Location: Corn Host Plant Resistance ResearchTitle: A two-dimenstional proteome reference map of the aflatoxigenic fungus Aspergillus flavus Author
Submitted to: Proteomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/22/2013
Publication Date: 5/1/2013
Citation: Pechanova, O., Pechan, T., Rodriguez, J., Williams, W.P., Brown, A. 2013. A two-dimensional proteome map of the aflatoxigenic fungus Aspergillus flavus. Proteomics. 13:1513-1518. DOI 10.1002/pmic.201100659. Interpretive Summary: Aspergillus flavus is a widely distributed fungal pathogen that infects corn and other important agricultural commodities and contaminates them with aflatoxins. Aflatoxins are highly toxic and pose a serious health hazard to animals and humans when contaminated food and feed are consumed. Aflatoxins also have carcinogenic effects. The field of filamentous fungal proteomics is relatively young; however, several studies have been reported for Aspergilli. Proteomic studies of A. flavus are now facilitated by the availability of its genomic sequence. From the results of proteomic studies, we present the first proteome profile and 2-D proteome reference map of the whole cell mycelial extract of A. flavus wild-type strain NRRL 3357. The description of A. flavus proteome provides insight into its basic biology and a basis for future proteomic investigations. These investigations are important in understanding the associations between A. flavus and its plant hosts and ultimately in reducing aflatoxin contamination of food crops such as corn.
Technical Abstract: The description of A. flavus proteome provides insight into its basic biology and a basis for its future proteomic investigations. Aspergillus flavus is a widely distributed fungal pathogen that infects important agricultural commodities (maize, tree nuts, etc.) and contaminates them with aflatoxins that it produces as secondary metabolites. Aflatoxins are highly toxic and pose a serious health hazard to animals and humans via digestion of contaminated food and feed. They also have carcinogenic effects. The field of filamentous fungal proteomics is relatively young; however, several studies have been reported for the Aspergilli genus. Moreover, proteomic studies of A. flavus are now facilitated by the availability of its genomic sequence. We present the first proteome profile and 2-D proteome reference map of the whole cell mycelial extract of A. flavus wild-type strain NRRL 3357 that was obtained by 2-DE and MALDI-TOF-MSMS. Aspergillus flavus NRRL 3357 was cultured in Potato Dextrose broth at 28oC for 6 days. Mycelia were harvested, frozen in liquid nitrogen and stored at -80oC. For proteomic studies, 500 mg of mycelia from each triplicate growth was cryogenically ground to a fine powder, and proteins were extracted using TCA/acetone precipitation. Proteins were resolved using 2-D PAGE, and reproducible spots were excised, in-gel digested, and spotted on ABI 01-192-6-AB MALDI plate. Mass spectrometry was performed using ABI 4700 MALDI TOF/TOF mass spectrometer. Proteins were identified using Result Dependent Analysis of ABI GPS Explorer V3.6 software and MASCOT search algorithm against the Aspergillus flavus subset of UniProtKB database. Gene Ontology annotations for identified proteins were retrieved from the AgBase database. From 773 reproducible protein spots present on 2-D gels, 677 (87.6%) were identified and they corresponded to 538 distinct protein species that ranged in molecular masses between 5.8 and 213.6 kDa and isoelectric points from 4.1 to 11.4. Functional classification based on Gene Ontology annotations revealed that 471 proteins (87.7%) had assigned a total of 3980 GO terms. With regard to Molecular Function, the most terms belonged to catalytic activity (30.4%) and binding (19.7%) followed by nucleotide binding (11.8%), hydrolase (13.0%) and transferase activities (11.8%). For Biological Process, central metabolic pathways such as metabolism of carbohydrates, lipids, nucleic acid/nucleotides, amino acids, and proteins contributed with 7.8%, 0.4%, 7.7%, 5.7% and 5.2% of GO terms, respectively. Enzymes from glycolysis and TCA cycle dominated carbohydrate metabolism category and were among the most abundant spots on 2-D gels. A substantial number of terms belonged to biosynthesis (18.1%), including proteins from translation (ribosomal proteins, aminoacyl-tRNA synthetases, etc.), and biosynthesis of nucleotides, amino acid and vitamins. Approximately 6.7% of terms were allocated to transport and they contained porins, carriers and subunits of membrane-associated proteins from respiratory chain. Fewer terms (4.5%) were categorized into catabolism, and included proteases and subunits of proteasome complexes. Distribution of A. flavus proteins among cellular compartments revealed that majority of proteins had assigned general terms cell (17.9%), intracellular (16.9), cytoplasm (14.1%) and protein complex (10.6%). Mitochondrion, nucleus, ribosome, membrane and cytoskeleton accounted for 3.2%, 4.0%, 3.3%, 4.7% and 1.7% of GO terms, respectively. For this study, A. flavus was grown on PDB media, in which it has been known to produce aflatoxins. We indeed detected and identified O-methyltransferase A (omtA) and AflK/ vbs/ VERB synthase, two of the key enzymes in aflatoxin synthesis pathway. Proteins ver-1, norA, and aflatoxin B1-aldehyde reductase GliO-like are other gene candidates possibly related to aflatoxin metabo