Biochem. J. (2006) 394, 125–134 (Printed in Great Britain)
125
doi:10.1042/BJ20051199
Functional properties and differential mode of regulation of the nitrate
transporter from a plant symbiotic ascomycete
Barbara MONTANINI*1 , Arturo R. VISCOMI*, Angelo BOLCHI*, Yusé MARTIN†, José M. SIVERIO†, Raffaella BALESTRINI‡,
Paola BONFANTE‡ and Simone OTTONELLO*2
*Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, 43100 Parma, Italy, †Instituto Universitario de Enfermedades Tropicales y Salud Pública, Departamento de
Bioquı́mica y Biologı́a Molecular, Grupo del Metabolismo del Nitrógeno, Universidad de La Laguna, E-38206, La Laguna, Spain, and ‡Dipartimento di Biologia Vegetale, Università di
Torino and Istituto per la Protezione delle Piante (Sezione di Micologia), Consiglio Nazionale delle Ricerche, 10125 Torino, Italy
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 µM
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.
INTRODUCTION
some exceptions (e.g. nit-10), are usually clustered with NR and
NIR genes and are generally induced by nitrate and repressed
by reduced nitrogen sources such as ammonium and glutamine
[3,4,9,10].
Particularly interesting, although so far very little explored,
are nitrate transporters from plant symbiotic fungi, such as the
ectomycorrhizal ascomycete Tuber borchii addressed in the present study. In these organisms, which colonize most tree species in
temperate forests, soil-retrieved nitrogen not only serves for selfnutrition, but also is a key player in the mutualistic nitrogen/carbon
trade established with the plant host. This bidirectional nutrient
exchange takes place in specialized symbiotic structures, called
ectomycorrhizae, which connect the extraradical fungal mycelium
to the root system, thereby dramatically expanding the effective
surface area for nutrient soil exploration. Investigations carried out
over the last few decades have provided ample evidence as to the
contribution of ectomycorrhizae to plant nitrogen status and have
highlighted their crucial ecological role in nitrogen cycling within
forest ecosystems ([11,12] and references therein). This renewed
perception of the ecological importance of ectomycorrhizae has
revived efforts to elucidate the molecular processes underlying
nitrogen assimilation and the related starvation stress responses
in ectomycorrhizal fungi [13–15]. A number of genes coding for
Nitrate is a major source of nitrogen for a variety of organisms,
including mycorrhizal fungi and their plant hosts, and it is often
the nutrient that limits their growth. On a global scale, reduced
nitrogen derived from nitrate assimilation is estimated to exceed,
by approx. two orders of magnitude, the amount of nitrogen that is
assimilated via nitrogen fixation. Prior to reductive assimilation
through the action of NR (nitrate reductase) and NIR (nitrite
reductase), nitrate is internalized via an energy-dependent uptake
process by specific plasma membrane transporters. A large
group of NTs (nitrate transporters), from both prokaryotes and
eukaryotes, belong to the MFS (major facilitator superfamily)
and, specifically, to the NRT2 family of NT-encoding genes [1–3].
The most well characterized fungal members of this family are
NrtA (formerly known as CrnA) and NrtB from Aspergillus
nidulans [4–6] and Ynt1 from Hansenula polymorpha [7,8]. They
share a similar membrane topology, in which two sets of six
TM (transmembrane) helices, with an NIN CIN (N-terminus intracellular/C-terminus intracellular) orientation, are connected by
a large cytosolic loop. Two NT genes have been identified in
A. nidulans [6], whereas only one is present in Ha. polymorpha
[3] and Neurospora crassa (nit-10; [9]). Fungal NT genes, with
Key words: gene regulation, Hansenula polymorpha, mycorrhiza,
nitrate/nitrite transport, nitrogen deficiency, Tuber borchii NRT2
family transporter (TbNrt2).
Abbreviations used: EST, expressed sequence tag; GST, glutathione S-transferase; MFS, major facilitator superfamily; NCBI, National Center for
Biotechnology Information; NIN/OUT , N-terminus intracellular/extracellular; NIR, nitrite reductase; NR, nitrate reductase; NS, nitrate signature; NT, nitrate
transporter; ORF, open reading frame; SSM, synthetic solid medium; TbNrt2,Tuber borchii NRT2 family transporter; TM, transmembrane.
1
Present address: UMR INRA/UHP 1136, Interactions Arbres/Micro-organismes, Université Henri Poincaré-Nancy I, 54506 Vandoeuvre-les-Nancy,
France.
2
To whom correspondence should be addressed (email s.ottonello@unipr.it).
The nucleotide sequences reported in this paper have been submitted to the GenBank® , DDBJ and EMBL databases under the accession numbers
AF462038 (TbNrt2 coding region) and AY786418 (TbNrt2 upstream non-coding region).
c 2006 Biochemical Society
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B. Montanini and others
ammonium transporters and nitrogen assimilation enzymes have
been isolated from these organisms [16–22]. In contrast, only
one gene coding for an as yet functionally uncharacterized NT
has been identified so far in a mycorrhizal fungus, the basidiomycete Hebeloma cylindrosporum [23]. At variance with the
requirement for both nitrogen catabolite derepression and nitratemediated induction commonly displayed by nitrate assimilation
components, the mRNA for this NT was found to be up-regulated
by nitrogen deprivation in the absence of nitrate. Such a mode
of regulation, which is shared by the NR and NIR genes of
He. cylindrosporum [16,23], has thus far been documented only in
this organism. This raises a question as to whether it represents a
unique feature of this species or an adaptive trait of plant symbiotic
fungi in general, and whether additional molecular features may
distinguish the NTs (and other nitrate assimilation components)
of symbiotic fungi from their saprotrophic counterparts.
