The gene YEF3 function encoding translation elongation factor eEF3 is partially conserved across fungi

Introduction Translation is a fundamental process of life. In eukaryotes, the elongation step of translation is highly conserved and is driven by eukaryotic translation elongation factors (eEF)1A and eEF2. A significant variation of the elongation is the activity of eukaryotic elongation factor (eEF) 3 in Saccharomyces cerevisiae encoded by the gene yeast elongation factor (YEF3) with orthologs in all fungal species, a few algae, and some protists. In S. cerevisiae, YEF3 is an essential gene and eEF3 plays a critical role in translation elongation, as it promotes binding of the ternary complex acylated-Transfer RNA (tRNA)—eEF1A—Guanosine-5'-triphosphate (GTP) to the aminoacyl (A) site of the ribosome, the release of uncharged tRNAs after peptide translocation, and ribosome recycling. Even though YEF3 was discovered more than 40 years ago, eEF3 has been characterized almost exclusively in S. cerevisiae. Methods We undertook an in vivo genetic approach to assess the functional conservation of eEF3 across phylogenetically distant fungal species. Results We found that eEF3 from Zygosaccharomyces rouxii and Candida glabrata (both belonging to phylum Ascomycota), Ustilago maydis (phylum Basidiomycota), and Gonapodya prolifera (phylum Monoblepharomycota), but not Aspergillus nidulans (phylum Ascomycota), supported the growth of S. cerevisiae lacking the endogenous YEF3 gene. We also proved that eEF3 is an essential gene in the ascomycetes C. glabrata and A. nidulans. Discussion Given that most existing knowledge on fungal translation has only been obtained from S. cerevisiae, our findings beyond this organism showed variability in the elongation process in Fungi. We also proved that eEF3 is essential in pathogenic fungi, opening the possibility of using eEF3 as a target to fight candidiasis.

Introduction: Translation is a fundamental process of life.In eukaryotes, the elongation step of translation is highly conserved and is driven by eukaryotic translation elongation factors (eEF) A and eEF .A significant variation of the elongation is the activity of eukaryotic elongation factor (eEF) in Saccharomyces cerevisiae encoded by the gene yeast elongation factor (YEF ) with orthologs in all fungal species, a few algae, and some protists.In S. cerevisiae, YEF is an essential gene and eEF plays a critical role in translation elongation, as it promotes binding of the ternary complex acylated-Transfer RNA (tRNA)-eEF A-Guanosine-'-triphosphate (GTP) to the aminoacyl (A) site of the ribosome, the release of uncharged tRNAs after peptide translocation, and ribosome recycling.Even though YEF was discovered more than years ago, eEF has been characterized almost exclusively in S. cerevisiae.

Introduction
Translation plays a central role in gene expression in all forms of life (Hershey et al., 2019).The elongation step of translation consists of the decoding of a messenger ribonucleic acid's [[m]RNA] genetic information into protein by the ribosome and translation factors.In eukaryotes, this step requires the action of eukaryotic translation elongation factors (eEF) eEF1A and eEF2, as well as the nucleotide exchanger eEF1B (Dever et al., 2018).Elongation starts when an acylated-tRNA (also known as aminoacyl-tRNA, aa-tRNA) is brought to the ribosomal A site as part of the ternary complex aa-tRNA-eEF1A-GTP.Conformational changes in the ribosomal decoding center drive codon-anticodon pairing between aa-tRNAs and mRNA codons.Formation of the peptide bond is carried out at the ribosome peptidyl transferase center (PTC), between the incoming amino acid of the acylated-tRNA and the one positioned in the peptidyl (P) site (peptidyl-tRNA).Afterward, a peptide bond is formed, and eEF2 drives tRNA translocation from the A-P site to the P-E site so that the deacylated-tRNA is positioned at the exit (E) site and the peptidyl-tRNA is in the P site.GTP hydrolysis triggers eEF1A-GDP release, the uncharged tRNA leaves the exit (E) site, and the ribosome moves three nucleotides in the 5′-3′ direction to position the next codon at the empty A site. eEF1A is then recycled to its active form (eEF1A-GTP) by eEF1B.This cycle is repeated until an mRNA stop codon is placed at the free A site, which triggers peptide release from the ribosome to terminate translation.The ribosomal subunits dissociate, release the mRNA, and may enter into a new round of initiation (Choi et al., 2018;Dever et al., 2018).A significant variation in translation elongation in eukaryotes is the requirement of eukaryotic elongation factor 3 (eEF3) in the budding yeast S. cerevisiae.During the elongation cycle, eEF3 facilitates (1) the release of deacylated-tRNAs from the ribosomal E site after peptide translocation (Ranjan et al., 2021); (2) binding of the ternary complex to the A site (Triana-Alonso et al., 1995;Andersen et al., 2006); and (3) plays a role in the recycling process, as it promotes the disassembly of post-termination complexes into their components (Kurata et al., 2010).YEF3, which encodes eEF3, is an essential gene in S. cerevisiae (Qin et al., 1990).The orthologs of YEF3, termed eEF3 genes, have been found in silico in all fungal species and some algae but not in animals, plants, or the vast majority of protists (Mateyak et al., 2016;Murina et al., 2019).
Despite its ubiquitous presence across the fungi kingdom and the fact that 48 years have passed since its discovery (Skogerson and Wakatama, 1976), YEF3 and eEF3 have been genetically, biochemically, and structurally characterized only in the yeast S. cerevisiae.The function of YEF3 orthologs in species other than S. cerevisiae has been scarcely studied, and it is not known whether the eEF3 gene is essential in different fungal species.In this study, we demonstrate that the function of YEF3 is partially conserved across phylogenetically distant fungi.We also show that eEF3 is an essential gene in Candida glabrata and Aspergillus nidulans, fungal species with enormous medical and research relevance.

