Figure 6. BUD-6 accumulation during conidiogenesis. (A) BUD- 6-GFP accumulated at septation sites in developing macroconidio- phores (arrowhead). Scale bars, 10 mm. (B) In cytologically separated, but physically still attached conidia, BUD-6 fluorescence persisted at the cell poles; either at both or only at one pole in case of the terminal conidium. Scale bars, 10 mm. (C) In addition to strong fluorescence at the cell poles, bright clusters of BUD-6-GFP also accumulated at other locations of the cell cortex. Scale bar, 5 mm. doi:10.1371/journal.pone.0030372.g006 terminal conidium (Figure 6B). Strongly fluorescent clusters of BUD-6-GFP of unknown function were also distributed at the cell cortex of attached macroconidia at this stage (Figure 6C). With progressing age of the developing colony the percentage of conidia retaining cortical BUD-6 clusters decreased (Figure S3). Table 2. N. crassa strains used in this study. Neurospora Crassa Polarisome Architecture Characterization of Dbud-6 Homokaryotic Dbud-6 strains were successfully generated through backcrossing of the heterokaryotic Dbud-6 mat a gene deletion strain obtained from the FGSC with a Dsad-2 mat A strain incapable of meiotic silencing (Table 2), and with the selection of ascospore progeny on hygromycin B medium. For 15 out of the 20 selected clones (75%), the phenotypic change cosegregated with hygromycin B resistance, indicating that the resulting mutant phenotype was caused by the targeted gene deletion and not due to unintended rearrangements introduced during sexual repro- duction elsewhere in the genome. Absence of the native bud-6 locus from the genome was confirmed by PCR (Figure S1). The homokaryon selection procedure using crossing has been repeated once, and confirmed by vegetative selection of single conidia. Both selection strategies resulted in hygromycin B resistant colonies with identical mutant phenotypes. BUD-6 was required for normal colony development, maintenance of hyphal morphology, septum formation and conidiogenesis Due to multiple morphological defects, loss of BUD-6 resulted in mutant strains unable to establish normal colony architecture (Figure 7A and B). Defects included, hyperbranching, lack of hyphal differentiation - including primary and secondary branches and fusion hyphae - lack of septa, and incomplete conidiogenesis. In comparison to the wild type, branching frequency was more than doubled in Dbud-6 mutants (Figure 7C), and radial colony extension was decreased by a factor of 10 or more (Figure 7D), resulting in very dense, slow growing and aconidiate colonies. FM4–64 staining of Dbud-6 hyphae confirmed the absence of septa and revealed another highly interesting phenotype; the absence of the Spk (Figures 8B–E). Despite the apparent loss of organized assembly of the polarized tip growth apparatus, evident e.g. by the lack of a nuclear exclusion zone (Figure 8D), which normally appears distal to the Spk in the wild type (Figure 8C) [60], or frequent apical branching (Figure 8E), polarized extension of hyphae, however, was still possible. In contrast to most other membranes, such as the plasma membrane, endocytic vesicles or vacuolar membranes, application of the lipophilic dyes FM1–43 or FM4–64 does not stain the nuclear envelope in N. crassa [54,61]. Therefore, nuclei appear negatively stained as black subspherical Strain Wild type N150 Dmus51 Dbud-6 (heterokaryon) Dbni-1 (heterokaryon) Dsad-2 Dbni-1 Dspa-2 Dspa-2 NCLAP5-1 NECL46-9 NECL47-5 NECL48-8 bni-1-sgfp doi:10.1371/journal.pone.0030372.t002 Genotype wt; mat A Dmus-51::bar+; his23; mat A Dbud-6::hph+, Dmus-51:: bar+bud-6+mus51; mat a Dsad-2::hph+, mat A Dbni-1::hph, Dmus-51:: bar+bni-1+mus51; mat a Dbni-1::hph+, Dmus-51:: bar+, mat a Dspa-2::hph+, mat A Dspa-2::hph+, mat a his23+Pccg-1::spa-2-sgfp; mat A Dmus-51::bar+; his-3+::Pccg-1::bud-6-sgfp; mat A Dmus-51::bar+; his-3+::Pccg-1::bud-6-mchfp; mat A Dbud-6::hph+, mat a Dbni-1::hph+, his-3+::Pccg-1::bni-1-sgfp; mat a Source FGSC # 9013 FGSC # 9717 FGSC # 11424 Shiu et al., 2006 FGSC # 11490 This study FGSC # 11140 FGSC # 11141 Araujo-Palomares et al., 2009 This study This study This study Justa-Schuch et al., 2010 PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e30372