<p>The specific objectives of this proposal are: </p>
<p>1) Development of a multiple-oligonucleotide microarray for the whole-genomes of the fungal plant pathogens Fusarium oxysporum, F. verticillioides, and F. graminearum; </p>
<p>2) Functional analysis of genes required for pathogenicity and host specificity; and </p>
<p>3) Development of an integrated database for comparative genomic studies.</p>
<p>NON-TECHNICAL SUMMARY:<br/> Fusarium species are perhaps the most important fungal plant pathogens worldwide. Together these microbes cause economically important disease on nearly every species of cultivated plant. Many species also pose a threat to human health and food safety by contaminating agricultural products with harmful mycotoxins. Due to their importance to agriculture, whole genome sequences have been developed for the Fusarium pathogens, F. graminearum (cause of scab disease of wheat and barley) F. oxysporum (tomato wilt) and F. verticillioides (corn kernel, root and stalk rot). The research undertaken by this proposal takes advantage of the relatedness of the three fungal genomes and will allow an unprecedented ability to predict genes and how these genes are expressed in plant pathogenic fungi. A database will be constructed allowing public display and
analysis of these data. By understanding the genes and gene expression patterns exhibited by these microorganisms while they are causing disease, we may be able to develop novel, stable, and environmentally sensible disease management practices aimed at interfering with the essential processes of pathogenesis. These management strategies may involve chemical or physical disruption of the fungal developmental pathways vital to causing disease.
<p>APPROACH: <br/>Having DNA sequence data for three pathogenic Fusarium species 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). 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 will 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. We hypothesize that conserved patterns of gene expression may reflect evolutionary constrain based on their functional significance to the developmental process being studied. For plant infection (Objective 2), 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 3) will be constructed.
<p>PROGRESS: 2008/02 TO 2013/01<br/>Target Audience: During this reporting period we reached target audiences of other plant scientists, plant pathologistsand plant improvement professionals through publications and web accessable databases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training: Lokesh Kumar (master student): developed a computational program to identify potential transcription factor binding sites based on the aligned sequence of F. graminearum with F. oxysporum, F. verticillioides and F. solani species and generated a list of genes associated with each potential binding site. Combining the expression data that will be generated through this study, the information can be used to investigate the potential functional association of the binding sites and genes have the binding
sites in their promoter regions (see publication Kumar et al., 2010). Wilfried Jonkers (postdoctoral): created deletion mutations in orthologs of the SGE1, ZIF1 and ZCF1 genes in F. graminearum and/or F. oxysporum and is conducting functional analysis of these transcription factors by comparing their expression profiles in mutant and wild type and by analyzing the differences in expression patterns between species. He has taken the lead in writing the first manuscript describing the design and performance of the new, multi-fungal genome microarray. Wende Liu (postdoctoral) Xiaoying Zhou (graduate student) Undergraduate students: Cynallyhia Indriago (Coker College), Bryan Gmusungu (Coker College), Sam Egel (Purdue), Samantha Strangeland (University of Minnesota). Microarray analysis with the cpkA and Fac1 mutants was conducted by Cynallyhia Indriago and Bryan Gmusungu. How have the
results been disseminated to communities of interest? http://www.broadinstitute.org/annotation/genome/fusarium graminearum/MultiHome.
<p>PROGRESS: 2008/02/01 TO 2009/01/31<br/>OUTPUTS: A custom Affymetrix GeneChip microarray has been designed and manufactured containing all gene sequences from the three fungal genomes, Fusarium graminearum, F. verticillioides and F. oxysporum. Through the interactions with the Affymetrix supporting teams and the Broad Institute Expression profiling platform, the PIs have developed a detailed project plan including sample collection, experimental conditions, imagine scanning, data processing and analysis. DNAs from all organisms and RNAs collected in vitro to validate the chip have been collected. Mutants of F. graminearum and F. verticillioides that are deleted for genes encoding the FMK1 MAP kinase, adenylate cyclase, and catalytic subunits of PKA have been generated and phenotypically characterized. We also have requested and received the Gbb1 (mutants deleted for the
G-beta subunit of trimeric G-protein gene) deletion mutants of F. graminearum and F. verticillioides from Dr. Yin-Won Lee at Seoul National University and Dr. Won-Bo Shim at Texas A&M University, respectively. RNA samples have been isolated from these mutants and are ready for microarray analysis. PARTICIPANTS: Dr. Kistler has isolated RNAs and DNAs to be used to validate the microarray and has had contributed to the design of the array. Dr. Ma has been the team leader on bioinformatics input on array design. Dr. Jin-Rong Xu has contributed to array design and has overseen generation of fungal mutants. Graduate student Xiaoying Zhou has conducted mutagenesis experiments and gained laboratory training in molecular biology and genomics. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.