Databases search
We used 1,044 amino acids of the S. cerevisiae eEF3 protein to perform Basic local alignment search tool (BLAST) searches at: http://blast.ncbi.nlm.nih.gov/ and www.uniprot.org/blast/.eEF3 from the species selected for this study is listed in Table 1.

Genomic DNA extraction and plasmid construction
eEF3 DNA from Gonapodya prolifera and A. nidulans was in vitro synthesized with the preferential codon usage of S. cerevisiae into the pUC57-simple and pESC-TRP vectors, respectively, and purchased from GeneScript (Supplementary Table 1).Z. rouxii genomic DNA was purchased from the American Type Culture Collection (ATCC).Genomic DNA isolation from S. cerevisiae, C. glabrata, and Ustilago maydis was carried out using the Wizard Genomic DNA Purification Kit.The cloning for plasmid construction was achieved according to standard techniques (Sambrook and Russel, 1999).
An in-frame human influenza hemagglutinin (HA) protein tag sequence was introduced at the 3'-end of all eEF3 DNAs.Polymerase chain reaction (PCR)-amplified DNA bands were cloned into the pTZ57R/T vector to create the respective pTZ57-eEF3-HA constructs (Supplementary Table 2).Clones without artifactual mutations were further subcloned onto the p301-TRP1 vector to obtain the corresponding p301-eEF3-HA constructs.Recoded eEF3-HA DNAs (GeneScript) were subcloned from pUC57-simple onto the p301-TRP1 vector.
For pVT-U-YEF3 construction, S. cerevisiae YEF3 was PCRamplified using genomic DNA as a template and cloned into the vector pVT-U (Vernet et al., 1987).To construct the plasmid pYC12-eEF3, C. glabrata eEF3 was PCR-amplified using genomic DNA as a template and cloned onto the vector pTZ57R/T.A DNA clone without artifactual mutations was further subcloned onto vector pYC12 (Yáñez-Carrillo et al., 2015).The C. glabrata eEF3 KO-cassette eEF3 ::NATMX was created consisting of 1,000 nucleotides upstream (genomic left border, LB) of the eEF3 AUG start codon followed by the Nourseothricin resistance gene (NATMX) DNA and 1,000 nucleotides downstream of the eEF3 gene stop codon (genomic right border, RB; Supplementary Table 4).The eEF3 KO-cassette was further cloned into pTZ57R/T to create the plasmid pTZ57-cassette.The NATMX gene DNA was subcloned from the vector pAG25 (Goldstein and McCusker, 1999)

Phenotypic rescue of the YEF deletion in S. cerevisiae
Protocols for yeast growth and gene deletion were according to Nosek and Tomaska (2013) and Xiao (2014).All strains used in this study are listed in Table 3.

