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First published online July 10, 2003; 10.1104/pp.103.020438 Plant Physiology 132:2023-2033 (2003) © 2003 American Society of Plant Biologists The Arabidopsis NHL3 Gene Encodes a Plasma Membrane Protein and Its Overexpression Correlates with Increased Resistance to Pseudomonas syringae pv. tomato DC30001Departments of Stress and Developmental Biology (A.V., D.S., J.L.) and Secondary Metabolism (B.H.), Institute of Plant Biochemistry, D–06120, Halle/Saale, Germany; and Biocenter of the Martin-Luther-University, D–06120, Halle/Saale, Germany (G.H.)
The Arabidopsis genome contains a family of NDR1/HIN1-like (NHL) genes that show homology to the nonrace-specific disease resistance (NDR1) and the tobacco (Nicotiana tabacum) harpin-induced (HIN1) genes. NHL3 is a pathogen-responsive member of this NHL gene family that is potentially involved in defense. In independent transgenic NHL3-overexpressing plant lines, a clear correlation between increased resistance to virulent Pseudomonas syringae pv. tomato DC3000 and enhanced NHL3 transcript levels was seen. These transgenic plants did not show enhanced pathogenesis-related gene expression or reactive oxygen species accumulation. Biochemical and localization experiments were performed to assist elucidation of how NHL3 may confer enhanced disease resistance. Gene constructs expressing amino-terminal c-myc-tagged or carboxyl-terminal hemagglutinin epitope (HA)-tagged NHL3 demonstrated membrane localization in transiently transformed tobacco leaves. Stable Arabidopsis transformants containing the NHL3-HA construct corroborated the findings observed in tobacco. The detected immunoreactive proteins were 10 kD larger than the calculated size and could be partially accounted for by the glycosylation state. However, the expected size was not attained with deglycosylation, suggesting possibly additional posttranslational modification. Detergent treatment, but not chemicals used to strip membrane-associated proteins, could displace the immunoreactive signal from microsomal fractions, showing that NHL3 is tightly membrane associated. Furthermore, immunofluorescence and immunogold labeling, coupled with two-phase partitioning techniques, revealed plasma membrane localization of NHL3-HA. This subcellular localization of NHL3 positions it at an initial contact site to pathogens and may be important in facilitating interception of pathogen-derived signals.
Most plants do not succumb to the many existing potential pathogens, which may be ascribed to the existence of nonhost resistance in plants (Nürnberger and Scheel, 2001  ). Acquisition of virulence factors has enabled some pathogen
strains to overcome this plant species resistance
(Van't Slot and Knogge, 2002).
In response to those pathogens capable of infection, plants have evolved a
repertoire of counter-active defense mechanisms
(Nürnberger and Scheel,
2001). One of the best studied defense mechanisms is the
genetically defined race-specific resistance that is determined by products of
resistance (R) genes that recognize pathogen-specific avirulence
(Avr) proteins (Takken and Joosten,
2000).
It was initially proposed that the R proteins act as receptors for
pathogen-encoded Avr proteins to initiate a signal activating plant defense
responses (Keen, 1990
NDR1 (nonrace-specific disease resistance) is required for proper
function of a subclass of R genes that includes RPM1
(Century et al., 1997 Here, we assessed the effect of NHL3 overexpression on the in planta growth of pathogenic bacteria in transgenic plants. To further understand the possible function of NHL3, we localized the epitope-tagged NHL3 proteins in microsomal preparations of tobacco and Arabidopsis leaves. We could show that NHL3 is a glycosylated plasma membrane protein. The NHL3 location and the correlation between NHL3 overexpression and bacterial resistance support the hypothesis that this gene contributes to disease resistance against pathogenic bacteria.
NHL3 Overexpression Correlates with Resistance against Virulent Bacteria
NHL3 has only weak sequence homology (41% similarity/27% identity) to NDR1.
