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Our first objective is to investigate oxylipin recognition by Aspergillus receptors encoded by the genes gprA and gprD. We propose to build a simple colorimetric bioassay for receptor/ligand interactions: a yeast reporter strain into which we can insert Aspergillus G protein-coupled receptors (GPCRs), and look for their response to host and fungal oxylipin derivatives via activation of a yeast reporter gene. We will begin by testing two GPCRs (GprA and GprD) from A. nidulans in the yeast reporter to confirm and expand our previous results, which suggested that these are likely plant oxylipin receptors. <P>Our second objective is to query A. flavus GPCRs for activation by oxylipins. Homologs of the A. nidulans genes, gprA and gprD, are present in A. flavus. We will clone these homologs into the yeast reporter and test their response to pure oxylipin compounds, endogenous oxylipin extracts, and extracts from maize lines altered in their oxylipin profiles.<P> This work is funded by a seed grant to enable foundation-building for future studies. The ultimate goal of understanding Aspergillus GPCR/oxylipin interactions is the eventual design of strategies for control of aflatoxin contamination of seed. <P>At least two outcomes of this work will seed future work (beyond the scope of this proposal) by the PD. First, the currently proposed work will generate a bioassay by which Aspergillus membrane receptors could be screened in high-throughput fashion against large chemical libraries to identify compounds that disrupt oxylipin binding.<P> Second, very little is understood about GPCR/ligand pairs for filamentous fungi, outside of mating factors and their cognate pheromone receptors. <P>We wish to further the understanding of signal perception in fungi - specifically, in the important pathogen A. flavus.
Non-Technical Summary: Aspergillus is a fungal species that infects nuts and grains, and contaminates them with a carcinogenic/lethal secondary metabolite called aflatoxin. Aflatoxin biosynthesis is regulated in part by chemical signals called oxylipins that are secreted by both Aspergillus and its plant hosts. This proposal focuses on the perception of oxylipin signals by Aspergillus. We propose to build "reporter strains", comprising Aspergillus membrane receptors expressed in yeast cells, which will reveal oxylipin/receptor interactions via color changes in the yeast reporter cells. We will use these reporters to i) characterize recognition of fungal oxylipins by membrane receptors in Aspergillus, and ii) characterize recognition of plant oxylipins by membrane receptors in Aspergillus. Our ultimate goal is to understand seed/Aspergillus crosstalk at the molecular level, and use our results from this work in future studies, in order to identify other natural and synthetic compounds that activate or block Aspergillus oxylipin receptors. Such compounds may be candidates or leads for treatments whereby to control aflatoxin contamination of food and feedstuffs. <P> Approach: Many functional assays exist for testing GPCRs against potential ligands. We will employ a functional assay which has been successfully used by others to identify ligands of GPCRs from filamentous fungi. This assay exploits the pheromone mating signal transduction pathway from the yeast S. cerevisiae. In S. cerevisiae MATa cells, perception of alpha-factor pheromone by the alpha-factor receptor Ste2p (a GPCR) activates a MAP kinase cascade leading to expression of genes for cell cycle arrest, mating, and karyogamy. Our reporter strains will replace the native Ste2p gene with Aspergillus GPCR cDNAs for GprA and GprD. We will then treat the yeast reporter strains with oxylipins that we hypothesize are ligands for each Aspergillus GPCR in question: i) the plant oxylipins 9S-HPODE and 13S-HPODE, ii) purified native Aspergillus oxylipins, and iii) oxylipin extracts from wild type and LOX-deleted maize lines. Activation of the yeast reporter strains will be assessed via beta-galactosidase activity of a Ste2p-responsive reporter gene, FUS1-lacZ. Verification of positive results will be done by measuring one, or both, of two hallmark events in G protein signaling pathway activation: G-protein activation and cAMP accumulation. We will perform standard biochemical assays to measure either cAMP accumulation or G protein activation i) on GPCRs isolated within yeast reporter strains, and ii) in native A. flavus (WT vs. GPCR mutants), to verify putative GPCR activation by oxylipin ligands.