1.10.4S
EFFECTS OF G-PROTEIN ALPHA AND BETA SUBUNIT GENE DISRUPTION AND MUTAGENESIS ON COMPLEX FUNGAL BIOLOGICAL PROCESSES

S GAO, S KASAHARA and DL NUSS

Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, Maryland, 20742-4450, USA

Background and objectives
G-protein-mediated signal transduction is involved in the regulation of complex biological processes, including vegetative growth, pigment production, asexual sporulation, mating and virulence in the chestnut blight fungus Cryphonectria parasitica. Each of these processes was reduced or eliminated as a result of the targeted disruption of cpg-1, a gene encoding the G-protein Gi alpha subunit CPG-1 [1]. A second G alpha subunit, CPG-2, was found to be dispensable for these functions. These studies have been extended by the cloning and targeted disruption of a C. parasitica G-protein beta subunit gene and the determination of the effect of CPG-1 mutagenesis on fungal phenotype using a recently developed complementation system.

Results and conclusions
Disruption of a C. parasitica G beta gene also resulted in reductions in pigmentation, sporulation and virulence, but caused an increase rather than a decrease in vegetative growth [2]. Stable transformation of a cpg-1 disruptant with a cDNA copy of the cpg-1 coding region under the control of the cpg-1 promoter completely restored normal phenotype. Advantage was taken of this complementation system to examine the effect of specific CPG-1 mutations on the complex fungal processes regulated through G-protein signal transduction. The differential effect of independent mutations of CPG-1 N-terminal putative acylation sites on fungal phenotype will be presented as an example of the utility provided by filamentous fungi as experimental systems for general studies on Gi alpha function, accumulation and subcellular localization.

References
1. Gao S, Nuss DL, 1996. Proceedings of the National Academy of Sciences, USA 93, 14122-14127.
2. Kasahara S, Nuss DL, 1997. Molecular Plant-Microbe Interactions 10, 984-993.