The APSES protein Sok2 is a positive regulator of sporulation in Ashbya gossypii.

Ashbya gossypii is a homothallic, flavinogenic, filamentous ascomycete that starts overproduction of riboflavin and fragments its mycelium quantitatively into spore producing sporangia at the end of a growth phase. Mating is not required for sporulation and the standard homothallic laboratory strain is a MATa strain. Here we show that ectopic expression of Saccharomyces cerevisiae MATα2 in A. gossypii completely suppresses sporulation, inhibits riboflavin overproduction and downregulates among others AgSOK2. AgSok2 belongs to a fungal-specific group of (APSES) transcription factors. Deletion of AgSOK2 strongly reduces riboflavin production and blocks sporulation. The initiator of meiosis, AgIME1, is a transcription factor essential for sporulation. We characterized the AgIME1 promoter region required for complementation of the Agime1 mutant. Reporter assays with AgIME1 promoter fragments fused to lacZ showed that AgSok2 does not control AgIME1 transcription. However, global transcriptome analysis identified two other essential regulators of sporulation, AgIME2 and AgNDT80, as potential targets of AgSok2. Our data suggest that sporulation and riboflavin production in A. gossypii are under mating type locus and nutritional control. Sok2, a target of the cAMP/protein kinase A pathway, serves as a central positive regulator to promote sporulation. This contrasts Saccharomyces cerevisiae where Sok2 is a repressor of IME1 transcription.

Developmental Growth Control Exerted via the Protein A Kinase Tpk2 in Ashbya gossypii.

Sporulation in Ashbya gossypii is induced by nutrient-limited conditions and leads to the formation of haploid spores. Using RNA-seq, we have determined a gene set induced upon sporulation, which bears considerable overlap with that of Saccharomyces cerevisiae but also contains A. gossypii-specific genes. Addition of cyclic AMP (cAMP) to nutrient-limited media blocks sporulation and represses the induction of sporulation specific genes. Deletion of the protein kinase A (PKA) catalytic subunits encoded by TPK1 and TPK2 showed reduced growth in tpk1 but enhanced growth in the tpk2 strain; however, both mutants sporulated well. Sporulation can be blocked by cAMP in tpk1 but not in tpk2 strains. Similarly, TPK2 acts at a second developmental switch promoting the break in spore dormancy. In S. cerevisiae, PKA phosphorylates and inhibits Msn2/4. The transcript profiles of the tpk1 and msn2/4 mutants were very similar to that of the wild type under sporulation conditions. However, deletion of the single A. gossypii MSN2/4 homolog generated a specific sporulation defect. We identified a set of genes involved in spore wall assembly that was downregulated in the msn2/4 mutant, particularly DIT2, suggesting that poor spore viability may be due to lysis of spores. Our results reveal specific functional differences between the two catalytic PKA subunits in A. gossypii and identified Tpk2 as the key A kinase that transduces developmental decisions of growth. Our data also suggest that Msn2/4 is involved only at a late step of sporulation in A. gossypii and is not a major regulator of IME1.

Fungal model systems and the elucidation of pathogenicity determinants.

Fungi have the capacity to cause devastating diseases of both plants and animals, causing significant harvest losses that threaten food security and human mycoses with high mortality rates. As a consequence, there is a critical need to promote development of new antifungal drugs, which requires a comprehensive molecular knowledge of fungal pathogenesis. In this review, we critically evaluate current knowledge of seven fungal organisms used as major research models for fungal pathogenesis. These include pathogens of both animals and plants; Ashbya gossypii, Aspergillus fumigatus, Candida albicans, Fusarium oxysporum, Magnaporthe oryzae, Ustilago maydis and Zymoseptoria tritici. We present key insights into the virulence mechanisms deployed by each species and a comparative overview of key insights obtained from genomic analysis. We then consider current trends and future challenges associated with the study of fungal pathogenicity.

Major contribution of the Ehrlich pathway for 2-phenylethanol/rose flavor production in Ashbya gossypii.

