Download The symbiotic ion channel homolog DMI1 is localized in the nuclear

The Plant Journal (2007) 49, 208–216
doi: 10.1111/j.1365-313X.2006.02957.x
The symbiotic ion channel homolog DMI1 is localized in the
nuclear membrane of Medicago truncatula roots
Brendan K. Riely1, Géraldine Lougnon2, Jean-Michel Ané2 and Douglas R. Cook1*
Department of Plant Pathology, University of California, One Shields Avenue, Davis, CA 95616, USA, and
2
Department of Agronomy, University of Wisconsin, 1575 Linden Drive, Madison, WI 53706, USA
1
Received 7 August 2006; accepted 14 September 2006.
*For correspondence (fax 530 754 6617; e-mail [email protected]).
Summary
5
Legumes utilize a common signaling pathway to form symbiotic associations both with rhizobial bacteria and
arbuscular mycorrhizal fungi. The perception of microbial signals is believed to take place at the plasma
membrane, activating a cascade that converges on the nucleus where transcriptional reprogramming
facilitates the symbioses. Forward genetic strategies have identified genes in this signaling pathway including
Medicago truncatula DMI1 (Doesn’t Make Infections 1) that encodes a putative ion channel. Although the DMI1
homologs from Lotus japonicus, CASTOR and POLLUX, were recently reported to be localized in plastids, we
report here that a functional DMI1::GFP fusion is localized to the nuclear envelope in M. truncatula roots when
expressed both from a constitutive 35S promoter and from a native DMI1 promoter. Localization may be
mediated in part by sequences located within the amino-terminus of DMI1. This region of DMI1 is required for
symbiotic signal transduction, and its replacement with a bona fide plastid transit peptide from the glutamine
synthetase 2 gene does not restore DMI1 function. These new data place DMI1 in the nuclear envelope in close
proximity to the origin of Nod-factor-induced calcium spiking.
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Keywords: nucleus, Medicago truncatula, nodulation, ion channel, nuclear localization signal, symbiosis.
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4
Introduction
Symbiotic associations between plants and microbes allow
many plants to grow under nutrient-limiting conditions.
More than 80% of land plants can enter into symbioses with
arbuscular mycorrhizal (AM) fungi, thereby facilitating the
uptake of inorganic nutrients (Harrison, 2005); whereas legumes can enter into an additional symbiosis with soil bacteria called rhizobia, thereby gaining access to reduced
nitrogen through nitrogen fixation (Soltis et al., 1995).
Morphologically the two symbioses appear quite distinct,
although they share a number of features including microbial infection of the host plant, transcriptional activation of a
common subset of plant genes, and formation of an intracellular microbe–plant interface where nutrient exchange
occurs. Most notably, both of these plant–microbe collaborations commence with the exchange of molecular signals
within the rhizosphere, a situation that is best understood in
the case of symbiotic nitrogen fixation.
Legume roots secrete flavonoids, which trigger the synthesis of a bacterial signal molecule known as Nod factor.
Nod factors are lipochitooligosaccharidic molecules com208
posed of b-1,4-linked N-acetyl-glucosamine residues harboring various substituents along the sugar backbone
(D’Haeze and Holsters, 2002). The nature of these substituents varies by bacterial species, and is the basis for the often
high degree of host specificity observed in legume–rhizobium interactions. Nod factors from compatible rhizobia
reorient root hair growth to entrap bacteria and facilitate
their entry into developing root hairs. Nod factors also
initiate a developmental program within the host, creating a
new meristematic zone in the root cortex from which a
nodule develops. Bacterial infection and nodule development converge when the invading bacteria traverse a plantderived infection thread into the developing nodule, where
they are released into the cytoplasm of specialized host cells
of the nodule central tissue. Within these host cells the
differentiated bacteria, surrounded by a symbiotic membrane of host origin, function as facultative organelles for
nitrogen fixation.
Genetic analyses in several legume species have identi8 fied mutants that are unable to form root nodules (Nod–),
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd
1
DMI1 is localized in the nuclear membrane 209
and which are blocked at various points in the symbiotic
interaction (reviewed by Riely et al., 2006). A subset of Nod–
mutants are also unable to form symbiotic associations with
AM fungi, indicating that these genes participate in a
common symbiotic pathway (Catoira et al., 2000; Kistner
et al., 2005). The overlap of the two pathways suggests that
rhizobia may have co-opted part of the signaling pathway
from the more ancient AM symbioses to evolve the younger
nitrogen-fixing symbiosis in legumes. Many of these common symbiotic genes have recently been cloned and phylogenetic analyses have identified orthologs in non-legume
species. The presence of these genes in non-legume species
is consistent with this model (Zhu et al., 2006).