eEF deletion in C. glabrata
To delete C. glabrata eEF3, C. glabrata strain ura3 ::Tn903NeoR (Cormack and Falkow, 1999) was transformed with the pYC12-eEF3 and plated onto a minimal medium (yeast nitrogen base + glucose, SD) supplemented with a dropout lacking uracil (casamino acids, CAA; Takara Bio Inc.) as an auxotrophic marker.The resulting yeast genotype was ura3 ::Tn903NeoR <pYC12-eEF3>.To delete the C. glabrata eEF3 gene, the C. glabrata strain ura3 ::Tn903NeoR <pYC12-eEF3> was transformed with 600 ng of the C. glabrata eEF3 KO-cassette (which contains NATMX gene that confers resistance to nouseothricin) and grown in Yeast extract peptone dextrose (YPD) medium supplemented with the antibiotic clonNAT (200 µg/mL) to select the C. glabrata strain ura3 ::Tn903NeoR, eEF3 ::NATMX <pYC12-eEF3>.Several isolated colonies growing in this medium were randomly selected, and their genomic DNA was isolated.The correct deletion of the eEF3 gene by the cassette insertion was further corroborated by PCR.We obtained five colonies (numbered 4-8) of the desired genotype.Colonies were plated onto a minimal medium (SD + CAA) + uracil ./fmicb. .eEF deletion in A. nidulans A. nidulans was cultured on supplemented minimal medium (MM) using standard procedures described by Hill and Käfer (2001).For the deletion of eEF3, we followed the heterokaryonrescue method as described by Osmani et al. (2006).The flanking regions of eEF3 Open reading frame (ORF) were amplified with specific primers (Supplementary Table 4).The genomic left border (LB) was amplified with primers eEF3-P1 and P3, and the genomic RB was amplified with primers P5 and P8.The Aspergillus fumigatus pyrG gene was used as an auxotrophic marker, and it was amplified from the plasmid pFN03 by using the primers pyrG-F and pyrG-R (Osmani et al., 2006).The three DNA fragments LB, RB, and pyrG were joined together with the PCR-fusion method using nested primers P2 and P7 (Szewczyk et al., 2006).The resulting PCR product was transformed as described previously in the pyrG − auxotrophic strain GR5 (Waring et al., 1989).Transformants were screened by PCR for the homologous integration event.

Microscopy
Bright field, differential interference contrast (DIC), and fluorescence images were taken at room temperature with an Axiophot Microscope (Zeiss), Germany.For GFP imaging, spores were incubated overnight on cover slides in 0.5 ml of liquid MM supplemented with 2% glycerol (de-repression of the alcA promoter).Images were taken the next day with a 63X immersion objective.For imaging of the KO-transformants, spores were grown in liquid or solid (0.8% agarose) MM supplemented with uracil and uridine (+UU) or without uracil and uridine (-UU).Images with the bright field 10X objective were taken directly from Petri dishes.Images were collected and analyzed with the Zen Software (Zeiss), Germany.

Immunoblotting
Yeast cells were grown to O.D. = 1, pelleted, and further lysed on a frozen mortar in 100 mM KCl, 20 mM HEPES-KOH pH 7.6, 0.2 mM EDTA, 10% Glicerol, 0.1% Tritón X-100, 7 mM β-Mercaptoetanol and an EDTA-free Proteases Inhibitor Cocktail (Roche), Swiss.Protein concentration was determined using the Bradford Reagent (BioRad), United States according to the manufacturer's instructions.A volume of 20 mg of total protein lysate was resolved on 8% SDS-polyacrylamide gels and either stained with Coomassie blue (loading control) or blotted onto nitrocellulose membranes.Membranes were proved with a monoclonal anti-HA-HRP antibody (Santa Cruz, sc-7392).The expected size of proteins is shown in Supplementary Table 3.

