Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSof superoxide by transferring electrons from NADPH to oxygen. Neurospora crassa contains the NADPH oxidases NOX-1 and NOX-2 and a commonregulatory subunit NOR-1. NOX-2 is essential for ascospore germination, while NOX-1 is required for sexual and asexual development, polar growth andcell fusion. NOR-1 is essential for all these NOX functions. We have found that a functional NOR-1::GFP fusion is localized throughout the cytoplasm,enriched at the hyphal tip and sometimes in aggregates. This suggests that the functional NOX complexes are probably not localized at the plasmamembrane. Up to now NOX function in fungi has been evaluated in mutants that completely lack NOX proteins. We generated nox-1 alleles that result inNOX-1 proteins carrying substitutions of proline 382 by histidine or cysteine 542 by arginine, which affect NADPH-binding. Equivalent mutations inphagocytic Nox2/gp91phox do not affect protein stability but completely lack oxidase activity. P382H and C542R mutants did not produce sexual fruitingbodies and showed a decreased growth and differentiation of aerial mycelia, without affecting production of conida. These results indicate that sexualdevelopment depends on ROS production by NOX-1, whereas during asexual differentiation NOX-1 plays an important role independently of its catalyticactivity. Dnox-1, Dnor-1, P382H NOX-1 and C542R NOX-1 mutants were all able to produce some conidial anastomosis tubes (CATs) but they were unableto complete cell-cell fusion. All these mutants are also impaired in vegetative hyphae-hyphae fusion, which might explain the growth defects in Dnox-1 andDnor-1 strains. CATs production is delayed in the presence of antioxidant N- acetyl cystein (NAC) and Dsod-1 strains show an increase in CATs fusions. Theresults suggest that some ROS may be implicated in signaling CATs homing and vegetative fusion.181. DYNAMICS OF THE PROTEINS BUD-2 AND BUD-5 DURING CELL POLARIZATION IN NEUROSPORA CRASSA. E. Castro-Longoria, C. Araujo-Palomares, N.Cano-Domínguez. Microbiology Department, CICESE. Carretera Ensenada-Tijuana No 3918 Zona Playitas, C.P. 22860. Ensenada, B.C. México.Polarized growth in filamentous fungus requires an excellent and precise machinery to select specific sites where multiple protein complexes assembleto ensure the generation of highly polarized hyphae. One of these protein complexes is the Rsr1p/Bud1p-Bud2p-Bud5p module, which, in Saccharomycescerevisiae, has the function of selecting the proper site of budding. However, in filamentous fungi the function of this module is unknown. In this study, wecharacterized the intracellular localization and dynamics of protein homologues for BUD-2 and BUD-5 in the filamentous fungus Neurospora crassa.Preliminary results of in vivo confocal microscopy analysis shows that both BUD-2 and BUD-5 display distinct localization patterns in both mature hyphaeand germlings. In mature hyphae, BUD-2 localization is confined to the apical cytosol, occupying the core of the Spitzenkörper (Spk), while BUD-5 wasobserved in the apical region of the cells as a bright spot with higher intensity at the center base adopting a hand fan shape, partially colocalizing with theSpk. In contrast, BUD-2 in germlings was associated with the cell membrane and organized as a cap shape covering the apex of the cells, while BUD-5localization was observed in three different ways: as a bright spot at the apex of germinating spores, then as a cytosolic crescent-shape in longer germtubes and finally adopting a similar localization pattern as in mature hyphae. BUD-2 and BUD-5 also display distinct localization patterns during branchingand septum formation. BUD-2 participates in septum formation while BUD-5 was only involved during the initiation of lateral branches. The distinctcellular localization patterns of BUD-2 and BUD-5 suggest that although both proteins may be important for cell polarity establishment, they alsoparticipate in other morphogenetic processes in N. crassa.182. The role of calcium and calmodulin during cell fusion and colony initiation in Neurospora crassa. Chia-Chen Chang, Nick Read. Fungal Cell BiologyGroup, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3JH.Calcium is an ubiquitous signalling molecule which regulates many important processes in filamentous fungi including spore germination, hyphal growth,mechanosensing, stress