Some of the earliest responses of legume roots to
rhizobial Nod factors involve either the transport or the
diffusion of ions across root hair membranes. Calcium,
potassium and chloride are all rapidly mobilized in root hairs
responding to Nod factors (Felle et al., 1998, 1999; Shaw and
Long, 2003). Calcium is a known regulator of polarized tip
growth and may participate in redirecting root hair growth,
thereby leading to the entrapment of bacteria and subsequent infection (Esseling et al., 2003). Following root tip
depolarization, the nuclei of activated root hairs commence
the periodic release and re-uptake of calcium known as
calcium spiking (Ehrhardt et al., 1996). Calcium spikes have
been identified and extensively studied in many eukaryotic
systems, and the frequency and amplitude of calcium spikes
are believed to encode information eliciting changes in
cytoskeleton and gene expression (Oldroyd and Downie,
2004). Transcriptional reprogramming is a likely output from
Nod-factor-induced calcium spiking. In particular, a calcium/
calmodulin-dependant kinase capable of translating calcium
oscillations has recently been found to operate genetically
upstream of nodulation-specific transcription factors (Kaló
et al., 2005; Lévy et al., 2004; Mitra et al., 2004; Smit et al.,
2005).
Many Nod– mutants exhibit defects in some or all of these
ion movements implying that ion mobilization is required
for eliciting symbiotic responses (Shaw and Long, 2003;
Wais et al., 2000). It is therefore not surprising that mutants
deficient for both fungal and bacterial symbioses contain
defects in putative ion channels. The Medicago truncatula
DMI1 gene (Doesn’t Make Infections 1) encodes a protein
9 that is homologos with prokaryotic cation channels and is
essential for microbial symbioses (Ané et al., 2004). dmi1
mutant root hairs undergo typical growth deformation in
response to Nod factors, but exhibit neither the oscillations
in intracellular calcium nor the subsequent changes in gene
expression and activation of cell division that are characteristic of the host’s response to Nod factors (Catoira et al.,
2000; Shaw and Long, 2003; Wais et al., 2002). Despite the
overall homology with prokaryotic potassium channels,
DMI1 lacks the canonical GYG motif in the putative filter
region. As a consequence the nature of the ions mobilized by
DMI1, and therefore its role in Nod factor signal transduction
is currently unknown (Ané et al., 2004). Lotus japonicus
contains two close homologs of DMI1, CASTOR and POLLUX, both of which are required for infection and nodule
development (Imaizumi-Anraku et al., 2005). In a similar
manner as dmi1 mutants, castor and pollux mutants undergo root hair depolarization, but do not respond to Nod
factors with nuclear calcium spikes, and are also unable to
form AM symbioses. Interestingly, CASTOR and POLLUX
both contain putative chloroplast transit peptides, which are
proposed to target these proteins to plastids. The role of
plastids in symbiotic signal transduction is currently unknown (Imaizumi-Anraku et al., 2005).
In order to model Nod factor signaling mechanisms, it is
necessary to have a clear understanding of where the
various molecules mediating the signal reside. We investigated DMI1 localization in M. truncatula roots and found that
in contrast to CASTOR and POLLUX, DMI1 is localized to the
nuclei of root cells and root nodules. The N-terminus of
DMI1 does not contain a plastid-targeting transit peptide,
but instead contains a sequence that is sufficient to direct
proteins to the nucleus. The localization of DMI1 to the
nucleus, a site of Nod-factor-induced Ca2þ oscillations,
suggests DMI1 may play a direct role in conducting ions
within the nuclear compartment.
Results
Functional DMI1::GFP is localized to the nuclear periphery
Previous reports indicated that CASTOR and POLLUX, the
DMI1 homologs from L. japonicus, contain transit peptides
that target these proteins to root plastids. We analyzed the
DMI1 amino acid sequence in silico using multiple online
prediction programs and each gave conflicting results. Although ChloroP (http://www.cbs.dtu.dk/services/ChloroP/)
predicted a plastid localization for DMI1, WoLF PSORT
(http://wolfpsort.seq.cbrc.jp/) predicted DMI1 resides in the
plasma membrane, and the TargetP algorithm (http://
www.cbs.dtu.dk/services/TargetP) suggested DMI1 resided
in an unidentified non-plastid compartment (data not
shown). Given these discrepancies, we sought to determine
experimentally where DMI1 was localized in M. truncatula
roots. We cloned a DMI1::GFP fusion into the pRedRoot
vector under the control of the constitutive 35S promoter,
and transformed this construct into M. truncatula roots
(Figure 1a). pRedRoot contains the DsRED1 gene under the
control of the constitutive ubiquitin promoter, which enables transgenic roots to be identified under fluorescent
illumination (Limpens et al., 2004). Introduction of this construct into dmi1 mutants restored their ability to form root