Aroma alcohols of fermented food and beverages are derived from fungal amino acids catabolism via the Ehrlich pathway. This linear pathway consists of three enzymatic reactions to form fusel alcohols. Regulation of some of the enzymes occurs on the transcriptional level via Aro80. The riboflavin overproducer Ashbya gossypii produces strong fruity flavours in contrast to its much less aromatic relative Eremothecium cymbalariae. Genome comparisons indicated that A. gossypii harbors genes for aromatic amino acid catabolism (ARO8a, ARO8b, ARO10, and ARO80) while E. cymbalariae only encodes ARO8a and thus lacks major components of aromatic amino acid catabolism. Volatile compound (VOC) analysis showed that both Eremothecium species produce large amounts of isoamyl alcohol while A. gossypii also produces high levels of 2-phenylethanol. Deletion of the A. gossypii ARO-genes did not confer any growth deficiencies. However, A. gossypii ARO-mutants (except Agaro8a) were strongly impaired in aroma production, particularly in the production of the rose flavour 2-phenylethanol. Conversely, overexpression of ARO80 via the AgTEF1 promoter resulted in 50% increase in VOC production. Together these data indicate that A. gossypii is a very potent flavour producer and that amongst the non-Saccharomyces biodiversity strains can be identified that could provide positive sensory properties to fermented beverages.

Yap1-dependent oxidative stress response provides a link to riboflavin production in Ashbya gossypii.

Ashbya gossypii is a natural overproducer of riboflavin. Overproduction of riboflavin can be induced by environmental stress, e.g. nutritional or oxidative stress. The Yap-protein family has a well-documented role in stress response. Particularly, Yap1 has a major role in directing the oxidative stress responses. The A. gossypii YAP-family consists of only three genes in contrast to its closest relative Eremothecium cymbalariae, which has four YAP-homologs. Gene order at Eremothecium YAP-loci is conserved with the reconstructed yeast ancestor. AgYap1p is unique amongst Yap-homologs as it lacks the cysteine-rich domains (CRDs). AgYAP1 expression is inducible and GFP-AgYap1 localizes to the nucleus. Agyap1 mutants displayed higher sensitivity against oxidative stress - H(2)O(2) and menadione - and are strongly reduced in riboflavin production. High levels of cAMP, which also reduce riboflavin production, show a synergistic effect on this sensitivity. AgYAP1 and a chimera of AgYAP1 (with the DNA-binding domain) and ScYAP1 (with the CRDs) can both complement the Scyap1 oxidative stress sensitivity. This suggests that the DNA-binding sites of ScYap1 are conserved in A. gossypii. Expression of AgRIB4, which contains three putative Yap1-binding sites, assayed via a lacZ-reporter gene was strongly reduced in an Agyap1 mutant suggesting a direct involvement of AgYap1 in riboflavin production. Furthermore, our data show that application of H(2)O(2) stress leads to an increase in riboflavin production in a Yap1-dependent manner.

Forward genetics in Candida albicans that reveals the Arp2/3 complex is required for hyphal formation, but not endocytosis.

Candida albicans is a diploid fungal pathogen lacking a defined complete sexual cycle, and thus has been refractory to standard forward genetic analysis. Instead, transcription profiling and reverse genetic strategies based on Saccharomyces cerevisiae have typically been used to link genes to functions. To overcome restrictions inherent in such indirect approaches, we have investigated a forward genetic mutagenesis strategy based on the UAU1 technology. We screened 4700 random insertion mutants for defects in hyphal development and linked two new genes (ARP2 and VPS52) to hyphal growth. Deleting ARP2 abolished hyphal formation, generated round and swollen yeast phase cells, disrupted cortical actin patches and blocked virulence in mice. The mutants also showed a global lack of induction of hyphae-specific genes upon the yeast-to-hyphae switch. Surprisingly, both arp2 Delta/Delta and arp2 Delta/Delta arp3 Delta/Delta mutants were still able to endocytose FM4-64 and Lucifer Yellow, although as shown by time-lapse movies internalization of FM4-64 was somewhat delayed in mutant cells. Thus the non-essential role of the Arp2/3 complex discovered by forward genetic screening in C. albicans showed that uptake of membrane components from the plasma membrane to vacuolar structures is not dependent on this actin nucleating machinery.

Dual-colour fluorescence microscopy using yEmCherry-/GFP-tagging of eisosome components Pil1 and Lsp1 in Candida albicans.

PCR-based gene targeting technologies have previously been developed for Candida albicans molecular genetic manipulation. Modular marker plasmids for the functional analysis of C. albicans genes have been generated to delete genes, exchange promoters and tag genes with GFP. Here, we have embedded two fluorescent proteins encoded by Venus and yEmCherry into the pFA-plasmid series and demonstrate their usefulness in dual colour microscopy. To this end we analysed the localization of C. albicans homologues of Pil1 and Lsp1, which in S. cerevisiae are components of eisosomes. We find that Pil1/Lsp1-containing eisosomes are cortical protein complexes in C. albicans.Pil1 and Lsp1, tagged with either GFP or yEmCherry, strictly co-localized during all growth stages. Eisosomes, however, localized at distinct positions not overlapping with either cortical actin patches or the endocytosis marker protein Abp1 in yeast or the Spitzenkörper in hyphal cells. To demonstrate the use of Venus yellow fluorescent protein we performed time lapse microscopy of yeast and hyphal stages using a histone H4-Venus tag. As demonstrated, these additions to the toolbox enable a wide range of in vivo applications in C. albicans.

