Members of the NPF3 Transporter Subfamily Encode
Pathogen-Inducible Nitrate/Nitrite Transporters in Grapevine
and Arabidopsis
Regular Paper
Sharon Pike1,3, Fei Gao1,3, Min Jung Kim2,4, Sang Hee Kim1, Daniel P. Schachtman2,5 and
Walter Gassmann1,*
1
Division of Plant Sciences, Christopher S. Bond Life Sciences Center, and Interdisciplinary Plant Group, 1201 E. Rollins Rd., University of
Missouri, Columbia, MO 65211-7310, USA
2
Donald Danforth Plant Science Center, 975 N. Warson Rd., St. Louis, MO 63132, USA
3
These authors contributed equally to this work.
4
Present address: Examination Division of Food and Biological Resources, Korean Intellectual Property Office, Daejeon 302-701, Republic
of Korea.
5
Present address: Monsanto Company, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA.
*Corresponding author: E-mail, [email protected]; Fax, +1-573-884-9676.
(Received September 11, 2013; Accepted November 8, 2013)
Vitis vinifera, the major grapevine species cultivated for wine
production, is very susceptible to Erysiphe necator, the causal
agent of powdery mildew (PM). This obligate biotrophic
fungal pathogen attacks both leaf and berry, greatly affecting
yield and quality. To investigate possible mechanisms of nutrient acquisition by successful biotrophs, we characterized a
candidate NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER
FAMILY (NPF, formerly NRT1/PTR) member, grapevine
NFP3.2, that was up-regulated in E. necator-inoculated susceptible V. vinifera Cabernet Sauvignon leaves, but not in resistant V. aestivalis Norton. Expression in Xenopus laevis oocytes
and two-electrode voltage clamp measurements showed that
VvNPF3.2 is a low-affinity transporter for both nitrate and
nitrite and displays characteristics of NPF members from
other plants. We also cloned the Arabidopsis ortholog,
AtNPF3.1, and showed that AtNPF3.1 similarly transported
nitrate and nitrite with low affinity. With an Arabidopsis
triple mutant that is susceptible to E. necator, we found
that AtNPF3.1 is up-regulated in the leaves of infected
Arabidopsis similarly to VvNPF3.2 in susceptible grapevine
leaves. Expression of the reporter b-glucuronidase (GUS)
driven by the promoter of VvNPF3.2 or AtNPF3.1 in
Arabidopsis indicated that both transporters are expressed
in vascular tissue, with expression in major and minor veins,
respectively. Interestingly, the promoter of VvNPF3.2 allowed
induced expression of GUS in minor veins in PM-infected
leaves. Our experiments lay the groundwork for investigating
the manipulation of host nutrient distribution by biotrophic
pathogens and characterizing physiological variables in the
pathogenesis of this difficult to study grapevine disease.
Keywords: Arabidopsis thaliana Erysiphe necator Nitrate
transporters Powdery mildew Vitis aestivalis Vitis vinifera.
Abbreviations: dpi, days post-inoculation; GFP, green fluorescent protein; GUS, b-glucuronidase; NPF, NRT1 PTR family;
NRT, nitrate transporter; PAD4, PHYTOALEXIN DEFICIENT4;
PEN2, PENETRATION2; PM, powdery mildew; PTR, peptide
transporter; qRT-PCR, quantitative real-time PCR; SAG101,
SENESCENCE-ASSOCIATED GENE101; Va, Vitis aestivalis; Vv,
Vitis vinifera.
The nucleotide sequence reported in this paper has been
submitted to GenBank under accession number KF649633
(VvNPF3.2 cDNA).
Introduction
Nitrate transporters play an essential role in plants. Reduced
nitrate together with fixed carbon provide the building blocks
for amino acid and protein biosynthesis needed for plant growth,
development and defense against pathogens. Nitrate transporters in roots take up nitrate from the soil (Tsay et al. 2007).
When photosynthetic reductants are available, nitrate is transported to the leaf via xylem where it is reduced and assimilated
(Lillo 2008). Nitrate transporters are therefore needed to load
and unload xylem parenchyma for long-distance transport, to
import nitrate into the cytosol and to store excess nitrate in the
vacuole. Nitrate transporters also load phloem to remobilize nitrogen from older leaves (Fan et al. 2009).
