Metabolic Profiling of the Aspergillus flavus-Maize Interaction to Identify Fungal Pathogenicity and Host Plant Resistance Factors

<p>The goal of the proposed research is to determine the form and function of metabolites produced by uncharacterized Aspergillus (A.) flavus secondary metabolite (SM) gene clusters as well as the identification of primary and secondary metabolites (the metabolome) of maize that play a role in host resistance during the A. flavus-maize interaction. A systems approach utilizing genomics, transcriptomics and metabolomics will allow the identification of key maize genes/metabolites that represent resistance factors against A. flavus. Performing comparative metabolomics of infected resistant and susceptible maize lines will enable the identification of metabolites from maize that are unique (or present in greater amounts) only in the resistant lines. Using genomics/transcriptomics the gene(s) can be identified that is related to biosynthesis of the putative resistance-associated metabolite(s). RNA interference-mediated silencing or CRISPR/Cas9-based knock out of the gene(s) can be used to confirm that it is responsible for the observed resistance phenotype. Any confirmed resistance genes can then be used in genetic improvement of maize with enhanced resistance to A. flavus infection and aflatoxin contamination through marker-assisted breeding or through overexpression of resistance genes in transgenic maize lines. In a similar fashion we can also identify A. flavus metabolites that play a significant role in maize colonization. Using comparative metabolomics of fungal strains specialized to infect corn with those that are found mainly in the soil, it will be possible to identify key virulence-associated metabolites. Genes responsible for biosynthesis of the virulence-associated metabolites can be targeted for RNAi-mediated host-induced gene silencing. Studies will continue to identify metabolites produced by uncharacterized A. flavus SM biosynthetic gene clusters and the role that their associated metabolites play in the biology of the fungus. We will attempt to activate expression of silent gene clusters by growing A. flavus on a number of different substrates. We predict that by growing the fungus on different carbon and nitrogen sources we will turn-on some of these silent gene clusters thus allowing production and characterization of the cluster SMs. If we cannot find the nutritional conditions that activate a particular SM gene cluster of interest, we will use heterologous gene expression systems to identify the cluster metabolite. Genes encoding enzymes required for the first committed step in biosynthesis of the metabolite (usually a polyketide synthase or nonribosomal peptide synthetase)and associated genes (e.g. P450 oxidoreductases) will be introduced into the heterologous host using specialized plasmid vectors that allow for high level expression of the A. flavus genes of interest. The transformed heterologous host will be cultured, metabolites extracted and production of the precursor A. flavus SM will be analyzed by mass spectrometry and NMR techniques.</p>