Location: Food and Feed Safety Research2010 Annual Report
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.
3. Progress Report
The research group at Northern Illinois University (NIU) is investigating genetic regulatory mechanisms that control the detrimental impact of fungal species that are of agricultural importance, including Aspergillus flavus. One of our major interests is the study a novel regulatory system, called velvet (VeA), unique to fungi like Aspergillus flavus, with high potential to control plant diseases. We have demonstrated that velvet is vital for the production of aflatoxin, cyclopiazonic acid and aflatrem mycotoxins by Aspergillus flavus. We also showed that VeA is indispensable for the production of sclerotia, resistant structures formed by Aspergillus flavus. Furthermore, recent data indicated that VeA is necessary for maximum pathogenicity by the fungus. Virulence of Aspergillus flavus on peanut and corn seeds was reduced in the absence of the veA gene product. Our studies indicate that production of key enzymes necessary for infection of plant tissue is decreased if this veA gene is deleted compared to the control strains, particularly amylase (starch breakdown) activity. In addition, parallel functional genomic studies (studies to determine the functionality of genes) comparing Aspergillus flavus wild type (infact gene) with a veA deletion fungal strain were performed to elucidate veA-affected regulatory pathways that might be involved in controlling toxin production, sporogenesis (spore or seed formation), or formation of sclerotia (fungal survival structures). The results from this study indicated that genes involved in nitrogen metabolism are regulated by veA, including niaD, encoding a nitrate reductase (involved in nitrogen metabolism). In this experiment, niaD expression was significantly lower in the veA mutant compared to the wild-type. Microscopic examination of sclerotia showed that the niaD mutant produced fewer and smaller sclerotia than the wild-type strain. Analysis of conidial (spore) formation and aflatoxin production showed that the niaD mutant (where this gene is rendered non-functioanl) produced about 10-fold more conidia than the wild-type, but less aflatoxin. These results indicate that nitrogen metabolism plays a role in both secondary metabolism and development in Aspergillus flavus and that expression of some of the genes involved are veA-dependent. We are also charactering a new secondary metabolism gene cluster containing a gene whose expression, according to our microarray analysis, is veA-dependent. Other selected genes from these functional genomic studies will be characterized in the near future. In addition, we are evaluating the effect of osmotic and oxidative stress in Aspergillus flavus mycotoxin production. Research progress was monitored through teleconferencing, frequent email communications, and reports.