Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the Saccharomycotina CTG clade which has been studied over the last 40 years due to its biotechnological interest, biological control potential and clinical importance. Such a wide range of applications in various areas of fundamental and applied scientific research has progressively made C. guilliermondii an attractive model for exploring the potential of yeast metabolic engineering as well as for elucidating new molecular events supporting pathogenicity and antifungal resistance. All these research fields now take advantage of the establishment of a useful molecular toolbox specifically dedicated to C. guilliermondii genetics including the construction of recipient strains, the development of selectable markers and reporter genes and optimization of transformation protocols. This area of study is further supported by the availability of the complete genome sequence of the reference strain ATCC 6260 and the creation of numerous databases dedicated to gene ontology annotation (metabolic pathways, virulence, and morphogenesis). These genetic tools and genomic resources represent essential prerequisites for further successful development of C. guilliermondii research in medical mycology and in biological control by facilitating the identification of the multiple factors that contribute to its pathogenic potential. These genetic and genomic advances should also expedite future practical uses of C. guilliermondii strains of biotechnological interest by opening a window into a better understanding of the biosynthetic pathways of valuable metabolites.

Candida glabrata, an opportunistic fungal pathogen, accounts for 18–26 % of all Candida systemic infections in the US. C. glabrata has a robust oxidative stress response (OSR) and in this work we characterized the role of glutathione (GSH), an essential tripeptide-like thiol-containing molecule required to keep the redox homeostasis and in the detoxification of metal ions. GSH is synthesized from glutamate, cysteine, and glycine by the sequential action of Gsh1 (γ-glutamyl-cysteine synthetase) and Gsh2 (glutathione synthetase) enzymes. We first screened for suppressor mutations that would allow growth in the absence of GSH1 (gsh1∆ background) and found a single point mutation in PRO2 (pro2-4), a gene that encodes a γ-glutamyl phosphate reductase and catalyzes the second step in the biosynthesis of proline. We demonstrate that GSH is important in the OSR since the gsh1∆ pro2-4 and gsh2∆ mutant strains are more sensitive to oxidative stress generated by H₂O₂ and menadione. GSH is also required for Cadmium tolerance. In the absence of Gsh1 and Gsh2, cells show decreased viability in stationary phase. Furthermore, C. glabrata does not contain Saccharomyces cerevisiae high affinity GSH transporter ortholog, ScOpt1/Hgt1, however, our genetic and biochemical experiments show that the gsh1∆ pro2-4 and gsh2∆ mutant strains are able to incorporate GSH from the medium. Finally, GSH and thioredoxin, which is a second redox system in the cell, are not essential for the catalase-independent adaptation response to H₂O₂.

The mitochondrial (mt) genome of the sea anemone Metridium senile contains genes for only two transfer RNAs (tRNAs), tRNAf-Met and tRNATrp. Experiments were conducted to seek evidence for the occurrence of functional tRNAs corresponding to these genes and for the participation of nuclear DNA-encoded tRNAs in mt-protein synthesis. RNA sequences corresponding to the two mt-tRNA genes were located in mitochondria and it was shown that 3′-CC (and possibly A, but no other nucleotide) is added post-transcriptionally to the 3′ end of at least 50 % of mt-tRNAf-Met molecules and to a small fraction of the mt-tRNATrp molecules. Using specific oligonucleotide primers based on expected nuclear DNA-encoded tRNAs in a series of RACE experiments, we located the nuclear genes for tRNAGln, tRNAIle, tRNAi-Met, tRNAVal and tRNAThr. Data from Northern blot analyses indicated that mtDNA-encoded tRNAf-Met is limited to mitochondria but that nuclear DNA-encoded tRNAVal and tRNAi-Met are present in the cytoplasm and in mitochondria. These data provide direct evidence that in M. senile, mature, functional tRNAs are transcribed from the mtDNA-encoded tRNAf-Met and tRNATrp genes, and are consistent with the interpretation that both nuclear DNA-encoded tRNAVal and tRNAi-Met are utilized in mitochondrial and cytosolic protein synthesis.

The soil borne fungus, Fusariumvirguliforme, causes sudden death syndrome (SDS) in soybean, which is a serious foliar and root rot disease. The pathogen has never been isolated from the diseased foliar tissues; phytotoxins produced by the pathogen are believed to cause foliar SDS symptoms. One of these toxins, a 13.5-kDa acidic protein named FvTox1, has been hypothesized to interfere with photosynthesis in infected soybean plants and cause foliar SDS. The objective of this study is to determine if FvTox1 is involved in foliar SDS development. We created and studied five independent knockout fvtox1 mutants to study the function of FvTox1. We conducted Agrobacteriumtumefaciens-mediated transformation to accomplish homologous recombination of FvTox1 with a hygromycin B resistance gene, hph, to generate the fvtox1 mutants. Approximately 40 hygromycin-resistant transformants were obtained from 10⁶ conidial spores of the F. virguliforme Mont-1 isolate when the spores were co-cultivated with the A. tumefaciens EHA105 but not with LBA4044 strain carrying a recombinant binary plasmid, in which the hph gene encoding hygromycin resistance was flanked by 5′- and 3′-end FvTox1 sequences. We observed homologous recombination-mediated integration of hph into the FvTox1 locus among five independent fvtox1 mutants. In stem-cutting assays using cut soybean seedlings fed with cell-free F. virguliforme culture filtrates, the knockout fvtox1 mutants caused chlorophyll losses and foliar SDS symptoms, which were over twofold less than those caused by the virulent F. virguliforme Mont-1 isolate. Similarly, in root inoculation assays, more than a twofold reduction in foliar SDS development and chlorophyll losses was observed among the seedlings infected with the fvtox1 mutants as compared to the seedlings infected with the wild-type Mont-1 isolate. These results suggest that FvTox1 is a major virulence factor involved in foliar SDS development in soybean. It is expected that interference of the function of this toxin in transgenic soybean plants will lead to generation of SDS-resistant soybean cultivars.

