1a.Objectives (from AD-416)
Identify key genes involved in regulation of oxidative stress, nitric oxide production, and nitrogen metabolism in A. nidulans and A. flavus whose expression are dependent on the presence of regulatory genes such as veA. Determine role of these genes in regulation of biological activities such as fungal toxin production, morphogenesis, and virulence.
1b.Approach (from AD-416)
Data acquired from A. flavus whole genome microarray/two-hybrid studies will be used to identify key genetic components of signaling pathways that control aflatoxin production and fungal morphogenesis. Using both A. flavus and the model fungus, A. nidulans, gene inactivation studies will determine the role of novel as well as previously characterized genes involved in fungal response to oxidative stress on toxin production and morphogenesis. The role of VeA and other regulatory proteins on production of hydrolytic enzymes involved in fungal pathogenesis of crop plants will be determined using molecular and biochemical techniques.
This collaboration addresses work described in objectives 1, 2 and 3 of the project plan to identify the molecular mechanisms that regulate the fungus-plant interaction and the production of aflatoxin. One goal of this reporting period was to determine why starch degradation is reduced in the Aspergillus (A.) flavus veA mutant (fungus where veA gene is defective) as production of starch degrading enzymes is required for successful infection of plant tissues in corn. The goals of this project were substantially met as assays for various enzymes involved in starch metabolism showed that an alpha-amylase or possibly a glucoamylase (enzymes that breakdown starch) was not as active in the A. flavus veA mutant strain and this indicates that VeA may control expression of genes involved in amylase degradation. Further enzyme assays are being conducted to better elucidate the enzyme(s) whose production is dependent on VeA. A second goal was to determine the effects of osmotic and oxidative stress in A. flavus mycotoxin production and the role that VeA plays in governing these effects. The research goals of this project have been partially met as growth experiments showed that the A. flavus veA mutant exhibits differential growth and developmental characteristics compared to the wild-type A. flavus under conditions of osmotic or oxidative stress. Gene expression studies are underway to identify which key genes involved in osmotic and oxidative stress are differentially regulated in the veA mutant compared to wild-type. A third goal of this reporting period was to generate A. flavus strains that can be used to study the interaction of key regulators of A. flavus development and secondary metabolism such as the genes LaeA and VeA with other transcription factors (genes) such as AflR, NsdD and NsdC. This goal has been substantially met as all needed plasmid vectors (vehicles for transportating genes into cells) were constructed and transformed into A. flavus. Research is currently underway to determine which of these regulatory factors physically interact with one another. A fourth goal of this reporting period was to study the role of nitric oxide in A. nidulans development and toxin production. This goal was substantially met as we found that nitric oxide induces resistant structure formation as well as changes in the biosynthesis of mycotoxins. A manuscript has been submitted for publication regarding this work. Research progress was monitored through teleconferencing, frequent email communications and reports.