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Abstract
Denitrification is responsible for converting soluble nitrogen to gaseous forms as part of the nitrogen cycle. Denitrification has major implications for trends in intensive agriculture and global warming. Denitrification is primarily performed by specific types of microbes, including bacteria, archaea, and fungi. My research focused on fungal denitrification. I aimed to gain a holistic understanding of the denitrification pathway in the soil-borne maize pathogen Fusarium verticillioides. Prior to my involvement in the project, genes highly up-regulated ( 8-fold) in response to hypoxia and/or nitric oxide were found through transcriptome analysis of wild type F. verticillioides. This collection of genes was presumed to be directly or indirectly tied to fungal denitrification, nitrogen metabolism, and/or nitric oxide detoxification of reactive nitrogen oxides. My phylogenetic analyses revealed that fungal orthologs of dissimilatory nitrite reductase (DNI1) and nitric oxide reductase (NOR1) are nearly exclusive to the Ascomycota, and the ability to denitrify is limited to a select number of genera. Further, within the genus Fusarium, only select species complexes harbor denitrifiers. Prior to my arrival at UGA, some genes from the RNA-seq dataset hypothesized to be involved in denitrification were deleted using Agrobacterium-mediated fungal transformation. My research evaluated these deletion mutants for their potential to grow under low-oxygen conditions, emit N2O, produce fumonisins, and infect maize seedlings. Deletion of genes within the denitrification pathway had a marginal effect on fumonisin production and virulence. However, deletion of NOR1, a cytochrome P450 nitric oxide reductase, caused a significant decrease (99%) in N2O production from F. verticillioides. Adding FvNOR1 back into the deletion mutant background using protoplast complementation restored the wild type phenotype and conclusively demonstrated the genetic role of NOR1 in N2O production. Thus, NOR1 has been identified as a key target for mitigation of N2O emissions from agricultural fields where Fusarium species are commonplace. My research increases understanding of the denitrification pathway in ascomycetes, assesses the phylogenetic limitedness of denitrification in fungi, and identifies a novel target for inhibition of fungal-derived agricultural N2O emissions. My research results are anticipated to enable development of new solutions for mitigation of N loss and N2O emissions.