Location: Toxicology & Mycotoxin Research2016 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:
A total of 46 Fusarium (F.) verticillioides genes putatively encoding lactamases have been identified, and gene deletion mutants have been created for half of these. 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. Research is also underway indicating the quorum sensing activity of fungi and their secondary metabolites. Additional research supporting the unique physiological activity of fungi is that Fusarium species are among only a few fungi having a fully functional denitrification pathway. The F. verticillioides genes conferring denitrification have been identified and are being deleted so that we can fully characterize this physiologically important process of hypoxia-induced nitrate respiration and production of nitrous oxide (N2O). We have developed the in vitro assays to phenotype and characterize the deletion mutants. 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. This objective involves collaboration with the Mycotoxin Prevention and Applied Microbiology Research Unit, NCAUR, Peoria, Illinios. The genomes of multiple strains of Acremonium (A.) zeae, including NRRL 6415, are being sequenced to enhance our ability to identify common genes shared by A. zeae and F. verticillioides. We have already successfully determined that two strains of A. zeae, NRRL 13540 and NRRL 31242, have the FDB1 gene cluster. We have previously shown that this cluster was horizontally acquired by Colletotrichum graminicola from a progenitor of F. verticillioides. We will generate an A. zeae deletion mutant for a lactamase gene in its FDB1 cluster. 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. We are basing our strategy on a recent publication and have identified the target genes for silencing. We have not yet had an opportunity to generate the appropriate construct but are planning this in the near future.
1. Fungi are also capable of denitrification, and not just under low oxygen conditions. Bacteria have long been noted as the primary microbes responsible for denitrification, which is nitrate respiration under low oxygen resulting in release of atmospheric nitrogen (N2). In the past few years studies have shown that fungi may actually be the primary denitrifying microbes, and instead of releasing N2, fungi release the greenhouse gas N2O. Such emissions by fungi may explain agriculture’s significant contribution to global N2O levels. We have found that Fusarium (F.) species such as F. verticillioides are among a limited number of fungi having the full set of genes conferring denitrification and that these genes are highly expressed when the fungus is exposed to nitric oxide (NO). Further, NO induces these genes even when oxygen is available, indicating the pathway is not strictly a low oxygen respiration pathway but instead also functions to detoxify NO. We are fully characterizing these genes and will be seeking inhibitors of the encoded enzymatic functions as a strategy to reduce N2O emissions and also reduce the fitness of F. verticillioides. Such inhibition and reduced fitness should result in reduced production of fumonisin mycotoxins, thus resulting in corn that is safer for human and animal consumption.
2. Horizontal transfer of a gene cluster among three fungal pathogens of corn. ARS researchers in Athens, Georgia, and Peoria, Illinois, have discovered the presence and structural conservation of a gene cluster in Acremonium (A.) zeae, Colletotrichum (C.) graminicola, and Fusarium (F.) verticillioides that supports the hypothesis that corn, as the common host of these three fungi, is a driving force impacting the evolution of their genomes. Specifically they have shown the horizontal gene transfer of the FDB1 cluster between these distantly related fungi. Acquisition of the cluster presumably enhances their fitness by conferring the ability to degrade antifungal phytochemicals produced by corn. This discovery provides greater understanding of how these important plant pathogens have evolved and how corn-breeding efforts for increased production of these phytochemicals may have selected for these fungi and facilitated their frequency of plant infection.
3. Research under the advisement of ARS scientists in Athens, Georgia, has been recognized for its potential impact on soil-borne plant diseases and their control. A PhD student from the Department of Plant Pathology at the University of Georgia is conducting ARS-funded research related to Sub-objective 1.1 of our project plan, and this student has received a prestigious, highly competitive award from the Storkan-Hanes-McCaslin Research Foundation.
5. Significant Activities that Support Special Target Populations: