Functional properties and differential mode of regulation of the nitrate transporter from a plant symbiotic ascomycete

Abstract

Nitrogen assimilation by plant symbiotic fungi plays a central role in the mutualistic interaction established by these organisms, as well as in nitrogen flux in a variety of soils. In the present study, we report on the functional properties, structural organization and distinctive mode of regulation of TbNrt2 (Tuber borchii NRT2 family transporter), the nitrate transporter of the mycorrhizal ascomycete T. borchii. As revealed by experiments conducted in a nitrate-uptake-defective mutant of the yeast Hansenula polymorpha, TbNrt2 is a high-affinity transporter (K(m)=4.7 microM nitrate) that is bispecific for nitrate and nitrite. It is expressed in free-living mycelia and in mycorrhizae, where it preferentially accumulates in the plasma membrane of root-contacting hyphae. The TbNrt2 mRNA, which is transcribed from a single-copy gene clustered with the nitrate reductase gene in the T. borchii genome, was specifically up-regulated following transfer of mycelia to nitrate- (or nitrite)-containing medium. However, at variance with the strict nitrate-dependent induction commonly observed in other organisms, TbNrt2 was also up-regulated (at both the mRNA and the protein level) following transfer to a nitrogen-free medium. This unusual mode of regulation differs from that of the adjacent nitrate reductase gene, which was expressed at basal levels under nitrogen deprivation conditions and required nitrate for induction. The functional and expression properties, described in the present study, delineate TbNrt2 as a versatile transporter that may be especially suited to cope with the fluctuating (and often low) mineral nitrogen concentrations found in most natural, especially forest, soils.

Figure 1. DNA gel blot and 5′-flanking region analysis of TbNrt2

(A) T. borchii genomic DNA digested with HindIII (lane 1), BamHI (lane 2) or EcoRI (lane 3) was probed with a ³²P-labelled TbNrt2-derived DNA fragment (shown in B). The migration positions of DNA size markers run alongside are indicated on the left. (B) Restriction map of the TbNrt2 cDNA and localization of the adjacent NR (tbnr1) gene. The positions of the DNA probe (black bar) and of the antisense riboprobe (white bar) used for DNA gel-blot and RNase-protection analyses respectively, as well as the annealing positions of the four oligonucleotide primers used for tbnr1 localization and the head-to-head orientation of TbNrt2 and tbnr1, are indicated. Arrows indicate the position and orientation of the two primer pairs utilized to amplify the intergenic region interposed between tbnr1 and TbNrt2. (C) Sequence analysis of the TbNrt2–tbnr1 intergenic region (2187 bp). Nit2- and Nit4-like control elements are represented as grey and black boxes respectively. The sequence of the 200-bp region upstream of the TbNrt2 initiator ATG (shown in italic) is reported. The two main transcription start sites identified by primer extension analysis (arrowheads at positions −44 and −81), as well as the putative promoter region (boxed) and the TATA-box (underlined), are indicated.

Figure 2. Functional and expression analysis of TbNrt2 in Ha. polymorpha

(A) Immunoblot analysis of TbNrt2 in crude membrane preparations (10 μg of total protein) derived from the pYNR-EX-TbNrt2 transformed strain (Δynt1-TbNrt2) and from the YNT1-disrupted untransformed isogenic strain (Δynt1). The estimated molecular masses of the polypeptides recognized by the anti-TbNrt2 serum (54 and 40 kDa) and the migration positions of molecular-mass markers (in kDa) are indicated on the right. (B) Rescue of yeast growth in the presence of a limiting nitrate concentration. Serial dilutions of wild-type (WT), Δynt1 and Δynt1TbNrt2 cells were spotted on to YG agar containing 500 μM NaNO₃ as the sole nitrogen source and incubated for 3 days at 37 °C.

Figure 3. Nitrate and nitrite transport by TbNrt2

(A) Time-course of nitrate uptake. Transport assays were started with the addition of 100 μM NaNO₃ to cells previously exposed to nitrate and were conducted for the indicated times on Δynt1 (▲), WT (■) and Δynt1TbNrt2 (◆) strains. (B) Concentration-dependence of nitrate uptake by Δynt1TbNrt2 cells. The rate of nitrate uptake (v) by a TbNrt2 transformant (YM5) was assayed as in (A) at the indicated nitrate concentrations. (C) Nitrite uptake by TbNrt2. Transport assays, started with the addition of NaNO₂ to cells previously exposed to nitrate, were conducted for the indicated lengths of time on a Δynt1TbNrt2 transformant grown in YG medium (pH 6.0) containing 50 μM (◆), 25 μM (■) or 12.5 μM (▲) NaNO₂; untransformed Δynt1 control cells (▼) were assayed in parallel in the presence of 50 μM NaNO₂.

Figure 4. Topological model of TbNrt2

(A) Structural organization of the TbNrt2 polypeptide according to the consensus prediction of five prediction methods. Amino acid residues that are identical in the alignment of all available NRT2 fungal sequences are shaded grey; TM helices are labelled with Roman numerals. Amino acids corresponding to the MFS and NS motifs, and to the putative protein kinase C phosphorylation site (SPR) are enclosed in dark circles; the two arginine residues (located in TM helices II and VIII) that are positionally equivalent to those required for high-affinity nitrate transport by A. nidulans NrtA [39] are represented as white letters on a black background. (B) Helix boundaries predicted by concordant prediction programs. Boundaries chosen for the TbNrt2 topology model shown in (A) are in bold.

