CAA: A Proteomic Analysis of Hypothetical Small Secreted Proteins: Assigning Function in Important Fungal Genomes

许多真菌基因组测序项目都充满了假想的小基因和相应的小基因产物。这些蛋白质的大小为20-30 kDa或更小，约为一个蛋白质结构域的大小。本项目采用比较结构基因组学方法，对一种典型的生物营养型植物病原菌--灰霉病菌分泌的小分子假想蛋白的生物学功能进行了研究。之所以选择灰霉菌，是因为它作为一种毁灭性的植物病原菌具有重要的经济意义，也因为它在甜酒生产中作为一种经济上有益的有机体而被应用。灰霉病菌的基因组与核盘菌的基因组密切相关，核盘菌是一种‘白腐病’，危害400多种植物。这一信息证明了利用灰霉病菌和菌核菌基因组来加速国际灰霉病菌-菌核菌基因组联盟对这两个基因组的正确注释是合理的。对灰霉菌假想蛋白分泌的研究将为理解胞外蛋白在灰霉菌以及其他多种真菌的致病和存活中的作用提供相当大的知识回报。该项目将包括确定先前在灰霉病菌S蛋白分泌物的质谱图(MS)中鉴定的小假设蛋白的生物学功能。到目前为止，已经发现了9个小(30 KDa)的假想蛋白质。无论是蛋白质产物还是基因表达，都有待进一步研究。该项目将分为四个模块。第一个模块包括利用MS对来自灰霉病菌的假想蛋白质与其他真菌基因组(包括广泛的真菌数据库和致病、腐生和菌根真菌共生体的DOE-JGI真菌基因组序列)进行全基因组比较分析，以确定大小不超过30 kDa的保守的小假想蛋白质。第二个模块将涉及克隆和表达被确定为灰霉菌和核盘菌特异性决定簇的cDNA。通过计算机搜索，暗示丝状真菌之间可能的致病决定因素的其他B.cinerea cDNA也将被克隆，作为该模块的第二层。最后，将在消息级别生成每个基因的表达简档。第三个模块将为灰霉病菌的九个感兴趣基因创建敲除突变体。每个模块产生的所有信息都将被考虑，以确定每种蛋白质在灰霉病菌生物学中所起的生物学作用。广泛影响：该项目为本科生的跨学科培训提供了一个极好的框架。将特别注意雇用来自不同种族和性别的代表性不足的学生。目前正在探索一项新的教育计划，以便在佐治亚大学(UGA-CCRC)和位于亨茨维尔的阿拉巴马大学(UAH-结构生物学中心和生物科学系)之间进行技术知识的交流。这门课将以加州大学每年教授的蛋白质组学课程为基础，将增加关于蛋白质结晶技术的联合讲座，以及对真菌生物及其分泌蛋白质的研究。这门课程将允许UGA和UAH的学生学习最基本的蛋白质组学技术背后的理论。

Many fungal genome sequencing projects are riddled with hypothetical small genes with correspondingly small gene products. These proteins are 20-30 kDa or less and about the size of a single protein domain. In this project, a comparative structural genomics approach is used to elucidate the biological function of small hypothetical proteins secreted by a model biotrophic fungal plant pathogen, Botrytis cinerea. B. cinerea was selected because of its significant economic importance as a devastating plant pathogen and also for its application as an economically beneficial organism for the production of sweet wines. The genome of B. cinerea is closely related to that of Sclerotinia sclerotiorum, a ''white rot'', devastating 400+ species of plants. This information has justified using both the Botrytis and Sclerotinia genomes to speed the correct annotation of both genomes by the International Botrytis-Sclerotinia Genome Consortium. A study of the hypothetical protein secretions of B. cinerea would afford a considerable knowledge return toward understanding the role of extracellular proteins in both the pathogenesis and survival for B. cinerea as well as for a variety of other fungi. The project will involve determination of the biological function of small hypothetical proteins which have previously been identified in mass spectrophotometric (MS) profiles of B. cinerea''s protein secretions. So far, nine small (30kDa) hypothetical proteins have been found. Both the protein products and gene expression and will be further studied. This project will be divided into four modules. The first module consists of a genome wide comparative analysis of hypothetical proteins from B. cinerea with other fungal genomes (including the BROAD fungal databases and DOE-JGI fungal genome sequences of pathogenic, saprophytic and the mycorrhizal fungal symbionts) using MS, to identify conserved small hypothetical proteins of sizes 30 kDa or less. The second module will involve the cloning and expression of cDNAs identified as determinants specific to both B. cinerea and S. sclerotiorum. From the computer searches, other B. cinerea cDNAs alluding to possible pathogenicity determinants among filamentous fungi will also be cloned as a second tier for this module. Lastly, an expression profile for each gene will be generated at the message level. The third module will be creating knock-out mutants for the nine genes of interest in B. cinerea. All information generated from each module will be considered toward determining the biological role each protein serves in the biology of B. cinerea.Broader Impact: This project provides an excellent framework for interdisciplinary training of undergraduate students. Particular attention will be given for hiring underrepresented students from a variety of ethnicities and genders. A novel education program is being explored to allow for an interchange of technical knowledge between the University of Georgia (UGA-CCRC) and the University of Alabama in Huntsville (UAH-Center for Structural Biology and Department of Biological Sciences). The class will be based on the proteomics course already taught yearly at UGA, joint lectures will be added on protein crystallization techniques and a study of fungal organisms and their secreted proteins. This course will allow the students at both UGA and UAH to be taught the theory behind the most essential proteomics techniques.