Neurospora Crassa Polarisome Architecture Figure 7. Loss of BUD-6 resulted in a reduced colony extension rate and hyperbranching. (A) Hyperbranching and polar extension defects resulted in very slowly and extremely dense developing mycelial colonies of Dbud-6, in comparison to the wild type after 24 hours of incubation. (B) Hyphal morphology of wild type and Dbud-6 at the colony margin. Scale bars, 0.5 mm and 0.25 mm, respectively. (C) Quantification of branching frequency, which on average was more than doubled in the mutant compared to the wild type. (D) Comparison of average colony extension speed between Dbud-6 and wild type. doi:10.1371/journal.pone.0030372.g007 objects without a fluorescent border in the otherwise highly fluorescent membranous background of the cytoplasm. In some instances, conidiophore-like aerial hyphae could be observed in older parts of the mycelium which attempted to differentiate conidia through constriction, but due to the apparent block in cytokinesis were never completed (Figure 8F). Taken together, the phenotypic defects mirrored the multifunctional role of BUD-6 implicated by its subcellular localization: in apical caps in growing germ tubes, CATs and regenerating tips; as subapical cloud in mature hyphal tips and branches; at forming septa and persistent at septal pores, as cytokinetic rings during conidiogenesis; and at sites of VHF. Characterization of Dbni-1 A key effector of polarisome activity in yeast is the formin Bni1, which stimulates F-actin nucleation in a Bud6-dependent manner [32,41]. To investigate the functional relationship of the only formin homolog identified in Neurospora, BNI-1, to the filamentous fungal polarisome, we sought to characterize the corresponding gene deletion mutant (Table 2), and analyze the subcellular dynamics of BNI-1-GFP [62] in greater detail, and in relation to SPA-2 and BUD-6. Deletion of the formin gene bni-1 phenocopied Dbud-6 to a large extent Genetic deletion of bni-1 has been reported to be lethal because viable ascospores could not be recovered from sexual backcrosses between the Dbni-1 heterokaryon (FGSC11490) and the wild type [62]. In an alternative approach, we attempted to remove wild type nuclei carrying the native copy of bni-1 from the heterokaryotic gene deletion strain vegetatively, i.e. through PLoS ONE | www.plosone.org 9 January 2012 | Volume 7 | Issue 1 | e30372