The General Amino Acid Permease FfGap1 of Fusarium fujikuroi Is Sorted to the Vacuole in a Nitrogen-Dependent, but Npr1 Kinase-Independent Manner

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.

Fig 1. Identification of potential general amino acid permeases in F. fujikuroi.

(A) Maximum likelihood tree showing the phylogenetical relationship between the general amino acid permeases (GAP) from S. cerevisiae [36], C. albicans [37], N. crassa [38], P. chrysogenum [39], and 19 F. fujikuroi proteins with the highest sequence homology to S. cerevisiae Gap1. Branches show bootstrap values (%), scale bar indicates amino acid substitutions per site. The fungal GAP proteins branched together with eleven putative GAP sequences of F. fujikuroi (orange box) (B) Expression analysis of putative GAP-encoding genes in F. fujikuroi. Total RNA was isolated from the wild-type and the ΔAREA and ΔAREB deletion mutants grown for 3 days in ICI liquid cultures with 6 mM (-Gln) or 60 mM (+Gln) glutamine as single nitrogen source and used for Northern blot analysis. 18S rRNA was visualized as loading control.

Fig 2. FFUJ_09118 is a functional homologue of S. cerevisiae Gap1.

Nitrogen utilization growth assay of S. cerevisiae mutants gap1Δdip5Δ and gap1Δssy1Δ complemented with putative GAP-encoding genes of F. fujikuroi (FFUJ_01137, FFUJ_05331, FFUJ_09118, FFUJ_11370) and PcGAP1 of P. chrysogenum. Fresh cells of tested yeast strains were adjusted to an optical density of 1 and (A) plated on yeast minimal agar with 1 mM citrullin (Cit), 10 mM aspartate (Asp), 10 mM phenylalanine (Phe), 1 mM tyrosin (Tyr) or 10 mM isoleucine (Ile) or (B) a series of 10-fold dilutions (left to right) were dropped on yeast minimal agar with 1 mM ammonium sulfate ((NH₄)SO₄) or glutamate as sole nitrogen source. Plates were incubated for 4 days at 30°C.

Fig 3. FfGap1 has no impact on nitrogen-mediated expression of SM biosynthesis genes.

Total RNA was isolated from the wild-type and the ΔFfGAP1 mutant grown for 3 days in liquid ICI medium with 6 mM (-Gln) or 60 mM (+Gln) glutamine as single nitrogen source and used for Northern blot analysis. The blot was hybridized with the ent-copalyldiphosphate/ent-kaurene synthase gene (CPS/KS) of the gibberellin and the monooxygenase gene (BIK2) of the bikaverin biosynthesis cluster. 18S rRNA was visualized as loading control.

Fig 4. Subcellular localization of FfGap1 depends on nitrogen availability.

F. fujikuroi wild type transformed with a FfGap1-GFP fusion construct was cultivated in liquid ICI medium with 6 mM glutamine for 48 h. (A) Cells were observed by fluorescence (GFP) and brightfield microscopy (BF) before (- N) and 30 min and 300 min after addition of 12 mM glutamine (+ Gln). (B) Cells were stained with FM4-64 and observed 60 min after addition of 12 mM Gln by fluorescence (FM4-64, GFP) and brightfield (BF) microscopy.

Fig 5. Cross-species complementation of the S. cerevisiae npr1Δ mutant with F. fujiuroi Npr1-like kinases leads to partial restoration of growth on different nitrogen sources.

S. cerevisiae wild-type (WT) and npr1Δ mutant strains were transformed with FfNPR1-1, FfNPR1-2, FfNPR1-3 and ScNPR1. Fresh cells of tested yeast strains were adjusted to an optical density of 1 and plated on yeast minimal agar with different nitrogen sources: 1 mM glutamate (glt), 1 mM ammonium sulfate (aml), 3 mM ammonium sulfate (am3), 1 mM citrulline (cit1), 1 mM proline (pro), 1 mM arginine (arg), 1 mM valine (val), 1 mM urea, 1 mM tryptophane (trp). Plates were incubated for 3 to 7 days at 30°C.

Fig 6. F. fujikuroi Npr1-like kinases do not have an impact on Gap1 stability in yeast.

The S. cerevisiae npr1Δ mutant and npr1Δ transformed with FfNPR1-1, FfNPR1-2, FfNPR1-3 and ScNPR1 were cultivated in liquid minimal medium with low amounts of urea as sole nitrogen source. Membrane enriched protein extracts were used for Western blot analysis. Specific antibodies were used to detect Gap1 and the ammonium permease Mep2. Pma1 was detected as protein loading control.

Fig 7. Growth assay of ΔFfGAP1 and ΔFfNPR1-1, ΔFfNPR1-2 and ΔFfNPR1-3 single, double and triple mutants on different nitrogen sources.

Strains were grown on solid ICI minimal medium with either no nitrogen (-N) or the indicated concentrations of various nitrogen sources (A, B) or on CM complete medium (B) at 28°C for 4 days.

Fig 8. Npr1-like kinases do not influence intracellular sorting of FfGap1.

F. fujikuroi wild-type and ΔFfNPR1-1 and ΔFfNPR1-3 mutants, all transformed with a FfGap1-GFP fusion construct, were cultivated in liquid ICI medium with 6 mM glutamine for 48 h. Cells were observed by fluorescence (GFP) and brightfield microscopy (BF) before (- N) and 120 min after addition of 12 mM glutamine (+ Gln).

Fig 9. Conserved lysine residues influence sorting and protein stability of FfGap1.

F. fujikuroi wild-type transformed with the FfGap1-Gfp or the FfGap1(K7/15A)-Gfp fusion construct was cultivated in liquid ICI medium with 6 mM glutamine for 48 h. (A) Cells were observed by fluorescence (GFP) and brightfield microscopy (BF) before (- N) and 2 h and 5 h after addition of 12 mM glutamine (+ Gln). (B) Western blot analysis of protein extracts before (- N) and 2h after addition of 12 mM glutamine (+ Gln). Hybridization with an anti-actin (actin) antibody was used as protein loading control.