Thermal Cycler with PlatinumH Taq High Fidelity DNA polymerase (Invitrogen) according to the manufacturer’s instruc- tions. The gel-purified PCR product was digested with XbaI and PacI and subsequently ligated into accordingly linearized pMF272 [54] and pJV15-2 [55], respectively. In-frame integration of the BUD-6 encoding fragment in the resulting vectors pMY1 and pMY2 was verified by restriction digest analysis and sequencing. Ectopic expression of the fusion protein was under control of the glucose-repressible ccg-1 promoter which ensures low level expression on 2% sucrose-supplemented minimal medium [54,56]. Transformation and transformant selection Transformations were performed using a standard electropora- tion protocol for N. crassa [57]. To generate strains expressing BUD-6-GFP and BUD-6-mCherry, linearized (NdeI-digested) pMY1 and pMY2, respectively, were targeted to the his-3 locus of strain FGSC 9717 (mat A his-32 Dmus-51::bar+). Transformants were selected by recovery of histidine auxotrophy on selection medium, and screened for signal intensity and localization using laser scanning confocal microscopy (LSCM). From all selected transformants five BUD-6-GFP expressing strains and one BUD- 6-mCherry expressing strain displayed fluorescence signals suitable for subsequent live-cell imaging studies. Selected BUD-6-GFP and BUD-6-mCHFP transformants were named NECL46 and NECL47, respectively (Table 2). As the fluorescence signal of the mCherry fusion protein was considerably weaker and prone to rapid photobleaching, compared to the equivalent GFP fusion construct, most experiments were conducted with strain NECL46 (Table 2). Visualization of BNI-1 dynamics was performed using the complemented Dbni-1(BNI-1-GFP) strain kindly provided by Justa-Schuch et al. (2010). Fluorescent staining To visualize the plasma membrane and organelle membranes, mature hyphae and germlings were stained with 2 mM of the lipophilic marker dye FM4–64 (prepared from 200 mM stock in DMSO; Molecular Probes, Eugene, OR) as previously described in detail [58]. Live-cell imaging microscopy For live-cell imaging of mature hyphae, mycelium incubated overnight on solid VMM agar was prepared using the ‘‘inverted agar block’’ method [58] and imaged on an inverted LSCM (LSM 510 Meta, Carl Zeiss, Go¨ ttingen, Germany), using 636/ 1.4 Plan-Apochromat and 1006/1.3 Plan-Neofluar oil immersion objectives. Fluorescence signals were detected with the following settings: GFP (excitation 488 nm from an Argon laser, 545 nm dichroic mirror, emission 505–550 nm), mCherry (excitation 543 nm from an HeNe laser, 570 nm dichroic mirror, emission 574–691 nm) and FM4–64 (excita- tion 488 nm from an Argon laser, 570 nm dichroic mirror, emission 574–691 nm). Digital images were captured and analyzed using the implemented LSM 510 software (version 3.2; Carl Zeiss). A transmitted light detector permitted simultaneous recording of widefield phase contrast images with confocal fluorescence signals. Additionally, for the acquisition of some 3D time series a DeltaVision epifluorescence micro- scope (Applied Precision, Issaquah, WA) was used, and germlings prepared as described previously [26]. Final image and time series manipulation was performed using the ImageJ platform (http://rsbweb.nih.gov/ij/). Neurospora Crassa Polarisome Architecture Supporting Information Figure S1 Genetic verification of gene deletion mutants by PCR. (A) Colony PCR results from two isolated clones (NECL48-5 and NECL48-8, Table 2) confirming absence of the 3.2 kb fragment amplified from the bud-6 ORF in the wild type. The bands ,500 bp are likely to be unspecific products of the used oligonucleotides, as they show up equally in all three strains. (B) Multiplex PCR genotyping results confirming that through isolation of monosporic microcolonies wild type nuclei carrying the bni-1 gene have been removed from the heterokaryotic Dbni-1 strain (FGSC 11490, Dmus51 background), generating the homokaryon selected (HS) Dbni-1 strain. The wild type control (left panel) contains all gene loci except the KO cassette targeted to the bni-1 locus. The 700 bp fragment at the bottom of each lane was amplified from the actin locus and served as an internal reaction control for each individual PCR, particularly important for those reactions where no other product is expected due to the absence of the tested locus. The heterokaryotic gene deletion strain (middle panel) still contains the native bni-1 gene, but also a population of transformed nuclei harboring the KO cassette at this locus. As Dmus-51 strains were used as recipients of the KO cassette, the mus-51 gene is absent from any Dbni-1 gene deletion strain that has not been backcrossed to a wild type. In the vegetatively selected Dbni-1 homokaryon (right panel) the primer pairs detecting the 59 region of the bni-1 locus and parts of this ORF anywhere in the genome did not result any product, confirming complete absence of this locus from the genome in the selected mutant strains. (TIF) Figure S2 Ectopic expression of fluorescent BUD-6 fusion constructs did not interfere with colony develop- ment. (A) Colony morphology of wild type N150, NECL46-9 and NECL47-5 after 24 and 48 hours of growth on Vogel’s medium at 28uC. No differences with respect to general colony architecture or conidiation pattern were observed between wild type and transformants. (B) Colony extension rates were measured every two hours over a period of 24 h and statistically analyzed (C). No significant differences between the three strains could be observed. (TIF) Figure S3 Changes in the abundance of BUD-6 clusters in developing conidia. VMM slants were inoculated with the BUD-6-GFP expressing strain NECL46-9 (Table 2), and continu- ously incubated for 10 days at 28uC. At the indicated time points conidial samples were taken and observed using laser confocal microscopy. As this analysis was based on sampling only single random optical sections of fields of conidia the results are an underestimate of the number of clusters present (i.e. clusters outside the focal plane were not captured). Each optical section was taken at a random plane through the spores, and thus provides the average distribution pattern within the cell population at each time point. (A) Example image showing the scored pattern of BUD-6 cluster distribution in freshly harvested conidia. Scale bar, 10 mm. (B) The graph displays the percentage of cells showing the particular BUD-6 cluster distribution pattern at the indicated sampling times hours post inoculation (hpi). With colony development the number of conidia with cortical clusters at one or both cell poles decreases, whereas the number of cells containing intracellular clusters increases. Together, this data indicates that the changes in BUD- 6 cluster distribution are probably connected to conidial matura- tion, which causes the redistribution of BUD-6 from sites of cytokinesis to internal compartments. (TIF) PLoS ONE | www.plosone.org 20 January 2012 | Volume 7 | Issue 1 | e30372