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Research Project: Eliminating Fusarium Mycotoxin Contamination of Corn by Targeting Fungal Mechanisms and Adaptations Conferring Fitness in Corn and Toxicology and Toxinology Studies of Mycotoxins

Location: Toxicology & Mycotoxin Research

2017 Annual Report

1a. Objectives (from AD-416):
Objective 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. Sub-objective 1.1 – Identify and characterize hydrolytic lactamases conferring adaptive advantages to F. verticillioides. Sub-objective 1.2 – Determine if F. verticillioides produces quorum sensing or quorum sensing inhibitory compounds in vitro and during endophytic colonization of corn. Sub-objective 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). Objective 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. Sub-objective 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. Sub-objective 2.2 – Evaluate competitive interactions between F. verticillioides and A. zeae and profile their transcriptional and metabolic responses. Objective 3: Develop and improve control strategies for mycotoxin contamination by targeting fungal-specific enzymatic activities, using molecular technologies such as host-induced gene silencing. Sub-objective 3.1 – Develop and express RNAi silencing constructs for in vitro growth inhibition of F. verticillioides. Sub-objective 3.2 – Develop and transform into corn functional vector(s) for host-induced gene silencing (HIGS). Sub-objective 3.3 – Testing transgenic corn lines for resistance to F. verticillioides.

1b. Approach (from AD-416):
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.

3. Progress Report:
Fusarium (F.) verticillioides and other mycotoxigenic and plant pathogenic Fusarium species are incredibly well adapted to their hosts and general environments. They have developed complex metabolic strategies for competition, survival, and proliferation. For example, F. verticillioides possesses 46 genes putatively encoding lactamases that may confer tolerance to xenobiotic compounds. Using our OSCAR-based gene deletion strategy, we have created deletion mutants for most of these genes, and this library of mutants will allow us to test various lactam compounds for their antifungal effects and identify what genes may confer any resistance. We have identified lactam compounds that inhibit the growth of F. verticillioides, and these are being investigated further for their potential utility as antifungal treatments. Further, RNA-seq data are being analyzed for experiments involving exposure of F. verticillioides to various lactam compounds, including pyrrocidines, the antibiotic compounds produced by Acremonium (A.) zeae.RNA-seq data are also available from co-culturing F. verticillioides with A. zeae. In terms of microbial interactions, we have utilized a modified biosensor system to show 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 abilities 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 most are now deleted so that we can fully characterize this physiological process of hypoxia-induced nitrate respiration and production of nitrous oxide (N2O). The instrumentation for measuring N2O is not yet operational but is being configured. We have developed the in vitro assays to phenotype and characterize the deletion mutants under various nutrient and oxygen conditions, including hypoxia (5% oxygen) using a custom-built chamber housing a Bioscreen instrument for real-time, quantitative assessment of growth. Multiple RNA-seq datasets have been generated and are being analyzed. We have demonstrated the F. verticillioides denitrification genes are highly induced in response to nitric oxide (NO) exposure even when oxygen levels are normal, indicating this pathway is not strictly related to hypoxic respiration but is also responsible for NO detoxification. Experiments to evaluate the impact of denitrification on interactions with corn plants are scheduled to begin once in vitro characterization is complete. This objective involves collaboration with the Mycotoxin Prevention and Applied Microbiology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois. The genomes of multiple strains of A. zeae, including strain NRRL 6415, were sequenced to enhance our ability to identify common genes shared by A. zeae and F. verticillioides. We have successfully determined that two strains of A. zeae, NRRL 13540 and NRRL 31242, have a FDB1 gene cluster that is clearly syntenic with the FDB1 cluster of F. verticillioides. We have previously shown that this cluster was horizontally acquired by Colletotrichum graminicola from a progenitor of F. verticillioides. It now appears A. zeae is also part of the evolutionary history of this gene cluster. We have attempted to generate an A. zeae deletion mutant for the lactamase gene in its FDB1 cluster, but to-date this has not been successful. We may attempt this again with a different strain. Based on our characterization of this gene in F. verticillioides, we expect the A. zeae mutant to be sensitive to a lactam antifungal phytochemical produced by corn. In addition, we are currently analyzing the A. zeae genomes for secondary metabolite gene clusters with the goal of identifying candidate clusters responsible for biosynthesis of the pyrrocidines. Competitive interactions between F. verticillioides and A. zeae are being studied, including the impact of the antifungal pyrrocidine metabolites produced by A. zeae. A single RNAi silencing construct targeting the three Cyp51 genes of F. verticillioides has been made, and in vitro testing will begin soon to demonstrate inhibition of growth. This silencing plasmid and a plasmid for over-expression of the triple Cyp51 sequence have been provided to our collaborator at the Donald Danforth Plant Science Center (St. Louis, Missouri) where transgenic corn lines are being generated. The over-expression plasmid will be used to test for any phenotypic interference in the resulting transgenic lines of corn.

4. Accomplishments
1. Reduction of nitrate and reactive nitrogen compounds and the impact on fumonisin mycotoxin contamination of corn. Historically, bacteria have long been noted as the primary microbes responsible for denitrification, which under low oxygen results in release of atmospheric nitrogen. In the past few years, studies have shown that fungi may actually be the primary denitrifying microbes, but instead of releasing nitrogen, fungi release the major greenhouse gas nitrous oxide. Such emissions by fungi may explain agriculture’s significant contribution to high global nitrous oxide levels. ARS researchers in Athens, Georgia found that Fusarium verticillioides, a fumonisin mycotoxin producing pathogen of corn, is among a limited number of soil fungi having the full set of denitrification genes that can convert nitrate into nitrous oxide and whose overall fitness may be related to this conversion. The scientists have functionally characterized the responsible genes with the ultimate goal of identifying inhibitors of denitrification as a strategy to reduce global nitrous oxide emissions and reduce the fitness of F. verticillioides. Such inhibition and reduced fitness should also result in reduced production of fumonisin mycotoxins, thus resulting in corn that is safer for human and animal consumption.

Review Publications
Bolton, S.L., Mitchell, T.R., Brannen, P.M., Glenn, A.E. 2017. Assessment of mycotoxins in Vitis vinifera wines of the Southeastern United States. American Journal of Enology and Viticulture. doi:org/10.5344/ajev.2017.16089.
Bacon, C.W., Hinton, D.M., Mitchell, T.R. 2016. Quorum signaling mycotoxins: A new risk strategy for bacterial biocontrol of Fusarium verticillioides and other endophytic fungal species? Journal of Agricultural and Food Chemistry. doi:10.1021/acs.jafc.6b03861.
Gold, S.E., Glenn, A.E., Paz, Z. 2017. Rapid deletion production in fungi via Agrobacterium mediated transformation of OSCAR deletion contructs. Journal of Visualized Experiments. 124:e55239. doi:10.3791/55239.
Blacutt, A.A., Mitchell, T.R., Bacon, C.W., Gold, S.E. 2016. Bacillus mojavensis RRC101 lipopeptides provoke physiological and metabolic changes in the course of antagonism against Fusarium verticillioides. Molecular Plant-Microbe Interactions. 29(9):713-723. doi:10.1094/MPMI-05-16-0093R.