Characterization of α-factor pheromone and pheromone receptor genes of Ashbya gossypii.

The genome of Ashbya gossypii contains homologs of most of the genes that are part of the Saccharomyces cerevisiae pheromone-signal transduction cascade. However, we currently lack understanding of a potential sexual cycle for this pre-whole genome duplication hemiascomycete. The sequenced strain bears three identical copies encoding MATa. We show that the syntenic A. gossypii homolog of MFα1 (AFL062w) does not encode a mature α-factor peptide, but identified another gene, AAR163c, which encodes a candidate α-specific mating pheromone and is thus reannotated as AgMFα2. The expression of the AgSTE2α-factor receptor in an Scste2 S. cerevisiae MATa strain resulted in dosage-dependent growth arrest upon exposure to A. gossypiiα-factor, which indicated that the pheromone response was effectively coupled to the S. cerevisiae signal transduction cascade. Comparison of α-pheromones and α-pheromone receptors showed greater conservation between Eremothecium cymbalariae and S. cerevisiae than between A. gossypii and E. cymbalariae. We constructed A. gossypii strains deleted for the STE2 and STE3 pheromone receptors. These strains showed no phenotypic abnormalities and an ste2, ste3 double mutant is still able to sporulate. The deletion of STE12 as the downstream target of pheromone signalling, however, led to a hypersporulation phenotype.

Candida albicans Vrp1 is required for polarized morphogenesis and interacts with Wal1 and Myo5.

Recently, a link between endocytosis and hyphal morphogenesis has been identified in Candida albicans via the Wiskott-Aldrich syndrome gene homologue WAL1. To get a more detailed mechanistic understanding of this link we have investigated a potentially conserved interaction between Wal1 and the C. albicans WASP-interacting protein (WIP) homologue encoded by VRP1. Deletion of both alleles of VRP1 results in strong hyphal growth defects under serum inducing conditions but filamentation can be observed on Spider medium. Mutant vrp1 cells show a delay in endocytosis - measured as the uptake and delivery of the lipophilic dye FM4-64 into small endocytic vesicles - compared to the wild-type. Vacuolar morphology was found to be fragmented in a subset of cells and the cortical actin cytoskeleton was depolarized in vrp1 daughter cells. The morphology of the vrp1 null mutant could be complemented by reintegration of the wild-type VRP1 gene at the BUD3 locus. Using the yeast two-hybrid system we could demonstrate an interaction between the C-terminal part of Vrp1 and the N-terminal part of Wal1, which contains the WH1 domain. Furthermore, we found that Myo5 has several potential interaction sites on Vrp1. This suggests that a Wal1-Vrp1-Myo5 complex plays an important role in endocytosis and the polarized localization of the cortical actin cytoskeleton to promote polarized hyphal growth in C. albicans.

Analysis of flocculins in Ashbya gossypii reveals FIG2 regulation by TEC1.

For 95% of the Ashbya gossypii protein-encoding genes there is a Saccharomyces cerevisiae homolog. Out of these 90% are arranged in a conserved, syntenic, gene order. Interestingly, A. gossypii adhesins, encoded by homologs of S. cerevisiae FLO-genes, are found in non-syntenic positions. A. gossypii contains only a small set of adhesins: two FLO5, a FLO11 and a FIG2 homolog, but no FLO1, FLO9, or FLO10 homolog. Here we present the functional analysis of the A. gossypii adhesins and their potential transcriptional regulators SFL1, FLO8, and TEC1. Deletion of individual classes of FLO-genes did not reveal any phenotype. Lack of SFL1 or FLO8 showed reduced growth. The expression of adhesins in different strain backgrounds was tested using promoter-lacZ-fusions. We found that SFL1 acts as a suppressor of one of the FLO5 genes and FLO8 but particularly of FIG2. Interestingly, FIG2 expression was abolished in a tec1 mutant. We identified three potential Tec1-binding sites in the FIG2-promoter by similarity to S. cerevisiae Tec1-binding sites. The AgCHT2 promoter, which regulates a sporulation specific chitinase, also harbours potential Tec1-binding sites. Consequently, expression of CHT2 was not detected in a tec1 strain. This suggests that Tec1- binding sites are conserved between A. gossypii and S. cerevisiae even though there are different Tec1 target genes in each of these organisms.

Functional analysis of Candida albicans genes encoding SH3-domain-containing proteins.

