2008 Annual Report 1a.Objectives (from AD-416) Comparative functional genomics of plant pathogenic Fusarium species. Objective 1. Design, generate and validate a whole genome microarray for the fungal plant pathogens F. graminearum, F. verticillioides and F. oxysporum. Objective 2. Define shared genes and patterns of gene expression found in all species as well as those that are specific to particular host – pathogen interactions, host and tissue specificity. Objective 3. Define genes and patterns of gene expression unique to fungal reproductive development as well as those that are specific to particular spore states. Objective 4. Develop a comparative and functional genomics database. 1b.Approach (from AD-416) With the development of whole genome sequence assemblies for F. graminearum, F. oxysporum, F. verticillioides, and F. solani, we have described conserved genes and other structural elements. For this proposal, these sequence data and the improved accuracy of gene predictions resulting from comparative analysis will allow for the construction of a highly accurate, genome-wide microarray for the three most closely related Fusarium species (Objective 1). Further objectives seek to extend comparative analysis by looking for conserved patterns of gene expression among the three fungi under a variety of environmental conditions including during plant infection (Objective. 2)and sporulation (Objective 3). The significance of conserved non-coding regions will be tested by their correlation with genes exhibiting conserved expression patterns. Physical proximity of conserved co-expressed genes may be used to define functional gene clusters. Because phenotypic similarities and differences exist among the species, comparative gene expression patterns can be placed in a biological context. Thus conserved patterns of gene expression among species will be discovered during, for example, fungal challenge of compatible hosts or during ascospore development. We hypothesize that conserved patterns of gene expression may reflect evolutionary constrain based on their functional significance to the developmental process being studied. For both plant infection (Objective. 2)and reproduction (Objective 3), we intend to test this hypothesis by deleting selected genes such as stage-specific transcription factors, to determine their effect on fungal development. To make results from proposed study and comparative analyses accessible for the community users, a coherent database incorporating comparative structural and functional data (Objective. 4)will be constructed. 3.Progress Report The project was initiated in January, 2008 and planning has been undertaken to design a second generation microarray for determining fungal gene expression during growth in plants and in culture. The expression levels of thousands of fungal genes from more than eight fungal species will be monitored over time. We have created mutations in several identified genes in order to determine their function and role in spore germination and pathogenesis. Progress on the project goals has been coordinated in 2008 by two conference calls with the microarray design team from Affymetrix, Inc., and monthly phone calls with the cooperator.