Results
eEF from phylogenetically distant species cross-complements YEF in S. cerevisiae We analyzed whether the function of eEF3 is conserved in distant fungal species.We retrieved YEF3 ortholog sequences from the different databases and six species were selected from across four phyla, namely Z. rouxii, Candida glabrata, and A. nidulans (all three belonging to the phylum Ascomycota), U. maydis (belonging to the phylum Basidiomycota), and G. prolifera (belonging to the phylum Monoblepharomycota; Wijayawardene et al., 2020).
Through homologous recombination and plasmid shuffling experiments, we performed phenotypic rescue assays (Altmann et al., 1989), consisting of heterologous complementation of the S. cerevisiae YEF3 plasmid with the eEF3 DNA from the species mentioned above.We generated the haploid S. cerevisiae strain yef3::KANMX, <pVT-U-YEF3>, in which we deleted the YEF3 gene with the KANMX gene and introduced the constitutively expressed S. cerevisiae YEF3 DNA in the vector pVT-U (Figure 1A).This strain was afterward transformed with the eEF3 DNA from the ascomycetes C. glabrata and A. rouxii, which contain an in-frame HA tag in the p301 vector (Figure 1A).In the case of A. nidulans, the eEF3 DNA with an in-frame HA tag was cloned into the high-copy pESC-TRP vector (Figure 1A).Both vectors are under the inducible galactose (Gal10) promoter.To verify the eEF3-HA protein expression, we performed Western blotting analysis of total protein extracts using an anti-HA antibody (Figure 1B).Subsequently, S-gal plates were plated in S-gal medium supplemented with 5-FOA to expel the plasmid pVT-U-YEF3.Therefore, yeast growth depends solely on the expression of p301-eEF3-HA or pSEC-TRP-eEF3-HA in the case of A. nidulans.We observed that eEF3-HA DNA from C. glabrata, Z. rouxii, and S. cerevisiae (positive control), but not the empty vector, supported the growth of S. cerevisiae lacking the endogenous YEF3 gene (Figure 1C).Although the eEF3-HA cDNA from A. nidulans was cloned in a high-copy vector, eEF3-HA was barely expressed compared to the other HA-eEF3 (Figure 1B), and the low eEF3-HA synthesized was not able to support the growth of S. cerevisiae lacking the endogenous YEF3 gene.The growth curves of the strains that rescued the function of S. cerevisiae YEF3 showed similar growth patterns (Figure 1D).
We next analyzed eEF3 from distant non-Ascomycota phyla using the same genetic approach and verified the expression of eEF3-HA proteins encoded by the plasmid p301-TRP1 (Figure 2A) by performing Western blotting experiments (Figure 2B).We observed that U. maydis and G. prolifera eEF3-HA also rescued the function of S. cerevisiae YEF3 (Figure 2C).The growth curves were similar for all the organisms that did complement (Figure 2D).

eEF is an essential gene in C. glabrata and A. nidulans
Genetic experiments demonstrated that YEF3 is an essential gene in S. cerevisiae (Qin et al., 1990).However, whether the YEF3 orthologs are essential in other species has not been investigated.To address this question, we performed chromosomal recombination and plasmid shuffling experiments to delete eEF3 with a KO-cassette with a selection marker.Its homologous integration disrupts the original gene, which is substituted by the resistance cassette.
For C. glabrata, we constructed the eEF3 KO-cassette eEF3 ::NATMX, consisting of the NATMX marker that confers resistance to the antibiotic cloNAT, flanked by regions of the eEF3 gene (Figure 3A, upper).Simultaneously, the C. glabrata eEF3-HA DNA in the vector pYC12, which constitutively expresses eEF3, was introduced in cells of the strain ura3 ::Tn903NeoR, eEF3 ::NATMX to generate the strain ura3 ::Tn903NeoR, eEF3 ::NATMX, <pYC12-eEF3-HA> in which we can control the lack of eEF3.To corroborate the correct homologous integration of the eEF3 KO-cassette, we amplified a fragment of DNA that spans from within NATMX to the adjacent region of this cassette.We found the integration of the cassette in several independent transformants, except in the wild-type cells (Figure 3A, lower).
We next performed a growth assay in the presence of 5-FOA that was selected for cells that had lost the pYC12-eEF3 plasmid.Serial dilutions at a 1:10 ratio were plated onto MM supplemented with uracil (growth control; Figure 3B) and on MM containing uracil and 5-FOA (Figure 3C).Since endogenous eEF3 is deleted and the plasmid pYC12-eEF3 was expelled in the presence of 5-FOA, the absence of growth of ura3 ::Tn903NeoR, eEF3 ::NATMX cells in the latter medium indicates that eEF3 is an essential gene in C. glabrata (Figure 3C).We obtained identical results in three independent colonies in which a successful deletion of endogenous eEF3 was carried out.
We also assessed whether eEF3 of A. nidulans is essential.To address this, we next performed the heterokaryon rescue technique (Osmani et al., 2006) that allows the production of polynucleated protoplasts with two genetically distinct types of nuclei, i.e., one possesses the deleted gene and the other contains the wild-type allele.For this aim, we constructed an eEF3 KO-cassette that consisted of the A. fumigatus pyrG + gene as an auxotrophic marker and flanking regions of the eEF3 open reading frame.We transformed the pyrG −− auxotrophic strain GR5 caused by the pyrG89 mutation that requires UU to grow (Waring et al., 1989) with the eEF3 KO-cassette that complements the UU auxotrophy.In this method, the homologous integration event at the eEF3 locus occurs in only one of the nuclei, and a multinucleated heterokaryon strain is created containing both wild-type and eEF3 nuclei.Thus, the heterokaryon grows on selective minimal media lacking (-UU) and on non-selective minimal media (+UU).The conidia are mononucleated, and the GR5 grows on +UU but not on -UU minimal media.In contrast, conidia containing the eEF3 KOcassette can grow in selective -UU minimal media too.If the eEF3 cassette is integrated at the eEF3 locus and the deletion is lethal, the spores of the transformed conidia cannot grow in any medium.
To verify the homologous integration of the selection marker, we amplified a fragment of DNA that spans from pyrG to the adjacent region of the eEF3 KO-cassette (Figure 4A).We found homologous integration of the eEF3 KO-cassette in all the tested transformants except transformant 3.As expected, no amplification was obtained in the wild-type cells (negative control).We observed that transformant numbers 4, 7, and 9 did grow on +UU but not on -UU (Figure 4B) media, indicating that only the wild-type conidia germinated on +UU.Spores were further cultivated overnight in liquid selective -UU or non-selective +UU minimal media.Microscopic analyses showed that the eEF3 spores germinated in selective minimal media -UU and produced up to three germination tubes, but they could not grow further (Figure 4B inset and Figure 4C).The growth of transformants 1, 2, 5, 6, and 8 can be explained by an additional heterologous integration event of the selection marker.We conclude that eEF3 is an essential gene in A. nidulans.