Nitrate transporter families are large, vary by plant species
and are not well characterized. The NITRATE TRANSPORTER1/
PEPTIDE TRANSPORTER FAMILY (NPF, formerly NRT1/PTR)
members transport not only nitrate and peptides as their family
name implies, but also diverse substrates such as glucosinolates
and the plant hormones auxin and ABA (Krouk et al. 2010,
Plant Cell Physiol. 55(1): 162–170 (2014) doi:10.1093/pcp/pct167, available online at www.pcp.oxfordjournals.org
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NPF3s: pathogen-inducible NO3–/NO2– transporters
Kanno et al. 2012, Léran et al. 2013). Nitrate transporters within
the NPF that have been characterized by expression in Xenopus
laevis oocytes mainly mediate nitrate transport with low affinity
(millimolar range). However, at least one nitrate transporter,
AtNPF6.3 (CHL1, AtNRT1.1), displays dual low and high affinity
in oocytes (Liu and Tsay 2003), although the function of this
transporter in high-affinity nitrate uptake in planta is unclear
(Glass and Kotur 2013). In addition to nitrate, AtNPF6.3 transports auxin (Krouk et al. 2010). Nitrite, the product of cytosolic
nitrate reduction, was shown to be transported by Cucumis
sativus (cucumber) NPF3.1 (CsNrt1-L) (Sugiura et al. 2007).
The complex system of characterized nitrate transporters
that load and unload nitrate during long-distance transport
in Arabidopsis (Dechorgnat et al. 2011, Wang et al. 2012, and
references therein) represents only a small fraction of the 53
predicted NPF and seven high-affinity NRT2 transporters (Tsay
et al. 2007).
Powdery mildew (PM) is caused by a biotrophic pathogen that
must obtain nutrients from a living plant throughout its life cycle.
In grapevine, PM is caused by the ascomycete Erysiphe necator.
Both the leaf and berry of Vitis vinifera, the major species cultivated for wine production, are very susceptible to PM, greatly
affecting yield and quality (Stummer et al. 2005, Gadoury et al.
2012). The contribution of individual plant nutrient transporters
to fungal infection is unknown, although PM is a disease that one
might expect to be enhanced by increased nitrate transport. It has
long been known that overfertilization with nitrate increases the
severity of mildews in many crop plants (Marschner 2012). It has
also been shown that high nitrogen supply increased PM infection
in several grapevine cultivars (Keller et al. 2003).
Here, to investigate possible mechanisms of nitrogen acquisition by biotrophic fungi, we functionally characterized a
candidate NPF transporter, VvNPF3.2, that was up-regulated
in E. necator-inoculated susceptible V. vinifera Cabernet
Sauvignon leaves, but not in resistant V. aestivalis Norton in a
previous transcriptomic analysis of grapevine responses to
PM (Fung et al. 2008). Expression in Xenopus oocytes and
two-electrode voltage clamp measurements showed that
VvNPF3.2 and its previously uncharacterized Arabidopsis
ortholog, AtNPF3.1 (At1g68570), are low-affinity transporters
for both nitrate and nitrite and display biophysical characteristics of known NPF transporters. VvNPF3.2 and AtNPF3.1
appear to have overlapping but spatially distinct localization
in major and minor veins. Interestingly, the VvNPF3.2 promoter
caused inducible b-glucuronidase (GUS) expression in minor
veins after PM infection. We propose that E. necator may be
manipulating the host leaf into becoming a sink for nitrate.
Results
An NPF transporter up-regulated by PM infection
in susceptible grapevine leaves
To identify PM-responsive transporter candidate genes that are
up-regulated in PM-infected grapevine leaves at time points
when the fungus could acquire nutrients from the plant, we
examined previously published Vitis GeneChip data from a
comparison of PM-inoculated resistant Norton and susceptible
Cabernet Sauvignon (Fung et al. 2008). In that study, secondary
hyphae observed microscopically indicated that fungal
haustoria were most probably developed and obtaining host
nutrients by 24 h post-inoculation. No transporters were significantly up-regulated in the resistant cultivar Norton (data
not shown). In contrast, in leaves of susceptible Cabernet
Sauvignon, Arabidopsis orthologs of amino acid transporters,
an oligopeptide transporter and a putative nitrate transporter
were up-regulated by 24 h after inoculation. The putative
nitrate transporter VvNPF3.2 (Vitis GeneChip probe set ID
1613896_at corresponding to the gene GSVIVT01025795001
in the PN40042 genome), an NPF member, was up-regulated
at 12, 24 and 48 h post-inoculation (data not shown).
To verify the transcript levels of Vitis NPF3.2, quantitative
real-time PCR (qRT-PCR) was performed on cDNA that had
been synthesized from the same RNA samples collected for the
Vitis GeneChip data (Fung et al. 2008). Because nitrate metabolism in leaves changes in response to light and circadian
rhythms (Lillo 2008), we chose 0, 24 and 48 h after PM inoculation as time points to compare mRNA levels for these Vitis
genes. VvNPF3.2 transcript levels in PM-infected leaves were not
significantly up-regulated as compared with mock-inoculated
leaves immediately after infection. However at 24 h, VvNPF3.2
transcript levels were significantly greater in PM-inoculated
leaves than in mock-inoculated leaves, whereas by 48 h any
additional changes in VvNPF3.2 mRNA levels were masked by
apparent non-specific inoculation-induced changes (Fig. 1). In
Norton, differences between VaNPF3.2 mRNA levels in mockand PM-inoculated samples were not significant at any of the
time points (data not shown). These data partially confirm the
results obtained by GeneChip hybridization, and suggest that
VvNPF3.2 may play a role during host colonization by PM.
