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.

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.

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.

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.

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.

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.

Reliable computation of roots in analytical waveguide modeling using an interval-Newton approach and algorithmic differentiation.

For the modeling and simulation of wave propagation in geometrically simple waveguides such as plates or rods, one may employ the analytical global matrix method. That is, a certain (global) matrix depending on the two parameters wavenumber and frequency is built. Subsequently, one must calculate all parameter pairs within the domain of interest where the global matrix becomes singular. For this purpose, one could compute all roots of the determinant of the global matrix when the two parameters vary in the given intervals. This requirement to calculate all roots is actually the method's most concerning restriction. Previous approaches are based on so-called mode-tracers, which use the physical phenomenon that solutions, i.e., roots of the determinant of the global matrix, appear in a certain pattern, the waveguide modes, to limit the root-finding algorithm's search space with respect to consecutive solutions. In some cases, these reductions of the search space yield only an incomplete set of solutions, because some roots may be missed as a result of uncertain predictions. Therefore, we propose replacement of the mode-tracer approach with a suitable version of an interval- Newton method. To apply this interval-based method, we extended the interval and derivative computation provided by a numerical computing environment such that corresponding information is also available for Bessel functions used in circular models of acoustic waveguides. We present numerical results for two different scenarios. First, a polymeric cylindrical waveguide is simulated, and second, we show simulation results of a one-sided fluid-loaded plate. For both scenarios, we compare results obtained with the proposed interval-Newton algorithm and commercial software.

Analysis of the cell wall integrity pathway of Ashbya gossypii.

Fungal cells are exposed to rapidly changing environmental conditions, in particular with regard to the osmotic potential. This requires constant remodeling of the cell wall and, therefore, the cell wall integrity (CWI) MAP-kinase pathway plays a major role in shaping the fungal cell wall to protect from adverse external stresses. To provide a comprehensive functional analysis of the Ashbya gossypii CWI pathway we generated a set of ten deletion mutants in conserved components including the cell surface sensors AgWSC1 and AgMID2, a putative Rho1-guanine nucleotide exchange factor, AgTUS1, the protein kinase C, AgPKC1, the MAP-kinases AgBCK1, AgMKK1 and AgMPK1, and transcription factors known to be involved in CWI signaling AgRLM1, AgSWI4 and AgSWI6. Deletion of AgPKC1 shows a severe growth defect with frequent tip cell lysis. Deletion of components of the MAP-kinase module generates a pronounced colony lysis phenotype in older regions of the mycelium. Cytoplasmic leakage was assayed using alkaline phosphatase and ß-galactosidase release assays. This indicated that the lysis phenotypes of CWI pathway mutants may be useful to facilitate the isolation of riboflavin from A. gossypii. Remarkably, the Agwsc1 mutant showed a strong (up to 8-fold) increase of riboflavin in the growth medium compared to the parental strain.

Molecular determinants of sporulation in Ashbya gossypii.

Regulation of development and entry into sporulation is critical for fungi to ensure survival of unfavorable environmental conditions. Here we present an analysis of gene sets regulating sporulation in the homothallic ascomycete Ashbya gossypii. Deletion of components of the conserved pheromone/starvation MAP kinase cascades, e.g., STE11 and STE7, results in increased sporulation. In kar3 mutants sporulation is severely reduced, while deletion of KAR4 as well as of homologs of central Saccharomyces cerevisiae regulators of sporulation, IME1, IME2, IME4, and NDT80, abolishes sporulation in A. gossypii. Comparison of RNAseq transcript profiles of sporulation-deficient mutants identified a set of 67 down-regulated genes, most of which were up-regulated in the oversporulating ste12 mutant. One of these differentially expressed genes is an endoglucanase encoded by ENG2. We found that Eng2p promotes hyphal fragmentation as part of the developmental program of sporulation, which generates single-celled sporangia. Sporulation-deficient strains are arrested in their development but form sporangia. Supply of new nutrients enabled sporangia to return to hyphal growth, indicating that these cells are not locked in meiosis. Double-strand break (DSB) formation by Spo11 is apparently not required for sporulation; however, the absence of DMC1, which repairs DSBs in S. cerevisiae, results in very poor sporulation in A. gossypii. We present a comprehensive analysis of the gene repertoire governing sporulation in A. gossypii and suggest an altered regulation of IME1 expression compared to S. cerevisiae.

