| Cell fusion plays a vital role in the life cycle of the filamentous fungus, Neurospora crassa. During the sexual life cycle, cell fusion is required for the mating between the female mating structure protoperithecia and the asexual spore conidia of the opposite mating type. During the asexual life cycle, cell fusion can occur between conidia and vegetative hyphae cells, which is critical for the formation of an interconnected hyphal network. N. crassa depends on the interconnected hyphal network to transport nutrient within the colony to support both asexual and sexual development. Examination of classical hyphal fusion mutants showed that defect in hyphal fusion usually gives rise to two easily observable morphology defect phenotypes, the lack of female mating structure, the protoperithecium, and a flat conidiation pattern. Hyphal fusion is a general survival strategy for many filamentous fungi, however the mechanism in regulating cell fusion is poorly understood. With well-developed molecular and genetic tools (genome-wide deletion library, gene expression profiling, vast plasmid collections), N. crassa provides a very good platform to study cell fusion.;In this study, we took advantage of the available gene deletion library and performed a morphology-based genome-wide screen looking for cell fusion mutants. After examination of over 10,000 single gene deletion mutant strains, we identified 25 cell fusion genes, which, for the first time, allowed us to systematically look at the regulation of cell fusion. Among the 25 genes, 12 genes have been previously shown as needed for cell fusion. The study confirmed the involvement of four signaling modules during cell fusion, the MAK-1 MAP Kinase pathway, the MAK-2 MAP Kinase pathway, a calcium signaling pathway and the STRIPAK complex. This raised interesting questions about how the newly identified cell fusion genes regulate cell fusion.;In order to address these questions, we used molecular cloning techniques to label 7 new cell fusion genes with either GFP, RFP or HA tags. We were most interested in their protein expression patterns and their localizations inside the cell. We also looked at MAK-1 and MAK-2 activation in the gene deletion mutants to determine whether the mutants were affected in the activation of these 2 pathways. We demonstrated that HAM-6, HAM-7 and HAM-8 are cell type-specific proteins which function upstream in the MAK-1 pathway regulating cell fusion. The other four proteins provide more general functions in regulating N. crassa growth.;Finally, we looked at the GPI-anchored cell wall protein HAM-7. In collaboration with others, we were able to confirm that HAM-7 function as MAK-1 MAP kinase pathway sensor in regulating hyphal fusion. |