Discussion
In this study, we demonstrated that eEF3 from the fungal species Z. rouxii, C. glabrata, U. maydis, and G. prolifera supports the growth of S. cerevisiae lacking endogenous YEF3.We could not determine whether or not A. nidulans eEF3 complements the lack of eEF3 in S. cerevisiae.The low expression level of A. nidulans eEF3 that we systematically observed might be the result of DNA recombination, protein degradation, or both, as the protein might be harmful to S. cerevisiae.eEF3 from other Ascomycota species, including Schizosaccharomices pombe (Mateyak et al., 2018) and Candida albicans (Di Domenico et al., 1992;Myers et al., 1992), and the basidiomycete yeast Cryptococcus neoformans (Blakely et al., 2001), also cross-complement S. cerevisiae YEF3.eEF3 from a fungus-like species, namely the parasitic oomycete Phytophthora infestans, also cross-complements S. cerevisiae YEF3 (Mateyak et al., 2018).

Rational design of fungicides: eEF in the spotlight
The biotechnological and clinical importance of fungal infections has been dramatically growing.However, the control of fungal pathogens is currently limited due to the small number of effective fungicides and the lack of suitable targets (Kim et al., 2020).Here, we have shown that eEF3 is an essential gene in C. glabrata and A. nidulans, two ascomycetes with strong medical and research relevance, respectively (Turner and Butler, 2014;Glöckner and Cornely, 2015;Park et al., 2017;Chen et al., 2021;Hernandez-Ramirez et al., 2021).This is the first study showing the essentiality of the eEF3 gene beyond S. cerevisae.Since no plant or mammalian eEF3 ortholog exists, our observations open for the first time the possibility of using rationally designed drugs to target eEF3 to fight human fungal infections by these species, as well as other infections by harmful fungal species (Sturtevant, 2002).These drugs would be an alternative to the current antifungal drugs, which are based on azoles that disrupt cell walls but display inefficient results overall.

