Myctoxin contamination of food and feed is a worldwide problem causing human and animal diseases as well as death. Losses to growers exceed $1 billion annually. In the US the two major mycotoxin-producing fungi on maize are Aspergillus flavus and Fusarium verticillioides. Aflatoxins produced by A. flavus and fumonisins produced by F. verticillioides commonly contaminate of maize in North Carolina and the southeastern US. <P>The overall goal of this project is to develop stable and economically sustainable control practices for these diseases. Despite breeding efforts over the last 30 years, no commercial maize genotypes have adequate resistance to A. flavus or F. verticillioides or to the accumulation of the toxins they produce. A limitation to commercial plant breeding for resistance to aflatoxin contamination is the lack of a way to rapidly identify resistant genotypes and easily follow this resistance in breeding populations developed to move resistance into agronomically desirable genotypes. <P>The focus of the outlined research is to better characterize the host parasite interactions between these two fungi and maize seeds to develop scorable traits for resistance and to identify candidate genetic markers for use in marker assisted breeding. <P>The specific objectives are: 1) Characterize infection and colonization of maize kernels by A. flavus and F. verticillioides; 2) Characterize tissue-specific host responses to wild type and characterized mutants of A. flavus and F. verticillioides; 3) Analyze gene expression at the maize seed-A. flavus interface; 4) Evaluate seeds from Near Isogenic Line (NIL) populations of maize for susceptibility to A. flavus; 5) Profile the extracellular proteins produced during pathogenesis of maize kernels.
Non-Technical Summary: Mycotoxins are toxic and carcinogenic compounds produced by fungi growing on food and feed. Mycotoxin contamination is a worldwide problem causing losses exceeding 1 billion dollars annually. The major mycotoxins on maize are aflatoxins produced by Aspergillus flavus and fumonisins produced by Fusarium verticillioides. Losses in the US to aflatoxin contamination alone results in direct losses of 200 million dollars annually. This loss is compounded by indirect losses due to contaminated by-products, such as distillers grains. Plant resistance is the most economical strategy for resistance to plant diseases. Unfortunately, 30 years of breeding efforts has not resulted in maize genotypes with adequate resistance to fumonisin or aflatoxin accumulation. A limitation to commercial plant breeding for resistance is the lack of a way to rapidly identify resistant genotypes and easily follow this resistance in breeding populations developed to move resistance into agronomically desirable genotypes. The focus of the outlined research is to understand the dynamics of the disease in maize seed and locate the important defenses, either physical or chemical, that can be enhanced by plant breeding. <P> Approach: We will carefully follow the colonization of developing maize kernels by A. flavus and F. verticillioides. Maize inbred B73 will be planted in plots and grown under standard cultural practices, including the application of insecticides. Each ear will be hand pollinated. Individual kernels will be inoculated by wounding kernels in the early dough stage of development (R4). Inoculated kernels will be removed and treated one of three ways depending on the objective. One subset will be fixed in ten percent Buffered Formalin Phosphate Fixative for histological studies. Another subset will be quick frozen in liquid nitrogen and stored at negative 80C until either extracted for DNA quantification of fungal growth or used for Laser Capture Microscopy and RNA sequencing. To assess fungal growth in endosperm and embryo tissue, the embryo will be removed from the seed and growth will be quantified in both tissues using specific primers for A. flavus. Kernels will be fixed using ten percent Buffered Formalin Phosphate fixative followed by paraffin embedding, sectioning, and staining with Periodic Acid Schiff (PAS). We will utilize a Carl Zeiss PALM Laser Microbeam system for micro-dissection and capture of both fungal and maize tissue. We will use the Picopure RNA isolation procedure for isolation of RNA. We propose to use the Illumina Genome Analyzer II to sequence captured RNA from the LCM studies outlined above. To profile the extracellular proteins produced during pathogenesis of maize kernels we will use the vacuum infiltration and centrifugation techniques to remove the apoplastic wash fluid. Sample preparation will be based on a diethylamine extraction adapted for analysis of proteins by LC/MS/MS without the need for precipitation or ultrafiltration cleanup. Peptide analyses will be performed with a Thermo Surveyor liquid chromatograph coupled to a Thermo LTQ linear ion trap mass spectrometer. Proteins in the extracts will be identified by searching tandem mass spectra against protein databases in FASTA format using Bioworks Browser software (version 3.3, Thermo). Data from all the studies above will the analyzed with the goal of identifying key genes involved in the interaction of maize seeds with the two mycotoxin producing fungi. These genes will then be given to plant breeders to determine if they can be associated with resistance to mycotoxin contamination.