Homology of the VvNPF3.2 transporter
We isolated a full-length VvNPF3.2 cDNA from a Cabernet
Sauvignon cDNA library using the PN40024 V. vinifera
genome sequence as a reference. Among predicted proteins,
VvNPF3.2 showed the highest amino acid sequence similarity to
C. sativus CsNPF3.2, Arabidopsis AtNPF3.1, characterized below,
and the uncharacterized Oryza sativa (rice) predicted protein
OsNPF3.1 (Sugiura et al. 2007, Tsay et al. 2007). We compared
the amino acid sequence of VvNPF3.2 with these orthologs
(Supplementary Fig. S1). Although VvNPF3.2 and CsNPF3.2
show 72% amino acid sequence identity, there are pronounced
differences in their N- and C-termini. In particular, VvNPF3.2
and the Arabidopsis and rice orthologs seem to be lacking a
chloroplast targeting signal predicted for CsNPF3.2 (Sugiura
et al. 2007). VvNPF3.2 has 58% and 53% identity to AtNPF3.1
and OsNPF3.1, respectively, as reported for the identity of
CsNPF3.2 to these two (Sugiura et al. 2007). Computational
prediction of subcellular localization using iPSORT (Bannai
et al. 2002) predicts plasma membrane localization for
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Fig. 1 VvNPF3.2 mRNA levels increase in PM-infected grapevine
leaves. Quantitative real-time PCR analysis of NPF3.2 transcript
levels in mock- and PM-inoculated leaves of V. vinifera Cabernet
Sauvignon at 0, 24 and 48 hours post-inoculation (hpi). Values represent averages from three biological replicates normalized with 60SRP
transcript levels (Gamm et al. 2011), and error bars denote the standard error. An asterisk denotes a statistically significant difference
between VvNPF3.2 transcript levels in mock- and PM-inoculated
leaves at 24 hpi (*P < 0.05); ns, not significant. This experiment was
repeated twice with similar results.
VvNPF3.2, AtNPF3.1 and OsNPF3.1. We experimentally verified
the plasma membrane localization (and absence of chloroplast
localization) of AtNPF3.1 and VvNPF3.2 by transient expression
in Nicotiana benthamiana (Supplementary Fig. S2).
VvNPF3.2 is a low-affinity nitrate/nitrite
transporter
Electrophysiological experiments were performed to determine
the substrates transported by VvNPF3.2. As an NPF, it could be
expected to transport nitrate, dipeptides or amino acids with
low affinity. As an ortholog with very high similarity to
CsNPF3.2, it could be expected to transport nitrite with high
affinity. Co-transporters can be efficiently characterized by expressing cRNA in Xenopus oocytes and measuring changes in
current when a substrate is added. Using two-electrode voltage
clamp to control membrane voltage during the measurement,
we clamped at –40 mV to prevent inducing the endogenous
oocyte nitrate transporter when 10 or 20 mM nitrate was
applied. To minimize the confounding contributions of other
ions, we used a bath solution containing only 0.15 mM CaCl2,
mannitol and MES/Tris buffer (Huang et al. 1999). Under these
conditions, oocytes injected with VvNPF3.2 cRNA had significantly more negative (inward) current than uninjected control
oocytes when 1, 5, 10 or 20 mM HNO3 was applied in the bath
solution at pH 5.5 (Fig. 2). Nitrate-incited currents increased
with increasing concentration; however, plots of measurements
up to 10 mM were approximately linear, indicating that
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Fig. 2 VvNPF3.2 transports nitrate with low affinity. Each oocyte was
exposed to increasing concentrations of HNO3 at pH 5.5 and a holding
potential of –40 mV. Results are averages of difference currents ± SE.
For each concentration, n = 6–17 VvNPF3.2-expressing and 6–20 uninjected oocytes from 3–9 different Xenopus frogs were used. Asterisks
denote significant differences in current levels between uninjected
and VvNPF3.2-injected oocytes as determined by Student’s t-test
(*P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0005).
transport was not saturated. More than half of the measurements showed saturation by 20 mM HNO3, and the concentration at which currents were half-maximal (Km) was calculated
to be 23 ± 8 mM (n = 6, average ± SD). This low substrate affinity is consistent with VvNPF3.2 being an NPF member.
Uptake of uncharged or negatively charged substrates is
usually coupled to uptake of one or more protons, resulting
in uptake of a net positive charge that is recorded as a
pH-dependent inward current. Indeed, VvNPF3.2-mediated
currents were significantly more negative at pH 5 than at pH
7 (Fig. 3), suggesting that, like other characterized nitrate transporters, VvNPF3.2 is a proton-coupled nitrate transporter.
