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Abstract

The mating-type locus in Neurospora crassa controls mating and sexual development. The fusion of reproductive structures of opposite mating-type, A and a, is required to initiate sexual reproduction. However, the fusion of hyphae of opposite mating-type during vegetative growth results in growth inhibition and cell death, a process that is mediated by the tol locus. Mutations in tol are recessive and suppress mating-type associated heterokaryon incompatibility; heterokaryon incompatibility is a mechanism that prevents the formation of vigorous heterokaryons between genetically dissimilar individuals. In this study, the functional domain for heterokaryon incompatibility in the A mating-type protein, specifically MAT A-l, is mapped to a leucine-rich repeat. The tol gene encodes a putative 1011-amino acid polypeptide with a coiled-coil domain and a leucine-rich repeat (both essential for heterokaryon incompatibility). It contains a region of similarity with two het genes (HET-6 and HET-E). Repeat-induced point mutations in tol result in mutants that are wild-type during vegetative growth and sexual reproduction, but which allow opposite mating-type individuals to form a vigorous heterokaryon. Transcript analyses show that tol mRNA is present during vegetative growth but absent during a cross. These data suggest that tol transcription could be repressed in order to allow the co-existence of opposite mating-type nuclei during the sexual reproductive phase, tol is expressed in a mat A, mat a, A/a partial diploid and in a mating-type deletion strain, indicating that M AT A - l and MAT a-l are not absolutely required for transcription or repression of tol. These data suggest that TOL may interact with MAT A-l and/or MAT a-l to form a death-triggering complex. To understand how tol may mediate mating-type incompatibility, attempts have been made to isolate TOL-interacting proteins {top) by yeast 2-hybrid system. Five N. crassa mycelial cDNA-pGAL4 AD clones are shown to have plasmid-dependent interaction with TOL in yeast. One of them is a homologue of Schizosaccharomyces pombe vip-1, which is a p53- related protein. The tol gene is apparently conserved in many different Neurospora species. One particular allele, tol7 from N. tetrasperma, is of special interest. tol[sub T] is a natural tot in the regard that it does not mediate mating-type incompatibility. Evidence provided in this thesis suggests that two loci closely linked to tol[sub T] may mediate non-allelic vegetative incompatibility and sexual incompatibility, respectively.