Postgenomic gene-function analyses with Candida albicans are hindered by its constitutive diploidy and the lack of a sexual cycle. Rapid generation of mutant strains can be achieved using PCR-based techniques for directed gene alterations. Here, we report the analyses of nine C. albicans genes that encode Src Homology 3-domain proteins. Phenotypic analyses included the potential of the mutants to form hyphal filaments, maintain a polarized actin cytoskeleton or the ability to generate large vacuoles in the germ cells and in subapical compartments. The C. albicans homologs of the Saccharomyces cerevisiae BBC1, BOI2, BUD14, FUS1, HSE1, PIN3, RVS167, RVS167-2 and SHO1 were all found to be nonessential. Deletion of RVS167 resulted in a strain with a decreased ability to form hyphal filaments. The number of cortical actin patches was increased in Deltarvs167 strains and their distribution was depolarized in both mother and daughter yeast cells and along the hyphae during filamentous growth stages. Polarization of patches could be restored upon reintroduction of the wild-type gene. Deletion of BOI2 was found to generate a defect in vacuolar fusion in hyphae. In contrast to a deletion in the Deltawal1 gene, Deltaboi2 cells formed abundant hyphae, indicating that fragmented vacuoles do not inhibit filamentation. Placing BOI2 under control of the MAL2-promoter allowed the regulation of this phenotype depending on the growth conditions.

N-acetylglucosamine utilization by Saccharomyces cerevisiae based on expression of Candida albicans NAG genes.

Synthesis of chitin de novo from glucose involves a linear pathway in Saccharomyces cerevisiae. Several of the pathway genes, including GNA1, are essential. Genes for chitin catabolism are absent in S. cerevisiae. Therefore, S. cerevisiae cannot use chitin as a carbon source. Chitin is the second most abundant polysaccharide after cellulose and consists of N-acetylglucosamine (GlcNAc) moieties. Here, we have generated S. cerevisiae strains that are able to use GlcNAc as a carbon source by expressing four Candida albicans genes (NAG3 or its NAG4 paralog, NAG5, NAG2, and NAG1) encoding a GlcNAc permease, a GlcNAc kinase, a GlcNAc-6-phosphate deacetylase, and a glucosamine-6-phosphate deaminase, respectively. Expression of NAG3 and NAG5 or NAG4 and NAG5 in S. cerevisiae resulted in strains in which the otherwise-essential ScGNA1 could be deleted. These strains required the presence of GlcNAc in the medium, indicating that uptake of GlcNAc and its phosphorylation were achieved. Expression of all four NAG genes produced strains that could use GlcNAc as the sole carbon source for growth. Utilization of a GlcNAc catabolic pathway for bioethanol production using these strains was tested. However, fermentation was slow and yielded only minor amounts of ethanol (approximately 3.0 g/liter), suggesting that fructose-6-phosphate produced from GlcNAc under these conditions is largely consumed to maintain cellular functions and promote growth. Our results present the first step toward tapping a novel, renewable carbon source for biofuel production.

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.

A molecular toolbox for manipulating Eremothecium coryli.

The genus Eremothecium contains dimorphic and filamentous fungal species, most notably Eremothecium sinecaudum (Holleya sinecauda), a dimorphic plant pathogen, which was isolated from mustard seeds, and Eremothecium gossypii (Ashbya gossypii), a filamentous fungus, which is well known for its ability to produce riboflavin. In this study, we present the initial molecular characterization of another Eremothecium species classified as Eremothecium coryli. E.coryli is a dimorphic fungus. We have developed, based on previously described reagents, a transformation system for E. coryli using kanMX and NATMX3 as dominant selectable marker genes on freely replicating plasmids conferring resistance to the antibiotics G418 and nourseothricin, respectively. As reporter genes we could introduce lacZ and GFP, which were controlled either by the AgTEF1 promoter or by regulatable MET promoters derived from the A. gossypii and Saccharomyces cerevisiae MET3 genes. These newly established tools will allow a detailed comparison of different growth modes in filamentous or dimorphic species within the genus Eremothecium.

An Arf-GAP promotes endocytosis and hyphal growth of Ashbya gossypii.