ATP-binding cassette (ABC)-ATPases and the origin of eEF
The evolutionary origin of eEF3 is unknown.The existence of eEF3 orthologs in fungi, algae, some protists, and a few viruses suggests that the last eukaryotic common ancestor (LECA) possessed an eEF3 gene that was later lost in some taxonomic lineages, including metazoans, land plants, and different protists (Mateyak et al., 2016;Murina et al., 2019).Accordingly, S. cerevisiae eEF1A and eEF2 alone support translation with purified rat liver ribosomes in vitro.In contrast, rat liver eEF1A and eEF2 require yeast eEF3 to drive translation with yeast ribosomes (Skogerson and Engelhardt, 1977), suggesting that mammalian ribosomes evolved to catalyze protein synthesis in the absence of eEF3 (Mateyak et al., 2016), therefore losing the eEF3 gene.
Although there is no ATPase associated with translation elongation in mammals or plants, which is driven by the GTPases eEF1A and eEF2, some ABC ATPases play key roles in translation.This is the case of ABCE1, which interacts with release factors and drives the recycling step of translation in eukaryotes.In S. cerevisiae, GCN20 mediates ribosome-associated eIF2alpha kinase GCN2 during translation elongation under amino acid starvation (Vazquez de Aldana et al., 1995;Marton et al., 1997) and ARB1 controls ribosome biogenesis (Dong et al., 2005).In mammals, ABC50 interacts with eIF2 and associates with the ribosome to promote translation initiation (Tyzack et al., 2000).In bacteria, ABCF proteins bind the ribosome upon exposure to antibiotics.Etta occupies the ribosomal E site to displace drugs from the ribosome, and both VgaA and MsrE reset peptidyl transferase activity in response to antibiotic treatment (Gerovac and Tampé, 2019).Altogether, these phenomena show that ABC ATPases involved in translation are a common theme in life, supporting the notion that LECA might have possessed eEF3 activity that was lost later in evolution in some lineages.
Selenium is a trace element present in selenoproteins in the form of the amino acid selenocysteine (Sec).Most selenoproteins are enzymes containing Sec at the active site.Sec is encoded by the stop codon UGA, which is recoded and incorporated into polypeptides by the action of the Sec-specific elongation factor (eEFSec) and a specific tRNA (tRNA-Sec) as part of the Sec machinery (Labunskyy et al., 2014).Selenoproteins and the Sec machinery are essential for vertebrates and are commonly found in most algae (except red algae; Mariotti et al., 2015;Castellano, 2019;Liang et al., 2019;Jiang et al., 2020) but are missing in most insects and land plants.Selenoproteins are highly scattered throughout protists, which include many lineages lacking selenoproteins (Mariotti et al., 2015;Castellano, 2019).Sec was also lost at the root of Dikarya, which comprises the fungal phyla Ascomycota and Basidiomycota (Castellano, 2019;Mariotti et al., 2019).The Sec machinery genes were identified in nine fungal species belonging to three early-branching fungal groups, namely Chytridiomycota, Zoopagomycota, and Mucoromycota (Castellano, 2019;Mariotti et al., 2019).
Overall, the studies on eEF3, eEF1A, EFL, and eEFSec have unveiled a significant diversification of the translation elongation factors across eukaryotes.

Conclusion
The investigation of eEF3 across species within the fungal kingdom is crucial, not only because it shows variability in the translational process but also because of its potential and genetic research implications.Discoveries in this area not only contribute to the understanding of fundamental factors necessary for translation but also illuminate the processes by which such knowledge can be harnessed for the development of more effective products and technologies.Given that a significant portion of existing knowledge has only been obtained from S. cerevisiae, our study beyond this model organism revealed that eEF3 is essential in pathogenic fungi.This discovery holds promise for a breakthrough in antifungal research as this protein, absent in mammals and plants, could serve as a remarkably specific target.

FIGURE
FIGURE eEF from di erent Ascomycota species di erentially complements S. cerevisiae YEF .(A) The scheme of plasmids pVT-U-YEF containing the S. cerevisiae YEF (left), p -eEF -HA HA-tagged versions of eEF DNAs from di erent species used to perform the phenotypic rescues (right), and pSEC-TRP containing the A. nidulans eEF -HA.(B) Western blotting analysis using an anti-HA antibody (up) and Coomassie blue stain of total protein extracted from di erent species (down).(C) Phenotypic rescue using eEF from Ascomycota species.Experiment controls: Empty vector (p , negative control), S. cerevisiae (positive control).eEF DNAs from C. glabrata, Z. rouxii, and A. nidulans were tested.The media composition is shown.Plates S glu + ALH (p -eEF -HA or pSEC-TRP-eEF -HA expression is o ) and S gal + ALH (p -eEF -HA or pSEC-TRP-eEF -HA expression is on) are growth controls.Plates S gal + ALHU + -FOA have expelled pVT-U-YEF , and the growth relies only on p -eEF -HA or pSEC-TRP-eEF -HA (Phenotypic rescue).(D) Growth curves of yeast cells expressing eEF from S. cerevisiae, C. glabrata, or Z. rouxii.CG, Candida glabrata; ZR, Z. rouxii; SC, Saccharomyces cerevisiae; A, Adenine; L, Leucine; H, Histidine; U, Uracil; -FOA, -fluoroorotic acid hydrate.

