independent experiments. Error bars: data points.
in IM medium. An aliquot of the mixture was spotted onto a cellulose nitrate filter on an IM plate. To allow co-cultivation of 96 strains on a single rectangular cellulose nitrate filter on a plate with the same size as a microtiter plate in further experiments, only small volumes of co-cultivation mix can be applied onto the filter. Therefore, we used 8 µl aliquots. After incubation for 6 or 9 days at 22°C, the colonies were removed from the filter, resuspended in MY medium and aliquots applied onto MY plates suitable for selection of uracil prototrophic transformants. As shown in figure 4., AMT of BY4743 grown in 5 to 50 µl aliquots in a final volume of 200 µl YP in microtiter plates is possible using Agrobacterium carrying either pRAL7100 or pRAL7101. For both Agrobacterium strains the number of transformants is hardly affected by the amount of yeast cells used, at least for the amounts used in this
A B
experiment. Hence, for large scale experiments we chose to use 20 µl of overnight yeast culture into a final volume of 200 µl YPD. Similar experiments using Agrobacterium strains carrying pRAL7102, which is used for random T-DNA integration by non-homologous recombination, resulted in only small numbers of colonies, i.e. 10 to about 15 colonies per transformation as expected.
0 300 600 900 1200 1500 1800
5 10 20 30 40 50
Yeast overnight culture (µl) Number of transformants
0 300 600 900 1200 1500 1800
5 10 20 30 40 50
Yeast overnight culture (µl) Number of transformants
Figure 4. Effect of the amount of overnight culture used for the preculture of the yeast BY4743 in YPD on the AMT efficiency using a microtiter plate based protocol. Agrobacterium containing pRAL7100 (A) or pRAL7101 (B) was used. The number of uracil prototrophic transformants obtained after 6 ( ) and 9 ( ) days of co-cultivation are given. The data are from one representative experiment.
It has been reported that several S. cerevisiae mutants impaired in adenine biosynthesis are hypersensitive to AMT when the induction medium was deprived of adenine (Roberts et al., 2003). In order to investigate whether we can detect this increased AMT efficiency using our microtiter-based transformation protocol, we used an ade4 (ymr300c) deletion mutant together with the isogenic wild type strain BY4743 and the wss1 (yhr134w) deletion mutant as controls for transformation with Agrobacterium carrying plasmid pRAL7100. Transformation efficiency was about 8 fold higher for the ade4 deletion mutant (448 transformants) compared to that of the wild type strain (50 transformants) and the wss1 (65 transformants) disruption mutant.
In the procedure described above, after co-cultivation the cells were resuspended in MY medium and an aliquot was plated onto a selection plate. This procedure is not easily applicable for large number of strains. Therefore, we instead used swab to transfer the cells from the co-cultivation mixture to the selection plate. In initial experiments we showed that this procedure was working well, but that the number of transformants was up to 5-fold lower than with the original method. Nevertheless, for
A B
the screening of large numbers of yeast strains we preferred to transfer the colonies onto the selection plates using a swab (Figure 5). To investigate the applicability of the developed transformation protocol for the transformation of the collection of yeast deletion mutants maintained in 96-well microtiter plates, we applied the transformation protocol to the first two plates (plates 301 and 302) of the collection of homozygous diploid deletion strains. As a control, the wild type strain BY4743 and the ade4 deletion strain were added to the two empty wells of these plates. These deletion mutants were co-cultivated with Agrobacterium strains carrying plasmids pRAL7100, pRAL7101 and pRAL7102. The numbers of transformants obtained for each deletion strain using Agrobacterium with plasmids pRAL7100 and pRAL7101 present on these two plates are shown in Table 1 and 2. It is obvious that the transformation efficiencies vary considerably between the different strains. Moreover, the number of transformants varies from plate to plate as the wild type strain BY4743 gave 43 and 154 transformants with Agrobacterium with pRAL7100 when present on plates 301 and 302, respectively. Transformation efficiencies using Agrobacterium carrying the pRAL7102 plasmid were extremely low (data not shown). To determine the reproducibility of the large-scale AMT protocol, we selected six deletion mutants with the highest and three with the lowest transformation efficiencies observed after transformation with both Agrobacterium strains. The selected mutants were retested by transforming them using both the newly developed microtiter plate protocol and our standard method (Table 3). Using the microtiter plate protocol similar results were obtained for all of the strains. Using our standard protocol the increased AMT efficiency was confirmed for all six selected deletion strains. The decreased AMT efficiency could be confirmed for the three selected deletion strains, except that the decreased AMT efficiency was not found for the YAL045C deletion mutant in transformation by Agrobacterium carrying pRAL7100.
