Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSbody development and ascospore germination. Here, we present a functional characterization of the secreted a-CA CAS4. CAS4 seems to be involved inammonium metabolism but not in ascospore germination. The Dcas4 mutant displayed a slightly reduced vegetative growth rate and a delayed fruitingbodydevelopment. Based on real time PCR analysis cas4 is upregulated during the sexual development. Moreover, we present the phenotype of aquadruple mutant without any CAS genes. The complete CAS deletion strain (Dcas1/2/3/4) is able to grow under ambient air but the vegetative growthrate is drastically reduced and the mutant is only able to form thin hyphae. The mutant is even under elevated CO 2 levels (5 %) not able to form fruitingbodies. Heterologous expression in Saccharomyces cerevisiae demonstrated that CAS1 and CAS2 are active enzymes, but only CAS1 displays considerablein vitro activity. Furthermore, X-ray and gel filtration analyses revealed a tetrameric structure of CAS1 with a conserved histidine and two cysteine residuesin the active center.Elleuche and Pöggeler 2009: b-Carbonic anhydrases play a role in fruiting body development and ascospore germination in the filamentous fungusSordaria macrospora; PLoS ONE. 2009; 4(4): e5177.133. The Coprinopsis cinerea cag1 (cap-growthless1) gene, whose mutation affects cap growth in fruiting body morphogenesis, encodes the buddingyeast Tup1 homolog. H. Muraguchi, K. Kemuriyama, T. Nagoshi. Dept Biotechnology, Akita Prefectural Univ, Akita, Japan.We have mutagenized a homokaryotic fruiting strain, #326, of Coprinopsis cinerea and isolated a mutant that fails to enlarge the cap tissue on theprimordial shaft in fruiting. Genetic analysis of this mutant, cap-growthless, indicated that the mutant phenotype is brought about by a single gene,designated as cag1. The cag1 locus was mapped on chromosome IX by linkage analysis using RAPD markers mapped to each chromosome. The cag1 genewas identified by transformation experiments using BAC DNAs and their subclones derived from chromosome IX, and found to encode a homolog ofSaccharomyces cerevisiae Tup1. The Coprinopsis genome includes another Tup1 homologous gene, designated Cc.tupA. Expression levels of these twotup1 paralogs were examined using a real-time quantitative PCR method. Cc.tupA is predominantly expressed in vegetative mycelium. In contrast, in thecap tissue, transcript levels of cag1 are similar to that of Cc.tupA. Since it is known that S. cerevisiae Tup1 forms homotetramer, interactions of Cag1 withitself and Cc.TupA were examined using yeast two-hybrid system. Cag1 interacts with itself through the N-terminal region and with Cc.TupA. Like Tup1,which interacts with Cyc8, the N-terminal region of Cag1 also interacts with the N-terminal region of Cc.Cyc8, which contains tetratricopeptide repeats.Based on expression and yeast two-hybrid analyses of Cag1 and Cc.TupA, combined with information on S. cerevisiae Tup1, we speculate that, invegetative mycelium, Cc.TupA represses expression of genes required for cap growth, and Cag1, which might become expressed at the top of primordialshafts to produce the cap tissue and continue to be expressed in the cap tissue, might derepress and activate the expression through interaction withCc.TupA.134. Adaptation of the microtubule cytoskeleton to multinuclearity and chromosome number in hyphae of Ashbya gossypii as revealed by electrontomography. R. Gibeaux 1 , C. Lang 2 , A. Z. Politi 1 , S. L. Jaspersen 3 , P. Philippsen 2 , C. Antony 1 . 