nodules, indicating that this construct possessed activity
and that subsequent localization was therefore biologically
relevant (Figure 1b, c). We analyzed DMI1::GFP fluorescence
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2007), 49, 208–216
210 Brendan K. Riely et al.
(a)
35S GFP oct 35S:GFP
35S N
35S
DMI1
GFP oct 35S:DMI1:GFP
tDMI1 GFP oct 35S:tDMI1:GFP
35S N GFP oct 35S:NDMI1:GFP
35S N
35S cTP
GFP oct 35S:DMI1TM1:GFP
tDMI1 GFP oct 35S:GS2cTP:tDMI1:GFP
cTP GFP oct 35S:GS2cTP:GFP
(b)
C, N
NE
NE
N
NE, ER
P, NE
P
(c)
coding sequence and transformed this chimeric construct
into M. truncatula roots (Melo et al., 2003). The pattern of
10 GFP fluorescence in GS2cTP::GFP-expressing roots differed
significantly from that generated by DMI1::GFP. In contrast
to DMI1::GFP roots, we observed GS2cTP::GFP fluorescence in round structures approximately 1–2 lM in diameter
(Figure 2c, f), a pattern consistent with a plastid-localized
protein. Plastids were found both as discrete bodies
throughout the cytoplasm and were also observed in
clusters surrounding nuclei. However, we never observed
fluorescence in the nuclear rim itself.
The DMI1 amino terminus is sufficient, but not required,
11 to target DMI1 to the nucleus
Figure 1. Structure and function of constructs used in this study.
(a) Schematic diagram representing the chimeric GFP fusions. White bands in
DMI1 (Doesn’t Make Infections 1) represent transmembrane domains. Gray
denotes the plastid transit peptide from glutamine synthetase 2 (Melo et al.,
2003). Abbreviations indicate the observed localization of GFP fluorescence.
(b) Bright-field image of a root nodule on a dmi1 mutant plant transformed
with pRedRoot::35S::DMI1::GFP::oct plasmid.
(c) Fluorescent image indicating that the root in (b) is expressing the DsRED1
reporter gene. Abbreviations: C, cytoplasm; N, nucleus; NE, nuclear envelope;
ER, endoplasmic reticulum; P, plastid. Scale bar represents 1 mm.
in transformed, uninoculated roots using a confocal microscope. Fluorescence was primarily localized to the periphery
of nuclei in both root epidermal cells and root hairs (Figures 2b, e). The signal from GFP was typically homogeneous
although we occasionally observed patches around the
nuclear rim. (Supplemental Figure 1). Following inoculation
with Sinorhizobium meliloti, we did not observe differences
in DMI1::GFP localization in root hairs, root epidermal cells
(data not shown) or in root nodules (see below). As a marker
for plastid localization, we cloned the DNA sequence
encoding the transit peptide (TP) from the M. truncatula
glutamine synthetase 2 (GS2) gene to the 5¢ end of the GFP
If the 5¢ region of DMI1 encodes a transit peptide, as predicted
by ChloroP, then fusing this domain to GFP should result in
the accumulation of GFP in plastids as we observed with the
GS2cTP::GFP construct. ChloroP predicts that the N-terminal
69 amino acids of DMI1 constitute a plastid transit peptide.
We therefore fused the N-terminal 76 amino acids from DMI1
to GFP (NDMI1::GFP, Figure 1a) and expressed this construct
from the 35S promoter in M. truncatula roots. We observed
strong NDMI1::GFP fluorescence in the nucleus and little to
no fluorescence in the cytoplasm (Figure 3b). In contrast,
cells expressing GFP alone exhibited strong fluorescence
both in the nucleus and cytoplasm (Figure 3a). In neither case
12 did we observe fluorescence in plastids. To ensure that we
did not mistakenly omit key residues from the N-terminus of
DMI1, we generated an additional fusion between the GFP
and an N-terminal fragment encompassing the first 152
amino acids of DMI1 (DMI1TM1::GFP, Figure 1a). This construct contains both the putative transit peptide as well as the
first transmembrane domain of DMI1. In contrast to the
NDMI1:GFP construct, which we observed throughout the
nucleus, we observed DMI1TMI1::GFP primarily at the periphery of nuclei with weak fluorescence observed throughout
the cell in a pattern consistent with an endoplasmic reticulum
Figure 2. DMI1 (Doesn’t Make Infections 1) is
localized to the periphery of nuclei and not to
plastids. Medicago truncatula root epidermal
cells (a–c) or root hairs (d–f) expressing (a and
d) empty pRedRoot, (b and e) 35S::DMI1::GFP or
(c and f) GS2cTP::GFP were visualized on a
confocal microscope. Images represent merged
data from both the red and green channels. Bars
represent 10 lm. Red fluorescence indicates
propidium iodide staining of cell walls.