Table 3. Validation of AMT data of nine selected disruption mutants by re-testing using our standard protocol (Bundock et al., 1995) and the new microtiter plate-based protocol. In the standard protocol, but not in the new microtiter plate-based protocol, the yeast output is included. Transformation efficiencies are given relative to that of BY4743, transformed in the same experiments.
Relative AMT efficiency
pRAL7100 pRAL7101 Deletion strain
First screen by new protocol
Retest by standard protocol
Retest by new protocol
First screen by new protocol
Retest by standard protocol
Retest by new protocol
Incresead AMT
YLR085C (ARP6) 2.0 2.8 2.0 1.2 1.3 2.0
YAL056W (GPB2) 3.2 11.3 3.0 7.0 1.7 5.0
YLR025W (SNF7) 2.0 11.9 3.0 5.4 15.5 2.0
YLR055C (SPT8) 1.6 7.4 3.0 1.9 5.3 3.0
YAL011W (SWC3) 2.7 17.1 3.0 2.7 2.6 2.0
YGR122W 3.5 12.5 3.0 3.5 1.6 2.0
Decresed AMT
YAL040C (CLN3) 0.0 0.7 ≤0.2 0.0 0.3 ≤0.2
YLR058C( SHM2) 0.1 0.1 0.05 0.01 0.8 ≤0.2
YAL045C 0.0 1.9 ≤0.2 0.0 0.6 ≤0.2
Wt BY4743 1.0 1.0 1.0 1.0 1.0 1.0
Discussion
The availability of various collections of S. cerevisiae strains with systematic gene deletions, allows genome-wide searches for host genes involved in AMT. Presently, a method for large-scale AMT of yeast mutants has been described in which yeast colonies were directly replica plated onto a cocultivation plate with Agrobacterium (Roberts et al., 2003). We tested replica plating in our initial experiments, but in our hands it was neither very efficient nor reproducible. Yeast mutant strain collections are usually maintained in 96-well microtiter plates. Therefore, to perform large scale AMT of S. cerevisiae strain collections maintained in microtiter plates, we have developed a new protocol by modification of the protocol introduced by Bundock et
al. (Bundock et al. 1995). A schematic overview of the developed protocol is given in figure 5.
Using the developed protocol we were able to transform not only the BY4743 strain but also homozygous diploid deletion strains in the BY4743 background. We chose the collection of homozygous diploid deletion strains rather than the collection of haploid deletion strains as second site mutations will mostly stay hidden in diploid strains. The protocol is applicable for Agrobacterium strains carrying either the integrative plasmid pRAL7100 or the replicative plasmid pRAL7101. However, it is obvious that the transformation efficiencies vary substantially from experiment to experiment. Therefore, for screening collections of yeast strains it is necessary to include the wild type strain in each test allowing determination of the transformation frequency relative to the wild type strain. A major source of the variation may be that in large-scale transformations we did not correct the transformation efficiencies for the output of viable yeast cells as it is almost infeasible to determine this for all strains. Although these data are crude, retesting of the identified candidates by our standard protocol (Table. 3, Chapter 3 and 4) indicates that data are reliable. AMT efficiency depends on many factors such as yeast and Agrobacterium growth conditions, temperature, the pH of induction media and the ratio between Agrobacterium and yeast cells (Michielse et al., 2005). During our experiments the pH of induction medium appeared to be very critical and should be maintained between pH 5.25 and 5.35. In the large scale AMT protocol, a pH outside this range affected the growth of Agrobacterium, and resulted in very low transformation efficiency (data not shown). Recent work confirms that not only the virulence system of Agrobacterium is induced at acidic pH, but also the expression of a large number of Agrobacterium genes is influenced (Yuan et al., 2008). We found that using cellulose nitrate filters has a positive effect on AMT compared to co-cultivation directly onto IM plates (data are not shown). This may explain the low efficiency of AMT by replica plating directly on IM plates. In our experiments the co-cultivation was done for 6-7 days rather than for 2 days (Roberts et al., 2003) or for 9 days (Bundock 1999;
van Attikum 2003). Differences in optimal cocultivation time in the different protocols may be due to differences in temperatures during cocultivation (22°C versus 20°C) and/or differences in strains of yeast and Agrobacterium.