Nevertheless, suppression of NHL3 expression by virulent bacteria
(Varet et al., 2002
The transgenic line accumulating high NHL3 mRNA levels (Fig. 1A, line S13) showed significantly reduced bacterial growth 3 and 5 d postinfiltration (Fig. 1B). In contrast, the transgenic line S6, which does not overexpress NHL3, did not differ from the untransformed wild-type plants. The lines S7 and S5, accumulating intermediate levels of NHL3 mRNA, also showed an increased (but less dramatic) resistance to P. syringae pv. tomato DC3000 5 d postinfiltration (Fig. 1B). These observations were confirmed in at least three independent experiments. Notably, when the transgene expression levels were quantified by phosphorimaging of RNA gel blots, a clear correlation between transgene expression and resistance to virulent bacteria could be seen (Fig. 1B). Hence, high expression of NHL3 contributes quantitatively to resistance against P. syringae pv. tomato DC3000. However, when resistance test against two virulent Peronospora parasitica isolates (cv Noks and cv Waco) were performed, no difference in spore germination or sporulation was observed for these two isolates (data not shown). Thus, the enhanced resistance appears to be specific to the tested virulent P. syringae strain.
The overexpressing transgenic lines had no obvious phenotypic or
morphological differences from wild-type plants. This distinguishes these
lines from enhanced resistance mutants such as constitutive expressor of
pathogenesis-related (cpr) genes or mitogen-activated protein
kinase 4 (mpk4), which show some developmental defects or are dwarfs
(Petersen et al., 2000
Reciprocal experiments to study resistance phenotype through reducing
NHL3 expression (antisense gene constructs) or in gene knockouts were
also attempted. Seventy putative antisense lines were analyzed, but none
showed a convincing reduction of NHL3 mRNA levels after wounding
(note that NHL3 is also wound inducible;
Varet et al., 2002
Because our analysis above did not provide any clue toward understanding
the enhanced resistance brought about by overexpressing NHL3, we next
evaluated the localization of the protein. Similar to NDR1
(Century et al., 1997
An epitope tag strategy was chosen to study NHL3 localization because the specificity of two independent antipeptide antibodies generated for NHL3 could not be demonstrated. Gene constructs were created that expressed NHL3 with a C-terminal HA-epitope tag (NHL3-HA) or an N-terminal c-myc-epitope tag (c-myc-NHL3). Expression of these genes was driven by the dexamethasone (DEX)-inducible promoter (for NHL3-HA) or the constitutive 35S promoter (for c-myc-NHL3). For negative control, the corresponding empty vectors were also included. Leaves of tobacco were transiently transformed with Agrobacterium tumefaciens strains carrying these constructs (see "Materials and Methods" for details). Western-blot analysis of total protein extracts of tobacco leaves infiltrated with A. tumefaciens strains carrying the NHL3-HA construct and treated with DEX revealed three crossreacting bands (Fig. 3A). Only two crossreacting bands were detected in total protein extracts of leaves transformed with A. tumefaciens strains carrying the c-myc-NHL3 construct (Fig. 3B). For both constructs, the detected protein bands were located to the microsomal fraction and no signal was detectable in leaves infiltrated with the respective empty vectors (Fig. 3, A and B). A very weak signal was occasionally visible in the soluble fraction, possibly as a result of the high overexpression levels. The largest immunoreactive band of both constructs is about 37 kD (Fig. 3, A and B), which is approximately 10 kD larger than the predicted Mr of 27 for NHL3.
To ascertain that the unexpected size of the immunoreactive bands is not due to heterologous expression in tobacco, we conducted similar studies in the homologous system, Arabidopsis. Because A. tumefaciens-mediated transient expression in Arabidopsis was variable in our hands, we stably transformed Arabidopsis (ecotype Col-0) with the gene construct expressing NHL3-HA under the control of the DEX promoter. This construct was selected because it enables an DEX-inducible expression that reflects the temporal expression pattern of NHL3 by avirulent bacteria (A.Varet, unpublished data) and allows studies in the absence of pathogens. Westernblot analysis with the anti-HA antibody showed the presence of protein bands of approximately 37 kD in total and microsomal protein extracts, whereas no signal was detectable in DEX-treated leaves of plants transformed with the control empty vector (Fig. 3C). Notably, the increased size of the protein compared with the predicted size and its microsomal localization are conserved in Arabidopsis and tobacco. This might be indicative of posttranslational modification for NHL3.
