Location: Food and Feed Safety Research
Project Number: 6054-41420-009-003-N
Project Type: Non-Funded Cooperative Agreement
Start Date: Sep 21, 2018
End Date: Sep 20, 2023
Objective:
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.
Approach:
For identification of novel secondary metabolites from Aspergillus (A.) flavus, we will grow the fungus on different carbon and nitrogen source media in an effort to activate or increase expression of genes present in uncharacterized “silent” secondary metabolite gene clusters thus enabling production of the associated metabolite(s). Gene clusters that do not respond to growth of the fungus on these different media will be analyzed by cloning of specific cluster backbone genes (e.g. PKSs or NRPSs) and other key biosynthetic genes (e.g. P450s)into plasmid vectors. Vectors will be introduced into heterologous hosts such as Saccharomyces cerevisiae or Aspergillus nidulans. For both media-based and heterologous host-based studies, the strains will be cultivated, metabolites extracted and production of the metabolites initially analyzed by UHPLC-high resolution mass spectrometry followed by detailed analysis using multi-stage mass spectrometry (MSn) and nuclear magnetic resonance (NMR) to fully establish the structures of the new compounds.
To investigate maize and fungal metabolite changes upon maize-A. flavus interaction, LC-MS and GC-MS based metabolomics workflows comprising a standardized experimental setup for infection of maize kernels and fungal growth, and subsequent LC-MS/GC-MS data acquisition and processing will be applied. Resistant and susceptible maize lines and different A. flavus isolates (exclusively and not exclusively associated with the maize plant) will be included. A targeted approach will be applied to investigate whether specific primary metabolites are differentially abundant upon infection and depending on the maize line and A. flavus isolate. Investigation of the changes in secondary metabolites will be carried out using an untargeted metabolomics approach based on UHPLC-HRMS and multi-stage MS. In an initial phase, libraries of maize and A. flavus secondary metabolites will be made. Comprehensive profiling of A. flavus metabolites can be supported by stable isotope labelling strategies, i.e. fungus grown on carbon 13 (13C) and nitrogen 15 (15N) labeled grain and/or fungus grown on U-13C6-glucose according to Isotope Ratio Outlier Analysis (IROA) technology. We will use Venn diagrams as well as statistical and chemometric tools to detect specific A. flavus and maize metabolites produced upon infection.
