Neurospora Crassa Polarisome Architecture Figure 8. BUD-6 was required to organize the polarized growth apparatus at the hyphal tip. (A and B) Comparison of FM4–64 staining pattern of mature hyphae at the leading edge of the colonies in the wild type and Dbud-6 mutant confirmed the absence of septa in the mutant (arrows in A indicate septa in the wt), as well as the absence of the Spk at the hyphal tips of Dbud-6 (arrowheads in A point toward wt Spk). Scale bars, 5 mm. (C and D) Close up of the apical and subapical area of polarized growing mature hyphae of wt and Dbud-6. The arrowheads in C indicate the Spk, which shows up as a dark sphere in the phase contrast image and was brightly stained by FM4–64. No such structure was observed in hyphae of the Dbud-6 mutant. The squared bracket marks the subapical nuclear exclusion zone in the wild type, which is not established in the Dbud-6 mutant. Here, nuclei (arrows) reach further up into the hyphal tip (also seen in E). Scale bars, 5 mm. (E) Apical branching and lack of hyphal tip organization in Dbud-6. Scale bar, 5 mm. (F) Immature and malformed conidiophores in Dbud-6. Scale bar, 10 mm. doi:10.1371/journal.pone.0030372.g008 repeated isolation of monosporic microcolonies on selection medium. This method quickly, i.e. within one generation, resulted in strains with strong phenotypic defects very similar to those observed for Dbud-6 (Figure 9). Phenotypic alterations of the homokaryon-selected strains included, apical branching, the lack of hyphal differentiation, and the absence of septa, conidiophores and consequently conidia. PCR genotyping confirmed the correct exchange of the native gene locus against the knock-out cassette, as well as absence of bni-1 from the whole genome (Figure S1B). The strain purification and verification process was reproducible, suggesting that the observed phenotype was indeed the result of the exclusive deletion of the bni-1 locus, and not due to random mutations or other unintended alterations within the genomes of these strains. In line with findings in the Dbud-6 strain, FM4–64 staining of hyphae at the leading edge of Dbni-1 colonies confirmed the lack of septa, and most notably also included the lack of any functional organization of the tip apex; evident by the absence of the Spk and PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e30372