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<OL> <LI> Understand the molecular basis for fungal responses to conditions encountered
during invasion of crops in order to identify and modify the factors in corn and
other crops that could induce or impede aflatoxin formation or fungal development.
Accomplishment of objective 1 could lead to generic approaches to enhance resistance
in all crops vulnerable to aflatoxin contamination. <LI> Determine, by genetic and
physiological studies of diverse aflatoxin producing species, whether aflatoxin
production provides an adaptive advantage for fungal survival and invasion of crops,
particularly because many natural isolates of A. flavus do not produce aflatoxins.
<LI> Determine the molecular responses of aflatoxin producing fungi to stress factors,
particularly with regard to developing an understanding of the ability of the fungi
to adapt and produce toxins. <LI> Undertake and utilize newly available sequences of
genomic DNA from Aspergillus species to develop rapid and highly sensitive PCR based
tests to identify aflatoxigenic fungi and their toxins in contaminated food products.
Approach: <BR> (1) Microarray experiments will be used to identify candidate genes in A. flavus that
are turned on or off during a variety of environmental and nutritional conditions
that are known to alter gene expression affecting aflatoxin biosynthesis. The DNA
microarrays will exploit the available genomic resources of A. flavus ESTs and the
whole genome sequences, combined with statistical analysis of up and down regulated
signals on the chips. The candidate genes identified from microarrays (both A. flavus
EST and whole genome) will be verified or confirmed through RT-Q- PCR or other well
established methods. For further analysis, to identify specific genes, targeted gene
mutagenesis will be necessary to determine their biological function. Comparison of
the gene expression data under these aflatoxin-producing and non-producing conditions
will allow us to identify the specific genes expressed under aflatoxin-producing
conditions. <BR><BR>(2) The adaptive advantage of aflatoxin production in certain
environments will be determined by genetic and physiological studies. It will be
ascertained if gene cluster imparts some fitness advantage in some environmental
niches, particularly when aflatoxin production does not appear to be a virulence
factor for crop infestation. Isogenic isolates will be developed in A. flavus for
this experiment. Fungal viability will be measured for up to 12 months with these
isolates. The measure of longevity proposed in the present study as a measure of
fitness will be an important determinant for understanding aflatoxin production in
Aspergillus populations in crop soils. Comparisons of genomic sequences will be made
between toxigenic A. flavus found in agricultural fields and other Aspergillus
species (non-toxigenic and domesticated) to determine what genes are involved in
fungal virulence and toxin production of A. flavus, as well as its ability to survive
in the field. <BR><BR>(3) Stress cues that change the activity of proteins in the
biosynthetic pathway and gene transcription will be evaluated. The chromatin
structure of the gene cluster and adjacent regions will be studied after exposure to
aflatoxin inducing conditions. Parameters such as DNA methylation patterns, that
alter chromatin structure, will be explored in these adjacent regions to determine
how aflatoxin biosynthesis is turned on in the fungus. Understanding environmental
stress cues affecting aflatoxin gene expression may help to develop strategies to reduce aflatoxin contamination of corn under field conditions. It will be determined
if the drought tolerant varieties have lower levels of aflatoxin contamination
because the host-fungus interaction somehow alters the ability of the fungus to
initiate toxin production at the DNA level. <BR><BR>(4) The use of PCR to quantify the level
of toxigenic fungi in foods through the use of Taqman primer-probe assay will be
assessed to provide information on fungal bioburden, but not the level of aflatoxin
contamination. The test will be designed to measure the potential of the commodity to
become severely contaminated with the fungus (and consequently toxin) if stored under
conditions suitable for subsequent growth of the fungus.