The ADP-ribosylation factor (ARF) family of GTPases are highly conserved from yeast to human and regulate vesicle budding. Sec7 domain containing proteins stimulate the guanine nucleotide exchange on Arf proteins, while ARF-GTPase activating proteins stimulate the hydrolysis of GTP. Since vesicle trafficking is important for hyphal growth, we studied the Ashbya gossypii homolog of Saccharomyces cerevisiae ARF3 along with its putative GEF and GTPase-activating protein (GAP) encoded by YEL1 and GTS1, respectively. Deletion of YEL1 had no discernible phenotype and deletion of ARF3 had only a minor defect in vacuolar fusion. In contrast, deletion of GTS1 severely impaired hyphal growth, and mutants showed defects in the maintenance of polarity and the localization of cortical actin patches. The uptake of the lipophilic dye FM4-64 was delayed in gts1 hyphae, indicating a defect in endocytosis. Gts1 has several protein domains, of which the Arf-GAP domain is required for complementation of the gts1 mutant phenotype. GFP-tagged GTS1 under control of its endogenous promoter localized to the plasma membrane but was enriched at hyphal tips and septal sites corresponding to a role in polarized vesicle trafficking. Our results indicate that this ARF-GTPase module plays an important role for filamentous hyphal growth.

Early-Mid Pleistocene genetic differentiation and range expansions as exemplified by invasive Eurasian Bunias orientalis (Brassicaceae) indicates the Caucasus as key region.

Turkish Warty cabbage, Bunias orientalis L. (Brassicaceae) is a perennial herb known for its 250 years of invasion history into Europe and worldwide temperate regions. Putative centers of origin were debated to be located in Turkey, the Caucasus or Eastern Europe. Based on the genetic variation from the nuclear and plastid genomes, we identified two major gene pools in the Caucasian-Irano-Turanian region and close to the Northern Caucasus, respectively. These gene pools are old and started to diverge and expand approximately 930 kya in the Caucasus. Pleistocene glaciation and deglaciation cycles favoured later expansion of a European gene pool 230 kya, which was effectively separated from the Caucasian-Irano-Turanian gene pool. Although the European gene pool is genetically less diverse, it has largely served as source for colonization of Western and Northern Europe in modern times with rare observations of genetic contributions from the Caucasian-Irano-Turanian gene pool such as in North-East America. This study largely utilized herbarium material to take advantage of a biodiversity treasure trove providing biological material and also giving access to detailed collection information.

Development of brewing science in (and since) the late 19th century: molecular profiles of 110-130year old beers.

The 19th century witnessed many advances in scientific enzymology and microbiology that laid the foundations for modern biotechnological industries. In the current study, we analyze the content of original lager beer samples from the 1880s, 1890s and 1900s with emphasis on the carbohydrate content and composition. The historic samples include the oldest samples brewed with pure Saccharomyces carlsbergensis yeast strains. While no detailed record of beer pasteurization at the time is available, historic samples indicate a gradual improvement of bottled beer handling from the 1880s to the 1900s, with decreasing contamination by enzymatic and microbial activities over this time span. Samples are sufficiently well preserved to allow comparisons to present-day references, thus yielding molecular signatures of the effects of 20th century science on beer production. Opposite to rather stable carbohydrate profiles, some aldehydes reach up to 40-fold higher levels in the historic samples as compared to present-day references.

Chromosome number reduction in Eremothecium coryli by two telomere-to-telomere fusions.

The genus Eremothecium belongs to the Saccharomyces complex of pre-whole-genome duplication (WGD) yeasts and contains both dimorphic and filamentous species. We established the 9.1-Mb draft genome of Eremothecium coryli, which encodes 4,682 genes, 186 tRNA genes, and harbors several Ty3 transposons as well as more than 60 remnants of transposition events (LTRs). The initial de novo assembly resulted in 19 scaffolds, which were assembled based on synteny to other Eremothecium genomes into six chromosomes. Interestingly, we identified eight E. coryli loci that bear centromeres in the closely related species E. cymbalariae. Two of these E. coryli loci, CEN1 and CEN8, however, lack conserved DNA elements and did not convey centromere function in a plasmid stability assay. Correspondingly, using a comparative genomics approach we identified two telomere-to-telomere fusion events in E. coryli as the cause of chromosome number reduction from eight to six chromosomes. Finally, with the genome sequences of E. coryli, E. cymbalariae, and Ashbya gossypii a reconstruction of three complete chromosomes of an Eremothecium ancestor revealed that E. coryli is more syntenic to this ancestor than the other Eremothecium species.

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.

An indirect assay for volatile compound production in yeast strains.

Traditional flavor analysis relies on gas chromatography coupled to mass spectrometry (GC-MS) methods. Here we describe an indirect method coupling volatile compound formation to an ARO9-promoter-LacZ reporter gene. The resulting ß-galactosidase activity correlated well with headspace solid phase micro extraction (HS/SPME) GC-MS data, particularly with respect to the formation of rose flavor. This tool enables large-scale screening of yeast strains and their progeny to identify the most flavor active strains.