Molecular Dissection of a Specialized Peroxisomal Vesicle

Rockefeller University, New York NY

Investigators

Abstract

Filamentous fungi grow as a cellular syncytium where individual cells are interconnected by perforate crosswalls. Cell lysis in these systems triggers a rapid mechanism of septal pore sealing that is mediated by a dense-core vesicle known as the Woronin body. The Woronin body has been observed in over 50 species but its origin in the peroxisome and function associated with the cellular response to cell lysis have only recently been defined at the molecular level (Jedd and Chua, 2000). In the model system Neurospora crassa, the Woronin-body core is comprised of Hex1, a protein that is both necessary and sufficient for Woronin-body core formation. Deletion of the HEX1 gene eliminates the Woronin body from the cytoplasm and produces hyphae that bleed cytoplasm following cell lysis. Thus, Hex1 defines a novel peroxisomal vesicle that functions in the cellular response to cell lysis. The goal of this research is to understand basic mechanisms of Woronin body biogenesis and function. As part of this effort, the following specific aims will be addressed. i) Woronin-body associated proteins will be identified. The Woronin body will be biochemically purified and associated proteins identified by amino acid sequencing. Corresponding genes will then be cloned and analyzed to assess their role in the biogenesis and function of the Woronin body using genetic, biochemical and cell biological techniques. ii) The conservation of Woronin body structure and function will be determined by analyzing HEX1 and recently identified HEX1 alternative-splicing variants in several model ascomycetes using genetic and cell biological techniques. iii) A mechanism of Woronin body formation will be determined by analyzing the phenotype of Woronin-body-associated protein deletion strains and by reconstituting Woronin body formation in yeast cells using heterologous expression of Hex1 and protein interactants. In addition, the crystal structure of Hex1 will be determined and used to identify amino acid residues that mediate Hex1 self-assembly. Mutations in these residues will then be used to determine how Hex1 self-assembly influences Woronin body biogenesis. iv) The importance of the Woronin body for the pathogenicity of disease-causing fungi will be determined by deleting HEX1 from the genome of the rice blast fungus Magnaporthe grisea. The deletion strain will then be examined with respect to infection-related morphogenesis and host colonization. Results from this study will enhance our understanding of basic mechanisms governing vesicle formation and define a previously unrecognized pathway for protein sorting in the peroxisome. In addition, the possibility that the Woronin body functions as a pathogenicity factor in disease-causing fungi may define new strategies for combating fungal pathogens in both plants and humans.