Dissecting the mechanism of cell fusion and secretion in Neurospora crassa

PART I/nThe saprotrophic fungus, Neurospora crassa has evolved to be exceptionally good at secreting large amounts of enzymes necessary to degrade exogenous complex carbohydrates. It is well established that N. crassa transcriptionally regulates the synthesis of these enzymes in order to utilize plant cell wall components as a carbon source. However, downstream regulation of the secretory pathway likely improves the efficiency of secretion and plant cell wall degradation. The mechanisms through which this occurs are still obscure. To elucidate these unknown mechanisms, we conducted a forward genetic screen to identify and characterize defective trafficking mutants that cannot translocate cellulases in the ER or that improperly retain cellulases in the ER instead of secreting them into the extracellular environment. We generated a mutant library using random mutagenesis of a strain with a GFP tagged endoglucanase (EG-2), to screen via microscopy for mutants with mislocalized EG-2. From the screen, we identified a particularly interesting mutant (10C2) that has EG-2-GFP mislocalization to the ER, a growth defect and temperature sensitivity. We used bulked segregant analysis to identify the putative causal mutation. The potential gene of interest encodes a scaffolding protein that is known to interact with multiple different proteins in various organisms, thus exhibiting diverse cellular functions in different organisms. However, its function in N. crassa has not been explored. Preliminary analysis suggested that this mutation causes a general secretion defect, as opposed to one specific to the cellulolytic response./nPART II/nA large number of fusion genes have been identified in Neurospora crassa, however, functions of many of these genes are unknown. ham-7 is one of the fusion genes, whose encoded HAM-7 protein has no conserved domains, is bound to the cell wall by a glycosylphosphatidylinositol (GPI)-anchor, and appears to be a homolog of a new family of polysaccharide monooxygenases (PMO). We made point mutations of the conserved histidines, which is one of the defining characteristics of a PMO, in ham-7. The point mutants have a disruption in the catalytic activity of HAM-7 and phenocopy Δham-7: flat growth, female sterility and inability to undergo chemotropic interaction and cell fusion. Previous studies indicated that ham-7 may be playing a role in the cell wall integrity pathway, either involved in signaling or structure maintenance. Δham-7 showed similar sensitivity as wild type cells induced by cell wall stress agents, which suggested that ham-7 is more likely involved in signaling of cell fusion. We were not able to secrete HAM-7 by only truncating the GPI-anchor site, however, we were successful once we additionally removed three N-glycosylation cross-linkage sites. To understand the role of HAM-7 in the signaling pathway, we analyzed the differential expression levels of communication and fusion genes in Δham-7, Δmak-1 and Δmak-2 mutants. Our data indicated that ham-7 functions downstream of mak-1 and mak-2. Characterization of ham-7 will provide a better understanding of the proteins involved in signaling and remodeling of the cell wall in N. crassa.

Advisor: Glass, Louise N