Generation of new colonial mutants in Neurospora crassa using insertional mutagenesis

Filamentous fungi grow by a combination of the apical extension of tubular hyphae and the repeated creation of new hyphal branches. Neurospora crassa was used as a model organism to study the molecular biology of the tip growth process. Our approach was to transform a wild type strain with linear gene constructs which conferred resistance to hygromycinB (hphr). Relying on the propensity of transforming DNA to insert into the genomic DNA at ectopic sites, we selected for drug resistant, morphological mutants which exhibited restricted colonial growth. The collection of new morphological mutants included strains which formed restricted, yeast-like colonies as well as more intermediate, spreading colonial variants. For the three mutants described here, the morphological trait co-segregated with the drug resistant marker suggesting that the hphr construct had disrupted a gene required for apical growth. Southern blot analysis confirmed that hphr tag has integrated into genomic DNA. Microscopic characterization, including staining of nuclei and septa, and immunofluorescent labeling of microtubules revealed subtle defects in nuclear migration for two of these strains. After the linkage was confirmed, inverse PCR was used to recover flanking DNA from the rol-4 strain for sequencing. The rol-4 mutant resulted from the insertional inactivation of the Neurospora homolog of the ApsA gene shown previously in Aspergillus nidulans to be required for nuclear migration into conidiospores.