Neurospora Crassa Polarisome Architecture Figure 9. A Dbni-1 strain generated through vegetative homokaryon selection phenocopied growth defects of Dbud-6. (A and B) Wild-type like phenotype of the heterokaryotic Dbni-1 strain FGSC11490, including conidia and septa (arrows). Scale bars, 50 mm and 10 mm respectively. (C) The lack of septa in the homokaryotic Dbni-1 strain was confirmed by FM4–64 staining. Scale bar 50 mm. (D) FM4–64 staining also confirmed the absence of an organized apical tip growth apparatus, including the Spk. Scale bar, 10 mm. These defects closely resembled phenotypic key features of Dbud-6 (Figures 7 and 8). doi:10.1371/journal.pone.0030372.g009 nuclear exclusion zone (Figure 9C and D). Interestingly, the absence of septa resulted in the pronounced accumulation of vacuoles at the leading edge of a colony (Figure 10A and B). Despite the absence of differentiated fusion hyphae in the mature mycelium, Dbni-1 was able to undergo hyphal fusion. Unexpect- edly, hyphal fusion occurred at the very colony edge (Figure 10B, Movie S9); a feature not seen in the wild type, in which fusion is restricted to the subperiphery and beyond, but generally suppressed at the colony margin [63]. Upon closer inspection, we also found evidence for this phenotype at the colony periphery of Dbud-6 (Figure 10C). The only example of derepression of hyphal fusion at the colony periphery in the wild type, occurred as a result of interactions with conidial germlings (Figure 10D to F). The establishment of cytoplasmic continuity between conidial germlings and mature hyphae, involved either CATs protruded from germlings (Figure 10C), or the induction of fusion pegs from mature hyphae through the presence of germlings (Figure 10D and E). Taken together, the morphogenetic defects of Dbni-1 phenocop- ied those of Dbud-6 to a large extend, indicating that both proteins together operate in similar cellular contexts. To investigate these further we compared the subcellular dynamics of BNI-1-GFP with those of the other two polarisome components (BUD-6 and SPA-2) during the same set of morphogenetic processes. BNI-1 localized to sites of cell symmetry breaking, polarized germ tube growth, CAT-mediated cell fusion, vegetative hyphal fusion and septum formation During CAT-mediated cell fusion BNI-1-GFP dynamics followed those of BUD-6 and SPA-2 (Figure 2). The formin became recruited to homing CAT tips, focused at the attachment site and disappeared after cytoplasmic continuity was successfully established (Figure 11A, Movie S10). In contrast to BUD-6 and SPA-2, BNI-1 also became recruited to the cell cortex prior to cell symmetry breaking and remained associated with the tip of the forming protrusion (Figure 11A and B). VHF connections within the mycelia network of the Dbni-1::bni-1-gfp strain (Table 2) were indistinguishable from the wild type and thus BNI-1’s function in VHF was consistent with its role during CAT-mediated cell fusion (Figure 11A). The time sequence in Figure 11C shows that BNI-1- GFP labelled the inner perimeter of the opening fusion pore and disappeared immediately after cytoplasmic continuity was estab- lished. Again, following the dynamics observed for BUD-6 and SPA-2 (Figure 5), suggesting that during VHF all three proteins colocalize. As reported earlier for mature hypha [62], BNI-1 furthermore accumulated at incipient septation sites in conidial germlings, forming a constricting ring (Figure 11A). In polarized growing germ tubes BNI-1 accumulated in an apical crescent (Figure 11B). The unique insight from this set of experiments is that BNI-1, at least in the unicellular germling growth phase, is the only one of the three proteins that is clearly involved in the de novo establishment of new polarized growth sites. In mature hyphae BNI-1 localized to both an apical cap and the Spitzenko¨ rper core Further analyses in mature hyphae revealed that BNI-1 not only accumulated in apical clusters as already demonstrated [62], but moreover was a constituent of the Spk (Figure 12A). Relocation of BNI-1 clusters within the apical cap (Figure 12B) preceded Spk displacement and tip reorientation. Formation of a cortical BNI-1 crescent also coincided with lateral branch initiation (Figure 12B), during this event apical BNI-1 clusters associated with the apical plasma membrane transiently disappeared and extension of the main tip paused. This data suggests that targeted activation of the PLoS ONE | www.plosone.org 11 January 2012 | Volume 7 | Issue 1 | e30372