To test the applicability of the developed method for screening collections of deletion mutants, we screened 188 homozygous diploid deletion mutants, present on plates 301 and 302 (Giaever et al., 2002) and identified six mutants hypersensitive to AMT and three mutants hyposensitive to AMT. In a retest, using both the standard protocol and the new microtiter plate based protocol, the altered sensitivity of these mutants, except one, was confirmed (Table 3.), although the magnitude of this alteration is variable. Therefore, the method is very suitable for determining qualitative differences in AMT efficiencies of yeast strains present in collections of deletion strains.
Acknowledgments
We would like to thank Amke den Dulk-Ras and Raymond Brandt for their technical supports, and Martin Brittijn for drawing of the Figure 1.
Table 1. Microtiter plate based Agrobacterium-mediated transformation (AMT) of yeast deletion collection of BY4743 strain. (A) Systematic name of deletion strains maintained in plate no.301. (B) Number of transformants after AMT by integrative plasmid pRAL7100 (C) Number of transformants after AMT by replicating plasmid pRAL7101. (A) 1 2 3 4 5 6 7 8 9 10 11 12 A ADE4 YAL068C YAL067C YAL066w YAL065C YAL062W YAL061W YAL060W YAL059W YAL058W YAL056W YAL055W B YAL053W YAL051W YAL049C YAL048C YAL046C YAL045C YGR122W YAL042W YAL043C-a YAL040C YAL037W YAL036W C YAL035W YAL034C YAL031C YAL030W YAL029C YAL028W YAL027W YAL026C YAL023C YAL022C YAL021C YAL020C D YAL019W YAL018C YAL017W YAL015C YAL014C YAL013W YAL011W YGR121C YAL009W YAL008W YAL007C YAL004W E YAL005C YAL002W YAR002W YAR003W YAR014C YAR015W YAR018C YAR020C YAR023C YAR027W YAR028W YAR029W F YAR031W YAR030C YAR035W YAR037W YAR040C YAR042W YAR043C YAR044W YAR047C YLL001WYLL002WYLL005C G YLL010C YLL012W YLL013CYLL014W YLL015W YLL016W YLL017W YLL019CYLL020CYLL021W YLL023CYLL024C H Wild type YLL025W YLL026W YLL028W YLL029W YLL032CYLL038CYLL039CYLL040CYLL041CYLL042CYLL043W (B) 1 2 3 4 5 6 7 8 9 10 11 12 A 63 28 15 10 7 6 5 17 16 41 139 74 B16 13 5 4 22 2 153 12 20 0 28 16 C40 7 12 18 4 2 6 20 22 20 6 19 D 26 16 13 4 8 12 116 14 25 11 8 10 E 11 31 6 22 24 0 26 8 12 25 20 53 F 17 18 20 6 12 12 19 15 13 26 50 26 G 54 13 21 5 6 60 11 34 12 37 42 103 H 43 32 17 16 10 10 8 0 59 15 24 26 (C) 1 2 3 4 5 6 7 8 9 10 11 12 A 28 65 24 31 28 5 10 18 14 60 43 36 B8 11 11 11 49 0 128 5 4 1 4 15 C136 7 4 3 4 0 3 14 11 6 3 2 D 37 5 10 4 2 10 6 0 7 5 2 31 E 11 49 4 36 9 0 10 6 1 1 10 17 F 26 19 12 2 3 8 1 11 1 8 22 16 G 25 5 8 7 3 41 2 20 3 0 7 21 H 6 7 9 15 7 2 7 0 8 3 22 36