Figure 5. Phylogenetic relationships among fungal NRT2 transporters

A radial phylogenetic tree was constructed with neighbour-joining on the basis of the alignment of fungal NRT2 polypeptides homologous with TbNrt2; branches are drawn to scale (the scale bar corresponds to 0.1 changes per site). The NCBI accession numbers of the sequences used for tree construction are: CAB60009 (He. cylindrosporum Nrt2), P22152 (A. nidulans CrnA), AAL50818 (A. nidulans NrtB), CAD28427 (A. fumigatus CrnA), CAD71077 (N. crassa Nit-10), CAA11229 (Ha. polymorpha Ynt1), AF462038 (T. borchii TbNrt2), PC.9.52.1 (Phanerochaete chrysosporium; Joint Genome Institute), XP_401464 (Ustilago maydis), XP_380592 (Gibberella zeae).

Figure 6. Nitrogen-status-dependent modulation of the TbNrt2 mRNA

(A) Time course of TbNrt2 up-regulation in nitrogen-starved T. borchii mycelia. TbNrt2 mRNA levels were determined by RNase protection assays conducted on mycelia grown for 10 days on complete SSM (t₀) and then transferred for the indicated lengths of time to nitrogen-free SSM. A T. borchii β-tubulin (β-Tub) antisense riboprobe was included in all assays as an internal standard. The bands shown, which correspond to protection products of the TbNrt2 and β-Tub riboprobes, were visualized by autoradiography and quantified by phosphorimaging. Relative transcript abundance values (reported below each lane) were calculated by dividing the volumes of the TbNrt2 signals by the volumes of the corresponding β-Tub signals, followed by normalization with respect to TbNrt2 abundance in unshifted (t₀) controls. (B) TbNrt2 modulation following supplementation of nitrogen-starved mycelia with various nitrogen sources. Results obtained from RNase protection assays conducted on mycelia cultured for 21 days in nitrogen-free medium and then shifted for the indicated lengths of time to modified SSMs containing either NH₄Cl (NH₄⁺), L-glutamine (Gln), KNO₃ (NO₃⁻), or L-proline (Pro) (each at a 4 mM final concentration) as the sole nitrogen sources are shown. Data analyses and quantification were performed as in (A). Relative TbNrt2 transcript levels, normalized with respect to nitrogen-sufficient 10-day-old mycelia, are reported on the y-axis; relative TbNrt2 abundance in unshifted mycelia, nitrogen-deprived for 21 days, is indicated by the horizontal line.

Figure 7. Nitrogen-source-dependent regulation of TbNrt2 and tbnr1

(A) Results obtained from RNase protection assays conducted on mycelia cultured for 10 days in ammonium-containing SSM and then shifted for the indicated lengths of time to modified SSMs containing either KNO₃ or KNO₂ as the sole nitrogen sources. Data analyses and quantification were performed as in Figure 6(A). (B) RNA gel-blot analysis of mycelia cultured for 10 days in ammonium-containing SSM and then shifted for 5 days to SSM lacking any source of nitrogen (-N), to SSM containing either NH₄Cl (NH₄⁺), glutamine (Gln), proline (Pro) or nitrate (NO₃⁻) as the sole nitrogen sources, or to a modified SSM containing KNO₃ along with each of the indicated nitrogen compounds (NH₄⁺, Gln or Pro); all nitrogen compounds were added at a final concentration of 4 mM. The same blot was separately hybridized with either a TbNrt2 or a tbnr1 probe as indicated. Methylene-Blue-stained 28 S rRNA bands, used as loading controls, are shown below each lane. (C) Immunoblot analysis of TbNrt2 protein levels in crude membrane preparations (20 μg of total protein for each sample) derived from mycelia cultured for 10 days in ammonium-containing SSM and then shifted for the indicated lengths of time to either the same medium (NH₄⁺), to SSM lacking any source of nitrogen (-N) or to SSM supplemented with 4 mM KNO₃ (+NO₃⁻); no immunopositive signal was detected upon hybridization with pre-immune serum.

Figure 8. Immunofluorescence localization of TbNrt2 in ectomycorrhizae

(A) Immunodetection of TbNrt2 in Hartig net hyphae (h), close to root cortical cells (c), within sectioned tips from T. borchii/C. incanus mycorrhizae analysed by confocal microscopy. Arrows point to the green line labelling, associated with the fungal membrane, observed in transversally sectioned hyphae; arrowheads point to the fluorescent green signal detected in tangentially sectioned hyphae (bar=10 μm). Under the confocal microscopy analysis conditions used to visualize the FITC-labelled secondary antibody, the fungal wall displayed a characteristic red autofluorescence. (B) Transmitted-light view of the same sample shown in (A); symbols and magnification are the same as in (A). (C) Immunodetection of TbNrt2 (arrows) associated with the fungal membrane of developing mantle hyphae (h) ensheathing root cortical cells (c); visualization conditions and magnification are the same as in (A). (D) Low-magnification image (bar=25 μm) of a control section visualized by confocal microscopy in the absence of the primary antibody. The red autofluorescence is associated with plant and fungal cell walls; cc indicates the central cylinder, the other symbols are as specified in (A). A higher magnification image (bar=13 μm) of a different section, visualized by confocal microscopy using the pre-immune serum in place of anti-TbNrt2 antibody, is shown in the inset; no specific signal was detected in Hartig net or in mantle-forming hyphae (h).