Subcellular localization of the fatty acyl reductase involved in pheromone biosynthesis in the tobacco budworm, Heliothis virescens (Noctuidae: Lepidoptera).

Sex pheromone components are produced in specialized glands of female moths via well-characterized biosynthetic pathways, where a Fatty Acyl Reductase (FAR) is often essential for producing the specific ratio of the different pheromone components. The subcellular localization and membrane topology of FARs is important for understanding how pheromones are synthesized and exported to the exterior for release. We investigated the subcellular localization of HvFAR from the noctuid moth Heliothis virescens by producing recombinant fusion proteins with green fluorescent protein (GFP) in yeast. A C-terminally tagged construct was localized to the endoplasmic reticulum (ER) and retained full reductive activity on a broad range of saturated and unsaturated fatty acyl precursors. In contrast, an N-terminally-tagged construct was poorly expressed in the cytoplasm and was not enzymatically active, indicating that HvFAR requires a free N-terminal for both proper targeting and catalytic activity. A series of truncations of the N-and C-termini of HvFAR was conducted based on in silico-predicted hydrophobic domains and transmembrane regions. The N-terminally truncated protein was found in the cytoplasm and did not retain activity, emphasizing the importance of the N-terminal for FAR function. In addition, the orientation in the membrane of the C-terminus-tagged HvFAR-GFP construct was analyzed using a fluorescence protease protection (FPP) assay, implying that the C-terminal of HvFAR is orientated towards the cytoplasm. These results, together with previous data on the localization of desaturases, confirm the importance of the ER as a subcellular site of pheromone production.

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.

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.

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.

The Ashbya gossypii fimbrin SAC6 is required for fast polarized hyphal tip growth and endocytosis.

Ashbya gossypii has been an ideal system to study filamentous hyphal growth. Previously, we identified a link between polarized hyphal growth, the organization of the actin cytoskeleton and endocytosis with our analysis of the A. gossypii Wiskott-Aldrich Syndrome Protein (WASP)-homolog encoded by the AgWAL1 gene. Here, we studied the role of AgSAC6, encoding a fimbrin in polarized hyphal growth and endocytosis, and based on our functional analysis identified genetic interactions between AgSAC6 and AgWAL1. SAC6 mutants show severely reduced polarized growth. This growth phenotype is temperature dependent and sac6 spores do not germinate at elevated temperatures. Spores germinated at 30°C generate slow growing mycelia without displaying polarity establishment defects at the hyphal tip. Several phenotypic characteristics of sac6 hyphae resemble those found in wal1 mutants. First, tips of sac6 hyphae shifted to 37°C swell and produce subapical bulges. Second, actin patches are mislocalized subapically. And third, the rate of endocytotic uptake of the vital dye FM4-64 was reduced. This indicates that actin filament bundling, a conserved function of fimbrins, is required for fast polarized hyphal growth, polarity maintenance, and endocytosis in filamentous fungi.

Genome evolution in the eremothecium clade of the Saccharomyces complex revealed by comparative genomics.

We used comparative genomics to elucidate the genome evolution within the pre-whole-genome duplication genus Eremothecium. To this end, we sequenced and assembled the complete genome of Eremothecium cymbalariae, a filamentous ascomycete representing the Eremothecium type strain. Genome annotation indicated 4712 gene models and 143 tRNAs. We compared the E. cymbalariae genome with that of its relative, the riboflavin overproducer Ashbya (Eremothecium) gossypii, and the reconstructed yeast ancestor. Decisive changes in the Eremothecium lineage leading to the evolution of the A. gossypii genome include the reduction from eight to seven chromosomes, the downsizing of the genome by removal of 10% or 900 kb of DNA, mostly in intergenic regions, the loss of a TY3-Gypsy-type transposable element, the re-arrangement of mating-type loci, and a massive increase of its GC content. Key species-specific events are the loss of MNN1-family of mannosyltransferases required to add the terminal fourth and fifth α-1,3-linked mannose residue to O-linked glycans and genes of the Ehrlich pathway in E. cymbalariae and the loss of ZMM-family of meiosis-specific proteins and acquisition of riboflavin overproduction in A. gossypii. This reveals that within the Saccharomyces complex genome, evolution is not only based on genome duplication with subsequent gene deletions and chromosomal rearrangements but also on fungi associated with specific environments (e.g. involving fungal-insect interactions as in Eremothecium), which have encountered challenges that may be reflected both in genome streamlining and their biosynthetic potential.

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.