Another hallmark of most NPF nitrate transporters characterized in Xenopus is their selectivity for nitrate over dipeptides
and amino acids. We therefore performed selectivity experiments. Average currents in VvNPF3.2-injected oocytes at pH
5.0 were not significantly different from the control when
10 mM amino acids (P > 0.05) or dipeptides (P > 0.1–0.4)
were applied (Fig. 4). However, 10 mM HNO3 incited currents
in these batches of oocytes that were highly significantly different from uninjected control oocytes (P < 0.0001), indicating
that VvNPF3.2 selectively transports nitrate.
CsNPF3.2 was reported to transport nitrite (Sugiura et al.
2007), prompting us to test nitrite as a possible VvNPF3.2 substrate. Nitrite could not be applied as HNO2, necessitating its
addition as a salt. To minimize the effect of the additional
positive ion, we alternated solutions containing identical concentrations of sodium glutamate or sodium nitrite and subtracted the difference in elicited currents. Preliminary
experiments performed at pH 5 to give maximum transport
showed that 0.1 mM NO2– did not elicit currents in VvNPF3.2injected oocytes that were significantly different from currents
in control oocytes (P > 0.1; data not shown). Further experiments performed at pH 5.35 to permit a constant pH with
increasing sodium glutamate showed that significant inward
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NPF3s: pathogen-inducible NO3–/NO2– transporters
Fig. 3 VvNPF3.2-mediated nitrate transport is pH dependent. Current
was measured for each oocyte at a holding potential of –40 mV at pH
5, 6 and 7, without and with 10 mM HNO3. Results, corrected for
change caused by pH alone, are averages of difference currents ± SE.
For each concentration, n = 8 VvNPF3.2-injected and n = 8 uninjected
oocytes from three different Xenopus frogs. An asterisk denotes a
statistically significant difference between currents elicited by nitrate
at pH 5 in VvNPF3.2-injected and uninjected oocytes as determined by
Student’s t-test (*P < 0.05); ns, not significant.
Fig. 4 VvNPF3.2 transport is selective for nitrate. Currents induced by
10 mM HNO3 or the indicated amino acids and dipeptides were measured at a holding potential of –40 mV at pH 5 for each oocyte. Results
are averages of difference currents ± SE. HNO3 was the final substrate
in each series. Currents elicited in VvNPF3.2-injected and uninjected
oocytes are significantly different (*****P < 0.0001); ns, not significantly different (P > 0.05) as determined by Student’s t-test. For
each substrate, oocytes are from 3–5 different Xenopus frogs, and
averages are from the following numbers of measurements: Ala-Asp
(n = 9 VvNPF3.2-expressing oocytes; n = 8 uninjected oocytes), Gly–
Asp (n = 9; 8), Asp (n = 6; 6), Asp–Asp (n = 4; 4), Asp–Glu (n = 3; 4),
Glu (n = 5; 5), Glu–Glu (n = 5; 3), HNO3 (n = 15; 12).
transport is evident at 5, 10 and 20 mM nitrite in VvNPF3.2injected oocytes (Fig. 5). We concluded that VvNPF3.2 is a
low-affinity grapevine nitrate/nitrite transporter.
AtNPF3.1 is induced by E. necator in the
susceptible Arabidopsis pen2-1 pad4-1 sag101-2
triple mutant and encodes a low-affinity nitrate/
nitrite transporter
To explore further general features of nutrient transporter induction by biotrophic pathogens, we investigated whether
AtNPF3.1 is induced in Arabidopsis by E. necator during PM
infection similarly to VvNPF3.2. Because wild-type Arabidopsis
displays non-host resistance and cannot be colonized by
Fig. 5 VvNPF3.2 also transports nitrite with low affinity. Results are
averages of difference currents ± SE induced by the indicated concentrations of nitirite at pH 5.35 and a holding potential of –40 mV. For
each concentration, n = 7–8 VvNPF3.2-expressing and n = 7 uninjected
oocytes isolated from two Xenopus frogs. Asterisks denote significant
differences in current levels between uninjected and VvNPF3.2injected oocytes as determined by Student’s t-test (*P < 0.05;
**P < 0.01; *****P < 0.0005).
E. necator, we used the pen2-1 pad4-1 sag101-2 triple mutant
for infections. This mutant was reported to have lost non-host
resistance and to have become susceptible to non-adapted
fungal crop pathogens (Lipka et al. 2005). The pen2 mutation
allows the fungus to penetrate the host cuticle and the pad4
and sag 101 mutations prevent induced plant defenses subsequent to initial attempts at fungal colonization. Consistent with
this, we found that E. necator is able to infect pen2-1 pad4-1
sag101-2 plants and reproduce asexually (Supplementary
Fig. S3A). qRT-PCR was performed on samples from pen2-1
pad4-1 sag101-2 mutant leaves inoculated with E. necator or
mock-inoculated leaves. At 48 h post-inoculation, AtNPF3.1 was
significantly induced in E. necator-inoculated leaves as compared with the mock-inoculated leaves (Supplementary
Fig. S3B). This suggests that AtNPF3.1 in Arabidopsis and
VvNPF3.2 in grapevine could function similarly during fungal
colonization.
