Location: Toxicology & Mycotoxin Research
2019 Annual Report
Objectives
1. Determine the evolutionary history and molecular genetics of metabolic and developmental features enhancing the fitness of mycotoxigenic Fusarium (F.) species, including such areas as xenobiotic tolerance, denitrification, and nitric oxide detoxification and the contribution to greenhouse gas emission.
1.1. Identify and characterize hydrolytic lactamases conferring adaptive advantages to F. verticillioides.
1.2. Determine if F. verticillioides produces quorum sensing or quorum sensing inhibitory compounds in vitro and during endophytic colonization of corn.
1.3. Evaluate denitrification by Fusarium species and its impact on competitive fitness, in planta production of mycotoxins, and the production of the potent greenhouse gas, nitrous oxide (N2O).
2. Evaluate the influence of a common niche on the evolution and adaptation of two co-occurring, seed-borne, metabolically active maize endophytes, Acremonium (A.) zeae and Fusarium (F.) verticillioides.
2.1. Utilize comparative genomics to determine if F. verticillioides and A. zeae share gene clusters or other features that correlate to corn as their common host.
2.2. Evaluate competitive interactions between F. verticillioides and A. zeae and profile their transcriptional and metabolic responses.
3. Develop and improve control strategies for mycotoxin contamination by targeting fungal-specific enzymatic activities, using molecular technologies such as host-induced gene silencing.
3.1. Develop and express RNAi silencing constructs for in vitro growth inhibition of F. verticillioides.
3.2. Develop and transform into corn functional vector(s) for host-induced gene silencing (HIGS).
3.3. Testing transgenic corn lines for resistance to F. verticillioides.
4. Determine the interactions between fumonisin exposure and dietary factors on fetal and postnatal development using animal models to provide basic information for ongoing translational human studies.
5. Determine the efficacy of cooking methods to detoxify mycotoxins in co-contaminated corn using an in vivo rodent bioassay approach incorporating biomarkers.
Approach
1. Lactamase genes in Fusarium (F.) verticillioides confer resistance to environmental lactam-containing antibiotic compounds. F. verticillioides metabolites impact quorum sensing related activities. Fusarium species, notably F. verticillioides, have an active denitrification pathway that is linked to nitric oxide detoxification.
2. Association of F. verticillioides and Acremonium (A.) zeae with the common host (corn) resulted in the two fungi sharing highly homologous genes or gene clusters. F. verticillioides and A. zeae antagonistically interact with distinct transcriptional and metabolic reprogramming.
3. Construct silencing vectors, express in vitro, and conduct assays exposing F. verticillioides to the RNAi transcripts. Silencing constructs having in vitro efficacy will be transformed into corn. Lines of transformed corn will be screened for reduced infection, disease, and fumonisin accumulation.
4. Compare dose-response in mouse strains sensitive (LM/Bc) and insensitive (SWV) to neural tube defect induction by fumonisin B1. Compare dose-response for neural tube defect induction and selected gene expressions in fumonisin B1-exposed mice fed folate deficient or folate sufficient diets. Compare neonatal growth rates in offspring of mice fed diets containing fumonisins.
5. Determine the efficacy of alkaline cooking (nixtamalization) to detoxify corn
contaminated with aflatoxin alone or co-contaminated with aflatoxin and fumonisin
using a rat feeding bioassay.
Progress Report
Soilborne plant pathogenic fungi are well-adapted to their environments, which include their host(s) and the soil in which they reside. These fungi have developed complex metabolic strategies for competition, survival, and proliferation. For example, Fusarium verticillioides possesses 46 genes putatively encoding lactamases that are hypothesized to confer tolerance to antifungal compounds produced by the host plant (corn) or more frequently to competitor microbes in the cornfield soil environment. Using a gene deletion strategy developed by the Unit, deletion mutants for most of these lactamase genes have been created, and this library of mutants has allowed the test of various lactam compounds for their antifungal effects and the identification of what genes confer resistance to the lactams. This research aids the identification of potential antifungal treatments.
Regarding microbial interactions, a modified biosensor system was utilized by ARS researchers to demonstrate that mycotoxins and other fungal secondary metabolites can inhibit bacterial quorum sensing, thus limiting the growth and other biological features of the bacteria. Quorum sensing is a microbial mechanism used to synchronize all physiological activities within a population in order to enhance the ecological fitness capabilities of that organism, and we now have greater understanding of how F. verticillioides may modulate bacterial populations.
