of the primers and templates used for amplification. In some cases, an overlapping PCR strategy was used to introduce modifications in the original gene sequence (e.g. eliminate a predicted transcription termination site) or to generate gene fusions (e.g. ALA1::GFP under the control of the ALA1 promoter). In these cases, a first round of PCR amplifications was carried out to obtain overlapping fragments containing the desired modifications. In a second round, 1–2 mL of each of these PCR reactions were mixed together and used as template with primers that amplify the full- length product. The pENTRTM/D-TOPOH Cloning Kit and the Zero BluntH TOPOH PCR Cloning Kit for Sequencing were used for introducing the PCR fragments into the GatewayH-compatible vector pENTRTM/D-TOPOH (Invitrogen) and pCR4HBlunt- TOPOH vector (Invitrogen), respectively. All PCR products were fully sequenced in these plasmids before further cloning. Tables S3 and S4 contain a summary of all yeast and plant plasmids, respectively, generated in this work. For expression in yeast, the cDNA sequence corresponding to ALA1 (At5g04930) was PCR amplified both in untagged and hemaglutinin (HA) epitope-tagged forms and cloned into pENTRTM/D-TOPOH. GatewayH technology was then used to transfer the ALA1-codifying sequences to a GatewayH-compatible version of yeast plasmid pRS423-GAL [35] and its derivatives containing RGSH6:ALIS gene fusions [26]. These plasmids bear a bi-directional GAL1-10 promoter fusion that allows for coordinat- ed expression of two genes, as well as a HIS3 cassette for selection of yeast transformants in media lacking histidine. A modified version of ALA1 lacking a predicted transcription termination site, ALA1noTT (see above and Figure S2), was generated by overlapping PCR and cloned into yeast plasmids as above. In order to increase protein expression from the GAL1-10 promoter in pRS423-GAL [35], we modified this promoter to include the leader sequence of the yeast PMA1 gene. Pma1p is a yeast plasma membrane H+-ATPase, which can represent up to 15% of the total protein at this membrane [36]. This high level of expression is dependent on the strong constitutive PMA1 promoter. A piece of 238 bp of this PMA1 promoter from the transcription start point to the start codon [37] was fused to the GAL10 side of the GAL1-10 promoter in plasmid pRS423-GAL using an overlapping PCR strategy (see Tables S1 and S2 for details). A RGSH10 epitope and a Tobacco Etch Virus (TEV) protease cleavage site separated by a 3-glycine linker were engineered at the end of the PMA1 leader sequence in order to allow for immunological detection of expressed proteins. The PCR product cloned in pCR4HBlunt-TOPOH (Invitrogen) was digested with restriction enzymes BamHI and EcoRI and transferred to pRS423-GAL treated with the same enzymes, generating plasmid pMP4062. A version of this plasmid not containing the epitope tag (pMP4075) was generated by PCR amplification of the promoter- leader fusion with unmodified primers (see Tables S1 and S2 for details) and cloning as above. For further optimization of ALA1 expression in yeast, another modified version of the gene, yeALA1 (for a description see above and Figure S2), was produced by overlapping PCR and cloned into the pCR4HBlunt-TOPOH vector. After full sequencing, yeALA1 was transferred to yeast plasmids pMP4062 and pMP4075 using restriction enzymes EcoRI and SalI. In order to introduce other versions of the ALA1 gene in these modified yeast plasmids, the clones containing ALA1 and ALA1noTT in the pRS423-GAL backbone (pMP3560 and pMP3647, respectively, see Table S3) were digested with AgeI and BlpI. After DNA electrophoresis on agarose gels, the approximately 9 kb long fragment was isolated using a Gel AdvancedTM Gel Extraction System (Viogene, Taiwan). This eliminates part of the promoter Plant Plasma Membrane P4 ATPase and a fragment of around 170 bp of the ALA1 gene. The linearized plasmids were transformed into a wild type yeast strain (see below) together with a PCR fragment containing the full length GAL1-10 promoter::PMA1 leader fusion and the first 250 nt of the ALA1 gene both in tagged and untagged versions (see Tables S1 and S2 for details on the PCR strategy). After homologous recombination, plasmid DNA was extracted from the yeast cells by heating at 95uC for 4 min in the presence of 0.2% sodium dodecyl sulfate (SDS). After removal of cellular debris by centrifugation, 1 mL of the supernatant was used for electroporation of E. coli cells. Amplified plasmids were tested by restriction and sequenced to ensure the integrity of the DNA construct. Further yeast transformations for functional characterization (see below) were carried out with these sequenced plasmids. Introduction of ALA1 and ALA1noTT (both in HA-tagged and untagged versions) into yeast plasmids already containing RGSH6-tagged versions of ALIS (see above) had proven to be a tedious task and mutations in both the ALA1 and the ALIS gene sequences were often occurring during the cloning process, suggesting a toxic effect of these constructs for E. coli cells. In order to ease the cloning process, a two-plasmid transformation in yeast was used for co-expression of new ALA1 constructs with ALIS genes. For this purpose, plasmids containing RGSH6:ALIS in the pRS423-GAL backbone [26] were digested with BamHI and SacI and the 1-kb fragments corresponding to the tagged ALIS were ligated into pRS426-GAL [35] treated with the same enzymes. This plasmid contains the bi-directional GAL1-10 promoter and a URA3 cassette for selection of transformants on media lacking uracil. For expression of ALA1 in planta, the genomic DNA fragments corresponding to ALA1 and 2 kb of its adjacent 59UTR sequences were cloned by PCR from a total genomic DNA preparation (see Tables S1 and S2 for details). After cloning into pCR4HBlunt- TOPOH vector as described above, the new plasmids were used as templates in an overlapping PCR to generate an ALA1 promoter::ALA1 genomic DNA::GFP fusion, that bears artificial restriction sites XhoI and BstEI at the 59- and 39-ends, respectively. The DNA fusion digested with XhoI and BstEI was cloned into pCAMBIA1302 (CAMBIA, Brisbane, Australia) digested with SalI and BstEI. To clone ALA1 under the control of a double 35 S promoter, which should increase expression levels, new PCR amplifications were carried out to clone the genomic DNA fragment into vector pENTRTM/D-TOPOH as described above. Two individual PCR products containing or lacking a stop codon were obtained. GatewayH technology was used to further clone these products into pMDC43 or pMDC83 [28] to generate N- or C-terminal end fusions of GFP to ALA1. Yeast Strains and Media Wild type S. cerevisiae strain w303-1a (Mat a ade2-1 can1-100 his3- 11,15 leu3,112 trp1-1 ura3-1) was used for cloning different versions of ALA1 into yeast plasmids by homologous recombination (see above). Functional complementation was carried out employing S. cerevisiae mutant strain ZHY709 (MATa his3 leu2 ura3 met15 dnf1D dnf2D drs2::LEU2; [38]). Cells were grown at 30uC in standard rich medium with glucose (YPD) or galactose (YPG), or selective SD or SG media [39] containing Yeast Synthetic Drop-out Medium Supplement without the required amino acids (Sigma). Solid media were added 2% agar [40]. Papuamide B (Flintbox, Lynsey Huxham), duramycin (Sigma-Aldrich), and miltefosine (Calbio- chem) were added to rich synthetic SD or SG media to final concentrations 0.05 mg/mL, 1.5 mM and 2.5 mg/mL, respectively. PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e33042