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Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2007), 49, 208–216
1
DMI1 is localized in the nuclear membrane 211
Figure 3. The N-terminus is sufficient, but not
40 required, to target DMI1 to the nucleus. Confocal
images of Medicago truncatula root epidermal
cells expressing the following:
(a) GFP alone;
(b) the N-terminal 76 amino acids of DMI1
(Doesn’t Make Infections 1) fused to GFP;
(c and d) the N-terminal 152 amino acids of DMI1
including the first transmembrane domain fused
to GFP;
(e) truncated tDMI1:GFP lacking the N-terminal
76 amino acids of DMI1;
(f) GS2cTP::tDMI1::GFP on which the aminoterminus of DMI1 has been replaced with the Nterminus of glutamine synthetase 2. Images
represent merged data from both the red and
green channels. Arrowheads in
(d) highlight putative endoplasmic reticulum.
Bars represent 10 lm. Red fluorescence indicates
propidium iodide staining of cell walls.
13 (ER)-localized protein (Figure 3c,d). We did not observe
DMI1TM1::GFP in plastids.
These data indicate that the N-terminus contains a nuclear
localization signal (NLS), which targets DMI1 to the nucleus.
In order to determine if this domain is essential for DMI1
localization and function, we deleted the 76 amino acids from
the N-terminus of DMI1 and introduced a codon for a new
initiator methionine. The truncated DMI1 (tDMI1) was fused
to GFP and expressed under the control of the 35S promoter
in dmi1 roots. tDMI1::GFP manifested a similar pattern of
localization as did DMI1::GFP, in that it was localized to the
periphery of the nucleus both in root epidermal cells
(Figure 3e) and in root hairs (data not shown). These data
indicate that the N-terminus of DMI1 is sufficient, but not
14 required, for localization to the nucleus. We inoculated plants
constitutively expressing tDMI1::GFP and DMI1::GFP with
S. meliloti. In contrast to DMI1::GFP transgenic roots, which
efficiently formed root nodules, tDMI1::GFP roots were
completely Nod– even after a prolonged incubation with
S. meliloti (>1 month). These data indicate either that the
N-terminal domain of DMI1 contains sequences essential for
function, or that removal of these amino acids disrupts the
protein structure sufficiently to inhibit DMI1 activity.
The GS2 transit peptide does not restore function to
truncated DMI1
In contrast to DMI1, CASTOR and POLLUX are both reported
to function in root plastids (Imaizumi-Anraku et al., 2005).
Furthermore, ChloroP predicts that DMI1 also contains a
transit peptide that should target it to plastids. We therefore
wanted to know if we could artificially target DMI1 to plastids
and if re-localization could restore the function of the
tDMI1::GFP construct. We replaced the N-terminus of DMI1
with the transit peptide from GS2 and expressed the
GS2cTP::tDMI1::GFP fusion in M. truncatula roots. The vast
majority of GS2cTP::tDMI1::GFP signal was in plastids
(Figure 3f), consistent with the activity of the GS2 transit
peptide. However, weak signal was also observed around the
nucleus, consistent with the previously established capacity
of tDMI1::GFP to localize to the nucleus (Figure 3e). As with
GS2cTP::GFP, GS2cTP::tDMI1::GFP plastids were observed
both adjacent to the nucleus and dispersed throughout
the cytoplasm. These results differ from those obtained with
the full-length DMI1::GFP, and indicate that when given
an appropriate plastid targeting sequence DMI1 can be
efficiently targeted to the plastids.
In order to determine if GS2cTP could restore function to
tDMI1::GFP, we transformed dmi1 mutants with the
GS2cTP::tDMI1::GFP fusion and inoculated transgenic roots
with S. meliloti. As with tDMI1::GFP, and in contrast to
DMI1::GFP, GS2cTP::tDMI1::GFP did not complement the
dmi1 mutation as plants expressing this construct were
completely Nod– (n ¼ 10).
DMI1:GFP expressed from its native promoter is also
localized to the nucleus
To rule out the possibility that the observed pattern of localization was caused by over-expression from the 35S promoter, we analyzed DMI1::GFP function and localization
following expression from its native promoter. As a prelude
to subcellular localization of DMI1::GFP, we sought to
establish the tissue specificity of DMI1 promoter activity. As
shown in Figure 4a, M. truncatula nodules expressing the
uidA gene under the control a 1.8-kb DNA DMI1 promoter
fragment exhibited GUS activity in the distal regions of the
nodule. These results are consistent with in situ hybridizations performed by Limpens et al. (Limpens et al., 2005) using
a DMI1 probe. We fused GFP to the C-terminus of a genomic
clone of the DMI1 gene that contained the 1.8 kb of DNA upstream from the ATG (gDMI1) and 500 bp downstream of the
stop codon. dmi1 mutants expressing this construct formed
root nodules upon inoculation with S. meliloti, indicating that
the gDMI1::GFP fusion retained biological activity (Figure 4b,
c). We examined the distal region of root nodules expressing
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Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2007), 49, 208–216
212 Brendan K. Riely et al.
Figure 4. DMI1::GFP expressed from the DMI1 (Doesn’t Make Infections 1) promoter is also localized in the nucleus.
