Filamentous Ascomycete Ashbya Gossypii Research Articles

Cre-loxP-based system for removal and reuse of selection markers in Ashbya gossypii targeted engineering

The filamentous ascomycete Ashbya gossypii is amenable to genetic manipulation and is an excellent model system for studying eukaryotic cell biology. However, the number of selection markers in current use for both targeted gene integration and disruption in this fungus are very limited. Therefore, the Cre-loxP recombination system was adapted for use in A. gossypii and its effectiveness in recycling marker genes was demonstrated by constructing both single and double deleted Agura3 and Agade1 auxotrophic strains free of exogenous markers. In spite of its wide use, this is the first report in which the Cre-loxP system was applied to A. gossypii, opening new perspectives for targeted engineering of this fungus with several promising biotechnological applications.

Regulation of exit from mitosis in multinucleateAshbya gossypiicells relies on a minimal network of genes

In Saccharomyces cerevisiae, mitosis is coupled to cell division by the action of the Cdc fourteen early anaphase release (FEAR) and mitotic exit network (MEN) regulatory networks, which mediate exit from mitosis by activation of the phosphatase Cdc14. The closely related filamentous ascomycete Ashbya gossypii provides a unique cellular setting to study the evolution of these networks. Within its multinucleate hyphae, nuclei are free to divide without the spatial and temporal constraints described for budding yeast. To investigate how this highly conserved system has adapted to these circumstances, we constructed a series of mutants lacking homologues of core components of MEN and FEAR and monitored phenomena such as progression through mitosis and Cdc14 activation. MEN homologues in A. gossypii were shown to have diverged from their anticipated role in Cdc14 release and exit from mitosis. We observed defects in septation, as well as a partial metaphase arrest, in Agtem1Δ, Agcdc15Δ, Agdbf2/dbf20Δ, and Agmob1Δ. A. gossypii homologues of the FEAR network, on the other hand, have a conserved and more pronounced role in regulation of the M/G1 transition. Agcdc55Δ mutants are unable to sequester AgCdc14 throughout interphase. We propose a reduced model of the networks described in yeast, with a low degree of functional redundancy, convenient for further investigations into these networks.

Evolution of multinucleated Ashbya gossypii hyphae from a budding yeast-like ancestor

In the filamentous ascomycete Ashbya gossypii polarity establishment at sites of germ tube and lateral branch emergence depends on homologues of Saccharomyces cerevisiae factors controlling bud site selection and bud emergence. Maintenance of polar growth involves homologues of well-known polarity factors of budding yeast. To achieve the much higher rates of sustained polar surface expansion of hyphae compared to mainly non-polarly growing yeast buds five important alterations had to evolve. Permanent presence of the polarity machinery at a confined area in the rapidly expanding hyphal tip, increased cytoplasmic space with a much enlarged ER surface for generating secretory vesicles, efficient directed transport of secretory vesicles to and accumulation at the tip, increased capacity of the exocytosis system to process these vesicles, and an efficient endocytosis system for membrane and polarity factor recycling adjacent to the zone of exocytosis. Morphological, cell biological, and molecular aspects of this evolution are discussed based on experiments performed within the past 10 y.

A plasmid collection for PCR-based gene targeting in the filamentous ascomycete Ashbya gossypii

PCR-based gene targeting with heterologous markers is an efficient method to delete genes, generate gene fusions, and modulate gene expression. For the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, several plasmid collections are available covering a wide range of tags and markers. For several reasons, many of these cassettes cannot be used in the filamentous ascomycete Ashbya gossypii. This article describes the construction of 93 heterologous modules for C- and N-terminal tagging and promoter replacements in A. gossypii. The performance of 12 different fluorescent tags was evaluated by monitoring their brightness, detectability, and photostability when fused to the myosin light-chain protein Mlc2. Furthermore, the thiamine-repressible S. cerevisiae THI13 promoter was established to regulate gene expression in A. gossypii. This collection will help accelerate analysis of gene function in A. gossypii and in other ascomycetes where S. cerevisiae promoter elements are functional.

The anaphase-promoting complex/cyclosome is required for anaphase progression in multinucleated Ashbya gossypii cells.

Regulated protein degradation is essential for eukaryotic cell cycle progression. The anaphase-promoting complex/cyclosome (APC/C) is responsible for the protein destruction required for the initiation of anaphase and the exit from mitosis, including the degradation of securin and B-type cyclins. We initiated a study of the APC/C in the multinucleated, filamentous ascomycete Ashbya gossypii to understand the mechanisms underlying the asynchronous mitosis observed in these cells. These experiments were motivated by previous work which demonstrated that the mitotic cyclin AgClb1/2p persists through anaphase, suggesting that the APC/C may not be required for the division cycle in A. gossypii. We have now found that the predicted APC/C components AgCdc23p and AgDoc1p and the targeting factors AgCdc20p and AgCdh1p are essential for growth and nuclear division. Mutants lacking any of these factors arrest as germlings with nuclei blocked in mitosis. A likely substrate of the APC/C is the securin homologue AgPds1p, which is present in all nuclei in hyphae except those in anaphase. The destruction box sequence of AgPds1p is required for this timed disappearance. To investigate how the APC/C may function to degrade AgPds1p in only the subset of anaphase nuclei, we localized components and targeting subunits of the APC/C. Remarkably, AgCdc23p, AgDoc1p, and AgCdc16p were found in all nuclei in all cell cycle stages, as were the APC/C targeting factors AgCdc20p and AgCdh1p. These data suggest that the AgAPC/C may be constitutively active across the cell cycle and that proteolysis in these multinucleated cells may be regulated at the level of substrates rather than by the APC/C itself.