We expressed AtNPF3.1 in Xenopus oocytes and measured the
currents resulting from nitrate and nitrite application. HNO3 at 1,
5 and 10 mM and pH 5 caused significantly (P < 0.05) more
current in the AtNPF3.1-injected oocytes than in the uninjected
ones (Fig. 6). Four out of five oocytes showed saturation of the
transporter at 10 mM HNO3, with a Km of 9 ± 3 mM (n = 4,
average ± SD). No significant levels of inward current were
induced by 0.1 mM NO2 at pH 5 or 1 mM NaNO2 at pH 5.35
in injected oocytes as compared with controls (data not shown).
In contrast, 5, 10 and 20 mM NaNO2 induced inward currents in
injected oocytes that were highly significantly different from controls (Fig. 7). We concluded that AtNPF3.1 is also a low-affinity
nitrate/nitrite transporter.
Expression patterns of VvNPF3.2 and AtNPF3.1
To study VvNPF3.2 and AtNPF3.1 expression in planta,
stable transgenic Arabidopsis plants expressing promoter:GUS
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S. Pike et al.
Fig. 6 AtNPF3.1 transports nitrate with low affinity. Each oocyte was
exposed to increasing concentrations of HNO3 at pH 5.0 and a holding
potential of –40 mV. Results are averages of difference currents ± SE.
For each concentration, 8–9 AtNPF3.1-expressing oocytes and 5–6
uninjected oocytes, isolated from at least three different Xenopus
frogs, were measured. Asterisks denote significant differences in current levels between uninjected and AtNPF3.1-injected oocytes as
determined by Student’s t-test (*P < 0.05); ns, not significant.
Fig. 7 AtNPF3.1 transports nitrite with low affinity. Results are
averages of difference currents ± SE at pH 5.35 and a holding potential
of –40 mV. For each concentration, 9–10 AtNPF3.1-expressing and
six uninjected oocytes, isolated from two different Xenopus frogs,
were measured. Asterisks denote significant differences in current
levels between uninjected and AtNPF3.1-injected oocytes as determined by Student’s t-test (***P < 0.005; ******P < 0.000005); ns, not
significant.
constructs were generated in the Col-0 and pen2-1 pad4-1
sag101-2 backgrounds. In 2- to 4-week old Arabidopsis plants,
VvNPF3.2pro:GUS was strongly expressed in petioles and
large veins of both expanding and expanded leaves, with
slight expression in the main root, suggesting an involvement of
VvNPF3.2 in long-distance transport (Fig. 8A; Supplementary
Fig. S4A, C). AtNPF3.1pro:GUS was strongly expressed in expanded leaves along minor veins, and is clearly not expressed
in midveins, petiole or seedling roots (Fig. 8B; Supplementary
Fig. S4B, D). Expression was limited to smaller veins and
became stronger as leaves aged, perhaps indicating that
AtNPF3.1 functions in nitrogen remobilization.
We further used VvNPF3.2pro:GUS-expressing pen2-1 pad4-1
sag101-2 triple mutants to test for changes in VvNPF3.2 expression in response to E. necator. In Fig. 1, we showed
that VvNPF3.2 mRNA increased by 24 h after E. necator infection of Cabernet Sauvignon leaves. As shown in Fig. 8D,
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Fig. 8 Expression of VvNPF3.2 and AtNPF3.1 in transgenic Arabidopsis
Col-0 and pen2-1 pad4-1 sag101-2 leaves. GUS activity was visualized
as blue color. (A) Mature leaf containing VvNPF3.2pro:GUS. (B) Mature
leaf containing AtNPF3.1pro:GUS. Scale bars = 1 mm. (C, D) The
VvNPF3.2 promoter is activated by grapevine PM. (C) Mockinoculated leaves of transgenic Arabidopsis pen2-1 pad4-1 sag101-2
containing the VvNPF3.2pro:GUS fusion construct. (D) Leaves inoculated with E. necator. Mock- and E. necator-inoculated leaves were
from the same plant and stained for GUS activity at the indicated
days post-inoculation. Images are representative of three independent
transgenic lines, and experiments were repeated twice with similar
results.
VvNPF3.2pro:GUS activity steadily increased after triple mutant
Arabidopsis leaves were inoculated with E. necator as compared
with the mock-inoculated control (Fig. 8C), indicating that the
promoter of VvNPF3.2 was responsive to E. necator in this
heterologous system. By 5 days post-inoculation (dpi), the
PM-inoculated midveins stained more strongly than the
mock-inoculated midveins and more than at 0 dpi (Fig. 8D).
At 10 dpi, the GUS activity had even expanded to the minor
veins (Fig. 8D), possibly suggesting redirection of nitrogen distribution by E. necator. Induction of AtNPF3.1pro:GUS expression
by E. necator in transgenic Arabidopsis was not tested because
the mature leaf displayed a high level of GUS activity in the
absence of infection.
