Abstract
Secondary metabolite production of the phytopathogenic ascomycete fungus Fusarium fujikuroi is greatly influenced by the availability of nitrogen. While favored nitrogen sources such as glutamine and ammonium are used preferentially, the uptake and utilization of nitrate is subject to a regulatory mechanism called nitrogen metabolite repression (NMR). In Aspergillus nidulans, the transcriptional control of the nitrate assimilatory system is carried out by the synergistic action of the nitrate-specific transcription factor NirA and the major nitrogen-responsive regulator AreA. In this study, we identified the main components of the nitrate assimilation system in F. fujikuroi and studied the role of each of them regarding the regulation of the remaining components. We analyzed mutants with deletions of the nitrate-specific activator NirA, the nitrate reductase (NR), the nitrite reductase (NiR) and the nitrate transporter NrtA. We show that NirA controls the transcription of the nitrate assimilatory genes NIAD, NIIA, and NRTA in the presence of nitrate, and that the global nitrogen regulator AreA is obligatory for expression of most, but not all NirA target genes (NIAD). By transforming a NirA-GFP fusion construct into the ΔNIAD, ΔNRTA, and ΔAREA mutant backgrounds we revealed that NirA was dispersed in the cytosol when grown in the presence of glutamine, but rapidly sorted to the nucleus when nitrate was added. Interestingly, the rapid and nitrate-induced nuclear translocation of NirA was observed also in the ΔAREA and ΔNRTA mutants, but not in ΔNIAD, suggesting that the fungus is able to directly sense nitrate in an AreA- and NrtA-independent, but NR-dependent manner.
Highlights
The phytopathogenic ascomycete fungus Fusarium fujikuroi produces a broad spectrum of interesting secondary metabolites (SM) including the phytohormones gibberellins (GA) and various pigments and mycotoxins
We identified the main functional components involved in nitrate uptake and assimilation, including the genes encoding the nitrate reductase (NR), nitrite reductase (NiR), the main nitrate transporter NrtA and the transcription factor (TF) NirA and studied their expression in response to the presence of nitrate, nitrite, and glutamine
This genomic arrangement is different from the one in A. nidulans, where NIAD, NIIA, and nitrite transporter (NRTA) are located on the same chromosome in close proximity forming a co-regulated gene cluster (Cove, 1979; Brownlee and Arst, 1983; Johnstone et al, 1990)
Summary
The phytopathogenic ascomycete fungus Fusarium fujikuroi produces a broad spectrum of interesting secondary metabolites (SM) including the phytohormones gibberellins (GA) and various pigments and mycotoxins. We have shown that the global nitrogen regulators AreA and AreB play major roles in regulating nitrogen-controlled SM clusters (Michielse et al, 2014). Addition of preferred nitrogen sources, such as glutamine, to cultures led to rapid down-regulation of several nitrogen-repressed SM genes, e.g., the GA biosynthetic genes. Cultures supplied with nitrate showed a significant. Regulation of Nitrate Assimilation in Fusarium fujikuroi delay in repressing these clusters, probably due to the time-consuming conversion of nitrate to glutamine (Wagner et al, 2013). The ammonium permease (MepB) and the glutamine synthetase (GS) were shown to be involved in sensing ammonium and glutamine, respectively, nothing is known about potential nitrate sensors. We wished to determine the molecular mechanisms involved in nitrate sensing, uptake, and gene regulation networks in F. fujikuroi
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