In the present study, we report the functional, structural and
expression analysis of TbNrt2 (T. borchii NRT2 family transporter), the NT of T. borchii, a plant symbiotic ascomycete
that grows in a nitrification-proficient soil habitat, is reportedly
beneficial to its host plants and is closely related evolutionarily
to the fungi whose NTs have been the most well characterized
so far. TbNrt2 is a plasma-membrane-localized high-affinity NT
that is bispecific for nitrate and nitrite, and expressed in free
living mycelia as well as in root-contacting hyphae. It responds
positively to nitrate supplementation, but also to nitrogen starvation alone, thus pointing to nitrate-independent up-regulation as a
functional signature of NTs from symbiotic fungi. Interestingly,
however, TbNrt2 regulation differs from that of the adjacent NR
gene, which was found to be up-regulated in a strictly nitratedependent manner, as in most nitrate utilizing organisms. The
possible ecophysiological significance of this differential mode
of regulation is discussed.
EXPERIMENTAL
Biological materials
T. borchii Vittad. mycelia (isolate ATCC 95640) were grown in the
dark at 23 ◦C on a SSM (synthetic solid medium), either complete
or deprived of a single nutrient, as described previously [18].
Single nitrogen sources were utilized for all shift experiments,
except those involving nitrate, which was also supplied in combination with NH4 Cl, glutamine or proline.
Isolation of the TbNrt2 cDNA
A cDNA library constructed in the excisable phage vector Uni-Zap
XR from 20-day-old T. borchii mycelia (a gift from Dr A. Viotti,
Istituto di Biologia e Biotecnologia Agraria, CNR, Milano, Italy)
was used as a source of template DNA (100 ng/reaction) for PCR
amplifications. The oligonucleotides 5′ -GCCCTGCGCTTCTTCATCGG-3′ (plus) and 5′ -AAATGGCCGGCATGACGAAG-3′
(minus), designed on a highly conserved region of fungal and plant
NTs characterized previously [1], were used for the amplification
of the TbNrt2 cDNA. Amplification reactions were conducted
under the following ‘touch-down’ PCR conditions: 2 min of
initial denaturation at 94 ◦C, 20 cycles of 30 s denaturation at
94 ◦C and 30 s annealing at progressively lower temperatures
(from 60 to 51 ◦C) with a decrease of 1 ◦C every other cycle,
followed by 30 additional cycles at an annealing temperature
of 50 ◦C. The DNA fragment obtained from such amplification
was cloned into the pGEM-T-easy vector (Promega), sequenceverified, and used as a probe for plaque-hybridization analysis of
the T. borchii cDNA library [24]. A cDNA of 1770 bp (TbNrt2)
was thus identified, excised to produce plasmid pBlueScript-SK
c 2006 Biochemical Society
TbNrt2, sequenced on both strands and compared with known
NT genes using BLASTX. Standard PCR conditions were used
to map the upstream located tbnr1 gene (GenBank® accession
number AF533362). The tbnr1–TbNrt2 intergenic sequence
was amplified by PCR using 50 ng of genomic DNA as the
template and the 5′ -GACGAGTGATCCATGATGATG-3′ (plus)/
5′ -ATAAGAGACTCAACTGCATGTG-3′ (minus) oligonucleotides as primers. The resulting amplicon (1660 bp) was cloned into
the SmaI site of pBlueScript-SK (Stratagene) and sequenced on
both strands. The tbnr1–TbNrt2 intergenic sequence (see Figure 1C) was assembled manually by overlapping the above
1644 bp sequence (between positions −1641 and + 3 of TbNrt2;
numbered with respect to the ATG start site) with part of the
5′ -UTR (untranslated region) of tbnr1 (positions − 2187/− 1453,
with respect to the TbNrt2 ATG).
Functional assays in the yeast Ha. polymorpha
A 1523-bp-long fragment containing the TbNrt2 ORF (open
reading frame; 1503 bp in length) plus 13 bp and 7 bp of 5′ - and
3′ -flanking DNA respectively, was cloned into the SalI and SpeI
sites of the pYNR-EX vector, bearing the promoter and terminator
elements of the Ha. polymorpha NR (YNR1) gene [25], to
generate pYNR-EX-TbNrt2. The wild-type strain NCYC495 of
Ha. polymorpha and its derivative strains harbouring a disrupted
version of either the NT gene (ynt1::URA3 LEU2) or the
NR gene (ynr1::URA3), or both (ynt1::URA3 ynr1::URA3)
[3,7] were used in the present study. Yeasts were grown with
orbital shaking in liquid YG medium [0.17 % yeast nitrogen base
without amino acids and ammonium sulphate (Difco), supplemented with 2 % (w/v) glucose] plus the indicated nitrogen
sources. Yeast transformation was carried out as described previously [26] with 1 µg of pYNR-EX-TbNrt2 DNA, linearized with
BstII to target integration into the LEU2 locus [25]. The ynt1
status of recipient cells and their transformation by pYNR-EXTbNrt2 were verified by PCR analysis using the following sets
of primers: 5′ -CGGAATTCACATGTGATAGTGTT-3′ (plus)/
5′ -GCACATGTAGCAAAGTCC-3′ (minus) for the YNT1 locus;
and 5′ -GCAGCAATGATACAT-3′ (plus)/5′ -TATCCAACTTGCGCG-3′ (minus) for the YNR1 locus.
