The overall goal of this research is to identify and characterize molecular mechanisms that regulate the biosynthesis of mycotoxins during the infection of crop species by plant pathogenic fungi. This proposal focuses on the following two objectives: <OL><LI> Identify kernel factors and properties that influence mycotoxigenesis. <LI> Identify and characterize fungal genes that regulate mycotoxin biosynthesis.
Non-Technical Summary: Fungi produce a remarkable number of toxic secondary metabolites, collectively referred to as mycotoxins. Of considerable concern to human health are carcinogenic mycotoxins. Mycotoxins can also be teratogenic, neurotoxic, immunosuppressive, nephrotoxic, hematopoietic, and dermonecrotic, and can be acute or chronic. Mycotoxins also cause serious illnesses in many animals, particularly poultry, swine, horses, and, to a somewhat lesser extent, cattle. Contamination of cereal grains with mycotoxins is a common problem throughout the world, and the removal or detoxification of mycotoxins from contaminated grain is difficult and costly. A molecular-level understanding of the interactions between mycotoxigenic fungi and their hosts would enable the development of new strategies to control the accumulation of mycotoxins in agricultural commodities. We anticipate discovering genes that are involved in perceiving the kernel environment, transcription factors that regulate the expression of mycotoxin biosynthetic genes, global regulators of secondary metabolism, components of signal transduction pathways that link primary metabolism to secondary metabolism, and genes belonging to novel, previously undescribed families. <P> Approach: Experiments will be performed with A. flavus, F. graminearum, and F. verticillioides because they produce mycotoxins that profoundly affect human health, each is tractable to a wide variety of molecular techniques including targeted gene disruption, and all three have a wealth of supporting resources including commercially available microarrays for gene expression, collections of defined and/or tagged mutants, and fully sequenced genomes. Objective 1. Experiments will focus on the effect of starch composition, kernel development, pH, and kernel tissue on the production of aflatoxins by A. flavus and trichothecenes by F. graminearum. For F. graminearum, experiments will be performed in parallel on corn and wheat kernels. Additionally, we will focus on exploring genetic diversity in the host species. We will obtain ancestral species and undomesticated relatives of corn and wheat from repositories of genetic diversity and test their ability to support mycotoxin biosynthesis. We will also evaluate diversity lines of wheat and corn, which include commercial lines and exotic germplasm, to identify factors and properties that influence mycotoxigenesis. When kernel components and properties that impact mycotoxigenesis have been identified, we will obtain genetically defined mutants, as available, to corroborate our findings. Objective 2. We will combine techniques in conventional and molecular genetics with functional genomics. We will screen tagged mutants of the three fungi to identify isolates with toxin-related phenotypes. For each species, we will identify: positive regulators of mycotoxin biosynthesis, by screening for isolates that produce low levels of mycotoxins under favorable conditions; negative regulators, by identifying isolates that produce higher than normal levels of toxins under unfavorable conditions; and genes involved in related aspects of pathogenesis by observing which mutants grow poorly on kernels. Mycotoxin analyses will be performed by liquid or gas chromatography (HPLC or GC) to increase accuracy and sensitivity. For each mutant of interest, the tagged gene underlying the phenotype will be identified by either plasmid rescue or genome-walker PCR techniques. Microarrays will be used to identify genes whose expression is induced concurrently with the induction of mycotoxin biosynthetic genes. For each species, the top five candidate genes will be disrupted via homologous recombination and evaluated for toxin-related phenotypes. Finally, after the preliminary phenotypic and molecular characterizations of both the tagged and targeted mutants, genes identified as regulators of mycotoxin biosynthesis will be studied in greater detail at the molecular level, with the specific approaches depending on the types of genes that are identified.