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

本项目主要研究植物致病性真菌麦氏黑穗病菌（Ustilago maydis）交配能力受损的突变体的产生和特征。交配涉及到从酵母样生长到丝状生长的转变，然后沿着信息素梯度生长。突变体将为理解这种复杂发育现象的遗传基础提供工具。具体地说，利用紫外线辐射或化学诱变剂乙基甲磺酸诱变可以在马氏菌的单倍体细胞中诱导突变。通过形成宏观可见的感染结构，将对诱变细胞进行筛选，以确定其与兼容细胞类型交配的能力。这些结构的产生，被称为Fuz+反应，证明了细胞交配和形成感染丝的能力，这种感染丝随后会侵入植物组织。以前试图在美国分离交配突变体的尝试只集中在那些与兼容的野生型细胞完全不育（Fuz-）的突变体。这些研究还将通过筛选与交配受损突变体mim1杂交时无法交配的突变体来扩展。mim1突变降低了细胞与相容伴侣融合的效率。初步结果表明，有许多突变体，虽然它们是Fuz+，完全能够与野生型伴侣交配，但它们是Fuz-，不能与mim1突变体交配。因此，mim1突变体可以分离出许多交配受损突变体，否则这些突变体将无法被检测到。交配受损的突变体将以多种方式表现出来。几种显微技术，包括将细胞滴在覆盖有琼脂水的载玻片上进行交配试验，将被用来确定突变体在交配的哪个阶段被阻断。在以下几个步骤中被阻断的突变体，包括：交配丝的形成和生长，交配丝向兼容的伴侣细胞的取向，交配丝的融合，以及感染丝的形成，都应该被隔离。在可能的情况下，突变体将与兼容的野生型细胞杂交，并用于感染玉米植株。对这些杂交后代的表型分析将允许确定所涉及的基因座的数量，它们与其他已知基因的联系，以及它们的遗传模式。具有有趣表型的突变体将在分子水平上使用含有野生型马氏菌基因组DNA的质粒文库进行补充，并对互补DNA进行测序和分析。获得的序列将与数据库（如Genbank）中的序列进行比较，以寻找与已知基因的同源性。这种突变体狩猎结合了经典和分子遗传学的元素，以及在一个非常有趣的发育系统中进行形态表征的各种技术。该研究将主要由本科生进行，并由pi共同指导。该项目旨在为学生提供使用综合了遗传、分子和观察方法的技术来研究发展的经验。初步结果表明，突变体筛选和表征策略是合理的，这表明该项目将为酵母丝二态性和趋化性的调控机制提供深入的见解。这些行为发生在多种真菌中，包括许多重要的植物病原体。

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.