Cold-active lipases are of significant interest as biocatalysts in industrial processes. We have identified a lipase that displayed activity towards long carbon-chain-p-nitrophenyl substrates (C12–C18) at 25 °C from the culture supernatant of an Antarctic Penicillium expansum strain assigned P. expansum SM3. Zymography revealed a protein band of around 30 kDa with activity towards olive oil. DNA fragments of a lipase gene designated as lipPE were isolated from the genomic DNA of P. expansum SM3 by genomic walking PCR. Subsequently, the complete genomic lipPE gene was amplified using gene-specific primers designed from the 5′- and 3′-regions. Reverse transcription PCR was used to amplify the lipPE cDNA. The deduced amino acid sequence consisted of 285 residues that included a predicted signal peptide. Three peptides identified by LC/MS/MS analysis of the proteins in the culture supernatant of P. expansum were also present in the deduced amino acid sequence of the lipPE gene suggesting that this gene encoded the lipase identified by initial zymogram activity analysis. Full analysis of the nucleotide and the deduced amino acid sequences indicated that the lipPE gene encodes a novel P. expansum lipase. The lipPE gene was expressed in E. coli for further characterization of the enzyme with a view of assessing its suitability for industrial applications.

The cell wall defines the shape and provides osmotic stability to the yeast cell. It also serves to anchor proteins required for communication of the yeast cell with surrounding molecules and other cells. It is synthesized as a complex structure with β-1,3-glucan chains forming the basic network to which β-1,6-glucan, chitin and a number of mannoproteins are attached. Synthesis, maintaining and remodeling of this complex structure require a set of different synthases, hydrolases and transglycosidases whose concerted activities provide necessary firmness but at the same time flexibility of the wall moiety. The present state of comprehension of the interplay of these proteins in the yeast cell wall is the subject of this article.

The relative concentrations of ions and solutes inside cells are actively maintained by several classes of transport proteins, in many cases against their concentration gradient. These transport processes, which consume a large portion of cellular energy, must be constantly regulated. Many structurally distinct families of channels, carriers, and pumps have been characterized in considerable detail during the past decades and defects in the function of some of these proteins have been linked to a growing list of human diseases. The dynamic regulation of the transport proteins present at the cell surface is vital for both normal cellular function and for the successful adaptation to changing environments. The composition of proteins present at the cell surface is controlled on both the transcriptional and post-translational level. Post-translational regulation involves highly conserved mechanisms of phosphorylation- and ubiquitylation-dependent signal transduction routes used to modify the cohort of receptors and transport proteins present under any given circumstances. In this review, we will summarize what is currently known about one facet of this regulatory process: the endocytic regulation of alkali metal transport proteins. The physiological relevance, major contributors, parallels and missing pieces of the puzzle in mammals, yeast and plants will be discussed.

Transport across the plasma membrane is the first step at which nutrient supply is tightly regulated in response to intracellular needs and often also rapidly changing external environment. In this review, I describe primarily our current understanding of multiple interconnected glucose-sensing systems and signal-transduction pathways that ensure fast and optimum expression of genes encoding hexose transporters in three yeast species, Saccharomyces cerevisiae, Kluyveromyces lactis and Candida albicans. In addition, an overview of GAL- and MAL-specific regulatory networks, controlling galactose and maltose utilization, is provided. Finally, pathways generating signals inducing posttranslational degradation of sugar transporters will be highlighted.

Like other microorganisms, free-living Candida albicans is mainly present in a three-dimensional multicellular structure, which is called a biofilm, rather than in a planktonic form. Candida albicans biofilms can be isolated from both abiotic and biotic surfaces at various locations within the host. As the number of abiotic implants, mainly bloodstream and urinary catheters, has been increasing, the number of biofilm-associated bloodstream or urogenital tract infections is also strongly increasing resulting in a raise in mortality. Cells within a biofilm structure show a reduced susceptibility to specific commonly used antifungals and, in addition, it has recently been shown that such cells are less sensitive to killing by components of our immune system. In this review, we summarize the most important insights in the mechanisms underlying biofilm-associated antifungal drug resistance and immune evasion strategies, focusing on the most recent advances in this area of research.

Protein phosphatase 2A is a subgroup of widely conserved serine/threonine phosphatases and plays diverse roles in transcription, translation, differentiation, cell cycle, and signal transduction in many organisms. However, its roles in biotrophic and hemi-biotrophic phytopathogenic fungi remain to be investigated. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that was defective in vegetative hyphal growth. In the mutant, the T-DNA fragment was found to be inserted in the promoter region of a putative serine/threonine protein phosphatase 2A catalytic subunit (PP2Ac) gene MoPPG1. Deletion of MoPPG1 leads to severe defects in vegetative hyphal growth and conidiation. Conidia of the ∆Moppg1 null mutants were misshaped, and most of them were two-celled. The deletion mutants of MoPPG1 did not penetrate into host plant cells and failed to cause any disease lesions on rice leaves. Interestingly, significant reduction was found in the ∆Moppg1 null mutants in expression levels of several Rho GTPase family genes including MgCDC42, MgRHO3, and MgRAC1, which were important for pathogenesis of M. oryzae. Taken together, our results indicated that PP2Ac plays vital roles in asexual development and plant infection by regulating Rho GTPases in the rice blast fungus and perhaps other plant pathogenic fungi.