Nitrate transport was measured by determining the rate of
extracellular nitrate depletion as described previously [8]. Similar
experimental conditions, except for the replacement of NaNO3
with NaNO2 and the use of a 25 mM Mes/Tris (pH 6.0) medium,
were used for nitrite-uptake assays. Chlorate sensitivity was determined by serial dilution growth tests conducted in parallel on wildtype, ynt1 disruptant and YNT1-disrupted pYNR-EX-TbNrt2transformed (ynt1TbNrt2) strains. Cells were spotted on to YG
agar containing 5 mM NaNO3 and 1 mM proline, plus increasing
concentrations of KClO3 (from 50 up to 200 mM); plates were
incubated at 37 ◦C and photographed after 3 days.
DNA and RNA analyses
Genomic DNA samples for gel-blot analysis (4 µg each) were
digested with BamHI, EcoRI and HindIII, and electrophoresed on 0.8 % agarose gels. A random priming labelling kit
(Amersham Biosciences) was used for 32 P-labelling of TbNrt2
hybridization probes. Blotting on to Hybond-N (Amersham Biosciences), pre-hybridization and high-stringency washing were
conducted according to the manufacturer’s instructions. Total
RNA for RNase protection and RNA gel-blot assays was isolated
and quantified as described previously [13]. A 32 P-labelled antisense riboprobe (332 nt) was prepared by in vitro T7 RNA polymerase transcription of FokI-digested pBlueScript-SK-TbNrt2.
High-affinity nitrate transporter from Tuber borchii
Saturating amounts of a T. borchii β-tubulin riboprobe (259 nt)
were added to all reactions as an internal standard. Hybridization (5 µg of total RNA/assay), RNase A/T1 digestion
and gel fractionation were carried out as described previously
[13]. Protected fragments were visualized by autoradiography and
quantified with a Personal Imager FX using the Multi-Analyst/
PC software (Bio-Rad Laboratories). Heat-denatured total RNA
(20 µg for each sample) fractionated on glyoxal-agarose gels,
transferred on to a nylon membrane and hybridized with a
32
P-labelled full-length TbNrt2 cDNA probe was used for highstringency RNA gel-blot analysis [24]. Blotting on to GeneScreen
Plus (PerkinElmer Life Science), pre-hybridization and washing
were conducted according to the manufacturer’s instructions. The
same radioactive labelling and hybridization procedures were
applied to tbnr1; a 405-bp DNA fragment (positions 1125–1529)
prepared by PCR was used as a probe. Superscript III reverse
transcriptase (Invitrogen) and total RNA (5 µg) from mycelia that
had been nitrogen-starved for 7 days were used for primer extension analysis [24]. Template RNA and a radioactively labelled
primer (5′ -ATAGGAAGGCAATCATGAATCCGAATGTGC-3′ ),
encompassing positions 112–142 of the TbNrt2 coding region,
were heat-denatured (7 min at 80 ◦C) and annealed at 42 ◦C
for 1 h. After addition of the enzyme and other reaction components, reverse transcription was allowed to proceed for 1 h at
50 ◦C. Extended cDNA fragments were ethanol-precipitated, electrophoretically fractionated on a denaturing sequencing gel and
compared with the products of a sequencing reaction carried out
with TbNrt2 DNA using the same primer as above.
DNA and protein sequence analysis
Six different programs, set to standard parameters and relying on
single sequence predictions, were used to analyse the membrane
topology of TbNrt2. These are available at the following
addresses: MEMSAT2 (http://bioinf.cs.ucl.ac.uk/psipred/; [31]);
TopPred2 (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.
html; [34]); HMMTOP (http://www.enzim.hu/hmmtop/html/adv
submit.html; [36]); TMHMM2 (http://www.cbs.dtu.dk/services/
TMHMM/; [35]); TMpred (http://www.ch.embnet.org/software/
TMPRED form.html; [32]); TMAP (http://www.mbb.ki.se/
tmap/; [33]). The SSpro8 program (http://www.igb.uci.edu/
tools/scratch/; [38]) was used to predict β-turn regions. Sequence
similarity searches were conducted with BLAST on the nonredundant (nr) protein database at the NCBI (National Center
for Biotechnology Information; http://www.ncbi.nlm.nih.gov/).
The fungal genome databases at MIT (Massachusetts Institute
of Technology; http://www.broad.mit.edu/index.html), MIPS
(Munich Information Center for Protein Sequences; http://mips.
gsf.de/) and JGI (Department of Energy Joint Genome Institute;
http://www.jgi.doe.gov/sequencing/), and the EST (expressed
sequence tag) database at the NCBI plus the EST collection
at COGEME (Consortium for the Functional Genomics of
Microbial Eukaryotes; http://www.cogeme.man.ac.uk/) were
used to search for TbNrt2 homologues. Predicted polypeptide
sequences were aligned with CLUSTAL W [27]. Phylogenetic
analysis was conducted with the neighbour-joining algorithm
implemented in CLUSTAL X [27a]. Phylogenetic trees were
visualized with TreeView [28]. The programs ‘motif search’
(http://www.genome.ad.jp/), relying on the TRANSFAC database, and NNPP/Eukaryotic (eukaryotic promoter prediction
by neural network) and TESS (search for transcription factor
binding sites), at the BCM (Baylor College of Medicine) Search
Launcher server (http://searchlauncher.bcm.tmc.edu/), were used
to search for putative control elements in the 5′ -flanking region
of TbNrt2.
