1a.Objectives (from AD-416):
A) Sequencing and analysis of the whole genomes of additional aflatoxin-producing Aspergillus (A.) strains including both L and S type strains from A. flavus and A. parasiticus for genome wide comparison on their genome structure, gene sets, and gene functions; B) Identify gene and sets of genes involved in genetic regulation on aflatoxin formation, sclerotia development and pathogenecity; and C) Using Next Generation sequencing technologies to study fungus – plant (crop) interaction and to identify genes and factors that confer resistance against fungal infection in crops such as peanut or corn for devising strategies to eliminate or reduce aflatoxin contamination in food and feed.
1b.Approach (from AD-416):
The genome size of Aspergillus (A.) strain is about 36 Mega basepairs. Sequencing and assembly will be done by newer Next Generation Sequencing platform and at J. Craig Venter Institute (JCVI). The sequence will be annotated and the putative coding regions will be identified with the help of the A. flavus and A. oryzae gene model. Comparative analysis of the Aspergillus strains will be made in reference to the A. flavus and A. oryzae genome using the advanced software available at JCVI.
Both crop (peanut) and fungus (A. flavus) samples under infection condition will be collected. Total ribonucleic acid (RNAs) will be isolated from these samples. Next Generation Sequencing technologies (such as RNA-Seq by Illumina) will be used to determine transcriptional levels of all the genes expressed under those controlled conditions at a specific time point during fungal infection. Analysis and comparison on fungus and resistant vs. susceptible crop varieties will be performed to identify gene(s) involved in infection in fungus and resistance in crop plant at JCVI in the cooperator’s laboratory.
The major objective of this project is to sequence all the deoxyribonucleic acid (DNA) in the Aspergillus (A.) flavus genome (all its DNA) and to prepare microarrays (glass slides with DNA spots corresponding to all genes of this fungus). These arrays are for use in further research in understanding the aflatoxin contamination process in crops with a view of controlling aflatoxin contamination in corn, cotton, peanut, and tree nuts. Examining all the genes and the genetic machinery involved in the production of the harmful (carcinogenic) compound aflatoxin by the fungus A. flavus on crops, the entire fungal DNA was determined through whole genome sequencing at J. Craig Venter Institute (JCVI) in collaboration with North Carolina State University. Primary analysis of the DNA indicated that the A. flavus genome size is about 36.8 Mega Base pairs. Comparing the whole genome between the fungus A. flavus and A. oryzae (a non-toxigenic food grade industrial organism) has been completed. It has demonstrated that the two genomes are quite similar in genome structure, gene distribution, and gene homology. However, each species contains a unique set of about 300 genes. The ones in A. flavus may contribute to aflatoxin production. Using glass slides containing all the genes of the fungus (microarray fabricated at JCVI), genome wide gene profiling experiments have been conducted under specific conditions that favor aflatoxin production in the fungus. Genes and gene clusters that are putatively involved in aflatoxin formation have been identified. Several research papers have been published and a few manuscripts are under preparation. A peanut microarray containing majority of the peanut expressed sequence tag (EST) sequences has been constructed. Gene profiling using peanut microarray under infected conditions identified hundreds of peanut genes that are expressed in the peanut variety that shows resistance against A. flavus infection. In parallel with microarray gene profiling experiments, we have used Next Generation Sequencing technologies to identify genes potentially involved in aflatoxin production and fungal virulence. Preliminary results revealed that the mechanism of aflatoxin production is regulated by temperature by affecting the ratio of the two regulatory proteins (namely aflR and aflS) that are responsible for turning on of aflatoxin production genes. Under high temperature, the expression of specific regulatory genes, aflR and aflS, is significantly reduced. The change in ratio of aflR to aflS in high temperature is the main reason for shutting off of aflatoxin production. The DNA sequence data obtained from this project have been submitted to National Center for Biotechnology Information (NCBI) GenBank (genetic sequence) database. The microarray data are also submitted to the NCBI Gene Expression Omnibus (GEO) database. A database web server containing A. flavus EST and whole genome databases has been established at the Mid South Area Genomics Center for free access by USDA/ARS scientists.