Table 2. Microtiter plate based Agrobacterium-mediated transformation (AMT) of yeast deletion collection of BY4743 strain. (A) Systematic name of deletion strains maintained in corresponding in plate no.302. (B) Number of transformants after AMT by integrative plasmid pRAL7100 (C) Number of transformants after AMT by replicating plasmid pRAL7101. (A) 1 2 3 4 5 6 7 8 9 10 11 12 A YLL045CADE4 YLL046C YLL047W YLL051CYLL052CYLL053CYLL054C YLL055W YLL056CYLL057CYLL058W B YLL060C YLL061W YLL062CYLL063C YLR001C YLR003C YLR004C YLR006C YLR011W YLR012C YLR013W YLR014C C YLR015W YLR016C YLR017W YLR018C YLR019W YLR020C YLR021W YLR023C YLR024C YLR025W YLR028C YLR042C D YLR043C YLR044C YLR046C YLR047C YLR048W YLR049C YLR053C YLR054C YLR055C YLR056W YLR057W YLR058C E YLR059C YLR061W YLR062C YLR063W YLR064W YLR065C YLR068W YLR070C YLR072W YLR073C YLR074C YLR077W F YLR079W YLR080W YLR081W YLR082C YLR083C YLR084C YLR085C YLR087C YLR089C YLR090W YLR092W YLR093C G YLR094C YLR095C YLR096W YLR097C YLR098C YLR099C YLR102C YLR104W YLR107W YLR108C YLR109W YLR111W H Wild type YLR112W YLR113W YLR114C YLR118C YLR119W YLR120C YLR121C YLR122C YLR123C YLR124W YLR125W (B) 1 2 3 4 5 6 7 8 9 10 11 12 A 133 186 150 58 74 27 65 13 55 76 46 67 B41 143 128 90 83 98 24 0 30 102 49 46 C74 142 97 93 131 110 58 27 38 300 141 89 D 91 103 120 65 54 44 51 43 250 124 108 14 E 92 68 50 97 124 140 89 75 56 73 72 64 F 124 75 111 125 53 87 300 200 19 87 63 80 G 81 124 97 101 94 120 128 101 129 105 131 92 H 154 114 42 67 70 200 77 78 130 163 160 96 (C) 1 2 3 4 5 6 7 8 9 10 11 12 A 41 143 23 22 33 12 27 24 42 61 42 73 B59 27 40 69 16 - 13 196 34 9 17 7 C39 24 29 39 19 49 40 258 58 700 184 100 D 53 17 238 21 48 26 20 33 250 23 32 1 E 66 35 51 54 109 75 46 43 40 41 38 127 F 25 41 41 78 26 39 155 147 47 30 58 84 G 72 40 35 28 23 23 34 45 37 38 51 180 H 129 109 170 23 39 78 47 17 42 65 68 123
Day 1
Day 2
Day 11
Overnight culture in YPD + G418
Yeast mutant collection in 96-well plates Agrobacterium strains
Overnight culture in LB + kan
In fresh YPD In Induction Medium + AS 6 hrs
Centrifuge, wash cells, resuspend yeast cells in 180 µl Agrobacterium culture
Co-cultivate 8 μl of each Yeast-Agrobacterium mix on cellulose nitrate filter on induction plate Incubate 6-7 days at 22˚C
Streak out the colonies on selective minimal media Incubate 3-5 days at 30˚C
Count the number of transformants
Repeat the transformation for the candidate mutants using the same protocol Confirm selected candidate mutants using the standard protocol
6 hrs
Day 8
Figure 5. Schematic presentation of developed microtiter-based protocol for
Agrobacterium-mediated transformation of yeast strain collections. AS, acetosyringone, kan, kanamycin.