127
Immunoblot and immunofluorescence analyses
A polypeptide corresponding to the VI–VII loop region of
TbNrt2 (amino acid positions 219–314; see Figure 4A) fused
to bacterial GST (glutathione S-transferase) within the pGEX4T-2 expression vector (Amersham Biosciences) was used as
antigen for anti-TbNrt2 antibody production. Standard procedures
were used for recombinant protein expression and purification
(GSTrap application note, Amersham Biosciences) and rabbit immunization. Crude membrane fractions for immunoblot analysis
were prepared from both Ha. polymorpha and T. borchii as described previously [8]. Balanced amounts of the various fractions,
containing the same amount of total protein quantified with the
Coomassie Brilliant Blue G-250 dye (Pierce Biotechnology, Inc.)
method, were first fractionated on 12 % (w/v) polyacrylamide
gels [8], transferred to Hybond-ECL® (Amersham Biosciences)
and analysed with horseradish-peroxidase-conjugated anti(rabbit IgG) antibodies and enhanced chemiluminescence reagents (Pierce Biotechnology) according to the manufacturer’s
instructions. Parallel reactions carried out on untransformed Ha.
polymorpha cells or with the use of the pre-immune serum were
used as specificity controls. Immunofluorescence analysis was
conducted on in-vitro-produced T. borchii/Cistus incanus mycorrhizae [29]. Root segments were fixed overnight at 4 ◦C with
4 % (v/v) paraformaldehyde in PBS (pH 7.4). After washing
with PBS, they were embedded in 8 % (w/v) low-melting agarose.
Sections (100 µm thick), prepared with a Balzer Vibratome series
1.000 apparatus, were incubated overnight at 4 ◦C with the antiTbNrt2 serum (diluted 1:500–1:1000) in 50 mM phosphate buffer
(pH 7.2) containing 1 % (w/v) BSA. Sections were washed three
times (15 min each) with 50 mM phosphate buffer (pH 7.2),
saturated for 30 min with 1 % (w/v) BSA in 50 mM phosphate
buffer, and incubated at room temperature in the dark for 3 h with
FITC-conjugated goat anti-(rabbit IgG) (diluted 1:80). Sections
were then washed as before, mounted and analysed with a Leica
TCS SP2 confocal microscope, using the 488 nm Ar laser band
to excite both FITC fluorescence and paraformaldehyde-induced
tissue autofluorescence. The two signals were discriminated with
wavelength-specific emission filters: 500–540 nm for FITC (falsecoloured in green) and 590–630 nm for tissue autofluorescence
(false-coloured in red). Labelling specificity was assessed by
replacing the primary antibody with either buffer alone or buffer
with pre-immune serum.
RESULTS
Isolation and sequence analysis of TbNrt2
Oligonucleotide primers designed on a conserved region of known
NTs from fungi, plants and the alga Chlamydomonas reinhardtii
were initially employed for PCR amplification experiments
conducted on a T. borchii mycelium cDNA library. A 159-bplong DNA fragment matching the sequences of the above NTs
was obtained and used as a probe for library screening. Five
hybridization-positive cDNAs were identified, the longest of
which, named TbNrt2 (1770 bp; GenBank® accession number
AF462038), was sequenced and found to contain a 1503-nt-long
ORF, starting with an initiator ATG and lying in a good sequence
context according to translation initiation rules in fungi [30]. The
predicted translation product of the TbNrt2 cDNA is 501 amino
acids in length with an estimated molecular mass of 54.2 kDa. A
single mRNA of approx. 1800 nt was revealed by a high-stringency RNA gel-blot analysis using the full-length TbNrt2
cDNA as a probe. An essentially single-band pattern, shown in
Figure 1(A), was also obtained when T. borchii genomic DNA,
digested with either HindIII, BamHI or EcoRI, was hybridized
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Figure 1
B. Montanini and others
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 32 P-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.
with a cDNA probe (159 bp) not containing recognition sites for
any of the above restriction enzymes. The fainter hybridizing band
observed in the HindIII digestion mixture did not change appreciably when the same blot was hybridized under low-stringency conditions (results not shown). While not excluding the
possible existence of NT paralogues, as in A. nidulans [6], the data
indicate that TbNrt2 is a single-copy gene in the T. borchii genome.
Also shown (Figure 1B) are the results of PCR amplification
experiments using mixed primer pairs derived from TbNrt2 and
from the NR gene of T. borchii (tbnr1; [20]). Only one of the
four mixed primer pairs used for these experiments (made up
by an upstream annealing, TbNrt2-derived oligonucleotide and by
an oligonucleotide complementary to the most upstream part of
tbnr1) led to successful amplification, with the production of an
approx. 1700-bp-long amplicon. Thus, even though no information on the genomic localization of the NIR gene is currently
available, it appears that at least two nitrate assimilation genes
are clustered in the genome of T. borchii. Further support for
the NT identity of TbNrt2 was provided by sequence analysis of its 5′ -flanking region (GenBank® accession number
AY786418), which was found to contain a number of putative
control elements resembling the binding sites of nitrogen statusdependent regulators in other filamentous fungi, especially the
Nit2 and Nit4 regulators of N. crassa [9,10] (Figure 1C).
Functional properties of the TbNrt2 transporter
The nitrate transport ability of TbNrt2 was tested by functional
complementation assays carried out in an NT-disrupted strain
c 2006 Biochemical Society
of the yeast Ha. polymorpha (ynt1), which is unable to grow
on media containing 0.5 mM nitrate as the sole nitrogen source
and to transport nitrate at concentrations lower than 0.5 mM [7].
The ynt1 strain was transformed with the integrative expression
vector pYNR-EX-TbNrt2 in which the TbNrt2 ORF is under
the control of the promoter and terminator elements of the Ha.
polymorpha NR (YNR1) gene [25]. Six transformants capable
of growing on 0.5 mM NaNO3 were randomly picked and their
YNT1-disrupted status, as well as their integrative transformation
with pYNR-EX-TbNrt2, was verified and validated by PCR
analysis (results not shown). One such transformant (named YM5)
was chosen for further analysis. The expression of TbNrt2 in the
ynt1 strain bearing the chimaeric pYNR-EX-TbNrt2 construct
was determined by immunoblotting using an antiserum raised
against the VI–VII loop of TbNrt2 (amino acids 219–314; see
Figure 4A). As revealed by the immunoblot in Figure 2(A), a
major polypeptide of 54 kDa along with a minor band of approx.