(a) A 10-lm-thick GUS-stained section of a root nodule expressing the uidA gene under the control of the 1.8-kb DMI1 promoter. Images (b–e) are confocal images of
hand-sectioned root nodules.
(b) GFP fluorescence is evident in the distal regions of nodules from dmi1 mutant plants expressing gDMI1::GFP (white star). This region corresponds to the GUSstained regions shown in (a) and is not evident in gDMI1 nodules that lack GFP (c). A higher magnification image of the section in (b) is shown in panel (d) with the
nuclei highlighted by arrowheads. 35S::DMI1::GFP is shown in panel (e) for comparison. The fluorescence observed in the nitrogen-fixing zone (asterisks in b and c)
is caused by autofluorescence. Scale bars represent 75 lm in (b and c) and 25 lm in (d and e). The blue color in (a) is caused by GUS staining, whereas the pink
colour is counterstain from ruthenium red.
either gDMI1 (Figure 4c) or gDMI1::GFP (Figure 4b) corresponding to the region where we observed GUS activity.
Despite high levels of auto-fluorescence commonly observed
in the central tissue of root nodules (Figure 4b,c), the distal
region of the nodule exhibited relatively low auto-fluorescence, enabling detection of DMI::GFP fluorescence. We observed weak, but reproducible, fluorescence in the distal
region of gDMI1::GFP nodules, and a lack of fluorescence in
the corresponding region of nodules complemented by gDMI1
that lacked GFP. Higher magnification revealed that fluorescence was primarily localized in spherical structures approximately 10 lm in diameter (Figure 4d), consistent with the
previously observed nuclear localization of DMI1 (Figure 2b,
e). Moreover, we observed similar patterns of fluorescence in
the distal region of nodules expressing 35S::DMI1::GFP (Figure 4e). Thus, both DMI-promoter- and 35S-promoter-driven
DMI1::GFP yield complementing proteins with similar subcellular distributions, supporting the conclusion that functional DMI1 resides in the periphery of root and nodule nuclei.
Discussion
Here we report that a DMI1::GFP chimeric protein is localized
in the nucleus of M. truncatula roots, and not in the plastids
as has been reported for the DMI1 homologs CASTOR and
POLLUX. Nuclear localization was observed using both a
constitutive 35S promoter, as well as the native DMI1 promoter, ruling out the possibility that ectopic expression of
DMI1 causes mislocalization. Expression of DMI1:GFP from
either promoter restored the ability of dmi1 mutants to
nodulate, indicating that the fusion protein is functional. We
provide further evidence that DMI1 localization is mediated,
in part, by amino acids present in the N-terminal domain
of the DMI1 protein. Fusing the predicted DMI1 transit
peptide to GFP causes the GFP to be localized almost
exclusively in the nucleus and no fluorescence was detected
in plastids. Similar results were obtained when NDMI1::GFP
was expressed in other legume and non-legume species
16 (G. Lougnon and J.M. Ané, unpublished results), confirming
the ability of the N-terminus of DMI1 to direct proteins to the
17 nucleus, but not to plastids. In contrast, expressing a GFP
fusion containing the transit peptide of the M. truncatula
GS2 protein from the same 35S promoter localized GFP
exclusively in plastids, and replacing the N-terminus of
DMI1 with the GS2cTP results in clear localization of
GS2cTP::tDMI1::GFP in plastids, a pattern not observed
when the full-length coding sequence of DMI1 is fused to
GFP. These data provide strong evidence that DMI1
18 functions in the nucleus.
It is likely that additional sequences outside of the DMI1
amino terminus also function in targeting DMI1 to the
nucleus, as a truncated DMI1::GFP construct is also localized
in the nuclear envelope. There are examples from the
19 literature of proteins that contain multiple, distinct NLSs.
For example, the Lamin B receptor (LBR) contains multiple
non-overlapping NLSs. LBR nuclear localization is mediated
by a bipartite NLS in the amino terminus, as well as amino
acids within and adjacent to the first transmembrane
domain (Soullam and Worman, 1995). As with DMI1, fusion
of the soluble amino terminus of LBR to GFP targets the
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Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2007), 49, 208–216
1
DMI1 is localized in the nuclear membrane 213
chimeric protein to nuclei, whereas the C-terminal domain,
containing eight transmembrane domains, is still targeted to
the inner nuclear envelope in the absence of its amino
terminus (Soullam and Worman, 1995).
Analysis of the DMI1 peptide using PredictNLS (http://
cubic.bioc.columbia.edu/predictNLS/) and WoLF PSORT
identified three potential sequences within the DMI1 peptide
that could function as nuclear localization sequences. Two of
these sequences (PLKKTK and SRKRRQ) reside within the Nterminus and may target NDMI1::GFP to the nucleus.