Discussion
In this report we describe the cloning and characterization of
the grapevine transporter VvNPF3.2 and its previously uncharacterized Arabidopsis ortholog, AtNPF3.1, the only Arabidopsis
representative of the NPF3 subfamily (Léran et al. 2013). Our
measurements of currents in response to nitrate by twoelectrode voltage clamp in Xenopus oocytes expressing
VvNPF3.2 and AtNPF3.1 indicate that both proteins transport
nitrate with low affinity, that VvNPF3.2 does not transport dipeptides and that VvNPF3.2 nitrate-responsive currents are dependent on low external pH. These results are consistent with
transport activities that have been reported for most of the predicted Arabidopsis NPF members characterized in Xenopus
Plant Cell Physiol. 55(1): 162–170 (2014) doi:10.1093/pcp/pct167 ! The Author 2013.
NPF3s: pathogen-inducible NO3–/NO2– transporters
oocytes and formerly classified as subgroup 4 NRT1 (Tsay
et al. 2007).
Gauging by the apparent affinity for VvNPF3.2 and AtNPF3.1
(Km 23 mM and 9 mM, respectively) and the availability of
nitrate, these transporters could transport nitrate in the plant.
Nitrate in the xylem sap of decapitated plants can be found at
concentrations of 1–20 mM (Glass and Siddiqi 1995), varying
with season of the year and time of day. Xylem sap of pressurized V. vinifera cv Riesling leaves was reported to contain
0.5–3 mM nitrate depending upon soil and fertilization
(Peuke 2000). It is possible that VvNPF3.2 and AtNPF3.1 can
be switched between low-affinity and high-affinity transport by
post-translational modifications, similarly to AtNPF6.3 (Liu and
Tsay 2003), or that they transport untested substrates with
higher affinity, similarly to AtNPF2 members, low-affinity
nitrate transporters that also transport glucosinolate defense
compounds with high affinity (Nour-Eldin et al. 2012). In that
study, AtNPF3.1 did not transport glucosinolates.
The high homology of VvNPF3.2 to CsNPF3.2 and the inward currents seen with VvNPF3.2- and AtNPF3.1-expressing
Xenopus oocytes in response to millimolar nitrite raises the
question of what biological function low-affinity nitrite transport may have in planta. The transport of nitrite, the product of
nitrate reduction, has been less studied than the more easily
measured long-distance low-affinity xylem nitrate transport.
Until recently, nitrite was usually considered to be toxic, present only in very low micromolar amounts and able to cross
membranes by diffusion (Heber and Heldt 1981). Because
HNO2 is a weak acid with a pKa value of 3.4, a significant portion
of nitrite would be protonated under acidic apoplastic conditions (pH 5–5.5) and at millimolar concentrations would permeate the membrane without the action of a transporter.
Nevertheless, expression of the CsNPF3.2 gene in rice permitted
vigorous growth on 30 mM KNO3 or 20 mM KNO2 as sole
sources of nitrogen (Sustiprijatno et al. 2006), suggesting that
VvNPF3.2 could have a protective function in overfertilized
soils. The similarity of induction times for nitrite and nitrate
uptake and the interaction of the two uptake systems suggest
they may use the same transporters in some plants (Aslam et al.
1992), while evidence for distinct high-affinity nitrate and nitrite transporters in Arabidopsis was recently reported (Kotur
et al. 2013). In studies of NPF transporters, nitrite is usually not
tested as a substrate, and it is possible that dual nitrate/nitrite
transport is a more common feature of this transporter class.
Localization of VvNPF3.2 and AtNPF3.1
While the affinity of a transporter for a substrate and its availability defines what can be transported, where and when the
transporter is expressed mainly determines its function. The
very low VvNPF3.2pro:GUS and absent AtNPF3.1pro:GUS expression in Arabidopsis roots in our experiments indicates that
probably neither transporter takes up nitrate or nitrite from
the soil. Other reports support the lack of AtNPF3.1 root
expression in 1- (Zhao et al. 2010) and 2-week old
Arabidopsis (Tsay et al. 2007). Both studies showed moderate
to high expression of AtNPF3.1 in stem and leaf tissue, consistent with our results. However, slightly older roots than those
used in our experiments showed moderate microarray expression (Zhao et al. 2010).
