Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSSaturday, March 16 2:00 PM–5:00 PMScrippsTropic Growth and FusionCo-chairs: Andre Fleissner and Nick ReadRole of the cell fusion gene idcA in fungal mutualism. Carla J. Eaton 1,2 , Cornelia Staerkel 1 , Barry Scott 1,2 . 1) Institute of Molecular BioSciences, MasseyUniversity, Palmerston North, New Zealand; 2) Bio-Protection Research Centre, Massey University, Palmerston North, New Zealand.Maintenance of the mutualistic association between the fungal endophyte Epichloë festucae and perennial ryegrass relies on a number of importantsignalling pathways including ROS signalling by the NADPH oxidase (Nox) complex, and the cell integrity- and stress-activated MAP kinase pathways.Perturbation of these signalling pathways leads to a dramatic switch from mutualistic to pathogenic-like association with perennial ryegrass. Interestingly,all E. festucae ‘symbiotic regulator’ genes identified to date are involved in vegetative cell fusion in other fungi, suggesting hyphal fusion may play animportant role in maintenance of the mutualistic association. To investigate this putative link, the role of the cell fusion gene IDC1 (ham-5) was examined.IDC1 is of particular interest as in addition to its role in cell fusion it is also linked to Nox signalling in Podospora anserina. Disruption of E. festucae idcAleads to a dramatic symbiotic switch from mutualistic to pathogenic-like association with perennial ryegrass. Infected plants are severely stunted anddisplay precocious senescence. Biomass of the DidcA mutant in planta is significantly increased relative to the wild-type strain, and hyphae extensivelycolonise host vascular tissues. Formation of intra-hyphal hyphae by the DidcA mutant in planta is also abundant, possibly due to defective hyphal fusion ordefects in septation. The importance of idcA for maintenance of mutualistic association with perennial ryegrass supports the hypothesis that hyphal fusionis required for establishment of an interconnected hyphal network essential for mutualism.Role of extracellular calcium in budding yeast cell fusion. Pablo S. Aguilar. Cell Membranes Laboratory, Institut Pasteur de Montevideo, Montevideo,Uruguay.The molecular details of membrane fusion during yeast mating are poorly understood. The tetraspanner protein Prm1 is one of the few knowncomponents that acts at the step of bilayer fusion. In its absence, mutant mating pairs lyse or arrest in the mating reaction with tightly apposed plasmamembranes. The absence of another tetraspanner, Fig1p, which controls pheromone-induced Ca 2+ influx, yields similar cell fusion defects. Althoughextracellular Ca 2+ is not required for efficient cell fusion of wild-type cells, cell fusion in prm1 mutant mating pairs is dramatically reduced when Ca 2+ isremoved. A genetic screen was conducted to uncover genes that promote mating-dependent lysis in the absence of extracelular Ca 2+ . The role of differentcandidates in relation to Prm1p will be reviewed here.The role of calcium and calmodulin during cell fusion and colony initiation in Neurospora crassa. Chia-Chen Chang, Nick Read. Fungal Cell Biology Group,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+ .LFD-1 is a component of the membrane merger machinery during cell-cell fusion in Neurospora crassa. Javier Palma-Guerrero, N. Louise Glass. Plant andMicrobial Biology Department, UC Berkeley, Berkeley, CA.Cell-cell fusion is an essential part of the development of most eukaryotic organisms, playing an important role both during sexual development andvegetative growth. In the filamentous fungus Neurospora crassa, cell fusion events occur during all stages of the life cycle. This, together with thesequenced and annotated genome and the genetic tools available, makes this fungus a good model system for dissecting cell fusion. Plasma membranemerger is the last step in the cell-cell fusion process, occurring after cell wall remodeling, and it is the definitive event that allows for cytoplasm mixingbetween the fusing cells. Although molecular mechanisms associated with intracellular membrane fusion are well characterized, the molecularmechanisms of plasma membrane merger between cells are poorly understood. Only one gene encoding a protein involved in this last step of cell fusionhad been previously identified in N. crassa: Prm-1, a deletion of which results in strains that show a »50% reduction in vegetative and sexual cell fusion.We have identified a second gene, lfd-1, which is also involved in plasma membrane merger in N. crassa. LFD-1 is a plasma membrane protein only presentin ascomycete filamentous fungi. N. crassa strains carrying a deletion of lfd-1 results in a reduction in both vegetative and sexual cell fusion, and having asimilar, but less severe, phenotype than a Prm-1 deletion strain. Strains carrying both Prm-1 and lfd-1 deletions indicate that LFD-1 acts independently ofPRM-1. Strains carrying Prm-1 or lfd-1 mutations result in increased cell lysis during cell-cell fusion, a phenotype that was enhanced by reducingextracellular calcium concentration. These results suggest that the lysis phenotype associated with cell fusion events is due to membrane damage causedby defects during membrane merger, and which may be repaired in a calcium dependent process. Our results indicate that both PRM-1 and LFD-1 areimportant, but non-essential components of the cell fusion membrane merger machinery.100