Further, we are investigating fungal denitrification, another unique physiological activity of Fusarium species found only in a few other fungal groups. The F. verticillioides genes conferring denitrification have been identified and are being deleted so that the mutants can be functionally studied to experimentally assess the role of the genes in the physiological process of hypoxia-induced nitrate respiration and production of nitrous oxide. A Headquarters-funded Research Associate was onboarded to work on this project along with a PhD student from the University of Georgia. We are continuing to develop and modify in vitro assays to phenotype and characterize the deletion strains under various nutrient and oxygen conditions using a custom-built enclosed system for controlled atmosphere studies that allow for real-time, quantitative assessment of growth. Research into identification of inhibitors of fungal denitrification have begun.
The genomes of multiple strains of Sarocladium zeae (formerly Acremonium zeae) were sequenced to enhance our ability to identify common genes shared by S. zeae and F. verticillioides since both fungi commonly infect corn kernels. Our hypothesis is that corn, as a common host, is a driving factor in the evolution of these fungal genomes. We have successfully determined that S. zeae possesses a gene cluster that is evolutionarily related to the same cluster of genes in F. verticillioides. One of the cluster genes (MBL1) in F. verticillioides was shown to confer resistance to antimicrobial chemicals produced by corn, including the lactam 2-benzoxazolinone (BOA). We earlier attempted to generate a S. zeae deletion mutant for its MBL1 gene to test if it plays a similar role in this fungus, but, while we generated hundreds of transformants, none had the deletion. To overcome this bottleneck, we employed a CRISPR-Cas9 approach for gene editing in S. zeae. This is a powerful new tool for genetic modifications. This approach was successful, and we have now shown that, indeed, the S. zeae MBL1 gene confers resistance to BOA in that fungus as well. Further, we have analyzed S. zeae genomes for secondary metabolite gene clusters with the goal of identifying candidate clusters responsible for biosynthesis of the pyrrocidine metabolites. Three clusters are good candidates and are being targeted for gene mutation studies and characterization with regard to pyrrocidine production. Further, competitive interactions between F. verticillioides and S. zeae are being studied, particularly with regard to using S. zeae as a biocontrol agent to reduce both the fumonisin contamination of corn kernels and the infection of kernels by F. verticillioides. Gene transcription and metabolite production data were generated for the interaction of these two fungi and will aid in our evaluation of S. zeae in a biocontrol strategy. We have also performed growth chamber co-inoculation studies to assess biocontrol potential. To-date, the results have been encouraging. Lastly, we have shown that pyrrocidines dramatically suppress fumonisin production by F. verticillioides. This has great potential as a new control strategy for minimizing fumonisin contamination of corn. More details are given below in this report, including an Invention Disclosure submitted to The National Chemical Patent Committee.
Accomplishments
1. Discovery of a new strategy for suppressing production of fumonisin mycotoxins. Fungi such as Fusarium verticillioides are major concerns for food safety because of the mycotoxins they produce, most notably the fumonisins that cause a number of species-specific animal diseases and may cause neural tube birth defects, stunting, and esophageal cancer in humans. Control of the fungus and preventing the contamination of food and feed with fumonisins are challenging goals, and current methods for control and prevention are limited. The economic impact of mycotoxins is difficult to estimate due to the multifaceted nature of plant disease combined with contamination of the crops with the mycotoxins, but studies suggest fumonisin results in $20 million to $200 million in losses annually. ARS researchers in Athens, Georgia, have discovered a group of chemical compounds that inhibit the production of fumonisins by F. verticillioides. These are the pyrrocidines produced by another corn kernel inhabiting fungus, Sarocladium zeae. This fungus does not cause any plant disease symptoms and does not produce any known mycotoxins. The potential use of pyrrocidines as inhibitors of fumonisin production could have immense impact on the safety and value of corn and its processed products by reducing and managing fumonisin contamination. This would be a significant contribution to food safety and provide needed economic relief to corn growers and processors.
Review Publications
Bacon, C.W., Hinton, D.M. 2019. Fungal and bacterial maize kernel interactions with the vertically transmitted endophytic state of Fusarium verticillioides. In: Verma S., White, Jr J., editors. Seed Endophytes. Switzerland, AG: Springer Cham. p.191-209. https://doi.org/10.1007/978-3-030-10504-4_10.
Gao, S., Gold, S.E., Wisecaver, J.H., Zang, Y., Guo, L., Ma, L., Rokas, A., Glenn, A.E. 2019. Genome-wide analysis of Fusarium verticillioides reveals inter-kingdom contribution of horizontal gene transfer to the expansion of metabolism. Fungal Genetics and Biology. 128:60-73. https://doi.org/10.1016/j.fgb.2019.04.002.
Hanlin, R.T., Owens, J.O., Icard, A.H., Glenn, A.E., Gonzalez, M.C. 2018. Observations on the biology of Ophiodothella angustissima. North American Fungi. 13(2):1-9.