40 kDa (most probably a degradation product of the larger polypeptide) were recognized by the anti-TbNrt2 serum in crude
membrane preparations derived from the ynt1TbNrt2 transformant, but not from the untransformed isogenic strain. The
molecular mass of the larger immunopositive polypeptide is
the same as that predicted for TbNrt2, thus indicating that the
pYNR-EX-TbNrt2 construct supports the synthesis of the putative
T. borchii NT in Ha. polymorpha cells. Importantly, the same
TbNrt2 transformant is capable of growing in the presence of a
nitrate concentration (0.5 mM) that is not sufficient for growth
of ∆ynt1 cells (Figure 2B). As shown in Figure 3(A), the YM5
strain is capable of taking up nitrate, albeit with an efficiency that
High-affinity nitrate transporter from Tuber borchii
Figure 2
129
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 (ynt1-TbNrt2 ) and from the
YNT1-disrupted untransformed isogenic strain (ynt1). 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),
ynt1 and ynt1TbNrt2 cells were spotted on to YG agar containing 500 µM NaNO3 as the
sole nitrogen source and incubated for 3 days at 37 ◦C.
is approx. 3-fold lower than that of wild-type cells. This reduced
transport rate may be due to the use of a heterologous promoter,
to a sub-optimal plasma membrane targeting and/or impaired
stability in the heterologous host, or to an intrinsically lower transport capacity (and/or affinity for nitrate ions) of TbNrt2 compared
with Ynt1. To distinguish between the latter two possibilities, we
determined the apparent kinetic parameters for nitrate transport
by TbNrt2. As shown in Figure 3(B), nitrate transport by the
ynt1TbNrt2 transformant was saturable, with apparent K m
and V max values (derived from Lineweaver–Burk and EadieHoffstee analyses of the data) of 4.7 +
− 0.8 µM and 0.18 +
−
0.01 nmol · min−1 · mg−1 of cells respectively. Although the V max
was difficult to compare with values reported previously
because of uncertainties concerning expression levels and plasma
membrane localization, the K m value, which was independent of
the number of active TbNrt2 molecules, appeared to be close to
those reported previously for Ha. polymorpha Ynt1 [8] and for
the high-affinity NrtB transporter from A. nidulans [6]. Therefore
sub-optimal accumulation and/or membrane targeting, rather than
an intrinsically reduced affinity for nitrate ions, are likely to be
responsible for the lower nitrate uptake activity of TbNrt2 in
Ha. polymorpha.
The nitrite transport capacity of TbNrt2 was examined next.
In Ha. polymorpha, it has been shown that, at pH 6.0, nearly
all nitrite transport takes place through Ynt1 in such a way that,
in a ynt1 strain, the transport of this ion was negligible compared
with that of the wild-type strain [8]. As revealed by the results
of nitrite transport assays conducted in the ynt1 mutant and
in the corresponding TbNrt2 transformant (Figure 3C), TbNrt2
restored nitrite transport capacity. Although the kinetic parameters
were not determined, the data indicate that the K m for nitrite
transport was also in the micromolar range. What was also apparent, however, was that the rate of nitrite uptake at 50 µM
NaNO2 was lower than that measured at 12.5 µM. Based on
previous results in Ha. polymorpha [8], this behaviour probably
reflects the cellular toxicity of nitrite, due to a concentrationdependent nitrite-induced uncoupling effect. Additional proof
of the nitrite transport capacity of TbNrt2 was obtained from
experiments (results not shown) that showed the ability of nitrate
to competitively inhibit such a process. The ability of TbNrt2
to take up chlorate, thus leading to cell poisoning, was also
examined. As revealed by serial dilution growth tests carried
out at increasing chlorate concentrations (50–200 mM), the
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
ynt1 (䉱), WT (䊏) and ynt1TbNrt2 (䉬) strains. (B) Concentration-dependence of nitrate
uptake by ynt1TbNrt2 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 ynt1TbNrt2 transformant grown in YG
medium (pH 6.0) containing 50 µM (䉬), 25 µM (䊏) or 12.5 µM (䉱) NaNO2 ; untransformed
ynt1 control cells (䉲) were assayed in parallel in the presence of 50 µM NaNO2 .
ynt1TbNrt2 transformant grew as well as ynt1 and wild-type
cells in the presence of chlorate concentrations up to 100 mM,
whereas all strains were incapable of growing in the presence of
200 mM chlorate (results not shown). Although not ruling out
the possibility that TbNrt2 may transport chlorate (albeit with an
exceedingly high K m ), the data clearly point to its unappreciable
contribution to chlorate toxicity in Ha. polymorpha.
Membrane topology and phylogenetic relationships of TbNrt2
Six different prediction programs (MEMSAT2, TMpred, TMAP,
TopPred2, TMHMM2 and HMMTOP) were used to construct a
model of TbNrt2, reliably predicting the localization and orientation of individual TM helices. Five of these programs predicted
12 TM helices with an NIN CIN topology, whereas one less
TM helix and an NOUT CIN (N-terminus extracellular/C-terminus
c 2006 Biochemical Society
130
Figure 4
B. Montanini and others
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.
intracellular) orientation was predicted by HMMTOP. Following
the ‘majority-vote’ criterion, which yielded reliable topologies
when applied to membrane proteins of known structure analysed
with different prediction methods [37], we considered as valid the
12 TM helices/NIN CIN model shown in Figure 4(A). As shown
in Figure 4(B), there was generally a good agreement between
the five concordant programs not only on the general predicted
topology, but also on the identification of putative TM regions.
