2007 Annual Report 1a.Objectives (from AD-416) To determine the molecular basis for the phenomenon of non-production of aflatoxin in certain members of the A. flavus group of the fungi with a view to understanding the global regulation of toxin synthesis and the convergent evolution of aflatoxin biosynthesis. 1b.Approach (from AD-416) The present proposal is centered upon previously isolated A. parasiticus sec- (for secondary metabolism minus) strains, that display altered morphology and sporulation. Recent work from our laboratory has shown that a mutation in aflR coding or promoter region is not responsible for the sec-phenotype. Yet, the aflR expression is lowered in the sec-strains. There is no expression of the aflatoxin pathway genes, and aflR overexpression does not reverse the sec- phenotype. Preliminary results have also revealed clear differences between the sec- and their parental sec+ (for secondary metabolism plus) total protein-profiles. Proteomics and microarray technology will be used to determine the differences between sec+ and sec- strains at the molecular level. These differences will provide insights into the global regulatory mechanisms governing aflatoxin synthesis. 3.Progress Report This report serves to document research conducted under a Specific Cooperative Agreement (SCA) between the Agricultural Research Service (ARS) and Xavier University, New Orleans, Louisiana. Additional details of research can be found in the report for the in-house project 6435-41420-005-00D, “Aflatoxin Control Through Targeting Mechanisms Governing Aflatoxin Biosynthesis in Corn and Cottonseed.” Aflatoxins are cancer-causing compounds produced by certain fungi (Aspergillus flavus) when they invade crops such as cotton, peanuts, corn, and treenuts. In this project, the mechanism of aflatoxin production in the fungus, Aspergillus flavus, was studied at the molecular level using previously isolated non-toxigenic A. parasiticus sec- strains (i.e. strains that have lost the ability to make compounds such as aflatoxins that are not required for growth or secondary metabolites). During the current period, this project focused on two specific aims: (1) To further analyze the possible role of regulatory genes that may control aflatoxin formation, namely laeA, aflR, and aflJ, on causing the sec- phenomenon, and (2) To conduct the microarray analysis of the sec- strains of both types: a) The sec- strains (called mac sec-) derived by physical manipulation (maceration) of the fungal mycelia (chain of cells) of the toxigenic (sec +) or b) sec- strains derived by treating the fungal mycelia with the compound 5-Azacytidine (called 5-Aza sec-). Reproducible data using various molecular techniques suggested that although the regulatory genes aflR, aflJ, and laeA are necessary for aflatoxin production, they are not sufficient. Additional factors are needed for complete regulation of the turning on and off of aflatoxin production. Additionally, for microarray analysis the following strains were used that have the same gene components (isogenic): wild type sec+, mac sec- and 5-Aza sec- strains derived from the parent strain called SU-1 were grown in defined liquid medium for two time points, 16 hours and 24 hours. High quality ribonucleic acid (RNA) was isolated from the frozen mycelia while aliquots of the remaining liquid medium were frozen for aflatoxin quantification. The entire experiment was conducted in six different batches. A total of 36 RNA samples (two time points or treatments, three strains = total 36 samples) were then used for microarray labeling and hybridizations. Preliminary results reveal clear differences between the three strains and importantly among the two types of sec- strains at the significance level of 0.0001%. Further analysis of these results is ongoing to determine which genes are affected either positively or negatively following the maceration or 5-Aza treatment of fungal mycelia. Progress by cooperators was monitored through requiring routine teleconferencing, meetings, and scientific presentations of information relating to the project at professional society meetings, conferences and the Annual Aflatoxin Elimination Workshop sponsored by industry stakeholders.