Jürgen Wendland

Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi.

Mitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium-calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.

New modules for PCR-based gene targeting in Candida albicans: rapid and efficient gene targeting using 100 bp of flanking homology region

The use of PCR‐based techniques for directed gene alterations has become a standard tool in Saccharomyces cerevisiae. In our efforts to increase the speed of functional analysis of Candida albicans genes, we constructed a modular system of plasmid vectors and successfully applied PCR‐amplified functional analysis (FA)‐cassettes in the transformation of C. albicans. These cassettes facilitate: (a) gene disruptions; (b) tagging of 3′‐ends of genes with green fluorescent protein (GFP); and (c) replacements of endogenous promoters to achieve regulated expression. The modules consists of a core of three selectable marker genes, CaURA3, CaHIS1 and CaARG4. Modules for C‐terminal GFP‐tagging were generated by adding GFP‐sequences flanked at the 5′‐end by a (Gly‐Ala)3‐linker and at the 3′‐end by the S. cerevisiae URA3‐terminator to these selection markers. Promoter exchange modules consist of the respective marker genes followed by the regulatable CaMAL2 or CaMET3 promoters at their 3′‐ends. In order to ensure a reliably high rate of homologous gene targeting, the flanking homology regions required a size of 100 bp of gene‐specific sequences, which were provided with the oligonucleotide primers. The use of shorter flanking homology regions produced unsatisfactory results with C. albicans strain BWP17. With these new modules only a minimal set of primers is required to achieve the functional analysis of C. albicans genes and, therefore, provides a basic tool to increase the number of functionally characterized C. albicans genes of this human pathogen in the near future. Copyright

Ashbya gossypii : a model for fungal developmental biology

Ashbya gossypii is a riboflavin-overproducing filamentous fungus that is closely related to unicellular yeasts such as Saccharomyces cerevisiae. With its close ties to yeast and the ease of genetic manipulation in this fungal species, A. gossypii is well suited as a model to elucidate the regulatory networks that govern the functional differences between filamentous growth and yeast growth, especially now that the A. gossypii genome sequence has been completed. Understanding these networks could be relevant to related dimorphic yeasts such as the human fungal pathogen Candida albicans, in which a switch in morphology from the yeast to the filamentous form in response to specific environmental stimuli is important for virulence.

PCR-based methods facilitate targeted gene manipulations and cloning procedures

Abstract. Genome sequencing of a large number of organisms has provided a wealth of previously uncharacterized genes. Rapid functional analysis of these genes relies on efficient methods for targeted gene disruption. Gene replacement requires homologous recombination at the target locus. The efficiency of homologous recombination largely depends on the size of the flanking homology regions provided with the disruption cassette. Therefore, the ratio of targeted versus random integration into the genome governs the choice of tools applicable in any organism. PCR-based methods for gene disruption were first reported in Saccharomyces cerevisiae. Over the past years, additional tools have been developed for epitope- or green fluorescent protein-tagging of genes and for promoter exchanges. The attractiveness of these tools led to the generation of PCR modules for use in a wide variety of bacterial and fungal species. The high capacity of in vivo recombination of Sac. cerevisiae and Escherichia coli may also be used for heterologous DNA manipulations. This facilitates the generation of disruption cassettes for organisms that cannot be transformed with very short flanks of target homology regions. Furthermore, laborious cloning procedures, e.g. the generation of point mutations or the deletion of internal domains of genes, can be simplified by using these organisms as workhorses which will advance the general genetic toolkit.

Genome Sequence of Saccharomyces carlsbergensis, the World’s First Pure Culture Lager Yeast

Lager yeast beer production was revolutionized by the introduction of pure culture strains. The first established lager yeast strain is known as the bottom fermenting Saccharomyces carlsbergensis, which was originally termed Unterhefe No. 1 by Emil Chr. Hansen and has been used in production in since 1883. S. carlsbergensis belongs to group I/Saaz-type lager yeast strains and is better adapted to cold growth conditions than group II/Frohberg-type lager yeasts, e.g., the Weihenstephan strain WS34/70. Here, we sequenced S. carlsbergensis using next generation sequencing technologies. Lager yeasts are descendants from hybrids formed between a S. cerevisiae parent and a parent similar to S. eubayanus. Accordingly, the S. carlsbergensis 19.5-Mb genome is substantially larger than the 12-Mb S. cerevisiae genome. Based on the sequence scaffolds, synteny to the S. cerevisae genome, and by using directed polymerase chain reaction for gap closure, we generated a chromosomal map of S. carlsbergensis consisting of 29 unique chromosomes. We present evidence for genome and chromosome evolution within S. carlsbergensis via chromosome loss and loss of heterozygosity specifically of parts derived from the S. cerevisiae parent. Based on our sequence data and via fluorescence-activated cell-sorting analysis, we determined the ploidy of S. carlsbergensis. This inferred that this strain is basically triploid with a diploid S. eubayanus and haploid S. cerevisiae genome content. In contrast the Weihenstephan strain, which we resequenced, is essentially tetraploid composed of two diploid S. cerevisiae and S. eubayanus genomes. Based on conserved translocations between the parental genomes in S. carlsbergensis and the Weihenstephan strain we propose a joint evolutionary ancestry for lager yeast strains.