The five programs, however, produced slightly different results as
to the precise localization of the borders of individual membranespanning helices. To identify the most likely borders of individual
TM helices, we again relied on a majority-vote criterion, and incorporated into the model of Figure 4(A) consensus border residues suggested by at least three of the five prediction methods.
For all those instances in which the above consensus criterion
was not applicable, we resorted to SSpro8, a neural network
program capable of predicting eight different types of secondary
structures in globular proteins. This allowed us to identify individual positions at which α-helices are expected to be interrupted by β-turns, a diagnostic feature of TM helix-connecting
loops in membrane proteins. In accordance with the general
topology of NRT2 transporters [1], the N- and C-termini of
TbNrt2 and its TM-helix-connecting loops are all quite short
(4–34 amino acids, together corresponding to less than 30 %
of the entire protein), except for the 93-amino-acid loop interposed between TM helices VI and VII. This large loop, along
with a fairly short C-terminal tail, is a typical signature of fungal
NRT2s [39]. It accounts for approx. 18 % of the entire protein by
itself and was thus used as an antigen for anti-TbNrt2 antibody
production (Figure 2A). Also apparent is the predomiance of
positively charged amino acid residues associated with both extracellular and intracellular loops (Figure 4A), the main exception
being the negative charge of the large VI–VII loop (a feature
c 2006 Biochemical Society
that suggests the existence of electrostatic interactions between
oppositely charged cytoplasm-facing regions of TbNrt2). The reliability of this model was also supported by the presence, within
such a loop (positions 233–235), of a putative protein kinase
C phosphorylation site (Ser/Thr-Xaa-Arg/Lys, where Xaa is any
amino acid) found previously in a topologically equivalent position in other NRT2 transporters [1]. Additional diagnostic sequencess were the two MFS (positions 90–99 and 364–372) and
NS (nitrate signature; positions 150–171 and 437–454) motifs typically associated with NRT2 transporters, and two positionally conserved arginine residues (within TM helices II and VIII) that are required for high-affinity nitrate transport by A. nidulans NrtA [39].
By referring to the topological model described above, the
TbNrt2 polypeptide was compared with homologous sequences
from other fungi. Amino acid residues that were conserved in all
NRT2 transporters (Figure 4A) were dislocated along the entire
sequence. Conservation was maximal in TM helices I, IV and
V (46–52 % identity), whereas it was nearly absent in loops
VII–VIII, IX–X, XI–XII and in the region interposed between the
end of helix XII and the C-terminus. As apparent in Figure 5, three
separate groups, corresponding to unicellular ascomycetes, such
as Ha. polymorpha, filamentous ascomycetes and basidiomycetes,
are discernible in the NRT2 cluster. TbNrt2 belongs to the
filamentous ascomycetes group, with NtrB from A. nidulans as
its closest homologue.
TbNrt2 expression in mycelia cultured under different nitrogen
nutritional regimens
RNase-protection and RNA gel-blot assays were conducted to
determine TbNrt2 mRNA levels in T. borchii mycelia subjected
to different nitrogen status perturbations. We initially examined
the response of TbNrt2 to nitrogen deprivation. As shown in
High-affinity nitrate transporter from Tuber borchii
Figure 5
131
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
Figure 6(A), TbNrt2 mRNA was up-regulated as early as 6 h from
transfer to nitrogen-free medium and increased further thereafter.
Similar results were obtained using RNA gel-blot analysis (results
not shown, but see Figure 7B).
The reverse response, i.e. TbNrt2 down-regulation following
resupplementation of various nitrogen sources, was investigated
next. As shown in Figure 6(B), this was generally faster
than nitrogen-starvation-induced up-regulation and was always
preceded by a transient increase in TbNrt2 mRNA levels.
Ammonium and glutamine displayed the strongest downregulating capacity. They reduced TbNrt2 expression to levels
lower than those of t0 (time 0) controls (i.e. unshifted mycelia
cultured for 21 days in nitrogen-free medium) in less than 6 h
and turned it off almost completely in approx. 5 days. A much
more persistent increase in TbNrt2 abundance was observed with
proline, a poor nitrogen source for T. borchii ([18,22], and below),
which failed to lower TbNrt2 expression below t0 control levels,
even after 5 days of supplementation. An intermediate response,
with a marked increase in TbNrt2 abundance up to 6 h, followed
by a gradual decrease thereafter, was observed with nitrate. The
transient up-regulation observed after ammonium or glutamine
supplementation was as yet unexplained. The more substantial
and long-lasting increase in TbNrt2 levels observed with the
other two nitrogen compounds may, however, reflect the extremely
poor nitrogen source capacity of proline (and thus the unrelenting
nitrogen deprivation status imposed by the supplementation of this
amino acid) and nitrate-mediated induction of the NT mRNA as in
other fungi [3,4,6,9]. We tested the latter hypothesis by measuring
TbNrt2 expression in nitrogen (ammonium)-sufficient mycelia
transferred for various lengths of time to a medium containing
4 mM NaNO3 (or NaNO2 ) as the sole source of nitrogen. As shown
in Figure 7(A), TbNrt2 mRNA was indeed up-regulated (approx.