Because NHL3 contains four predicted N-glycosylation sites
(Fig. 2), we tested if the
epitope-tagged proteins are glycosylated in tobacco and Arabidopsis.
Solubilized microsomal fractions obtained from transiently transformed tobacco
leaves and DEX-treated NHL3-HA Arabidopsis plants were incubated with
peptide N-glycosidase F (PNGase F), a glycoamidase that liberates
N-linked oligosaccharides from glycoproteins
(Tarentino and Plummer, 1987
The loading of high protein amounts or extended exposure of western blots revealed the presence of several larger bands in microsomal fractions isolated from transgenic NHL3-HA Arabidopsis (data not shown). Furthermore, deglycosylation procedure also gave rise to a size shift of these larger bands (data not shown). When the protein extracts were treated with the homobifunctional chemical crosslinker, DTSSP, before SDS-PAGE and western blotting, these larger immunoreactive bands appeared, whereas the intensity of the 37-kD band was reduced (Fig. 4C). Four bands were seen in the crosslinking experiments (Fig. 4C), but another two larger bands could be seen when noncrosslinked protein extracts were loaded in the absence of reducing agents (data not shown). Taken together, the sizes of these additional bands suggest that NHL3-HA could form oligomers or be a component of larger protein complexes. These complexes appear to be partially resistant to denaturation during SDS-PAGE. To further test the prediction that NHL3-HA is a transmembrane protein and is not merely membrane associated, we used various treatments to strip protein subclasses from microsomal fractions isolated from NHL3-HA transgenic Arabidopsis plants. Salt or alkaline treatment, known to be effective in extracting proteins that are peripherally associated with membranes, did not remove the tagged NHL3 protein from the microsomal fraction (Fig. 4D). Only treatment with the nonionic detergent, Triton X-100, could shift the immunoreactive signal to the soluble fraction (Fig. 4D). Thus, NHL3-HA is tightly associated with membranes.
Immunohistochemical experiments were performed to further pinpoint the subcellular localization of NHL3 in the NHL3-HA Arabidopsis plants. No signal was detectable in the sections of plants transformed with the empty control vector (Fig. 5A). In the transgenic NHL3-HA plants, immunodecoration could be clearly detected but was not seen in every cell. This was seen in two different transgenic lines and is unlikely to be a result of inefficient DEX uptake. It could be that some cell-specific silencing might have occurred. Nevertheless, when observed, the immunofluorescent signal was restricted to a thin layer at the periphery of cells (Fig. 5, B and C). When two adjacent cells were labeled, the signals were in two separate lines with the cell wall space unstained (see arrowheads in Fig. 5, B and C). The label was, in most cases, a spotty line indicating regions of high concentration of the epitope. No label was seen in chloroplasts, nuclei, or tonoplasts (Fig. 5, C and D). Little or no signal was seen in the cytoplasm that is expected to encompass organelles such as chloroplasts or nuclei (see Fig. 5, B and C). Hence, despite poor resolution of the thin cytoplasm, it appears that the immunoreactive signal is not found in internal membranes such as endoplasmic reticulum. Immunogold-labeled ultrathin sections for electron microscopy were used to verify this. Even at this high resolution, the cytoplasm in labeled epidermal Arabidopsis cells is still relatively thin compared with the large vacuole (Fig. 5E). Similar results were observed for two independent transgenic lines, and Figure 5E shows a representative section where the detected gold labels were primarily observed at the cell periphery adjacent to the cell wall. Due to a weak plasmolysis, the plasma membrane is detached from the cell wall (Fig. 5E), which further highlights the absence of signals in the cell wall or extracellular matrix. In some cases, the labels were detected in membrane protrusions toward the cell wall, indicating plasma membrane localization. Controls performed by immunodecoration of sections of leaves harboring the control empty vector did not exhibit labeling (data not shown).
To strengthen the microscopy data, microsomes prepared from DEX-treated
transgenic NHL3-HA plants were subjected to aqueous two-phase
partitioning. The efficiency of the partitioning process was confirmed with
antibodies for the plasma membrane marker, proton-ATPase AHA2
(Palmgren et al., 1991
NHL3 transcripts were previously shown to accumulate during several incompatible interactions, upon in planta expression of AvrRpt2, and also very rapidly but transiently after mechanical wounding (Varet et al., 2002 ).
