Molecular and Functional Analyses of poi-2 , a Novel Gene Highly Expressed in Sexual and Perithecial Tissues of Neurospora crassa

Abstract

The poi-2 gene is highly and specifically expressed in starved and sexual tissues of the filamentous fungus Neurospora crassa. It encodes a 27-kDa protein, as shown by in vitro transcription and translation. The POI2 protein contains a hydrophobic signal sequence at the amino terminus followed by novel 16 tandem repeats of 13 to 14 amino acid residues; all repeats are separated by Kex2 processing sites. Repeat-induced point mutation (RIP)-mediated gene disruption was used to generate poi-2 mutants, and the mutated sequences showed either one of two distinct patterns: typical RIPs (GC-to-AT transitions) or insertion-deletion (indel) mutations. Although the poi-2 strains contained numerous mutations, all retained intact open reading frames (ORFs) of various lengths. They showed greatly reduced vegetative growth and protoperithecial formation and low viability of their sexual progeny. All poi-2 mutants had similar defects in male fertility and the mating response, but the nature of female fertility defects varied and corresponded to the length of the residual poi-2 ORF. Mutants with ORFs of approximately normal length occasionally completed sexual development and produced viable ascospores, while a mutant with a severely truncated ORF was female sterile due to its inability to form protoperithecia. Thus, poi-2 is essential for differentiation of female reproductive structures and perithecial development as well as for normal vegetative growth. The POI2 protein is involved in the mating response, probably as a component in the pathway rather than as a pheromone.