Colletotrichum graminicola as a system for studying the molecular genetics of conidial development in phytopathogenic fungi

Like many fungal pathogens of plants, Colletotrichum graminicola is dispersed and infects through asexually derived spores called conidia. The objectives of my thesis research were to: (1) characterize conidial development in C. graminicola; (2) develop techniques for studying the molecular genetics of conidial development; and (3) use these techniques to study genes that may be involved in conidiation. My results demonstrate the C. graminicola produces two different types of conidia. The two types have distinct ontogenies and are induced by different environmental cues. Techniques were developed for recovery of nonconidiating cultures, cultures producing exclusively one or the other type of conidium, or cultures producing both types of conidia. The cultures were suitable for the isolation of state-specific RNA. To manipulate the fungus genetically, I developed a DNA-demiated transformation system. A $\beta$-tubulin gene cloned from a benomyl-resistant mutant of the fungus conferred benomyl resistance in wild-type strains. Integration of circular plasmids carrying this gene was random. However, linearized and circular plasmids carrying other C. graminicola sequences were targeted to their respective homologous regions. During the cloning of the $\beta$-tubulin gene, and studies on its integration, it became apparent that C. graminicola contains a second $\beta$-tubulin gene. Because of the role of different $\beta$-tubulin isotypes in conidial development in other fungi, I studied the two C. graminicola $\beta$-tubulin genes. I cloned and sequenced both genes (TUB1 and TUB2). The deduced amino acid sequence of TUB2 is very similar to those of other fungal $\beta$-tubulins; the TUB1 sequence is much less conserved. Hybridization of TUB1 and TUB2 to poly (A) $\sp+$RNA indicated that TUB2 mRNA was more abundant than TUB1 mRNA in conidiating and nonconidiating cultures. There appeared to be a two- to three-fold increase in the level of TUB2 transcript relative to that of TUB1 in cultures producing one type of conidium. Disruption of TUB2 by site-specific integration did not result in any obvious changes in the fungus, including the development and germination of conidia. The data suggest that the divergent TUB1 product can function in place of the conserved TUB2 product