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Transcription factors (TFs) have critical roles in all facets of fungal growth and development. They typically represent the culmination of signaling pathways, resulting in transcriptional reprogramming of one to many genes. Computational algorithms can be used to analyze genomic databases, categorize TFs into structural families, and perhaps designate putative functions. Not surprisingly, true biological functions of most TFs have not been experimentally tested. <P>
The scientific rationale of this project is that a critical need exists for systematic functional characterization of TFs in agriculturally important plant pathogenic fungus F. verticillioides. Generating gene knockout mutants is a highly effective experimental approach to study gene function, and we now have the molecular genetic and genomic tools to generate large numbers of mutants in a reasonable amount of time. <P>The PI's long-term goal is to advance the knowledge of TF function in agriculturally important pathogenic fungus F. verticillioides. To understand why Fusarium species are successful pathogens, it is critical to determine the specific function of as many TFs as possible. <UL> <LI> How do Fusarium TFs regulate genes required for pathogenesis, both temporally and spatially <LI>Which TFs regulate morphogenesis (e.g., conidiation and germination) during host colonization H<LI>ow do TFs integrate environmental cues to induce (or repress) secondary metabolism <LI>To what extent are the functions of TFs conserved among closely related fungal pathogens F. verticillioides provides a unique opportunity to study the molecular and evolutionary basis of fungal pathogenesis. </ul> To address these questions, we will pursue following objectives: <OL> <LI> Generate F. verticillioides TF knockout/overexpression mutants; <LI> Functional Characterization of TFs in F. verticillioides; <LI>Identify TF-DNA binding sites using ChIP-Seq analyses.
NON-TECHNICAL SUMMARY: Fusarium verticillioides causes stalk and ear rots of maize, and produces fumonisin mycotoxins that accumulate in kernels and threaten food safety. The goal of this project is to systematically characterize F. verticillioides transcription factors (TFs) by determining their involvement in pathogenesis-related processes, including growth, morphological differentiation, and secondary metabolism/mycotoxin biosynthesis. Throughout the fungal kingdom, TFs are key intermediaries in reprogramming gene expression. However, in F. verticillioides and many other plant pathogenic fungi, little is known about the function of most TFs. Expansive resources for functional genomics are available in F. verticillioides, including a sequenced genome and techniques for highthroughput gene disruption, making the fungus ideal for genome-wide investigations into TF function. In this project, key families of TF-encoding genes will be disrupted via homologous recombination. Phenotypic abnormalities in growth, pathogenesis, and metabolism will be documented for each mutant. Selected TFs will be analyzed with ChIP-Seq techniques to identify DNA binding sites throughout the genome. All mutants and data generated during this project will be made publicly available to the scientific community.
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APPROACH: <BR> Objective 1: Generate F. verticillioides TF knockout mutants. F. verticillioides has 629 putative TFs classified into 41 TF families, and the PI's goal is to disrupt key families of these genes during the course of this project. The PI anticipated generating approximately 30 mutants per year. We will use the genome sequence information to design primers for split marker KO constructs and subsequently use polyethylene glycol (PEG)-mediated transformation approach to create TF knockouts. Validation of gene knockouts will be done first by PCR followed by Southern analysis. We have extensive experience working with F. verticillioides, and the procedure for generating targeted gene knockouts is well established in our laboratory.<P> Objective 2: Functional characterization of TFs in F. verticillioides. A comprehensive data set will be established for each TF mutant that will include assessments of development, pathogenicity, and fumonisin production. Alterations in colony morphology, growth rate, and pigmentation will be assessed by observing TF mutants growing on various media. Stalk pathogenicity assays will be performed on 12-week-old B73 maize plants. The impact of the TF mutation on fumonisin production will also be determined. <P>Objective 3: Identification of TF-DNA binding sites using ChIP-Seq analyses. We will select a cross-representative sampling of candidate TFs from five major (bHLH, C2H2 zinc finger, MADS box, bZIP, and APSES) families, focusing on TFs that show visible aberrant phenotypes during functional characterization. We will generate and express TF genes with S-tag and FLAG-tag in F. verticillioides for purification. To verify that the candidate 'enriched regions' interact with the selected TF, we will use purified TF-FLAG tag in electrophoretic mobility shift assay. DNA fragments from the entire promoter regions of the 'enriched regions' will be obtained by PCR. The fragments will be end-labeled with 32P and incubated with various concentrations of TF. Binding will be evaluated by electrophoresis of the mixture on a 4% native polyacrylamide gel.