Septation and Cytokinesis in Fungi

Cytokinesis is the ultimate step of a cell cycle resulting in the generation of two progeny. Failure of correct cell division may be lethal for both, mother and daughter cells, and thus such a process must be tightly regulated with other events of the cell cycle. Differing solutions to the same problem have been developed in bacteria and plants while cytokinesis in animal and fungal cells is highly similar and requires a contractile ring containing actomyosin. Cytokinesis in fungi can be viewed as a three-stage process: (i) selection of a division site, (ii) orderly assembly of protein complexes, and finally (iii) dynamic events that lead to a constriction of the contractile ring and septum construction. Elaborate mechanisms known as the Mitotic Exit Network (MEN) and the Septation Initiation Network (SIN) have evolved to link these events, particularly the final steps of cytokinesis, with nuclear division. The purpose of this review was to discuss the latest developments in the fungal field and to describe the central known players required for key steps on the road to cell division. Differences in the cytokinesis of yeast-like fungi that result in complete cell separation in contrast to septation which leads to the compartmentalization of fungal hyphae are highlighted.

Fungal model systems and the elucidation of pathogenicity determinants

Highlights • History of seven fungal species used as models for studying development and pathogenicity.• Outline of central stages of their life cycle and their infection processes.• Molecular toolkits used to study different aspects of pathogenicity.• Insight gained from genome sequencing projects.• Current research trends and future challenges.

Candida albicans Sfl1 Suppresses Flocculation and Filamentation

ABSTRACT Hyphal morphogenesis in Candida albicans is regulated by multiple pathways which act by either inducing or repressing filamentation. Most notably, Tup1, Nrg1, and Rfg1 are transcriptional repressors, while Efg1, Flo8, Cph1, and Czf1 can induce filamentation. Here, we present the functional analysis of CaSFL1, which encodes the C. albicans homolog of the Saccharomyces cerevisiae SFL1 (suppressor of flocculation) gene. Deletion of CaSFL1 results in flocculation (i.e., the formation of clumps) of yeast cells, which is most pronounced in minimal medium. The flocs contained hyphae already under noninducing conditions, and filamentation could be enhanced with hypha-inducing cues at 37°C. Expression of SFL1 in a heterozygous mutant under the control of the CaMET3 promoter was shown to complement these defects and allowed switching between wild-type and mutant phenotypes. Interestingly, increased expression of SFL1 using a MET3prom-SFL1 construct prior to the induction of filamentation completely blocked germ tube formation. To localize Sfl1 in vivo, we generated a SFL1-GFP fusion. Sfl1-green fluorescent protein was found in the nucleus in both yeast cells and, to a lesser extent, hyphal cells. Using reverse transcription-PCR, we find an increased expression of ALS1, ALS3, HWP1, ECE1, and also FLO8. Our results suggest that Sfl1 functions in the repression of flocculation and filamentation and thus represents a novel negative regulator of C. albicans morphogenesis.

Ras1-induced hyphal development in Candida albicans requires the formin Bni1.

ABSTRACT Formins are downstream effector proteins of Rho-type GTPases and are involved in the organization of the actin cytoskeleton and actin cable assembly at sites of polarized cell growth. Here we show using in vivo time-lapse microscopy that deletion of the Candida albicans formin homolog BNI1 results in polarity defects during yeast growth and hyphal stages. Deletion of the second C. albicans formin, BNR1, resulted in elongated yeast cells with cell separation defects but did not interfere with the ability of bnr1 cells to initiate and maintain polarized hyphal growth. Yeast bni1 cells were swollen, showed an increased random budding pattern, and had a severe defect in cytokinesis, with enlarged bud necks. Induction of hyphal development in bni1 cells resulted in germ tube formation but was halted at the step of polarity maintenance. Bni1-green fluorescent protein is found persistently at the hyphal tip and colocalizes with a structure resembling the Spitzenkörper of true filamentous fungi. Introduction of constitutively active ras1G13V in the bni1 strain or addition of cyclic AMP to the growth medium did not bypass bni1 hyphal growth defects. Similarly, these agents were not able to suppress hyphal growth defects in the wal1 mutant which is lacking the Wiskott-Aldrich syndrome protein (WASP) homolog. These results suggest that the maintenance of polarized hyphal growth in C. albicans requires coordinated regulation of two actin cytoskeletal pathways, including formin-mediated secretion and WASP-dependent endocytosis.

Apical localization of actin patches and vacuolar dynamics in Ashbya gossypii depend on the WASP homolog Wal1p

Analysis of the Ashbya gossypii Wiskott-Aldrich syndrome-like gene AgWAL1 indicates that it is required for the maintenance of polarized hyphal growth. Growth and organelle dynamics of the wild type and of wal1 and other mutant strains were monitored by in vivo (fluorescence) time-lapse microscopy. Loss of WAL1 led to slow growth and defects in polarized growth that produced swellings in subapical regions, whereas formation of hyphal tips and dichotomous tip branching occurred as in the wild-type. Few actin cables in Agwal1 cells were found to insert into the hyphal tip, but specific clustering of cortical actin patches was observed in subapical regions of hyphal tips instead of at the hyphal apex. Distribution and movement of vacuoles was observed in vivo using FM4-64. In the wild type and in the slowly growing mutant strains bem2 and cla4, which lack a Rho-GTPase-activating protein and a PAK kinase, respectively, early endosomes appeared in the hyphal tip, whereas very few early endosomes and small vacuoles were found in the wal1 mutant hyphal tips, thus linking the cortical patch defect of wal1 hyphae with the distribution of endosomes. Vivid movement of vacuoles seen in the wild type and in the bem2 mutant in subapical regions was largely reduced in the wal1 and cla4 mutants. The tubular structure of mitochondria (as visualized by DIOC6 in vivo) was similar in the wild type and the wal1 mutant, although wal1 mitochondria appeared to be larger. Interestingly, mitochondria were found to insert into the hyphal tips in both strains. Our results indicate a function for Wal1p in filamentous fungi in coordinating actin patch distribution with polarized hyphal tip growth.