Characterization of the genes encoding tropomyosin and ARP4 in Neurospora crassa

This study has focused on the genes encoding tropomyosin and ARP4 in the filamentous fungus Neurospora crassa. Tropomyosin is an extended coiled-coil protein that binds to actin filaments and influences many aspects of F-actin. Repeat Induced Point (RIP) mutagenesis was used to produce a tropomyosin mutant. Mating with strains containing multiple copies of the tropomyosin gene produced 20% inviable ascospores and approximately 7% of all spores exhibited an abortive germination phenotype. Only one out of 120 viable progeny displayed a mutant phenotype suggesting that the N. crassa tpm gene is essential for germination of ascospores. Moreover, scanning electron microscopy (SEM) and light microscopy of the tpm -RIPed strain showed that it branches more frequently than wild type. Staining with FITC labelled mouse alpha-actin did not detect an apical actin distribution, instead actin positioned predominantly in subapical zones. The actin related proteins (ARPs) have primary sequence homology to actin. In the present study, an ARP in N. crassa was identified that has 30--35% amino acid identity to ARP4 from other species. N. crassa ARP4 contains several sequence insertions and one of these insertions is highly divergent and may represent a new isotype. Like other ARP4s, NcARP4 has a nuclear localisation signal (NLS) suggesting that it is taken up by the nucleus. One morphological mutant strain was detected among 90 RIP mutagenesis progeny and its arp4 gene had very few substitutions, suggesting that, arp4 is also an essential gene. Approximately, 20% of the arp4-RIPed germlings have nuclei localized at the extreme tips of hyphae, in comparison to the 8--10 mum distance of the apical nucleus in wild type.