Abstract
The rice pathogenic fungus Fusarium fujikuroi is well known for the production of a broad spectrum of secondary metabolites (SMs) such as gibberellic acids (GAs), mycotoxins and pigments. The biosynthesis of most of these SMs strictly depends on nitrogen availability and of the activity of permeases of nitrogen sources, e.g. the ammonium and amino acid permeases. One of the three ammonium permeases, MepB, was recently shown to act not only as a transporter but also as a nitrogen sensor affecting the production of nitrogen-repressed SMs. Here we describe the identification of a general amino acid permease, FfGap1, among the 99 putative amino acid permeases (AAPs) in the genome of F. fujikuroi. FfGap1 is able to fully restore growth of the yeast gap1∆ mutant on several amino acids including citrulline and tryptophane. In S. cerevisiae, Gap1 activity is regulated by shuttling between the plasma membrane (nitrogen limiting conditions) and the vacuole (nitrogen sufficiency), which we also show for FfGap1. In yeast, the Npr1 serine/threonine kinase stabilizes the Gap1 position at the plasma membrane. Here, we identified and characterized three NPR1-homologous genes, encoding the putative protein kinases FfNpr1-1, FfNpr1-2 and FfNpr1-3 with significant similarity to yeast Npr1. Complementation of the yeast npr1Δ mutant with each of the three F. fujikuroi NPR1 homologues, resulted in partial restoration of ammonium, arginine and proline uptake by FfNPR1-1 while none of the three kinases affect growth on different nitrogen sources and nitrogen-dependent sorting of FfGap1 in F. fujikuroi. However, exchange of the putative ubiquitin-target lysine 9 (K9A) and 15 (K15A) residues of FfGap1 resulted in extended localization to the plasma membrane and increased protein stability independently of nitrogen availability. These data suggest a similar regulation of FfGap1 by nitrogen-dependent ubiquitination, but differences regarding the role of Fusarium Npr1 homologues compared to yeast.
Highlights
In the phytopathogenic ascomycete Fusarium fujikuroi, the biosynthesis of a broad spectrum of secondary metabolites (SMs), such as the plant hormones gibberellins (GA) and the red polyketide pigments bikaverin and fusarubins [1,2,3], is strictly repressed, while the biosynthesis of mycotoxins such as fusarin C, fusaric acid, and apicidin F is induced by high nitrogen concentrations [4,5,6,7]
In order to identify a potential orthologue of the S. cerevisiae Gap1, we retrieved 99 predicted amino acid permeases (AAPs) of the recently sequenced genome of F. fujikuroi IMI 58289 [7] (S1 Table)
It has been shown that in F. fujikuroi, most of the 45 SM gene clusters are regulated by nitrogen availability [7]
Summary
In the phytopathogenic ascomycete Fusarium fujikuroi, the biosynthesis of a broad spectrum of secondary metabolites (SMs), such as the plant hormones gibberellins (GA) and the red polyketide pigments bikaverin and fusarubins [1,2,3], is strictly repressed, while the biosynthesis of mycotoxins such as fusarin C, fusaric acid, and apicidin F is induced by high nitrogen concentrations [4,5,6,7]. Uptake and eventually sensing of nitrogen by specific permeases play key roles in nitrogen metabolism [8,9]. Processes of nitrogen sensing and subsequent transduction of the signal to a nitrogen regulation network have been intensively studied in yeast. Some of the AAPs have been proposed to play a role as nitrogen sensors, e.g. the non-transporting amino acid carrier homologue Ssy1 [15] and the general amino acid permease Gap1 [16]. Yeast Mep, and its orthologue from Candida albicans for instance, are proposed to play a signaling role as they are required for the filamentation process occurring upon nitrogen limitation [17,18,19,20,21]
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