4-fold) as early as 24 h from exposure to nitrate and increased
further (up to approx. 7-fold) within the 5-day observation window
of this experiment. A smaller, but significant, up-regulation was
also observed upon shift to an nitrite-containing medium, whereas
no variation in TbNrt2 expression was observed following transfer
to the same ammonium-containing medium used for the initial
growth of mycelia (results not shown, but see below). An RNA
gel-blot experiment was then carried out to examine the effect of
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 (t 0 ) 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 (t 0 ) 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 NH4 Cl (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.
nitrate when supplied in combination with other nitrogen sources
and to compare the outcome of nitrogen starvation and nitrate
supplementation on both TbNrt2 and on the adjacent tbnr1 gene
(see Figure 1B). As shown in Figure 7(B), TbNrt2 expression
was similarly up-regulated following transfer to either nitrogenfree or nitrate-supplemented medium, but not to ammonium- or
glutamine-containing medium. Also apparent is the extremely
poor N-source capacity of proline; in its presence, TbNrt2 levels
were as high as in N-starved mycelia (Figure 7B, lanes Pro,
-N and NO3 − ) and it did not interfere with nitrate-induced
TbNrt2 up-regulation (Figure 7B, compare lane + NO3 − /Pro with
lanes + NO3 − /NH4 + and + NO3 − /Gln). Importantly, the data also
show the distinct response profile of the tbnr1 gene, which redily responded to nitrate supplementation but not to nitrogen
deprivation.
As shown by the immunoblot in Figure 7(C), derepression of the
TbNrt2 mRNA following nitrogen deprivation and its induction by
nitrate was accompanied by similar variations at the protein level.
The main difference (reproducibly observed in three independent
experiments) was that protein accumulation under both nitrogendeprivation and nitrate-supplementation conditions was usually
less persistent than transcript up-regulation.
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132
Figure 7
B. Montanini and others
Nitrogen-source-dependent regulation of TbNrt2 and tbnr1
Figure 8
Immunofluorescence localization of TbNrt2 in ectomycorrhizae
(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 NH4 Cl (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.
(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).
TbNrt2 localization in mycorrhizae
non-specific binding to, and detection of, some unrelated plant
antigen.
Anti-TbNrt2 antibodies were used to examine the expression of
the TbNrt2 transporter and its localization in T. borchii/C. incanus
ectomycorrhizae [29]. A fairly strong and specific TbNrt2-associated signal was detected by immunofluorescence, which also
revealed a differential labelling of the hyphae depending on
their location. Labelling was more pronounced in Hartig-netand mantle-forming (Figures 8A and 8C respectively) hyphae
that were in close contact with the roots than in extraradical hyphae. The green fluorescent signal was predominantly associated
with the inner part of the fungal wall, whose scaffold was revealed
by a red autofluorescence. This green line signal, which was easily
appreciated in transversally sectioned hyphae, can be attributed
to the fungal membrane (Figures 8A and 8C). By contrast, a
more extensive, but blurred, signal was observed in tangentially
sectioned hyphae (Figure 8A). No specific signal was detected
when the primary antibody was omitted (Figure 8D) or the
pre-immune serum was used in place of anti-TbNrt2 antibody
(Figure 8D, inset). Some labelling was occasionally observed in
the cytoplasm of plant cells with both the the anti-TbNrt2 antibody
and the pre-immune serum. This labelling, however, always appeared as an intense intracellular green spot which was easily
distinguishable from the fungus-associated signal, suggesting
c 2006 Biochemical Society
DISCUSSION
The TbNrt2 transporter
The predicted topology and secondary structure of TbNrt2 resemble those of NRT2 transporters from non-mycorrhizal fungi
characterized previously. The most prominent among these shared
features is the large VI–VII loop we used as antigen for antibody
production, the MFS and NS motifs, and two positionally
conserved arginine residues that are required for high-affinity
transport by A. nidulans NrtA [39]. Some less common features
were revealed by functional complementation assays in Ha.
polymorpha, which also highlighted the potentialities of this yeast
as a model system for the study of heterologous NTs. These
include the ability of TbNrt2 to support high-affinity transport of
nitrate and nitrite, but not chlorate ions. With the sole exception
of A. nidulans NrtA assayed in Xenopus oocytes [5], the nitrite
transport capacity of other fungal NTs has not, so far, been
determined directly. However, the observation that growth on
nitrite is not impaired in either an A. nidulans nrtA/ntrB double
mutant [6], or in an N. crassa nit-10 mutant [9], has been taken
High-affinity nitrate transporter from Tuber borchii
as an indication that none of the above NTs is capable of nitrite
transport. TbNrt2 is distinct from both Neurospora Nit-10 and
Aspergillus NrtA, but not NrtB, also with regard to chlorate
(and contaminating chlorite) tolerance. The lack of any influence
on chlorate sensitivity, compared with the corresponding NTdisrupted strain, is a common property of Ynt1 and TbNrt2, and
of its closest relative, NtrB (Figure 5). An amino acid residue
potentially involved in chlorate tolerance is Arg188 , located in the
outer loop interposed between TM helices V and VI (Figure 4A),
which is conserved positionally in TbNrt2, Ynt1 and NtrB, but not
in NtrA. The cellular and molecular tools described in the present
study, coupled with site-directed mutagenesis, will allow testing
of this hypothesis. Chlorate tolerance, resulting from the inability
to internalize this toxic ion, may be especially relevant if one
considers that: (i) chlorate is a soil pollutant which is also used
as a non-specific herbicide; (ii) there is an intimate association
(and metabolite exchange) between mycorrhizal fungi and their
host plants; and (iii) TbNrt2 is expressed in mycorrhizae, where
it preferentially accumulates in symbiotic hyphae that are in close
contact with the roots (Figure 8). Interestingly, a similar trait,
namely resistance to phosphinotricin {the active ingredient of
the herbicide glufosinate (4-[hydroxy(methyl)phosphinoyl]-DLhomoalanine)}, has been documented previously for T. borchii
glutamine synthetase [22].
