Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSthese results show a novel function for mannitol in fungal growth and sexual development.208. A small lipopeptide pheromone with limited proline substitutions can still be active. Thomas J. Fowler, Stephanie L. Link. Department of BiologicalSciences, Southern Illinois University Edwardsville, Edwardsville, IL.Mating in many fungi involves communication with lipopeptide pheromones. These signaling molecules activate G protein-coupled receptors located onthe surface of a compatible mating partner and initiate a mating response. Some of the mushroom fungi code for scores of different lipopeptidepheromones among the mating types. Despite their small predicted size of approximately eleven amino acids, these pheromones have very specificpheromone receptor targets for mate discrimination. In past heterologous expression and mating studies in Saccharomyces cerevisiae, we have maderandom amino acid substitutions in one pheromone, Bbp2(4), from the mushroom fungus Schizophyllum commune, and site-directed mutations in aclosely related pheromone, Bbp2(7). These studies indicated that the peptide portion of the pheromones can be more variable than expected. Within arandom mutagenesis study of Bbp2(4), it was noted that the imino acid proline could be substituted for several of the natural residues and an activemutant pheromone was still produced. In this study, the heterologous mating assay was employed to test the extent that proline residues might besubstituted into a pheromone before activity was no longer detected. Mature Bbp2(4) is predicted to be eleven amino acids with a farnesyl tail(DSPDGYFGGYC-farnesyl). Single substitutions of proline at several non-natural positions did not stop production of active pheromone, but substitutionswith proline at several previously identified critical amino acid positions led to negative results in the mating assays. Among the substitutions that do notdisrupt all activity are DSPPGYFGGYC-farnesyl and DSPDGYFGPYC-farnesyl. The three-dimensional conformations of proline-substituted peptides insolution were predicted with PEP-FOLD and viewed with JMOL. The conformational differences of small pheromones tolerated by one receptor aresurprising. Substitution of two or more prolines at adjacent non-natural positions in a single pheromone does inhibit production of an active pheromone inthe heterologous mating assay. At present, it cannot be determined if multiple proline substitutions inhibit pheromone processing, pheromone transport,or interaction with the receptor.209. Function of Ras proteins in fungal morphogenesis of Schizophyllum commune. E.-M. Jung, N. Knabe, E. Kothe. Department of Microbiology, FriedrichSchiller University, Jena, Germany.The white rot basidiomycete Schizophyllum commune has been used as a model organism to study mating and sexual development as well as analysis ofcell development. Subsequent to nutrient and pheromone recognition, intracellular signal transduction was regulated by different pathways and MAPKsignalling cascades. The S. commune genome encodes more than 30 putative signal transduction proteins of the Ras superfamily containing the Ras, Rho,Rab, Ran and Arf subfamilies. Phylogenetic investigation of Ras proteins from various basidiomycetes show that they cluster in two main groups. Highsequence similarities between these proteins in basidiomycetes suggesting an ancient duplication event. To investigate the function of the small G-proteins Ras1 and Ras2 mutants with constitutively active ras1 alleles as well as a DRasGap1 mutant were analyzed. They show phenotypes withdisorientated growth pattern, reduced growth rates and hyperbranching effects. The fungal cytoskeleton, composed of actin and microtubules has beeninvestigated by immunofluorescence microscopy to reveal whether Ras signaling influences the formation of cytoskeleton. The second Ras protein, Ras2,was detected by genome analysis. Its function is analysed in current studies.210. The developmental PRO40/SOFT protein participates in signaling via the MIK1/MEK1/MAK1 module in Sordaria macrospora. Ines Teichert 1 , EvaSteffens 1 , Nicole Schnab 1 , Benjamin Fränzel 2 , Christoph Krisp 2 , Dirk A. Wolters 2 , Ulrich Kück 1 . 1) General & Molecular Botany, Ruhr University Bochum,Bochum, Germany; 2) Analytical Chemistry, Ruhr University Bochum, Bochum, Germany.Filamentous fungi are able to differentiate multicellular structures like conidiophores and fruiting bodies. Using the homothallic ascomycete Sordariamacrospora as a model system, we have identified a number of developmental proteins essential for perithecium formation. One is PRO40 [1], thehomolog of Neurospora crassa SOFT, and this protein was employed for protein-protein interaction studies to gain insights into its molecular function.Data from yeast two hybrid experiments with PRO40 as bait show an interaction of PRO40 with the MAP kinase kinase (MAPKK) MEK1. MEK1 is a memberof the cell wall integrity (CWI) pathway, one of three MAP kinase modules present in S. macrospora. The S. macrospora CWI pathway consists of MAPkinase kinase kinase (MAPKKK) MIK1, MAPKK MEK1 and MAP kinase (MAPK) MAK1, with additional upstream components, protein kinase C (PKC1) andRHO GTPase RHO1. Data from tandem affinity purification - MS experiments with PRO40 and MEK1 as bait indicate that PRO40 forms a complex withcomponents of the CWI pathway. Analysis of single and double knockout mutants shows that PRO40, MIK1, MEK1 and MAK1 are involved in the transitionfrom protoperithecia to perithecia, hyphal fusion, vegetative growth, and cell wall stress response. Differential phosphorylation of MAPKs in a pro40knockout strain was detected by Western analysis. We propose that PRO40 modulates signaling through the CWI module in a development-dependentmanner. Further interaction studies and complementation analyses with PRO40 derivatives provide mechanistic insight into the function of PRO40domains during fungal development. [1] Engh et al. (2007) Eukaryot Cell 6:831-843.211. Map-based identification of the mad photosensing genes of Phycomyces blakesleeanus. Silvia Polaino Orts 1 , Suman Chaudhary 1 , Viplendra Shakya 1 ,Alejandro Miralles-Durán 2 , Luis Corrochano 2 , Alexander Idnurm 1 . 1) Cell Biology & Biophysics, University of Missouri-KC, Kansas City, MO; 2) Departamentode Genética, Universidad de Sevilla, Spain.Phycomyces blakesleeanus is a filamentous fungus, a member of the subphylum Mucoromycotina. The main reason for the presence of Phycomyces inlaboratories is its sensitivity to light. The fruiting bodies phototropism of Phycomyces has served as a model of response to blue light in fungi. In 1967, inthe laboratory of Nobel laureate Max Delbrück, the first sensory mutants were isolated. Analysis on these strains has enabled a proposed sensorytransduction pathway that describes the flow of information from the sensors to the effectors. There are ten mutants, called mad mutants, divided intotwo classes: those of type 1 are madA, madB, madC and madI, which are altered only in photoresponses but not in others tropisms of the sporangiophore.The mutants in the madA and madB genes are altered in all photoresponses (phototropism, photomorphogenesis, photocarotenogenesis andphotomecism). These two mad genes are the only ones that have been identified and their corresponding proteins interact to form the Mad complex, themain photoreceptor complex of Phycomyces. The mutants altered in the madC gene are only affected in the phototropism. The remaining mad mutantsare called type 2 and are altered in the phototropism and other responses of the sporangiophore, like gravitropism and avoidance. Phycomyces cannot bestability transformed with DNA. To identify the eight unknown mad mutants, a positional cloning approach was taken coupled to Illumina sequenceinformation. A genetic map was constructed between two wild type parents, and then mad mutants crossed to one of these parents. Through mapping,we have identified candidates for the madC, madD, madJ, madF and madI genes, with greatest follow up characterization in madC. The madC geneencodes a Ras GTPase-activating protein, implicating Ras in the light signal transduction pathway in fungi.172