Figure 1.

Deletion of A. gossypii SPB components. Overlays of DIC image and AgH4-GFP signals from cells grown for ∼12 h at 30°C. (A) A typical wild-type mycelium at this stage contains multiple branches and 50–100 nuclei. (B) Terminal phenotype of mutants lacking components of the γ-tubulin complex. (C) Terminal phenotype of mutants lacking half-bridge components. Some mutants shown in B and C arrest as small mycelium with up to 12 branches containing a few nuclei and did not arrest as germlings with one nucleus or two nuclei. This is due to a maternal effect, where remnants of the wild-type protein are packed into mutant spores (Gladfelter et al., 2006). In A. gossypii, targeted deletions are performed with young mycelium containing ∼20 haploid nuclei that initially generate heterokaryotic strains: mixtures of nuclei carrying either the wild type or the deletion allele. On starvation, haploid mononucleate spores are produced in the heterokaryotic hyphae, and a substantial part of the mutant spores contain wild-type (maternal) protein, which can account for early growth and nuclear division in otherwise fatal deletions. Bars, 10 μm. (D) Radial growth of wild-type and deletion mutants on solid medium during 7 d of incubation at 30°C. Bar, 1 cm. (E) Overlays of DIC image and AgH4-GFP signals from hyphae showing nuclear distributions in wild-type and mutant strains. Bars, 5 μm. (F) Distribution of distances between adjacent nuclei was plotted using distance measurements between the nuclei in the first 50 μm of at least 20 different hyphae for each strain (n > 200 for each strain). The center of the GFP signal was used as the central point of the nucleus. Confidence values (p) for the χ² test were calculated for each data set between wild-type and mutant distributions. (G) Distance between the hyphal tip and the first nucleus was determined. Error bars represent the SE of the mean, n > 60 for each strain.