Although the predicted molecular weight of the NDMI1::GFP
protein (approximately 35 kDa) is below the level that can
freely diffuse in and out of the nucleus, the fact that we do
not see appreciable accumulation of GFP in the cytosol
indicates either that it is being actively targeted to the
nucleus, or that it is retained in the nucleus following
diffusion. In either case, the observation that the N-terminus
of DMI1 contains signals that are sufficient to cause GFP
accumulation within the nuclear compartment suggests that
DMI1::GFP also resides within the nucleus, but in association
with the inner nuclear membrane. We are currently performing electron microscopy to more precisely define the
20 localization of DMI1 within the nuclear envelope.
WoLF PSORT and Prosite (http://www.expasy.org/prosite/)
identified an additional bipartite NLS-like sequence
(RRTESNKEDVPLKKRVA) between the second and third
transmembrane domains of DMI1. This third NLS may
explain why the truncated DMI1::GFP protein retained nuclear localization, and why we observed weak remnant
fluorescence of GS2cTP::dDMI1::GFP in the nuclear rim.
Alternatively unidentified sequences within the transmembrane domains may function in nuclear targeting, as has been
shown for LBR (Soullam and Worman, 1995). We are
currently undertaking additional mutagenesis studies to
precisely define, and functionally characterize, the putative
NLSs in DMI1. That truncated DMI1 did not retain biological
function indicates that the N-terminus contains residues
critical for either protein function, or for the maintenance of
the appropriate protein structure.
Previous reports indicate that other essential components
of the Nod factor signaling pathway, as well as certain
physiological responses to Nod factor, are also localized in
the nucleus. The putative L. japonicus nucleoporin Nup133,
the M. truncatula calcium- and calmodulin-dependent-pro21 tein kinase DMI3, and the transcription factors NSP1 and
NSP2 all have been shown to be localized either in or around
the nucleus (Kaló et al., 2005; Kanamori et al., 2006; Smit
et al., 2005). Furthermore, both the second phase of the early
biphasic ‘calcium flux’, and the intracellular oscillations of
calcium, termed ‘calcium spiking’, also originate from the
nuclear membrane (Ehrhardt et al., 1996; Shaw and Long,
2003). Notably, both of these calcium movements are absent
in dmi1 mutants (Shaw and Long, 2003; Wais et al., 2000). As
members of a common signaling pathway often share
similar subcellular localization, these findings provide additional circumstantial yet compelling evidence that DMI1
operates in the nucleus.
In contrast to these nucleus-associated signaling compo22 nents, the M. truncatula LysM receptors LYK3 (NFR1 in
23;24 L. japonicus), NFP (NFR5 in L. japonicus) and LRR receptor
kinases DMI2 (SYMRK in L. japonicus) all contain signal
peptides, which are likely to target them to the plasma
membrane and possibly the infection thread, as was recently
demonstrated experimentally for DMI2 (Endre et al., 2002;
Limpens et al., 2003; Limpens et al., 2005; Madsen et al.,
2003; Radutoiu et al., 2003; Stracke et al., 2002). Nod factor
binding by one or more receptor kinases at the host’s plasma
membrane is likely to initiate a pathway that activates nuclear
responses. Pharmacological studies suggest a G-protein and
lipid signaling pathway links Nod factor perception at the
plasma membrane to responses manifested in the nucleus
(Charron et al., 2004; den Hartog et al., 2001, 2003). The
25 heterotrimeric G-protein agonist Mas7 activates ENOD11
transcription in dmi1 and dmi2 mutants but not in dmi3
mutants, indicating that second messengers operate upstream of dmi3 and either downstream of dmi1and dmi2 or in
a parallel pathway that converges with the pathway upstream
of ENOD expression (Charron et al., 2004). Phospholipase
26 and inositol triphosphate (IP3) receptor inhibitors block
ENOD11 expression, implicating phospholipases and inositol tri-phosphates as potential second messengers. The
authors of this study postulated that Mas7 activates phospholipases leading to the production of either IP3 or IP6 and
the subsequent release of calcium from the ER (Charron
et al., 2004).
Calcium spiking has been intensively studied in animal
systems and is likely to utilize a two-step activation mechanism involving both second messengers such as IP3 and
localized calcium release, which may also be triggered by
one or more of the aforementioned second messengers
(Cancela et al., 2000). That Mas7 can elicit calcium spiking in
dmi1 mutants precludes DMI1 as a direct conduit for calcium
oscillations (Giles Oldroyd, John Innes Centre, Norwich, UK,
27 personal communication). However, DMI1 may yet play a
direct role in this response by triggering the initial calcium
flux. Although DMI1 shares overall similarity with prokaryotic potassium channels, it lacks the canonical signature
motif in the filter region indicative of a potassium channel
(Ané et al., 2004; Imaizumi-Anraku et al., 2005) and may
instead be mobilizing calcium from the nuclear envelope.