Because the former two treatments cause HR, one might speculate that the onset
and progressive development of HR results in cellular damage that subsequently
leads to a sustained, wound-like induction of NHL3 expression. In
such a scenario, NHL3 expression would be a secondary effect that has
no consequence for pathogen defense. However, NHL3 expression is also
strongly induced by a nonpathogen of Arabidopsis, P. syringae pv.
phaseolicola and a Type III secretion mutant of P. syringae
pv. tomato DC3000 (Varet et al.,
2002). Arabidopsis leaves infiltrated with either one of these
strains do not show any HR or necrosis, thus ruling out the secondary wounding
as a causal induction of NHL3 expression. We have previously
suggested that virulent P. syringae pv. tomato DC3000
probably suppresses NHL3 expression through a translocated Type III
effector protein (Varet et al.,
2002). This led us to propose that NHL3 is possibly important in
the defense response against P. syringae pv. tomato DC3000.
The NHL gene family shows similarities to the tobacco HIN1
gene (Dörmann et al.,
2000), and because HIN1 is often used as a marker for the
HR (Gopalan et al., 1996),
NHL3 expression could affect the HR and thus contribute to resistance
during pathogen attack. To address this possibility, we generated
NHL3-overexpressing transgenic lines, but did not observe increase in
HR-like necrosis under long-day or short-day conditions (data not shown).
However, we could demonstrate a subtle but distinct increased resistance to
virulent P. syringae pv. tomato DC3000
(Fig. 1B). Overexpression of
NHL2, which is most homologous to NHL3 among the
NHLs, was shown to lead to light-dependent speck-like lesion
development, but not to increased resistance
(Dörmann et al., 2000).
This indicates that NHL2 and NHL3 might be functionally distinct.
Alternatively, a different outcome could be caused by the different
starting bacterial inoculum used. In contrast to 2 x 106 cfu
mL–1 used by Dörmann et al.
(2000
Although enhanced resistance was seen in the overexpressing lines, no
obvious phenotypic or morphological alterations were observed. The mechanism
of the enhanced resistance to P. syringae pv. tomato DC3000
is not a result of increased HR or constitutive defense activation as no
elevated PR (PR1, PR2, and PDF1.2) gene expression
was observed. Furthermore, the lack of increased resistance to two strains of
virulent P. parasitica (data not shown) also argues against
pleiotrophic effects or constitutive systemic acquired resistance. Hence, the
overexpression of NHL3 leads to a resistance mechanism that is
distinct from the pathways in enhanced resistance mutants such as cpr1,
cpr5, cpr6, or mpk4, which are smaller in stature and show
constitutive PR gene expression (Clarke et al.,
2000
The lack of suitable antibodies does not permit us to follow up on previous
mRNA expression analysis to determine if the native NHL3 protein also
accumulates after pathogen infection. It is also conceivable that the observed
transcript accumulation (Varet et al.,
2002 Knowledge of NHL3 localization at the subcellular level will assist in the elucidation of its biochemical function and mode of action in conferring enhanced disease resistance when overexpressed. With the aid of two different epitope tags, we confirmed the computer-based prediction of membrane localization using agroinfiltrated tobacco leaves (Figs. 2 and 3). The presence of an epitope tag on either termini did not affect membrane localization of the NHL3 protein (Fig. 3, A and B). Moreover, the detection of the c-myc-NHL3 fusion protein indicates the absence of a cleavable N-terminal signal peptide. Conversely, the detection of the C-terminal HA-epitope tag suggests no processing of the C terminus, which occurs in certain proteins such as glycosyl-phosphatidyl-inositol-anchored membrane proteins. This membrane localization, at least for NHL3-HA, is not an artifact of heterologous expression in tobacco because it could be verified in transgenic Arabidopsis (Fig. 3C). If transgene expression of the HA-tagged protein in both plant species had led to an incorrect localization, high levels of immunoreactive signals would have additionally been found in soluble fractions. However, the NHL3-HA fusion protein was always found at higher levels in the microsomal fractions. Thus, we conclude that the tagged NHL3 is membrane localized.
