The peripheral fusion phenotype of Dbni-1 and Dbud-6 might be founded in the disruption of the apical tip growth apparatus. Apical dominance suppresses both the formation of branches at the leading edge of the mycelium [85], and prevents the development of fusion hyphae at the colony periphery [63]. Apical dominance was not clearly evident in the Dbni-1 and Dbud-6 mutants in which vegetative hyphae were much less differentiated and lacked distinct Spk. In both mutants this breakdown in apical dominance coincided with derepressed VHF at the colony edge. In the wild type, this repression of VHF could intriguingly be overcome in the presence of conidial germlings by some unknown mechanism. The physiological role of suppressed hyphal fusion must be the promotion of colony growth. Fusion at the leading edge would slow down colony extension and substrate exploration, and consequently lead to significantly delayed colony development that exactly matches the phenotypes of the two mutant mycelia. Viable Bni1 mutants have been generated in A. gossypii and C. albicans, and failed to form hyphae or develop abnormally swollen hyphae, respectively [86,87]. A conditional SepA mutant of A. nidulans was found to be aseptate, exhibited an aberrant growth pattern and developed abnormally wide hyphae, indicating defects in targeted exocytosis [74,88]. These morphogenetic alterations are in line with the severe phenotype of the Dbni-1 and Dbud-6 mutants of N. crassa. In budding yeast it has been observed that all four polarisome components (Spa2, Pea2, Aip3/Bud6 and Bni1), as well as the cell fusion proteins Fus1 and Fus2 are required to terminate mating projection growth prior to cell fusion (reviewed in [35]). Likewise, the termination of polarized tip growth of CATs and vegetative fusion hyphae is a prerequisite for successful cell fusion. A shared phenotype amongst fusion mutants of N. crassa is continued tip growth of fusion hyphae upon physical contact with other hyphae [89,90]. Spa2D cells of budding yeast showed a mating cell fusion defect similar to fus1D and fus2D [91]. To our knowledge, whether or not Bud6 is required for mating cell fusion has so far not been reported in the yeast literature. Fus1 has been found to regulate the opening and expansion of the fusion pore between mating yeasts [92]. As Fus orthologs do not exist in N. crassa it is tempting to speculate that some polarisome components might have this role in filamentous fungi. Clearly, more detailed functional studies are required to test this idea. The complex filamentous fungal polarisome In summary, this investigation has shown that filamentous fungal polarisome components show higher functional diversity than their homologs in yeasts. To elucidate further molecular details of the filamentous fungal polarisome it appears reasonable to focus the attention on polarisome-dependent processes, i.e. exclude septation and cytokinesis in this context. The complex arrangement of the polarisome in mature hyphal tips, comprising the subapical BUD-6 cloud, BNI-1 clusters within the Spk core and the apical cap, and SPA-2 partially overlapping with the Spk but also fanning outwards into the apical dome, is a novel architectural feature of the filamentous tip growth apparatus that deserves more detailed investigation. Alongside the search for novel components of the filamentous fungal polarisome, under- standing the individual functions of the three known proteins in greater detail, and especially in interaction with the actin cytoskeleton, poses interesting challenges for the future. Materials and Methods Strains and culture conditions Strains of Neurospora crassa used in this study are listed in Table 2. Unless otherwise stated, strains were cultured at 28uC on Vogel’s Neurospora Crassa Polarisome Architecture Minimal Medium (VMM) [47] supplemented with 2% sucrose and solidified with 1.5% agar when needed. The auxotrophic Dmus51 strain was grown on VMM agar supplemented with 0.5 mg/ml histidine. All experimental manipulations were ac- cording to standard techniques [48,49]. Preparation of conidial cell suspensions for the assessment of germling development and observation of CAT-mediated cell fusion were performed as described previously [26,50,51]. Homokaryon purification using crossing Homokaryotic Dbud-6 strains were generated by selecting ascospores obtained from a sexual cross between the heterokary- otic Dbud-6 mat a gene deletion strain and Dsad-2 mat A (Table 2) on hygromycin B medium (200 mg/ml), and evaluating the percentage of co-segregation of the mutant phenotype with hygromycin resistance. Absence of the native bud-6 locus from the genome of isolated Dbud-6 mutants was verified by PCR (Figure S1). Homokaryon purification using conidida The heterokaryotic Dbud-6 or Dbni-1 strains were grown on VMM containing 200 mg/ml hygromycin B until enough asexual spores (macro- and microconidia) have developed. Conidia were harvested in 1 ml sterile dH2O and adjusted by eye to yield a slightly cloudy, orange suspension. 150 ml of a 1:5000 diluted spore suspension were evenly distributed on fresh VMM supplemented with 200 mg/ml hygromycin B using glass beads, and subsequently incubated at 30uC overnight. Developing microcolonies derived from single conidia and showing a mutant phenotype were excised under the stereomicroscope using a sterile syringe needle, and transferred onto small (5 cm diameter) Petri dishes containing VMM with hygromycin B. These were incubated at 30uC, and colony development assessed over the following days. In case wt-like colonies developed, the procedure was repeated three times or until prominent phenotype changes occurred. Subsequently genomic DNA was extracted from selected clones and changes in the genome were verified by multiplex PCR as explained in detail elsewhere [52]. Construction of pMY1 and pMY2 The Neurospora crassa locus NCU08468.3, was identified by BLASTp analysis in the Broad Institute Genome database (http:// www.broadinstitute.org/scientific-community/data) as the sole homolog of the S. cerevisae Aip3/Bud6 locus SCRG04267.1. NCU08468.3 encodes a protein of 1001 bp length with an annotated actin-interaction protein domain. In the most recent annotation of the bud-6 locus (NCU08468.5) an additional 49 amino acid-encoding N-terminal extension has been identified, which however, is not conserved in the orthologous budding yeast locus, and the resulting gene product does not represent any specific protein domain. Extraction of genomic DNA from the N. crassa strain N150 (FGSC #9013) was carried out using a DNeasy Plant Extraction Kit (Qiagen, Inc.) For this, mycelium from a 48 h VMM liquid culture (28uC, 250 rpm, dark) was filtered and pulverized in liquid nitrogen, and processed through the protocol according to manufacturer’s recommendations. Standard PCR and cloning procedures [53] were used to generate carboxyterm- inal BUD-6-GFP and BUD-6-mCherry fusion proteins. The bud-6 gene was amplified by PCR from genomic DNA extracted from the N. crassa wild type strain N150 (FGSC #9013), using oligonucleotides Bud6-XbaI-F 59-GCTCTAGAATGGGTCCC- CAAGCTGGCAT-39 and Bud6-PacI-R 59-CCTTAATTAACT- CTTCCTCCTCTTCCTCC-39. The PCR reaction was per- formed in a NYX Technik Amplitronix A6 (ATC401 ApolloH) PLoS ONE | www.plosone.org 19 January 2012 | Volume 7 | Issue 1 | e30372