Like the ER the nuclear envelope is also a store for
intracellular calcium, which is released by IP3 (Gerasimenko
et al., 2003; Xiong et al., 2004). Although the localization of
DMI1 in the vicinity of calcium spiking is certainly tantalizing,
a direct role in calcium mobilization is speculative. Clearly
the biochemical activity of this putative ion channel must be
experimentally determined and our current model requires
rigorous testing.
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214 Brendan K. Riely et al.
It is difficult to reconcile the differences in localization
between DMI1 and those of CASTOR and POLLUX. The
responses of diverse legume hosts to their cognate Nod
factors and symbionts, and the proteins that mediate these
responses show remarkable conservation in both structure
and function. It therefore seems unlikely that DMI1 and
CASTOR/POLLUX would be localized in different parts of the
cell. Although we cannot rule out the possibility that these
proteins do indeed function in distinct subcellular compartments, we provide evidence that the nucleus is the proper
site of DMI1 function, and speculate that CASTOR and
POLLUX may function there as well. Indeed, WoLF PSORT
and PredictNLS identified potential NLSs in POLLUX corresponding to those in DMI1, whereas WoLF PSORT, but not
PredictNLS, identified a possible NLS in the N-terminus of
28 CASTOR (B.K. Riely, unpublished data). It will be interesting
to learn where CASTOR and POLLUX GFP fusions are
localized in Lotus roots, as previous experiments were
conducted in the epidermal cells of onion peels and pea
roots. However unlikely, it is also possible that a low level of
DMI1 protein may be localized in plastids, which is sufficient
for function but insufficient for detection by confocal microscopy. Although the fact that there is no precedent for the
involvement of plastids in symbiotic signal transduction
does not preclude a role for plastids in Nod factor signaling,
the localization of DMI1 and its homologs in the nucleus fits
the current paradigm of symbiotic signal transduction.
Experimental procedures
Plant material and rhizobium inoculations
All experiments were performed using either M. truncatula cv.
Jemalong A17 seedlings or the GY15-3F-4 null mutant of dmi1 (Ané
et al., 2004). Seeds were scarified in concentrated sulfuric acid for
8 min, surface sterilized in 12% sodium hypochlorite, imbibed in
sterile water and plated on 1% deionized water agar plates. Seeds
were subsequently vernalized for 48 h at 4C and germinated by
29 incubation at 22C overnight. All nodulation assays were performed
30 using a suspension of approximately 107 colony forming units ml)1
of S. meliloti strain ABS7M (pXLGD4).
Plasmid construction
To construct the pLP100::pDMI1 plasmid, we amplified the DMI1
31 promoter from bacterial artificial chromosome (BAC) Mth2-54A24
DNA using primers 5¢-CCGGTACCCACAGCGAAGAAAAGGGAATAAGA-3¢ and 5¢-GGCCCGGGTTTCTTTTCTTCACACTTTTAAAA 3¢,
digested the PCR product with HindIII and XmaI, and ligated the 1.8kb fragment into the corresponding sites in the pLP100 vector
(Boisson-Dernier et al., 2001) to make a transcriptional fusion with
the uidA gene.
32 The GFP coding sequence (CDS) was amplified from pSMRSGFP
(Davis and Vierstra, 1998) and cloned into either the pTEX or the
pTEX(H) vector (Frederick et al., 1998) to generate 35S::GFP. These
vectors contain a multiple cloning site flanked by the 35S promoter
and octopine synthase terminator, and differ only in that the 35S
cassette is flanked by HindIII sites in pTEX(H), whereas it is flanked
by EcoRI and HindIII sites in pTEX. DMI1 was amplified from DMI1
cDNA (Ané et al., 2004) and cloned onto the N-terminus of GFP to
generate 35S::DMI::GFP. The NDMI1 fragment used the reverse
primer 5¢-CCGGTACCTCTTCTTTTTCTGGAGGTGCTGCC-3¢ to amplify the region encoding the N-terminal 76 amino acids of DMI1
from DMI1 cDNA, which was cloned in frame to the N-terminus of
GFP to generate 35S::NDMI1::GFP. The primer 5¢-AAGGTACCATGCAACCACCCCCTCCTCCTTCC-3¢ was used to truncate
DMI1 thus removing the N-terminal 76 amino acids. This PCR
fragment was cloned in frame to the N-terminus of GFP to generate
35S::tDMI::GFP. The region of GS2 encoding the 56 amino acid
transit peptide was amplified from an A17 root cDNA library (Ané
et al., 2004) using 5¢-AAGGTACCATGGCACAGATTTTGGCTCCTTC3¢ and 5¢-CTGCAGCAGTGCTGACTCCATTAATC-3¢. This fragment
was cloned both into 35S::GFP and 35S::tDMI1::GFP to generate the
35S::GS2cTP::GFP and 35S::GS2cTP::tDMI1::GFP constructs,
respectively. The 35S::oct cassettes containing the various GFP
constructs were either amplified from pTEX using the primers 5¢AACCCGGGCCATGGAGTCAAAGATTCAAATAG-3¢ and 5¢-AACCCGGGACAATCAGTAAATTGAAC-3¢ and cloned into the XmaI
site in pRedRoot, or were cut out of pTEX(H) using HindIII and
cloned into the HindIII site in pRedRoot. These were subsequently
transformed into the Agrobacterium rhizogenes strain Arqua1(pCH32) via electroporation.
