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Title: The role of Aspergillus flavus veA in the production of extracellular proteins during growth on starch substrates

item DURAN, ROCIO - Northern Illinois University
item GREGERSEN, SCOTT - Northern Illinois University
item SMITH, TIMOTHY - Northern Illinois University
item BHETARIYA, P - Northern Illinois University
item Cary, Jeffrey
item Harris Coward, Pamela
item Mattison, Chris
item Grimm, Casey
item CALVO, ANA - Northern Illinois University

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/4/2014
Publication Date: 3/4/2014
Citation: Duran, R.M., Gregersen, S., Smith, T.D., Bhetariya, P.J., Cary, J.W., Harris-Coward, P.Y., Mattison, C.P., Grimm, C.C., Calvo, A.M. 2014. The role of Aspergillus flavus veA in the production of extracellular proteins during growth on starch substrates. Applied Microbiology and Biotechnology. 98(11):5081-5094.

Interpretive Summary: This work describes experiments that have been conducted in an effort to better understand the genetic mechanisms that control the ability of the aflatoxin producing fungus, Aspergillus flavus, to invade plants. Aflatoxins are toxic and carcinogenic compounds often produced by this fungus during growth on crops such as corn, peanuts, cottonseed, and treenuts. Because of the potential health risks, aflatoxin contamination of food and feed crops is also of great economic importance to farmers who cannot sell their crops due to strict domestic and international regulatory guidelines with regards to aflatoxin contamination. We have identified a gene, designated veA, from A. flavus that is required for normal fungal development and aflatoxin production. In this study, we have shown that when veA is inactivated there is a reduction in the ability of the fungus to break down starch and proteins found in plant tissues due to a reduction in the activity of glucoamylases and proteases, respectively. In addition, we identified a number of other proteins that are absent/reduced in the veA mutant strain that may also be involved in A. flavus’ ability to infect plants. This work adds new information to the growing list of genes in A. flavus, controlled by VeA, that are necessary for the fungus to successfully survive in the field, invade plants and produce aflatoxins. This in turn will help in devising strategies for eliminating A. flavus aflatoxin contamination of food and feed crops.

Technical Abstract: The aflatoxin-producer and opportunistic plant pathogenic, filamentous fungus Aspergillus flavus is responsible for the contamination of corn and other important agricultural commodities. In order to obtain nutrients from the host A. flavus produces a variety of extracellular hydrolytic enzymes. Interestingly, A. flavus amylase and protease activity are dependent on the global regulator veA, a gene known to regulate morphogenesis and secondary metabolism in numerous fungi. Analysis of starch degradation by fungal enzymes secreted into broths of starch- or corn kernel-based media showed a notable accumulation of glucose in samples of the A. flavus control strain while the deletion veA sample accumulated high levels of maltose and maltotriose and only a small amount of glucose. Furthermore, SDS-PAGE and proteomics analysis of culture broths from starch- or corn kernel-based media demonstrated differential production of a number of proteins that included a reduction in the amount of a glucoamylase protein in the veA mutant compared to the control strain, while an alpha-amylase was produced in greater quantities in the veA mutant. Quantitative-real time PCR and western blot analyses using anti-glucoamylase or alpha-amylase antisera supported the proteomics results. Additionally, an overall reduction in protease activity was observed in the veA mutant including production of the alkaline protease, oryzin, compared to the control strain. These findings contribute to our knowledge of mechanisms controlling production of hydrolases and other extracellular proteins during growth of A. flavus on starch-based substrates and could potentially be used in strategies to reduce host colonization and aflatoxin contamination of crops.