C-RUI: Identification of Genes Regulating Cell Wall Integrity in Aspergillus nidulans

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An essential determinant of fungal growth and development is the fungal cell wall. Little, however, is known about the mechanisms by which the wall is constructed or developmentally modified. The goal of this research is to characterize a newly identified set of genes (Cal genes) in the model filamentous fungus Aspergillus nidulans, whose primary phenotype (hypersensitivity to the wall-compromising agent Calcofluor) has been correlated with the presence of structural defects in cell walls. Presumably, the functional alleles of these genes play roles in synthesizing or transporting wall constituents, in wall maturation and modification, or in signal transduction pathways that regulate wall metabolism. The work will proceed in three phases. In Phase I the number of available cal mutants will be increased by standard genetic methodology (six cal mutants have been identified to date, but the screening shows no signs of saturation), and extensive phenotypic profiles will be established for all mutants with regard to effects on cell wall chemistry, timing and morphology of spore germination, hyphal morphology, cytokinesis (septal placement), and the mitotic cycle. The objective of Phase II is to identify each of several Cal genes by 1) transforming cal mutants with cosmid or plasmid vectors carrying genomic (wild type) DNA and sequencing those inserts that cause phenotypic rescue, 2) identifying the complementing genes through transposon-mediated inactivation of each candidate open reading frame, and 3) determining the phenotypic effect of null mutations via targeted gene replacement. In Phase III, Cal gene products will be localized within the cell, either by construction of fusions between Cal genes and a green fluorescent protein gene or by epitope tagging and immunocytochemistry (EM and LM).Many fungi play important roles as producers of commercial products or as pathogens of plants and animals, and our ability to manipulate fungi to our advantage is limited by how well we understand the basic mechanisms of their growth and development. This research will increase our knowledge of fungal growth and development by identifying novel genes that affect cell wall integrity. Because undergraduate college students will play active, central roles in all aspects of this research, the project will also advance undergraduate education in the sciences and provide talented young people with experience relevant to making a choice to pursue research careers.