Functional Genomics of Fusarium Graminearum, the Wheat and Barley Scab Fungus

We will collaborate with Affymetrix to develop a GeneChip containing all predicted F. graminearum genes and make it publicly available. Another objective is to analyze gene expression profiles of F. graminearum in different infection and colonization stages, in mutants defective in plant infection or toxin production, and in different developmental stages. Genes differentially expressed during specific infection or development processes or in response to mutants will be identified by microarray analyses. The third objective is to experimentally determine the biological functions of selected candidate genes identified in microarray experiments. Targeted deletion mutants will be generated for genes chosen on the basis of expression profiles and bioinformatics analyses.
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Fusarium graminearum, the cause of head blight disease of wheat and barley, has resulted in large economic loss to U.S. agriculture in the last decade. The fungus and the toxic compounds that it produces are hazards to food safety, as ingestion of infested grain is harmful to both humans and livestock. In order to learn more about how this fungus is able to cause disease, we have taken a genomics approach. Genomics is the science of heredity by which the overall genetic capacity (genome) of a living organism is studied by obtaining its total DNA sequence. This information will be useful to scientists who are seeking new ways to develop Fusarium resistant wheat and barley because it potentially provides details of many genes required by the fungus to cause disease.
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Objective 1: An Affymetrix GeneChip microarray will be constructed for the genome of F. graminearum. We will provide the sequences of predicted F. graminearum genes to Affymetrix. Each authentic or predicted F. graminearum gene will be represented on the GeneChip with a probe set comprised of 11 pairs of 25-mer probes. Each probe pair consists of a 'perfect match' oligonucleotide and a control 'mismatch' oligonucleotide that differs only at a single nucleotide substitution of the 13th base. For predicted F. graminearum genes with matching ESTs, probe sets will be within 600 bp of the 3' end of EST sequences. For those predicted ORFs without matching ESTs, sequences toward the stop codon will be preferentially used for designing probes. The intended Affymetrix 64K array format (synthesis area 10.8 mm) can accommodate 16,230 probe sets (357,070 probes).
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Objective 2: Microarray analyses will be used for identifying genes important for plant infection and colonization, toxin production, and sexual development in F. graminearum. For these experiments, we will identify genes differentially expressed during infection and colonization of wheat or during infection of wheat with non-pathogenic mutants. Gene expression will be profiled during key differentiation processes (hyphal differentiation and perithecium development) and mycotoxin production.
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Objective 3: Targeted disruption mutants of selected fungal genes will be generated. For genes selected for further functional characterization, corresponding genomic sequences, including both upstream and downstream flanking regions, will be downloaded from WI-CGR. Gene replacement constructs will be generated with the ligation-PCR approach and transformed into PH-1 protoplasts as described (Hou et al., 2002). Gene replacement mutants will be identified by screening with PCR and confirmed by Southern blot analyses. All the gene replacement mutants will be purified by single spore isolation. In general, knockout mutants will be assayed for possible defect in mycelial growth, secondary metabolism, sexual and asexual reproduction, ability to colonize corn silks and infect wheat heads. Detailed phenotype characterization of individual gene replacement mutants depends on specific candidate genes disrupted. Mutants with interesting phenotypes will be further analyzed by complementation or co-segregation analyses to confirm the relationship between observed phenotypes and genes deleted. All three labs involved in this project have extensive experience in fungal genetics and pathogenesis, and are familiar routine techniques and procedures involved in these experiments. Overall, the proposed research will develop and utilize functional genomic resources and/or approaches to identify genes important for F. graminearum pathogenicity, development, and secondary metabolism. Results from the proposed experiments will be helpful for understanding molecular mechanisms of fungal-plant interactions in scab disease development, and may ultimately lead to the development of more effective fungicides or disease control strategies.