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

RUI：玉米黑粉菌真菌交配受损突变体的分离和表征

• 批准号: 9807807
• 负责人: Karen Snetselaar
• 金额: $ 16.49万
• 依托单位: St Joseph's University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9807807&HistoricalAwards=false
• 关键词: RUI; Isolation; Characterization; Mating; Impaired

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.

Snetselaar 9807807该项目的重点是产生和表征植物病原真菌玉米黑粉菌的突变体，这些突变体的交配能力受损。交配涉及从酵母样生长到丝状生长的转变，然后沿着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，在U.用紫外线辐射或化学诱变剂甲磺酸乙酯处理玉米。将筛选诱变细胞与相容细胞类型交配的能力，如肉眼可见感染结构的形成所证明。这些结构的产生，称为Fuz+反应，证明了细胞交配并形成感染丝的能力，该感染丝随后将侵入植物组织。玉米专门集中在那些与相容的野生型细胞完全不育（Fuz-）的玉米上。这些研究还将通过筛选与交配受损突变体mim 1杂交时无法交配的突变体来扩展。mim 1突变降低了细胞与其相容伴侣融合的效率。初步结果表明，有许多突变体，虽然他们是Fuz+和完全能够与野生型伴侣交配，是Fuz-和无法与mim 1突变体交配。因此，mim 1突变体允许分离许多交配受损突变体，否则将无法检测到。交配受损突变体将以多种方式表征。将采用几种显微镜技术，包括在水琼脂覆盖的载玻片上使用细胞滴的交配试验，以确定突变体在交配的哪个阶段被阻断。应分离在多个步骤阻断的突变体，所述步骤包括：交配丝的形成和生长、交配丝向相容伴侣细胞的定向、交配丝的融合和感染丝的形成。 在可能的情况下，突变体将与相容的野生型细胞杂交并用于感染玉米植物。这些杂交后代的表型分析将允许确定所涉及的位点的数量，它们与其他已知基因的连锁，以及它们的遗传模式。玉米和互补DNA测序和分析。将获得的序列与Genbank等数据库中的序列进行比较，以搜索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，并在一个非常有趣的发育系统中使用各种形态学表征技术。这项研究将主要由本科生在PI的共同监督下进行。该项目旨在为学生提供使用整合遗传，分子和观察方法来研究发展的技术的经验。初步结果表明，突变体的筛选和表征策略是合理的，他们表明，该项目将提供深入了解的机制，管理酵母丝二型性和趋化性。这些行为发生在各种真菌中，包括许多重要的植物病原体。