title: The phenotype of a phospholipase C (plc-1) mutant in a filamentous fungus, Neurospora crassa.
abstract: In the filamentous fungus Neurospora crassa, phospholipase C may play a role in hyphal extension at the growing tips as part of a growth-sensing mechanism that activates calcium release from internal stores to mediate continued expansion of the hyphal tip. One candidate for a tip-localized phospholipase C is PLC-1. We characterized morphology and growth characteristics of a knockout mutant (KO plc-1) and a RIP mutated strain (RIP plc-1) (missense mutations and a nonsense mutation render the gene product non-functional). Growth and hyphal cytology of wildtype and KO plc-1 were similar, but the RIP plc-1 mutant grew slower and exhibited abnormal membrane structures at the hyphal tip, imaged using the fluorescence dye FM4-64. To test for causes of the slower growth of the RIP plc-1 mutant, we examined its physiological poise compared to wildtype and the KO plc-1 mutant. The electrical properties of all three strains and the electrogenic contribution of the plasma membrane H(+)-ATPase (identified by cyanide inhibition) were the same. Responses to high osmolarity were also similar. However, the RIP plc-1 mutant had a significantly lower turgor, a possible cause of its slower growth. While growth of all three strains was inhibited by the phospholipase C inhibitor 3-nitrocoumarin, the RIP plc-1 mutant did not exhibit hyphal bursting after addition of the inhibitor, observed in both wildtype and the KO plc-1 mutant. Although the plc-1 gene is not obligatory for tip growth, the phenotype of the RIP plc-1 mutant - abnormal tip cytology, lower turgor and resistance to inhibitor-induced hyphal bursting - suggest it does play a role in tip growth. The expression of a dysfunctional plc-1 gene may cause a shift to alternative mechanism(s) of growth sensing in hyphal extension.
authors.affiliation: York University, Biology Department, 4700 Keele Street, Toronto, ON M3J 1P3, Canada. Electronic address: planters@yorku.ca.
authors.forename: Roger R
authors.initials: RR
authors.lastname: Lew
authors.affiliation: York University, Biology Department, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Giblon
authors.affiliation: York University, Biology Department, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.
authors.forename: Miranda S H
authors.initials: MS
authors.lastname: Lorenti
mesh_terms: D002118:Calcium / Q000378:metabolism; D055724:Electrophysiological Phenomena; D004791:Enzyme Inhibitors / Q000494:pharmacology; D055786:Gene Knockout Techniques; D056726:Genetic Association Studies; D005838:Genotype; D025301:Hyphae; D009154:Mutation; D009492:Neurospora crassa / Q000166:cytology / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D010641:Phenotype; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D010738:Type C Phospholipases; D002118:Calcium
keywords: Electrophysiology; Fluorescence imaging; Hyphal tip growth; Neurospora crassa; Phospholipase C; Turgor