<OL> <LI>Role of histone deacetylase inhibitors in plant pathogenesis. <BR>1. Map and characterize the depudecin gene cluster <BR>2. Construct depudecin-minus mutants<BR> 3. Test the mutants for altered virulence on Arabidopsis wild type, Arabidopsis pad3 mutant, and several cabbage cultivars. <LI>Role of secreted Fusarium graminearum proteins in virulence and defense induction. <BR>1. Construct mutant strains by targetted gene disruption of representative secreted proteins and test for altered virulence. <BR>2. Chromatographically fractionate the secreted proteins to identify the protein or proteins that induce seedling death. <BR>3. Express in heterologous systems (such as E. coli) a representative sample of housekeeping proteins such as glyceraldehyde-3-dehydrogenase and enolase, and test whether plants respond to them by various assays.
Non-Technical Summary: Plant diseases cost growers and consumers billions of dollars every year, and therefore a constant need for new strategies to control plant pathogens is required. A better understanding of the mechanisms by which plant pathogens invade and destroy plant tissues would lead to the development of more effective and environmentally safe methods to control plant diseases. Fungi are the single most important group of plant pathogens. Known virulence mechanisms of fungi include secreted toxic proteins and small chemicals known as secondary metabolites. We are working on the biosynthesis, mode of action, and role in determining the outcome of plant/pathogen interactions for representatives of each class. One focus of study is the role in disease of the histone deacetylase inhibitor. <P> Approach: We will experimentally characterize the genes (mRNA start and stop sites, and introns) by 3' and 5' RACE. We will disrupt each putative cluster gene individually and test the mutants for depudecin production. We have already found that the PKS and the transcription factor are required for depudecin production. We will extend this to the two monooxygenases and a gene of unknown function adjacent to the PKS. Disruption of the transporter will probably be lethal if it is essential for self-protection (like all eukaryotes, A. brassicicola has HDACs) (Baidyaroy et al., 2002). The boundaries of the cluster will be experimentally determined using reverse transcriptase-PCR and Northern blot analysis of the transcription factor mutant. The boundaries of the cluster will be defined as the set of genes regulated by the transcription factor. Depudecin-minus mutants will be tested for altered virulence on cabbage and Arabidopsis using standard spore drop inoculation on intact and wounded leaves. To address whether any of the secreted proteins are virulence factors, we propose to disrupt (genetically mutate) approximately 50 of the proteins detected in vivo. An intrinsic obstacle to gene knockout studies is genetic redundancy. Gene replacement by double crossover homologous integration is a standard procedure for F. graminearum in our laboratory. Transformants will be checked for homologous integration by gene-specific and hph gene-specific PCR primers and Southern blotting. Transformants showing gene replacement will be tested for virulence on wheat heads at anthesis and on maize ears and maize silks (Reid and Hamilton, 1995; Voigt et al., 2005), Virulence will be assessed by the rating scale of Reid and Hamilton (1995). Virulence assays will be extended with microscopic analysis of the time course of infection (Guenther and Trail, 2005; Jansen et al., 2005). Identification of genes with a role in virulence will permit detailed follow-up experiments on the specific biochemical roles of such genes and their protein products. The heterologous, purified proteins will be tested for elicitor activity. There are many assays of pathogen elicitors, and we recognize that no one assay is ideal. We propose to begin with three general assays that are technically amenable to high throughput, have been used in our lab and others to identify elicitors, and can identify both host and non-host elicitors. These are medium alkalinization of Arabidopsis cell cultures (Kunze et al., 2004), Arabidopsis seedling mortality (Pfund et al., 2004), and necrosis following injection into wheat leaves (Bohland et al., 1997; Koga et al., 1998). Fungal proteins with activity in one assay will be tested in the others. Ultimately, these elicitors will be further characterized by established methods, e.g., identification of the active peptides, binding assays, identification of receptors, screening for Arabidopsis mutants or accessions that do not respond (Boller, 2005; Zipfel and Felix, 2005).