RUI: Isolation and Characterization of Mating Impaired Mutants in the Fungus Ustilago maydis

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Snetselaar9807807This project focuses on generating and characterizing mutants of the plant-pathogenic fungus Ustilago maydis that are impaired in their ability to mate. Mating involves a transition from yeast-like to filamentous growth, followed by growth along a pheromone gradient. The mutants will provide a tool for understanding the genetic basis of this complex developmental phenomenon. Specifically, mutations are being induced in haploid cells of U. maydis using either treatment with ultraviolet radiation or the chemical mutagen ethylmethanesulfonate. Mutagenized cells will be screened for their ability to mate with a compatible cell type, as evidenced by the formation of macroscopically visible infection structures. The production of these structures, termed the Fuz+ response, demonstrates the ability of the cell to mate and form an infection filament that would subsequently invade plant tissue.Previous attempts to isolate mating mutants in U. maydis have focused exclusively on those which are completely sterile (Fuz-) with the compatible wild-type cells. These studies will be extended by also screening for mutants that are unable to mate when crossed with a mating impaired mutant, mim1. The mim1 mutation reduces the efficiency at which cells fuse with their compatible partners. Preliminary results show that there are many mutants that, while they are Fuz+ and fully able to mate with wild-type partners, are Fuz- and unable to mate with the mim1 mutant. Thus, the mim1 mutant allows the isolation of many mating-impaired mutants that otherwise would not be detected. Mating impaired mutants will be characterized in a number of ways. Several microscopic techniques, including mating assays employing drops of cells on slides covered with water agar, will be employed to determine at what stage of mating the mutants are blocked. Mutants blocked at a number of steps including: mating filament formation and growth, orientation of the mating filament to the compatible partner cell, fusion of the mating filaments, and formation of the infection filament should be isolated. Where possible, mutants will be crossed with compatible wild-type cells and used to infect corn plants. Phenotypic analysis of the progeny from these crosses will allow the determination of the number of loci involved, their linkage to other known genes, and their patterns of inheritance.Mutants with interesting phenotypes will be complemented at the molecular level using a plasmid library containing genomic DNA from wild-type U. maydis and the complementing DNA sequenced and analyzed. The sequences obtained will be compared to those in databases such as Genbank to search for homologies to known genes.This mutant hunt combines elements of classical and molecular genetics with a variety of techniques for morphological characterization in an extremely interesting developmental system. The research will be carried out primarily by undergraduate students jointly supervised by the PIs. The project has been designed to provide students with experience using techniques that integrate genetic, molecular, and observational approaches to studying development. Preliminary results show that the mutant screening and characterization strategies are sound, and they indicate that this project will provide insight into mechanisms that govern yeast-filament dimorphism and chemotaxis. These behaviors occur in a variety of fungi, including many important plant pathogens.