Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSWednesday, March 13 3:00 PM–6:00 PMMerrill HallCell Signaling Involved in Fungal Development and PathogenesisCo-chairs: Naweed Naqvi and Stefanie PöggelerStability of a G protein alpha subunit in genetic backgrounds lacking the G beta subunit or a cytosolic guanine nucleotide exchange factor. Alexander V.Michkov, Katherine A. Borkovich. Plant Pathology and Microbiology, University of California, Riverside, Riverside, CA.Heterotrimeric G proteins consist of alpha, beta and gamma subunits. Regulation is accomplished through the alternation between binding of GDP(inactive form) and GTP (active form) by the alpha subunit and dissociation of the alpha subunit and beta-gamma dimer. GDP/GTP exchange is facilitatedby both cell surface G protein coupled receptors and cytosolic guanine nucleotide exchange factors (GEFs), such as RIC8. Neurospora crassa has three Galpha subunits (GNA-1, GNA-2 and GNA-3), one G beta (GNB-1), and one G gamma (GNG-1). Interestingly, mutants lacking gnb-1 or the cytosolic GEF ric8exhibit some defects in common with the gna-1 deletion mutant, which may be explained by the reduced GNA-1 protein levels observed in these mutants.Previous studies in our laboratory showed that levels of gna-1 mRNA are similar in wild type and mutants lacking gnb-1 or ric8, consistent with a posttranscriptionalmechanism. Using genetic and biochemical approaches, this study investigated the mechanism underlying regulation of GNA-1 stability inregards to GTP/GDP bound state and amount of protein (normal or overexpressed). The results demonstrate that levels of GNA-1 protein are not visiblyreduced over 36 hours in a wild-type background after halting translation using cycloheximide, suggesting GNA-1 is very stable in wild type. To checkstability of GDP or GTP bound GNA-1 in different backgrounds, we transformed mutants lacking the gna-1 gene and gnb-1 or ric8 with a wild type (gna-1 WT ) or constitutively active, GTPase-deficient gna-1 allele (gna-1 Q204L ). Overexpressing gna-1 WT (GDP bound) in a wild-type background increased the levelof GNA-1 protein ~ 3 fold, while overexpression in a gnb-1 mutant gave a nominal increase (~ 1.6x). Overexpressing gna-1 Q204L (GTP bound) in the Dgnb-1or Dric8 backgrounds led to ~ 2 fold higher levels of GNA-1 compared to wild type. In summary, GNA-1 is very stable in wild type, but stability decreasesdramatically in gnb-1 and ric8 deletion mutants. The GTP-bound G alpha protein is more stable in a gnb-1 mutant background than GDP-bound GNA-1protein.The Putative Guanine Nucleotide Exchange Factor RicA Mediates Upstream Signaling for Growth and Development in Aspergillus. Nak-Jung Kwon 1 , HeeSoo Park 2 , Seunho Jung 3 , Sun Chang Kim 4 , Jae-Hyuk Yu 1,2 . 1) Dept Bacteriology, University of Wisconsin, Madison, WI. USA; 2) Molecular and EnvironmentalToxicology Center, University of Wisconsin, Madison, WI, USA,; 3) Department of Bioscience and Biotechnology, and Center for Biotechnology Research inUBITA, Konkuk University, Seoul, Republic of Korea; 4) Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Dae-Jon,Republic of Korea.Heterotrimeric G proteins (G proteins) govern growth, development, and secondary metabolism in various fungi. Here, we characterized ricA, whichencodes a putative GDP/GTP exchange factor for G proteins in the model fungus Aspergillus nidulans and the opportunistic human pathogen Aspergillusfumigatus. In both species, ricA mRNA accumulates during vegetative growth and early developmental phases, but it is not present in spores. The deletionof ricA results in severely impaired colony growth and the total (for A. nidulans) or near (for A. fumigatus) absence of asexual sporulation (conidiation). Theoverexpression (OE) of the A. fumigatus ricA gene (AfricA) restores growth and conidiation in the DAnricA mutant to some extent, indicating partialconservation of RicA function in Aspergillus. A series of double mutant analyses revealed that the removal of RgsA (an RGS protein of the GanB Gasubunit), but not sfgA, flbA, rgsB, or rgsC, restored vegetative growth and conidiation in AnricA. Furthermore, we found that RicA can physically interactwith GanB in yeast and in vitro. Moreover, the presence of two copies or OE of pkaA suppresses the profound defects caused by DAnricA, indicating thatRicA-mediated growth and developmental signaling is primarily through GanB and PkaA in A. nidulans. Despite the lack of conidiation, brlA and vosAmRNAs accumulated to normal levels in the ricA mutant. In addition, mutants overexpressing fluG or brlA (OEfluG or OEbrlA) failed to restore developmentin the AnricA mutant. These findings suggest that the commencement of asexual development requires unknown RicA-mediated signaling input in A.nidulans.The Aspergillus nidulans MAPK module AnSte11-Ste50-Ste7-Fus3 controls development and secondary metabolism. Oezguer Bayram 1* , Oezlem SarikayaBayram 1 , Yasar Luqman Ahmed 2 , Jun-Ichi Maruyama 1,4 , Oliver Valerius 1 , Silvio Rizzoli 3 , Ralf Ficner 2 , Stefan Irniger 1 , Gerhard Braus 1 . 1) Institute ofMicrobiology & Genetics, Department of Molecular Microbiology and Genetics, Georg-August-Universität, Grisebachstr. 8, D 37077 Goettingen, Germany;2) Department of Molecular Structural Biology, Institute for Microbiology and Genetics, Georg-August-Universität, Goettingen; 3) European NeuroscienceInstitute, Deutsche Forschungsgemeinschaft Center for Molecular Physiology of the Brain/Excellence Cluster 171, 37077 Göttingen; 4) Department ofBiotechnology, The University of Tokyo, Tokyo, Japan.The sexual Fus3 MAP kinase module of yeast is highly conserved in eukaryotes and transmits external signals from the plasma membrane to the nucleus.We show here that the module of the filamentous fungus Aspergillus nidulans (An) consists of the AnFus3 MAP kinase, the upstream kinases AnSte7 andAnSte11, and the AnSte50 adaptor. The fungal MAPK module controls the coordination of fungal development and secondary metabolite production. Itlacks the membrane docking yeast Ste5 scaffold homolog but similar to yeast the entire MAPK module interacts with each other at the plasma membrane.AnFus3 is the only subunit with the potential to enter the nucleus from the nuclear envelope. AnFus3 interacts with the conserved nuclear transcriptionfactor AnSte12 to initiate sexual development and phosphorylates VeA which is a major regulatory protein required for sexual development andcoordinated secondary metabolite production. Our data suggest that not only Fus3 but even the entire MAPK module complex of four physicallyinteracting proteins can migrate from plasma membrane to nuclear envelope.27th Fungal Genetics Conference | 29