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斥着假设的小基因和相应的小基因产物。 这些蛋白质是20-30 kDa或更小，并且大约是单个蛋白质结构域的大小。在这个项目中，一个比较结构基因组学的方法是用来阐明小的假设蛋白分泌的模式生物营养型真菌植物病原体，灰葡萄孢的生物学功能。B。选择灰葡萄是因为其作为破坏性植物病原体的显著经济重要性，以及其作为经济上有益的生物体用于生产甜葡萄酒。 B的基因组。灰霉病与核盘菌（Sclerotinia sclerotiorum）密切相关，核盘菌是一种“白腐病”，破坏400多种植物。这些信息证明了使用葡萄孢属和核盘菌属基因组来加速国际葡萄孢属-核盘菌基因组联盟对两种基因组的正确注释是合理的。 B的假想蛋白分泌物的研究。灰霉病将为理解细胞外蛋白在B的发病机制和存活中的作用提供相当多的知识回报。灰霉病菌以及其他各种真菌。 该项目将涉及确定以前在B的质谱（MS）图谱中鉴定的小的假设蛋白质的生物功能。cinerea的蛋白质分泌物。到目前为止，已经发现了9个小分子（30 kDa）的假设蛋白，无论是蛋白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。 第一个模块由来自B的假设蛋白质的全基因组比较分析组成。灰霉病菌与其他真菌基因组（包括BROAD真菌数据库和致病性、腐殖性和菌根真菌共生体的DOE-JGI真菌基因组序列）的比较，以鉴定大小为30 kDa或更小的保守小假设蛋白。cinerea和S.菌核从电脑搜索，其他B。暗示丝状真菌中可能的致病性决定子的灰霉病菌cDNA也将被克隆作为该模块的第二层。 最后，将在信息水平上生成每个基因的表达谱。 第三个模块将为B中的九个感兴趣的基因创建敲除突变体。灰叶 将考虑从每个模块产生的所有信息，以确定每个蛋白质在B生物学中的生物学作用。更广泛的影响：该项目为本科生的跨学科培训提供了一个很好的框架。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。 一个新的教育计划正在探索，以允许格鲁吉亚大学（UGA-CCRC）和亨茨维尔的亚拉巴马大学（UAH-结构生物学中心和生物科学系）之间的技术知识交流。 该课程将基于UGA每年教授的蛋白质组学课程，联合讲座将增加蛋白质结晶技术和真菌生物及其分泌蛋白的研究。 本课程将允许学生在UGA和UAH被教导背后的最基本的蛋白质组学技术的理论。

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.