Mode of regulation of TbNrt2
More distinguishing was the mode of regulation of TbNrt2, which
was found to be induced by nitrate, as in most nitrate-using organisms [1–3,10], but also up-regulated through a nitrate independent derepression mechanism triggered by nitrogen starvation. An
exception to this nitrogen-deprivation-based control was the transient TbNrt2 up-regulation observed after ammonium or glutamine refeeding of extensively starved mycelia (Figure 6B). In
keeping with the almost complete depletion of the amino acid
pool revealed by GLC/MS analysis of mycelia grown for more
than 5 days on nitrogen-free medium (results not shown), this
may reflect rapid nitrogen assimilation and transient TbNrt2
synthesis due to delayed accumulation of repressive nitrogen
compounds in severely starved cells. What is also intriguing is
the co-existence (and apparent redundancy) of nitrate-dependent
induction in a system that is primarily controlled by nitrogensource depletion. TbNrt2 levels were nearly the same under both
conditions (− N and + NO3 − ; Figure 7B), but TbNrt2 up-regulation in response to nitrate supplementation was generally
faster than that brought about by nitrogen depletion (Figures 6A
and 7A), and even more so when nitrate was supplied to mycelia
starved previously (Figure 6B). Thus a possible advantage of this
dual mode of regulation may be a faster up-regulation of nitrate
transport when both conditions are met.
Although as yet uncharacterized from a functional point of
view, an NT displaying the same kind of nitrate-independent
up-regulation as TbNrt2 has been described recently in the mycorrhizal basidiomycete He. cylindrosporum [23]. Interestingly,
the occurrence of a similar mode of regulation has been inferred previously from physiological data obtained in nitrophilic
yeasts such as Sporobolomyces roseus, Candida nitratophila and
Rhodotorula glutinis [40,41] and in the mycorrhizal basidiomycete Rhizopogon roseolus [11]. The above fungi colonize
different environments and are not closely related evolutionarily
to each other, nor to T. borchii. Therefore nitrate-independent
up-regulation of nitrate transport does not represent a conserved
feature shared by a restricted group of phylogenetically related
species, nor does it seem to be related to a particular growth
environment. Instead, it appears to be a fairly ancient regulatory
133
strategy that predated the appearance of multicellular fungi and
is now found in filamentous symbiotic ascomycetes and basidiomycetes.
Nitrate-independent TbNrt2 up-regulation and the
symbiotic lifestyle
What may then be the functional implications of nitrate-independent up-regulation and the reason(s) for its selective occurrence in mycorrhizal fungi? The most obvious answer is that
this regulatory strategy is somehow related to the ability of these
organisms to sustain host-plant nitrogen nutrition even under
conditions in which the concentration of available nitrate may
not be sufficient to induce nitrate assimilation genes. Under these
conditions, nitrate-independent up-regulation would also solve
the problem of nitrate uptake under ‘pre-induction’ conditions
(i.e. the entrance of the very first nitrate ions that are taken up
before fully executed nitrate-dependent induction). This strategy
would enable the fungus, and its host plant, to cope with the low
and fluctuating (by more than four orders of magnitude) nitrate
concentrations found in most natural, especially forest, soils. In
addition, simultaneous up-regulation of nitrate and ammonium
transporters by the same nitrogen shortage stimulus [18,21,22]
may allow a more balanced nitrogen nutrition when both inorganic
nitrogen sources are available in limited amounts. Although the
estimated K m of TbNrt2 for nitrate (4.7 µM) is in the lower
range of the values reported so far for fungal NTs, the obvious
disadvantage of this mode of regulation would be the high
biosynthetic cost of nitrate assimilation component expression
(NT, NR and NIR) in the presence of exceedingly low nitrate
concentrations, far below such K m . Noteworthy, in this regard,
is the observation that the NR gene of T. borchii (tbnr1), at
variance with TbNrt2 and with the corresponding genes of He.
cylindrosporum (nrt2 and nar1; [16,23]), is expressed at basal
levels under nitrogen-deprivation conditions and requires nitratemediated induction as in most organisms. Basal tbnr1 expression
in nitrogen-deficient mycelia (revealed by the faint hybridization
signal in Figure 7B) would alleviate the biosynthetic cost of
NR overexpression in the presence of low (non-inducing) nitrate
concentrations. Under the same conditions, however, it would
provide enough enzyme for nitrate reduction, which has been
shown to be functionally coupled with nitrate uptake [42].
Regulatory variation is one of the main pathways for organism
evolution and specialization. It is tempting to speculate that the
regulatory diversity of nitrate assimilation genes highlighted in
the present study may be one of the factors that determine the
environmental adaptation capacity of mycorrhizal (and non-mycorrhizal) fungi. Experimental verification of this hypothesis, as
well as more detailed studies of nitrogen-metabolism regulation
under symbiotic conditions, will be facilitated by the recent development of a genetic transformation system for T. borchii [43].
We thank Dr Angelo Viotti (Istituto di Biologia e Biotecnologia Agraria, CNR, Milano,
Italy) for the gift of the T. borchii cDNA library. The help of Elisabetta Soragni and Daniela
Pecorari in an early stage of this study is also gratefully acknowledged. The present
study was supported by grants from the Consiglio Nazionale delle Ricerche, the Regione
Emilia-Romagna and the University of Parma (FIL2004) to S. O., from the Fondo per
gli Investimenti della Ricerca di Base (‘Genomica funzionale dell’interazione tra piante
e microrganismi’) to A. B. and P. B., and by grant BFU2004-01012 from Ministerio de
Educación y Ciencia, Spain, to J. M. S. Y. M. is a Fellow of the Ministerio de Educación y
Ciencia, Spain.
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