1) European Molecular Biology Laboratory, Heidelberg,Germany; 2) Biozentrum, Molecular Microbiology, University of Basel, CH 4056 Basel, Switzerland; 3) Stowers Institute for Medical Research, Kansas City,USA.The filamentous fungus Ashbya gossypii and the yeast Saccharomyces cerevisiae evolved from a common ancestor based on the high level of gene orderconservation. Interestingly, A. gossypii lost the ability of cell divisions and exclusively grows as elongating multinucleated hyphae. Using electrontomography we reconstructed the cytoplasmic microtubule (cMT) cytoskeleton in three tip regions with a total of 13 nuclei and also the nuclearmicrotubules (nMTs) of four mitotic bipolar spindles. Each spindle pole body (SPB) nucleates three cMTs on average, similarly to S. cerevisiae SPBs. 80% ofcMTs were growing as concluded from the structure of their plus-ends. Very long cMTs closely align for several microns along the cortex to generatedynein-dependent pulling forces on nuclei. The majority of nuclei carry duplicated side-by-side SPBs, which together emanate an average of six cMTs, inmost cases in opposite orientation with respect to the hyphal growth axis. Such cMT arrays explain why many nuclei undergo short-range back and forthmovements. Following mitosis, daughter nuclei carry a single SPB. The increased probability that all three cMTs orient in one direction explains the highrate of long-range nuclear bypassing observed in these nuclei. These results demonstrate how cMT arrays, despite a conserved number of microtubules,could successfully adapt to the demands of multinuclearity during evolution from mono-nucleated budding yeast-like cells to multinucleated hyphae. Themodelling of A. gossypii mitotic spindles revealed a very similar structure to mitotic spindles of S. cerevisiae in terms of nMT number, length distributionand three-dimensional organisation even though A. gossypii carries 7 and S. cerevisiae 16 chromosomes per haploid genome. Our results suggest that thenMT cytoskeleton remained largely unaltered during the evolution and that two nMTs attach to each kinetochore in A. gossypii in contrast to only one in S.cerevisiae.135. High resolution proteomics of spores, germlings and hyphae of the phytopathogenic fungus Ashbya gossypii. L. Molzahn 1,2 , A. Schmidt 2 , P.Philippsen 1 . 1) Biozentrum, Molecular Microbiology, University of Basel, CH4056 Basel, Switzerland; 2) Biozentrum, Proteomics Facility, University of Basel,CH4056 Basel, Switzerland.Growth of the filamentous ascomycete A. gossypii is regulated by a genome very similar to the Saccharomyces cerevisiae genome even though thegrowth modes of both organisms differ significantly. During the previous decade progress was made to better understand some of these differences. 1.Cytokinesis in A. gossypii is not coordinated with mitosis and cell separation does not occur due to loss of specific genes which most likely led to theevolution of multinucleated hyphae. 2. Short nuclear cycle times and dynein-dependent pulling forces excerted on nuclei by autonomous cMT arrays withfast growing microtubules maintain a high nuclear density also in fast growing hyphae. 3. Polar growth sites once established support permanent andconstantly accelerating polar surface expansion at hyphal tips at rates of up to 40mm2/min compared to 1mm2/min of yeast buds. Very efficientexocytosis and endocytosis could be documented in hyphal tips of A.gossypii. We want to understand on a system level the differences between bothorganisms and have started a proteomic approach. Total protein extracted from spores and developing A. gossypii hyphae was digested with trypsin,mixed with heavy isotope-labeled reference peptides and subjected to high resolution tandem MS analyses. We could identify 3900 proteins at eachdevelopmental stage. Significant quantitative changes of these proteins with respect to clusters of orthologous groups (COG) or gene ontology (GO) termswere identified during A.gossypii development and between log-phase growing S. cerevisiae cells and fast growing A. gossypii hyphae. Importantdifferences concern ribosome biogenesis and translation, mitochondria biogenesis and respiration, glycolysis and gluconeogenesis, chromatin remodeling,chaperones, cell wall biosynthesis and the first reaction in several biosynthetic pathways.136. Indoor Fungal Growth and Humidity Dynamics. Frank J.J. Segers 1 , Karel A. van Laarhoven 2 , Henk P. Huinink 2 , Olaf Adan 2 , Jan Dijksterhuis 1 . 1) Appliedand Industrial Mycology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, Netherlands; 2) Department of Applied Physics, Eindhoven University ofTechnology, Eindhoven, Netherlands.154