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.
The research group at Northern Illinois University is investigating genetic mechanisms that control the detrimental impact of fungal species that are of agricultural importance, including aflatoxin-producing Aspergillus (A.) flavus. Our major interest is the study of the global fungal regulatory gene VeA (or velvet) and connected regulatory networks. This regulator is unique to fungi and it is conserved in other fungal species, particularly in the group called Ascomycetes. VeA is known to play a role in regulation of fungal development, secondary metabolism, and virulence and therefore, it has a high potential to be used to control plant diseases caused by Aspergillus and other fungi. We have investigated the effects of oxidative stress (type of stress involving reactive oxygen) in A. flavus and the role that VeA plays in the response to this stress. Our study revealed a reduction in survivability in the veA mutant as the concentration of the reactive oxygen species increased in the medium, compared to the control strain. This result indicates that the presence of veA is necessary for a normal adaptive response under exposure to oxidative stress. Furthermore, ribonucleic acid (RNA) analysis of veA mutant and control strain under oxidative stress indicated alterations in expression profiles of several genes involved in the HogA oxidative response signaling pathway (a type of network of proteins that is activated in response to external stresses on a cell) in the absence of veA. A manuscript reporting this work is also being prepared for publication. Also during this reporting period the study of A. flavus NsdD and NsdC regulatory genes and their role in morphogenesis (biological process involving development) and aflatoxin production has been published in Eukaryotic Cell. This work addresses research objectives 2 and 3 of Project Plan 6435-41420-005-00D. Studies have continued on the connection between veA and starch degradation by A. flavus. It is known that starch degrading enzymes are necessary for successful infection of plant tissues in corn. We observed that amylase activity (a type of enzyme activity needed for starch degradation) is significantly reduced in the veA mutant compared to the control strain in starch medium and also in corn medium. We have also detected alterations of protease activity, another enzyme possibly involved in successful fungal invasion, in the veA mutant compared to an A. flavus control. Culture supernatants of the veA mutant and control strain growing on corn or starch medium were analyzed for differences in proteins secreted into the growth medium. An initial set of data has been obtained and proteins putatively associated with hydrolytic activity in A. flavus have been identified. This work addresses research Objective 2 of Project Plan 6435-42000-020-00D. Additionally, the goal of performing in silico analysis (performed via computer simulation) of A. flavus secondary metabolism gene clusters #27 and #39 has also been accomplished. This analysis included structural genomics and comparative genomics using genomic (entirety of an organism’s hereditary information) databases from other fungal species. A manuscript on this analysis is now being prepared for publication. Furthermore, we have identified gene clusters similar to A. flavus cluster #39 that are partially conserved in other Aspergillus species. In collaboration with SRRC scientists we have also identified the compound produced by gene cluster #27. The compound is a pigment that is found in mature sclerotia (over wintering bodies or survival structures of fungi) of A. flavus and it appears to contribute to survival of sclerotia exposed to intense sunlight or drought conditions. This work has been recently submitted for publication in Fungal Genetics and Biology. Another two A. flavus secondary metabolite gene clusters are currently under study, clusters #23 and #14. This work addresses research outlined in Objective 1 of Project Plan 6435-41420-005-00D.