Horizontal Genetic Transfer in Asexual Dimorphic Pathogenic Fungi Our long term goal is to determine if parasexuality or some form of horizontal genetic transfer such as an occasional sexual cycle plays a role in altering pathogenicity or drug resistance in asexual dimorphic pathogenic fungi. Such fungi include the human pathogen. Candida albicans and the plant pathogen Fusarium oxysporum. Dimorphic fungi can reproduce as yeast (single cells) or mycelial (filamentous strands of cells). For pathogenic fungi, the mycelial stage is required for penetration into the host. In the mycelial stage, hyphal anastomosis is also possible which would allow the pathogens to form heterokaryons and enable horizontal genetic transfer without sexual structures (parasexuality). We have been studying heterokaryon formation in Fusarium oxysporum f.sp cubense (Foc) because it shares many of the traits of C. albicans. It is however a more convenient model system that C. albicans because it can be grown in the mycelial stage in vitro while C. albicans requires a mucosal membrane to induce the mycelial stage. In this proposal, we will create auxotrophic drug resistant strains of Fox by transformation with a disrupted gene. These mutants will be paired with wild type strain from our mutant after stable or transient heterokaryon formation. If genes can be transferred from one strain to another under our laboratory conditions, it suggests that drug resistance and pathogenicity genes could be transferred from pathogens to non- pathogens in nature. Asexual fungi may also have the ability to occasionally go through a sexual cycle. We have identified on mating type locus in some of our strains. If we find Fox strains on the other mating type, we will try to resurrect the sexual cycle by making heterokaryons between sexual strains of opposite mating type. We have observed that although two different nuclei can exist in a cell, only one mitochondrial genome occurs when heterokaryons are made by protoplast fusion. We have determined what happens in the average heterokaryon by genetic and molecular assays. Now, we propose to determine what happens in a single heterokaryon by microscopic observation. We will label the nucleus and mitochondrion with organelle specific strains and with the gene fluorescent protein and follow their fate after heterokaryon formation. Because only one mitochondrial genome survives after heterokaryon formation, we have a unique opportunity to create strains with unusual combinations of nucleus and mitochondrion. Comparison of the growth and physiology of these strains to the original parental strains will reflect the importance of the interaction of mitochondrion and nucleus.
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