The mechanics and functional consequences of hyphal fusion in Neurospora crassa; from genes to nutrient and nuclear translocation
Cell-cell fusion is integral in many developmental processes in eukaryotic organisms. In filamentous fungi, such as Neurospora crassa, hyphal anastomoses within the mycelium allows for the development of an interconnected network, which is thought to be important for colony homeostasis. Hyphal anastomosis mutants have altered body plans, but it is unclear how this affects colony functions such as nuclear and nutrient movement. In this study we characterize two new hyphal anastomosis muntants, ham-3 and ham-4. In addition we explore nuclear movement and mechanisms of nuclear dispersal in wild type colonies as well as a fusion mutant soft. Finally, we determine the influence of vegetative hyphal fusion on the movement of amino acids within a mycelium. Chapter one is a detailed review of the genetics and cell biology of hyphal fusion as well as the link between hyphal architecture and functionality in filamentous fungi.
In chapter two, novel N. crassa hyphal anastamosis mutants ham-3 and ham-4, are identified and characterized. ham-3 encodes a striatin - like homolog and ham-4 encodes a protein that contains a forkhead-associated (FHA) domain. Deletions of both genes cause severe fusion frequency decreases in conidial germlings, similar to a previously identified fusion mutant ham-2. In both yeast and humans striatin homologs, ham-2 homologs, and proteins containing FHA domains, interact in a complex which is thought to be involved in intracellular signalling and trafficking. We show that ham-2, ham-3, and ham-4 all have similar fusion defects indicating they might act in the same complex or pathway. In addition to fusion defects, homozygous crosses of ham-2, ham-3, and ham-4 exhibit aberrant meiosis resulting in abnormally shaped ascospores. ham-2 and ham-3 are female sterile, but ham-4 is female fertile. All three mutants are male fertile, and can undergo trichogyne/condium sexual fusion as a female. We suspect that the HAM-2,3,and 4 interact in a larger complex with a phocein homolog, MOB-3, to facilitate vegetative fusion and sexual development in N. crassa.
In chapter three, profiles of nuclear movement in N. crassa colonies were measured and described. Filamentous fungi have the ability to harbour multiple, genetically distinct nuclei in a common cytoplasm. These nuclei have free movement due to open septal pores large enough for nuclei to move through. In other fungal systems, this leads to sectoring of the population and single nuclear origin for homogeneous regions of expanding colonies. In N. crassa wild type colonies, we found that genetically different nuclei are well mixed through out the greater mycelium maintaining diversity of nuclear lineages. The distribution and nuclear velocities in a colony are maintained by a pressure source from growing hyphal tips driving nuclei through interconnected hyphae with different conductivities. When an alternative pressure, such as osmotic pressure is added to the system, nuclei can reverse flow toward the pressure source in a profile almost the mirror image of the forward movement profile. In addition nuclei toward the center of hyphae move faster relative to nuclei at the edges of the hypha indicating a pressure driven flow. We find that hyphal fusion also affects the flow profiles of N. crassa colonies. Fusion mutant soft has a nuclear velocity profile that reflects a hierarchical branching architecture where larger "feeder" hyphae supply nuclei to a multitude of tips.
Finally, in chapter four, the role of hyphal fusion in resource translocation is assessed. Nutrient translocation in fungi is necessary for biomass recycling in forest ecosystems, mycorrhizal associations, and substrate degradation. Maintenance of an interconnected network is thought to be necessary for intracolony communication and homeostasis. We tested whether the frequency of hyphal fusion influenced the translocation of 15N and 14C labeled 2-amino isobutyric acid (AIB) in N. crassa colonies. Two fusion mutants, soft, that has no vegetative hyphal fusion and prm-1, which has about a 50% fusion frequency as compared to wild type, were used to test amino acid translocation. Both soft and prm-1 have similar maximum growth rates as wild type but only soft showed a severe defect in translocation of the tracers. In addition we found that there is little reverse translocation in all of the strains. All of the strains had a greater level of total percent N at the tips of the colonies compared to other parts of the colony and soft had a more negative natural abundance 13C. In addition we found that thought germlings readily fuse and share resources, colonies greater that 0.5 cm in radius, cease to share resources. Developmental age affects resource sharing between colonies.