Mycotoxins: Biosecurity, Food Safety and Biofuels Byproducts (NC129, NC1025)

Non-Technical Summary:<br/>
Aflatoxins (AFs) are group of mycotoxins that are highly toxic and carcinogenic. AFs can contaminate corn, cereals, peanuts and other oil-seed crops. The aflatoxigenic fungus Aspergillus flavus uses asexual spores as the primary means of dissemination and infecting hosts. This proposal aims at controlling both fungal dispersal and AF production. Recent studies have identified a novel class of fungus-specific regulators called velvet. The absence of certain velvet regulators causes multiple and severe defects in many fungi. In A. flavus, one of the velvet regulators VelB is essential for proper sporulation, spore viability, sclerotia (protective structures) formation and AF production. The central hypothesis of this proposal is that VelB is a DNA-binding protein regulating expression of various genes associated with sporulation and AF biosynthesis. This project will test this hypothesis via genetic, biochemical and genomic approaches. Results will reveal the mechanisms bridging sporulation and AF biosynthesis via identifying groups of genes that are controlled by VelB and defining the genetic networks regulating spore formation and AF production. As VelB (and other velvets) is specific to fungi, it can be an excellent broad-spectrum anti-fungal target, and outcomes will provide new insights into controlling AF contamination and fungal infestation in food and feed.
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Approach:<br/>
This project will investigate the molecular mechanisms of VelB (and VosA)-mediated regulation of sporulation, spore viability and aflatoxin production in the major toxigenic plant pathogen Aspergillus flavus. Expected results include better understanding of the functions of these novel fungus-specific regulators in governing dissemination and mycotoxin biosynthesis, identification of groups of genes that are controlled by the key regulator VelB, and defining the networks regulating spore formation and aflatoxin production. Understanding the mechanisms governing sporulation and toxin biosynthesis will reveal critical points in the regulation where targeted controls can be developed. Better understanding of how sporulation and toxin biosynthesis is regulated by a common controller in this fungus will not only lead to novel prevention strategies, but also advance our understanding of fungal pathogenesis and toxigenesis in general. Eventually, outcomes of the proposed studies will provide new insights into the development of safe and effective control strategies (e.g., new antifungal drugs or RNAi for velvet and/or their interactions) for fungal dispersion and AF contamination in fields with minimum effects on environmental quality and human health.
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Progress:<br/>
2012/10 TO 2012/12<br/>
OUTPUTS: In this project, we focus on revealing the regulatory mechanisms of sporulation and aflatoxin production in the major toxigenic fungus Aspergillus flavus. For the three month period in 2012, we investigated functions of the two velvet proteins VelB and VosA, which are key regulators of Aspergilli sporulation and secondary metabolism. Velvet regulators are conserved in most filamentous and dimorphic fungi. Based on A. nidulans and A. fumigatus velvet regulator research, we have identified these crucial regulators from A. flavus. Further expression analyses of key regulators, including abaA, brlA, velB, vosA, and veA, in A. flavus were conducted by Northern blotting during the lifecycle. These regulators show similar expression pattern during developmental process as A. nidulans. To further understand VelB and VosA function in A. flavus, we generated VelB and VosA deletion mutants by employing double-joint PCR. VelB deletion mutant shows significant reduction of conidiation capability and sclerotia formation in A. flavus, while VosA deletion mutant does not reveal differences of both phenotypes. In addition, we are working to generate VelB/VosA overexpression (multi-copy) mutants by inserting velB/vosA in the pRG3-AMA1 vector which contains the auxotroph marker pyr4 and the autonomous replicating sequence AMA1. Further phenotypic characterization and expression analyses throughout various stages of vegetative growth and post-developmental induction of A. flavus wild type and mutant strains will be carried out. This project conducts genetics, biochemical, and bioinformatic tools to facilitate the research of sporulation and aflatoxin production in A. flavus. Expected results include a thorough understanding of velvet regulators function, identification genes which are regulated by the velvet regulators, and defining the genetic networks regulating spore formation and aflatoxin production in A. flavus. PARTICIPANTS: Ming-yueh Wu, a second year PhD student Genetics, has been working on the project. This project involves conduction genetics, genomics, biochemistry, and bioinformatics research, and therefore provides a great opportunity to train a next generation scientist. TARGET AUDIENCES: Results generated from this work will be presented at national, international and regional meetings of relevant associations, i.e., The Genetics Society of America, Gordon Research Conference, and the Food Research Institute (FRI) annual meetings. The PI will interact with industry, government regulators, academia, and consumers on food safety issues and provide accurate, useful information and expertise through FRI annual meetings and Newsletters. The work will be submitted for publication in high-profile scientific journals. This project is also a fit for outreach science education. The different phenotypes of A. flavus mutants which are created in this project will be an attractive target for nearly all age groups and delight their interests in science. Students will be exposed to the beauty of genetics and the most up-to-date research concepts. PROJECT MODIFICATIONS: Not relevant to this project.
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IMPACT: Filamentous fungi, including their metabolites and enzymes, have been used by humans for benefits, including antibiotics, organic acids, pigments, and food additives. However, some filamentous fungi are pathogens, which result in agricultural loss, environmental damage, and adverse health effects on humans and animals. Because of the importance of filamentous fungi in human daily life, molecular tools have been developed to enable scientists to understand these microorganisms. The main reproductive mode of filamentous fungi is the formation of asexual spores. In some cases, the fungal secondary metabolites are highly related to development. Aspergillus flavus, an opportunistic pathogen of plants and humans, produces numerous secondary metabolites, including the most notorious, aflatoxin. Among mycotoxins, aflatoxin B1 is one of the most potent carcinogens and can contaminate oil-seed crops, such as corn, cereals, sorghum, and peanuts. Due to the carcinogenicity and toxicity, aflatoxins have been regulated by the USFDA since 1965. In 2003, mycotoxins, including aflatoxin, were estimated to cause a crop loss of $932 million per year in the United States. The cost of aflatoxin regulation and testing averages $466 million per year. Aside from economic loss, aflatoxins are also a threat to human life. Acute aflatoxicosis, associated with extremely high doses of aflatoxin, can leads to death in humans. Therefore, controlling both fungal dissemination and aflatoxin production is very important. Previous studies showed that fungal development and secondary metabolism are intimately associated via the activities of the novel velvet regulators. Velvet genes, including veA, velB, velC, and vosA, are highly conserved in many pathogenic fungi, and have been studied extensively in the model fungus Aspergillus nidulans. Understanding the function of these genes in A. flavus is of particular interest due to this species' agricultural and health impact, in particular its production of aflatoxin. In linking development and secondary metabolism the velvet genes are an ideal target for control strategies, as disruption of these genes can reduce the fungus's ability to spread and produce toxin. Here we investigated the roles of the velvet genes in Aspergillus flavus. The results show that the expression pattern of velvet regulators is similar to A. nidulans during the lifecycle, which implies that the velvet proteins' function may be highly conserved in all Aspergilli. Further functional analysis will be done by conducting phenotype and expression studies of the VelB/VosA deletion and overexpression mutants. To understand the regulation network of VelB and VosA, we will carry out a series of genomic studies. Understanding the mechanisms governing sporulation and aflatoxin biosynthesis will provide new insights into controlling detrimental activities of this agriculturally important fungus. Furthermore, this project will provide opportunities to promote excellence in science education and rigorous training of graduate and undergraduate students in the disciplines of microbiology, genetics and genomics.