Initial molecular characterization of a novel Rho-type GTPase RhoH in the filamentous ascomycete Ashbya gossypii

The organization of the actin cytoskeleton is of central importance in determining cell shape and morphogenesis. Rho-type GTP-binding proteins are known for their role to activate downstream effector proteins that act as scaffolds and direct the assembly of actin filaments. We have identified a novel Rho-type GTPase, RHOH, in the filamentous ascomycete Ashbya gossypii and also in one of its closest relatives, Holleya sinecauda both species belonging to the genus Eremothecium. RHOH is a paralog of AgRHO1 and was generated by tandem duplication in an evolutionary ancestor but is not found in Saccharomyces cerevisae. Deletion of RHOH was carried out in both A. gossypii and H. sinecauda and showed that this gene is non-essential. However, a cell wall defect was observed that led to increased cell lysis at the hyphal tip and an increased sensitivity against the cell wall agents calcofluor and SDS in the AgrhoH strain. These results suggested at least partially overlapping functions between RhoH and Rho1 without excluding any spilt functions of these proteins. Deletion of both the RHOH and RHO1 genes in A. gossypii exacerbated the mutant rho1 phenotype and resulted in germination deficient spores. This suggests that Rho1/RhoH -in contrast to Cdc42- are required at the early steps of spore germination.

Maximal polar growth potential depends on the polarisome component AgSpa2 in the filamentous fungus Ashbya gossypii.

We used actin staining and videomicroscopy to analyze the development from a spore to a young mycelium in the filamentous ascomycete Ashbya gossypii. The development starts with an initial isotropic growth phase followed by the emergence of germ tubes. The initial tip growth speed of 6-10 microm/h increases during early stages of development. This increase is transiently interrupted in response to the establishment of lateral branches or septa. The hyphal tip growth speed finally reaches a maximum of up to 200 micro/h, and the tips of these mature hyphae have the ability to split into two equally fast-growing hyphae. A search for A. gossypii homologs of polarisome components of the yeast Saccharomyces cerevisiae revealed a remarkable size difference between Spa2p of both organisms, with AgSpa2p being double as long as ScSpa2p due to an extended internal domain. AgSpa2 colocalizes with sites of polarized actin. Using time-lapse videomicroscopy, we show that AgSpa2p-GFP polarization is established at sites of branch initiation and then permanently maintained at hyphal tips. Polarization at sites of septation is transient. During apical branching the existing AgSpa2p-GFP polarization is symmetrically divided. To investigate the function of AgSpa2p, we generated two AgSPA2 mutants, a partial deletion of the internal domain alone, and a complete deletion. The mutations had an impact on the maximal hyphal tip growth speed, on the hyphal diameter, and on the branching pattern. We suggest that AgSpa2p is required for the determination of the area of growth at the hyphal tip and that the extended internal domain plays an important role in this process.

Determination of cell polarity in germinated spores and hyphal tips of the filamentous ascomycete Ashbya gossypii requires a rhoGAP homolog.

In the filamentous ascomycete Ashbya gossypii, like in other filamentous fungi onset of growth in dormant spores occurs as an isotropic growth phase generating spherical germ cells. Thereafter, a switch to polarized growth results in the formation of the first hyphal tip. The initial steps of hyphal tip formation in filamentous fungi, therefore, resemble processes taking place prior to and during bud emergence of unicellular yeast-like fungi. We investigated whether phenotypic similarities between these distinct events extended to the molecular level. To this end we isolated and characterized the A. gossypii homolog of the Saccharomyces cerevisiae BEM2 gene which is part of a network of rho-GTPases and their regulators required for bud emergence and bud growth in yeast. Here we show that the AgBem2 protein contains a GAP- (GTPase activating protein) domain for rho-like GTPases at its carboxy terminus, and that this part of AgBem2p is required for complementation of an Agbem2 null strain. Germination of spores resulted in enlarged Agbem2 germ cells that were unable to generate the bipolar branching pattern found in wild-type germ cells. In addition, mutant hyphae were swollen due to defects in polarized cell growth indicated by the delocalized distribution of chitin and cortical actin patches. Surprisingly, the complete loss of cell polarity which lead to spherical hyphal tips was overcome by the establishment of new cell polarities and the formation of multiple new hyphal tips. In conclusion these results and other findings demonstrate that establishment of cell polarity, maintenance of cell polarity, and polarized hyphal growth in filamentous fungi require members of &rgr;-GTPase modules.

Homologous recombination as the main mechanism for DNA integration and cause of rearrangements in the filamentous ascomycete Ashbya gossypii.

A slow and a fast growth phenotype were observed after transformation of the phytopathogenic fungus Ashbya gossypii using a plasmid carrying homologous DNA and as selectable marker the Tn903 aminoglycoside resistance gene expressed from a strong A. gossypii promoter. Transformations with circular plasmids yielded slowly and irregularly growing geneticin-resistant mycelia in which 1% of nuclei contained plasmid sequences. Occasionally, fast growing sectors appeared which were shown to be initiated by homologous integration of the transforming DNA. Transformants obtained with plasmids linearized within the homology region immediately exhibited fast radial growth. In all 28 transformants analyzed plasmid DNA was integrated homologously. Such apparent lack of nonhomologous recombination has so far not been observed in filamentous ascomycetes. In 14 transformants two to four tandemly integrated plasmid copies were found. They underwent several types of genetic changes, mainly in the older mycelium: excision of whole plasmid copies and rearrangements within the integrated DNA (inversions and deletions). These internal rearrangements involved 360-bp inverted repeats, remnants of IS-elements flanking the resistance gene, and 156-bp direct repeats, originating from the strong A. gossypii promoter. Improved vectors lacking sequence repetitions were constructed and used for stable one-step gene replacement in A. gossypii.