Cell fusion in Neurospora crassa
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The primary research aims of this thesis were the identification of novel cell fusion mutants of Neurospora crassa and the subsequent functional characterization of selected candidate proteins during conidial anastomosis tube (CAT)-mediated cell fusion by means of genetic, molecular, biochemical and live-cell imaging analysis. Chapter 1 provides a general introduction of the model organism and the cell fusion processes studied during different stages of the fungal lifecycle. Chapter 2 summarizes the materials and methods used. Chapter 3 introduces the comparative genomics screen conducted between appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae and hyphal fusion in Neurospora crassa. Novel cell fusion mutants were identified in MAP kinase signalling, redox-signalling and Rho-type GTPase signalling pathways, whereas no functional overlap in the cAMP response pathway between both species could be found. Chapter 4 demonstrates how newly developed fluorescent reporters for F-actin and activated Rho GTPases in filamentous fungi lead to novel insights into the dynamic rearrangement of the F-actin cytoskeleton and cortical activation of Rho GTPases during cell symmetry breaking, polarized tip growth and cell fusion. Chapter 5 focuses on the role of the cell wall integrity (CWI) MAP kinase pathway during cell fusion, and in particular, on the function of the terminal MAP kinase MAK-1 during CAT homing and fusion pore formation. Inhibitor studies indicated that MAK-1 kinase activity is required for its own recruitment to the fusion site already during homing and for cell wall remodelling during fusion pore enlargement between interacting cells. Chapter 6 presents ultrastructural scanning electron microscope (SEM) studies which indicate that defects in hyphal attachment, extracellular matrix deposition and cell wall remodelling prematurely abort morphogenesis of the female fruitbody. These findings are put into context with defects observed in mutants of components acting in related signalling pathways which appear to regulate non-self fusion events at later stages of sexual development leading to fertilization in N. crassa. Chapter 7 provides the first evidence for a role of NADPH-oxidase (NOX)-generated reactive oxygen species (ROS) in the regulation of morphogenetic changes required for CAT-mediated cell fusion. Redox-modification of signalling proteins might be involved in cell-cell chemoattraction. Chapter 8 provides a summary of the key findings and discusses future directions.