We have identified a gene mutation in Aspergillus nidulans, designated swoP (for swollen cell phenotype), which causes a morphological defect during spore germination and hyphal growth at the restrictive temperature of 42Â°C. Conidia of mutants swell to approximately 1.5 times the diameter of a non-mutant strain of identical genetic background and establish multiple points of polarity. Most polarized points swell isotropically, giving a multiple-budding appearance, at which point growth arrests in most cells. Cell wall weakness is indicated by a strong tendency of swollen cells to burst when observed under a coverslip. At 42Â°C, a very small minority of cells eventually produce colonies and asexual spores. Growth at 30Â°C is essentially normal, though hyphae are slightly wider than wild type and exhibit a minor steering defect. Cells grown at 42Â°C for up to 18 hours are capable of recovering normal hyphal morphology upon transfer to 30Â°C. When mutant hyphae growing at 30Â°C are transferred to 42Â°C, terminal compartments swell irregularly and may produce multiple budding points; subapical compartments also may swell and produce buds. The growth defect of swoP is complemented by the wild type allele of AN7462, which shows strong sequence homology to COG4, a component of the Golgi tethering complex associated with retrograde transport of COPI-coated vesicles. Sequencing of the COG4 allele of the swoP mutant reveals an adenine-to-cytosine mutation at base pair 2672, which results in a tyrosine-to-STOP substitution at amino acid position 780, truncating the plasmid containing this mutant sequence (instead of wild type) produces a partial remediation of the phenotype, but hyphal compartments still exhibit severe swelling defects. Meiotic mapping produces a ca. 12% recombination frequency between the swoP and AcuK loci, which is consistent with the chromosomal location of AN7462. We are currently working to localize the putative COG4 of A. nidulans via GFP-tagging and immunolocalization, as well as to demonstrate interactions between A. nidulans COG4 and other proteins involved in retrograde vesicle transport in the Golgi apparatus.
Full conference title:
9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 9th (2008)