FIGURE
FIGURE eEF from di erent phyla species di erentially complements S. cerevisiae YEF .(A) Scheme of plasmids pVT-U-YEF containing the S. cerevisiae YEF (left) and p -eEF -HA containing HA-tagged versions of eEF DNAs from di erent species used to perform the phenotypic rescues.(B) Western blotting analysis using an anti-HA antibody (up) and Coomassie blue stain of total protein extracted from di erent species (down).(C) Phenotypic rescue using eEF from species belonging to non-Ascomycota fungi.Experiment controls: Empty vector (p , negative control), S. cerevisiae (positive control).eEF DNAs from the basidiomycete U. maydis and the monoblepharomycete G. prolifera tested.The media composition is shown.Plates S glu + ALH (p -eEF -HA is o ) and S gal + ALH (p -eEF -HA expression is on) are growth controls.Plates S gal + ALHU + -FOA have expelled pVT-U-YEF , and the growth relies only on p -eEF -HA (Phenotypic rescue).(D) Growth curves of yeast cells expressing eEF from S. cerevisiae, U. maydis, or G. prolifera.UM, Ustilago maydis; GP, Gonapodya prolifera; SC, Saccharomyces cerevisiae; A, Adenine; L, Leucine; H, Histidine; U, Uracil; -FOA, -fluoroorotic acid hydrate.

FIGURE
FIGURE eEF is an essential gene in C. glabrata.[(A), upper] eEF KO-cassette was used to delete the endogenous eEF gene.LB, genomic left border; RB, genomic right border.A scheme of the amplified fragment of the eEF KO-cassette integrated onto the eEF locus is depicted.Arrows indicate the position of the used primers.(Lower) PCR analysis of independent KO-transformants (lanes -).(B, C) KO-strains that integrated the eEF KO-cassette.(B) Growth controls.(C) Lethality tests.The presence of -FOA triggers the expulsion of the pCY -eEF plasmid.The pYC -eEF loss leads to lethality phenotypes only in the strain ura ::Tn NeoR, eEF ::NATMX lacking endogenous eEF gene.The independent transformants , , and were tested, obtaining identical results.Genotypes in all cases are described.SD-CAA, minimal medium containing casamino acids; U, uracil; -FOA, -fluoroorotic acid hydrate.

FIGURE
FIGURE eEF is an essential gene in A. nidulans.(A) PCR analysis of the KO-strains.A scheme of the amplified A. fumigatus (Af) fragment of the KO-cassette integrated onto the eEF locus is depicted.Arrows indicate the position of the used primers.LB, genomic left border; RB, genomic right border (RB).(B) Spores of the wild-type strain (GR ) and several transformants with the integrated eEF KO-cassette were streaked out onto minimal medium (MM) plates with (+UU) or without (-UU) uracil and uridine.Inset: a microscopic picture of the spores taken directly from the Petri dish.(C) DIC microscopic pictures of the wild-type strain and transformant number .Spores were cultivated O/N on liquid MM with (+UU) or without (-UU) uracil and uridine at • C.
TABLE eEF from the fungal species selected for this study.
, and the different fragments of the C. glabrata eEF3 gene were PCR-amplified using genomic DNA and cloned onto the pTZ57R/T.All constructs were corroborated by Sanger sequencing.Plasmids are listed in Table2.
pESC-TRP-eEF3-HA nidulans A. nidulans eEF3-HA DNA cloned onto pESC-TRP1.This study p301-eEF3-HA prolifera G. prolifera eEF3-HA DNA cloned onto p301-TRP1 This study pMCB17apx pMCB17 version for fusion of Green fluorescent protein (GFP) to N-termini of proteins of interest in Aspergillus nidulans.Efimov et al., 2006 pSH114 A. nidulans eEF3 genomic sequence from ATG to stop in pMCB17apx.This study pFN03 It expresses the A. fumigatus pyrG marker.Aysha H. Osmani, Ohio State University TABLE Strains used in this study.