responses, circadian rhythms, and virulence. Transient increases in cytosolic free calcium ([Ca 2+ ] c) act as intracellular signals. As theprimary intracellular Ca 2+ receptor, calmodulin (CaM) converts these Ca 2+ signals into responses by regulating the activity of numerous target proteins. Wehave found that both Ca 2+ -free medium and two CaM antagonists (calmidazolium and trifluoperazine) selectively inhibit a form of cell fusion called conidialanastomosis tube (CAT) fusion that occurs during colony initiation in the fungal model Neurospora crassa. GFP labelled CaM localized as dynamic particlesassociated with the plasma membrane and moved around within the cytoplasm in both germ tubes and CATs. In particular, CaM showed a dynamicaccumulation at two growing tips of CATs that exhibit chemoattraction towards each other. CaM also localized at developing septa in germ tubes. The b-tubulin inhibitor, benomyl, reduced the movement of CaM in the cytoplasm. Moreover, the absence of extracellular Ca 2+ inhibited the recruitment of CaMto CAT tips as well as inhibiting CAT chemoattraction. The deletion of the myosin-5 (myo-5) gene caused the mis-localization of CaM in tips of growinggerm tube and CATs. This suggests that the movement of cytoplasmic CaM involves transport along microtubules, and the recruitment of CaM to tipsinvolves myosin-5 along F-actin and is dependent on extracellular Ca 2+ .183. Deletion of cAMP phosphodiesterase pde-2/acon-2 gene causes the enhanced osmotic sensitivity in os-1 and os-2 mutants of N. crassa. C. Kurata,M. Kamei, S. Banno, M. Fujimura. Dept Life Sci, Toyo Univ, Gunma, Japan.N. crassa has two putative cyclic nucleotide phosphodiesterases, PDE-1 (NCU00237) and PDE-2/ACON-2 (NCU00478). The pde-2 disruptants showed thenormal mycelial growth but lacked the ability to produce conidia, these phenotypes resembled those of the hah mutant which has a point mutation in thePKA (protein kinase A) regulatory subunit gene. The phenotypes of double mutants, pde-2;pkac-1 and hah;pkac-1 mutants, resembled those of the pkac-1mutant which shows slow growth and hyperconidiation. In contrast, hyperconidiation of the adenylyl cyclase cr-1 mutant was suppressed by the hahmutation but not by the pde-2 mutation. These results indicate that PDE-2 act as a major cAMP phosphodiesterase in cAMP-PKA pathway, its deletionleads to the hyper-activation of this pathway. Any mutants in cAMP-PKA pathway including pde-2 and hah mutants, did not show osmotic sensitivity.However, both pde-2 and hah mutations caused the enhanced osmotic sensitivity in os-1 (histidine kinase) and os-2 (MAP kinase) mutants, suggesting ofcross-talk between cAMP-PKA pathway and OS-2 MAP kinase pathway.184. Genetic analysis of GNB-1 and CPC-2 with the G alpha subunits in Heterotrimeric G protein signaling in Neurospora crassa. AMruta Garud. PlantPathology, UC, Riverside, Riverside, CA.Heterotrimeric G protein signaling is mediated by Gabg subunits. Neurospora crassa has three Ga subunits (GNA-1, GNA-2 and GNA-3), one Gb (GNB-1)and one Gg (GNG-1). The GNB-1 protein contains seven tryptophan-aspartate (WD) repeats, suggesting it assumes a beta propeller form. Genetic epistasishas been demonstrated between gnb-1 and the three Ga subunit genes. gna-3 is epistatic to gnb-1 for submerged culture conidiation, while gna-1 andgna-2 are epistatic to gnb-1 during aerial conidiation. In contrast, gnb-1 is epistatic to gna-2 and gna-3 during aerial hyphae development. Additionalproteins that have a 7-WD repeat structure have been implicated as Gb subunits in other fungi. The Cross Pathway Control (CPC-2) protein has a seven WDrepeat structure, and shares 70% similarity to the mammalian protein RACK-1. In Neurospora, CPC-2 was previously shown to play a role in general aminoacid control. Genetic epistasis with CPC-2 and the Ga proteins is being studied, using strains lacking cpc-2 and one Ga gene, as well as cpc-2 deletionmutants carrying constitutively activated, GTPase-deficient Ga alleles. It is seen that gna-3 is epistatic to cpc-2 during apical extension, aerial hyphae heightand asexual sporulation in submerged cultures. gna-1 and gna-2 demonstrate some functional independence. Yeast two hybrid assays show that CPC-2interacts with GNA-1 and GNA-3. Additional interactions are being examined using additional in vivo and in vitro methods to validate whether CPC-2 acts27th Fungal Genetics Conference | 165