The vascular expression we showed for VvNPF3.2pro:GUS
and AtNPF3.1pro:GUS has been reported for other NPF nitrate
transporters. AtNPF7.2 (AtNRT1.8), a long-distance transporter
that is expressed in xylem parenchyma of stems, leaves and also
roots (Li et al. 2010), is found in major veins, similarly to
VvNPF3.2pro:GUS. Expression of AtNPF2.13 (AtNRT1.7), which
was proposed to remobilize nitrate, increases in minor veins
of leaves as they age (Fan et al. 2009), similarly to
AtNPF3.1pro:GUS. With expression in N. benthamiana, we have
shown that AtNPF3.1 is located in the plasma membrane, befitting a role in loading or unloading nitrate in plant cells along the
vasculature. Interestingly, expression of VvNPF3.2pro:GUS in E.
necator-infected Arabidopsis strengthened between 0 and
10 dpi in major veins and even expanded into minor veins
near the end of this period. Transporter expression in main
leaf veins in the first days of infection would be expected to
move nitrate from stems or from storage in petioles. By 10 d,
the vast increase in fungal hyphae should cause reduced photosynthesis and local release of nitrate from vacuoles and catabolized proteins, necessitating more local distribution of
nitrate.
Biotrophs need to manipulate host metabolism and nutrient distribution in order to thrive (Chen et al. 2010). It has been
reported that the nutrient demands of the fungus during PM
infection affect the source–sink relationship in the plant, i.e.
nutrients that would normally go to young leaves or developing
seeds are instead exported to the fungus (Ayres et al. 1996,
Hall and Williams 2000, Chandran et al. 2010). The ability of
E. necator similarly to up-regulate VvNPF3.2 in grapevine leaves
and AtNPF3.1 in susceptible Arabidopsis leaves suggests that
the response of a susceptible plant to this pathogen generally
involves increased nitrate or nitrite transport. Because
E. necator and Golovinomyces orontii (PM of Arabidopsis)
appear to lack genes encoding inorganic nitrate transporters,
nitrate reductases and nitrite reductases (Spanu et al. 2010), it is
most likely that the plant increases nitrate transport to leaves,
reduces inorganic nitrate and provides the fungus with ammonium, amino acids and peptides. Consistent with this hypothesis, other V. vinifera transporters up-regulated 24 and 48 h after
PM infection include orthologs of the amino acid transporters
AtGAT1 and AtLHT1, and the oligopeptide transporter AtOPT4
(Fung et al. 2008). A proteomic study showing induction of
glutamine synthetase and alanine aminotransferases in PMinfected V. vinifera leaves by 48 h post-inoculation (Marsh
et al. 2010) also suggests increased organic nitrogen synthesis.
In conclusion, we anticipate that the Arabidopsis pen2-1
pad4-1 sag101-2 triple mutant will be a useful tool to elucidate
the effect of nitrate transport on the susceptibility of grapevine
to PM or other pathogens under different nutrient and environmental conditions. In particular, the effect of VvNPF3.2 and
AtNPF3.1 on susceptibility to PM and abiotic stress, nitrate
Plant Cell Physiol. 55(1): 162–170 (2014) doi:10.1093/pcp/pct167 ! The Author 2013.
167
S. Pike et al.
accumulation, growth and development could be tested with
relative ease in Arabidopsis as compared to grapevine.
Materials and Methods
RNA isolation and qRT-PCR
To verify transcriptional regulation of VvNPF3.2 and VaNPF3.2,
we used cDNA synthesized as described from the same RNA
used in the Vitis GeneChip hybridization (Fung et al. 2008).
To quantify transcripts of AtNPF3.1, total RNA was extracted
from Arabidopsis pen2-1 pad4-1 sag101-2 leaves with Trizol
(Invitrogen) at 0, 24 and 48 h after inoculation with E. necator
or from mock-inoculated triple-mutant leaves. cDNA was
synthesized from 1 mg of total RNA using an oligo(dT)15
primer and Moloney murine leukemia virus (MMLV) reverse
transcriptase (Promega). qRT-PCR was performed with an ABI
7500 system (Applied Biosystems) and SYBR GREEN PCR Master
Mix. Transcript levels were normalized using mRNA levels of the
60SRP (Gamm et al. 2011) and SAND gene (At2g28390) as internal standards for grapevine and Arabidopsis, respectively.
Primers used are listed in Supplementary Table S1.
Constructing transgenic Arabidopsis
VvNPF3.2– and AtNPF3.1–promoter GUS lines
The AtNPF3.1 promoter from Arabidopsis Col-0 (2,025 bp
upstream from the start codon) and the VvNPF3.2 promoter
from V. vinifera Cabernet Sauvignon genomic DNA (2,303 bp
upstream from the start codon) were cloned into the Gateway
entry vector pDONR201 and subcloned into binary vector
pYXT1 containing the GUS reporter gene. Transgenic
Arabidopsis promoter–GUS lines were generated by the floral
dip method (Clough and Bent 1998) using Agrobacterium
tumefaciens strain GV3101. Transformants were screened on
half-strength Murashige and Skoog medium (Sigma) containing
40 mg ml-1 kanamycin (Sigma). Single-locus homozygous transgenic lines expressing VvNPF3.2pro:GUS or AtNPF3.1pro:GUS were
selected by scoring for the segregation of kanamycin resistance
in the T2 and T3 generations.
