UBC Theses and Dissertations
Characterization of genes putatively involved in mating-type associated vegetative incompatibility in Neurospora crassa Hiltz, Megan Diane
Vegetative incompatibility is a ubiquitous phenomenon in many filamentous fungi. Neurospora crassa vegetative incompatibility involves the prevention of vigorous heterokaryons, mediated by genetic dissimilarity at eleven heterokaryon incompatibility (het) loci. During the vegetative phase, the mating-type (mat) locus functions as a het locus, while in the sexual phase, the mat locus confers mating identity and post-fertilization functions for completion of the sexual cycle, tol is an unlinked recessive mutation that suppresses mating-type vegetative incompatibility. TOL encodes a 1044 amino acid polypeptide that contains putative proteinprotein interaction domains. TOL contains a region called the SET domain that has similarity to HET-6 in N. crassa and HET-E in Podospora anserina (SET = HET-SIX, HET-E and TOL). Searches of the N. crassa genome yielded a large number of TOL-like sequences, which also share the SET domain and may indicate a novel motif region. BLASTP searches with TOL have identified similarity to Mms21p, a DNA repair protein in Saccharomyces cerevisiae. To identify additional proteins in the mating-type vegetative incompatibility pathway, TOL was used as bait in a yeast two-hybrid system. One of the putative pit genes (proteins that interact with TOL) was ncvipl, which has homology with vipl, an uncharacterized gene in Schizosaccharomyces pombe. RIP (repeat-induced point) mutational analyses of the ncvipl was conducted and progeny examined as to their effects on mating-type associated vegetative incompatibility. At the termination of this study, the influence of ncvipl remained unclear. Strains were constructed that will provide the tools for future analyses. A novel mutation was discovered that caused a partial suppression of mating-type vegetative incompatibility (Sup [mvi]) phenotype. This mutation appears to be linked to or in mat a, localizing the gene causing the Sup (mvi) phenotype to linkage group I. Partial diploid analysis and complementation assays failed to determine if the mutation was in mat a or not and demonstrated that the Sup (mvi) phenotype does not suppress mating-type vegetative incompatibility when the mating-type gene products are in the same nucleus. Further analysis is needed to determine the nature of the mutation and the full influence of the Sup (mvi) phenotype in a homoallelic condition.
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