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

University Of Alabama In Huntsville, Huntsville AL

Investigators

Abstract

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.