We amplified a genomic copy of DMI1 (gDMI1) from BAC Mth254A24 using the primers 5¢-GGGGACAAGTTTGTACAAAAAAGCAGGCTAAGCTTCAAACTCGAGAAATATGAG-3¢ and 5¢-GGGGACCACTTTGTACAAGAAAGCTGGGTCCCGGGGTACCTTCACCTGAGGCTAAAACAAC-3¢. These primers amplify a fragment from 1.8-kb
upstream of the ATG to the stop codon and introduce KpnI and
XmaI sites at the 3¢ end. This PCR product was recombined into the
Gateway compatible plasmid pDONR221 (Invitrogen, Carlsbad, CA,
33 USA). Primers 5¢-CAGGTACCTGACAGAAGAAAGGAATAG-3¢ and
5¢-TTCCCGGGTTCTTGGTCCAACGGTGTAATC-3¢ were use to am34 plify and clone 500 bp of DMI1 3¢untranslated region (UTR) into the
XmaI and KpnI sites leaving a unique XmaI site immediately in front
of the stop codon. Primers 5¢-AACCCGGGGGAGGTATGAGTAAAGGAGAAG-3¢ and 5¢-AACCCGGGTTATTTGTATAGTTCATCCATGCCATG-3¢ were used to amplify GFP, which was subsequently
cloned into the XmaI site. gDMI1, either with or without GFP, was
subsequently recombined into the Gateway compatible vector
pKGWRR harboring a constitutively expressed DsRED1 reporter
gene. (Smit et al., 2005) We electroporated these plasmids into the
Agrobacterium tumefaciens strain MSU440, which contains the Ri
plasmid (Sonti et al., 1995).
Generation of transgenic hairy roots
Hairy roots were generated essentially as described by BoissonDernier et al. (2001) using either the A. rhizogenes strain ArquaI
(pLP100 and pRedRoot) or MSU440 (pKGWRR plasmids), with the
exception that inoculated seedlings were sandwiched between filter
paper moistened with Fahraeus media (Fs). Transgenic hairy roots
expressing the DsRED1 marker were identified using a Zeiss fluor35 escent stereoscope fitted with the appropriate filter set (Zeiss,
Thornwood, NY, USA). Non-transformed roots were trimmed and
plants were transferred either to large filter plates of Fs media
without nitrate, or to pots containing Turface moistened with liquid
Fs without nitrate.
Histochemical analysis and microscopy
Staining for GUS activity was performed by incubating tissue in
Triton X-100, 50 mM NaPO4, pH 7.2, 2 mM potassium ferrocyanide,
ª 2006 The Authors
Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2007), 49, 208–216
1
DMI1 is localized in the nuclear membrane 215
2 mM potassium ferricyanide, 2 mM X-Gluc at 37C. GUS-stained
tissue was fixed in 5% glutaraldehyde, 0.1 M phosphate buffer, pH
7.2, under vacuum for 2 h before embedding in Technovit
(Heraeus Kulzer, Wehrhim, Germany) according to the manufacturer’s instructions. Sections (10-lm thick) were cut using a
Microm HM340 E rotary microtome (Microtom, San Leandro, CA,
36 USA) and counterstained in ruthenium red.
Transgenic roots used for confocal microscopy were stained with
37 propidium iodide (Sigma, St Louis, MO, USA) prior to mounting in
sterile water. Longitudinal sections of root nodules were generated
by hand. All samples were excited using the 488- and 568-nm lines
38 on a Krypton-Argon laser and observed using a Bio-Rad MRC 1024
39 confocal microscope (Zeiss).
Acknowledgements
We would like to acknowledge Dr Rene Geurts for the contributing
the pKGW-RR binary vector and the A. tumefaciens strain MSU440.
This work was funded by the University of Wisconsin Madison
Hatch Grant #WIS04872 and The US Department of Energy Biosciences Program Grant DE-FG02-01ER15200.
Supplementary Material
The following supplementary material is available for this article
online:
Figure S1. Bright patches on a DMI1::GFP nucleus. These patches
were always found associated with nuclei and, in contrast to
plastids, were not observed in the cytoplasm. Bars represent 10 lm.
Red fluorescence indicates propidium iodide staining of cell walls.
This material is available as part of the online article from http://
www.blackwell-synergy.com
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