Maintaining E. necator and inoculating leaves
Erysiphe necator was isolated and maintained as described in
Fung et al. (2008). To minimize damage from inoculation, transgenic pen2-1 pad4-1 sag101-2 lines expressing VvNPF3.2pro:GUS
or AtNPF3.1pro:GUS were infected with E. necator by transferring
10- to 12-day-old conidiospores onto the leaves with a fine
brush. Mock-inoculated Arabidopsis leaves were touched
with a clean brush with no E. necator spores.
Histochemical GUS assays
To visualize GUS expression, whole plants or freshly excised tissues expressing VvNPF3.2pro:GUS or AtNPF3.1pro:GUS were soaked
with substrate buffer (0.5 mM 5-bromo-4-chloro-3-indolyl glucuronide, 100 mM Tris, pH 7.0, 50 mM NaCl, 0.06% Triton
X-100, 3 mM potassium ferricyanide) and were incubated at
168
37 C for 16 h (Jefferson et al. 1987). Chl was cleared from stained
tissue by incubating in 70% ethanol. To visualize E. necator fungal
structures, leaves were stained with 0.05% aniline blue (Sigma) in
a lacto-glycerol solution (1 : 1 : 1, lactic acid : glycerol : water, by
vol.) (Vanacker et al. 2000). Imaging was performed using a
LEISTER Leica-M205-Stereoscope and a digital camera.
Agrobacterium-mediated transient expression and
confocal fluorescence microscopy
The green fluorescent protein (GFP) fusion binary constructs
were electroporated into A. tumefaciens strain GV3101. Bacteria
cultured overnight were harvested by centrifugation and resuspended in 10 mM MgCl2 with 100 mM acetosyringone (SigmaAldrich) and adjusted to an OD600 of 0.25–0.3. Agrobacterium
was incubated for 2 h at room temperature and infiltrated into
N. benthamiana leaves with a 1 ml needleless syringe. The
N. benthamiana plants were placed in an E-7/2 reach-in
growth chamber (Controlled Environments Ltd.) under a 16 h
light/8 h dark cycle at 25 C, 70% relative humidity. Tissues were
collected 3 d after infiltration for Western blot and confocal
microscopy. Plant tissues were viewed directly under a Zeiss
LSM 510 META NLO two-photon point-scanning confocal
system mounted on an Axiovert 200M inverted microscope
with a 40/1.2 C-Apochromat water immersion objective.
Yellow fluorescent protein fluorescence was excited by a
514 nm argon laser. Sample fluorescence was detected using a
500–550 nm band-pass emission filter.
Xenopus oocytes and two-electrode voltage clamp
VvNPF3.2 cDNA (1,815 bp with BamHI and EcoRI flanking restriction enzyme sites) and AtNPF3.1 cDNA (1,791 bp with ApaI
and XhoI flanking restriction enzyme sites) were generated by
PCR and cloned into the Xenopus expression plasmid pOO2
(Ludewig et al. 2002). Capped cRNA was synthesized by in vitro
transcription with a mMESSAGE mMACHINEÕ SP6 Kit
(Ambion, Inc.) according to the manufacturer’s instructions.
Oocytes were isolated, maintained and injected as described
(Pike et al. 2009). Two-electrode voltage clamp measurements
with additions of nitrate as HNO3 or of amino acids and dipeptides were conducted in a bath solution containing 230 mM
mannitol, 0.15 mM CaCl2 and 10 mM MES/Tris at pH 5.5
(Huang et al. 1999) or pH 5.0 as noted, 2–5 d after injection
with VvNPF3.2 or AtNPF3.1 cRNA. For measurements involving
nitrite, the pH was 5.35 and buffering with MES/Tris was
reduced to accommodate the buffering effect of sodium
glutamate. To control for the effect of added sodium, bath
solutions with sodium glutamate were alternated with bath
solutions containing equimolar sodium nitrite. Difference
current values were obtained by subtracting current levels
before substrate addition from current values after addition.
Accession numbers
AtNPF3.1, At1g68570; CsNPF3.2 (CsNitr1-L), Z69370; OsNPF3.1,
Os06g15370; VvNPF3.2, KF649633.
Plant Cell Physiol. 55(1): 162–170 (2014) doi:10.1093/pcp/pct167 ! The Author 2013.
NPF3s: pathogen-inducible NO3–/NO2– transporters
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the United States Department of
Agriculture-National Institute of Food and Agriculture [grants
No. 2008-38901-19367 to W.G. and D.P.S., and grant Nos. 200938901-19962 and 2010-38901-20939 to W.G.].
Acknowledgments
We thank Paul Schulze-Lefert and Volker Lipka for providing
pen2-1 pad4-1 sag101-2 Arabidopsis seeds, Wenping Qiu and Ru
Dai for providing E. necator inoculum and cDNA from samples
used in the Vitis GeneChip hybridization, the MU Molecular
Cytology Core for assistance with light and confocal microscopy, and Alissa Higgins for technical support.
Disclosures
The authors have no conflicts of interest to declare.
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