Deglycosylation experiments demonstrated that NHL3 is a glycosylated
membrane protein and glycosylation accounts partially for the larger size
observed in SDS-PAGE (Fig. 4, A and
B). Thus, the detection of two to three immunoreactive bands could
be a result of incomplete or different levels of glycosylation. Surprisingly,
treatment with PNGase F did not restore the mobility of the immunoreactive
protein to that of the calculated NHL3 molecular mass. Because PNGase F does
not hydrolyze N-glycans if they carry
Using different reagents, we could further show that NHL3 is not
peripherally bound but is tightly membrane associated
(Fig. 4D). Consistent with this
finding, all analysis programs predicted NHL3 to contain a transmembrane
stretch between amino acids 52 and 75, and therefore suggested it to be a type
III membrane protein (Fig. 2,
TM1). Type III membrane proteins lack cleavable signal peptide, have an
amino-terminal hydrophilic ectodomain (i.e. extracellular or lumenal depending
on localization), a single hydrophobic transmembrane stretch, and a
carboxyl-terminal cytoplasmic domain. Predictions of protein topology are
based on the charge difference rule where positive charges flanking the
transmembrane domain promote cytoplasmic retention and negative charges
promote translocation into the endoplasmic reticulum lumen
(Hartmann et al., 1989
Immunohistochemistry coupled with two-phase partitioning experiments
further allowed us to unequivocally pinpoint the subcellular localization of
the tagged NHL3 to the plasma membrane (Figs.
5 and
6). Intriguingly, many
R/Avr gene products are also localized to plant membranes and, in
particular, plasma membranes. RPM1, which confers resistance to P.
syringae expressing avrRpm1 or avrB
(Grant et al., 1995
Interestingly, the use of large amounts of protein in western blotting led
to the detection of larger complexes in membrane fractions. These appear to be
very stable interactions that can partially resist denaturation during
SDS-PAGE. The chemical crosslinking experiments
(Fig. 4C) further demonstrated
that these complexes are not due to trapping within membrane vesicle, but to
true proximity of the interacting proteins (note that DTSSP has a spacer arm
of only 12Å). The mobility of these crosslinked bands corresponds to
that of dimers, trimers, and larger oligomers. Thus, NHL3 may interact with
itself and/or with other proteins. In view of the enhanced resistance upon
overexpressing NHL3 (Fig.
1), it is tempting to speculate that it may possibly interact with
other signaling proteins required for resistance. As proposed by Grant and
Mansfield (1999
In summary, we have provided evidence that overexpression of NHL3,
which has sequence similarities to NDR1, can confer enhanced
resistance to virulent P. syringae pv. tomato DC3000 and
that it encodes a glycosylated plasma membrane protein. In conjunction with
our previous studies showing possible suppression of NHL3 expression
by virulent bacteria (Varet et al.,
2002
Plants and Growth Conditions Tobacco (Nicotiana benthamiana) plants were grown in the greenhouse at 22°C in a 14-h light/10-h dark cycle. Six- to 8-week-old plants were used for infiltration experiments. Arabidopsis ecotype Col-0 plants were grown in a phytochamber (Heraeus Voetsch, Balingen, Germany) at 22°C under short-day conditions (8-h light/16-h dark cycle) for infection experiments or under long-day conditions (16-h light/8-h dark cycle) for seed set in a potting mixture consisting of soil:sand (2:1).
The bacterial pathogen Pseudomonas syringae pv. tomato
DC3000 (Staskawicz et al.,
1987
An HA epitope was added to NHL3 by cloning its cDNA without the
translational stop codon into the XhoI and HindIII sites of
the pKHS-0 vector (a gift from Eric Marois and Ulla Bonas, Institute of
Genetics, Martin-Luther-University, Halle/Saale, Germany). The
NHL3-HA construct was then excised as an XhoI/SpeI
DNA fragment and was cloned into the DEX-inducible expression vector pTA7002
(Aoyama and Chua, 1997
The transient expression assays in tobacco were performed as described in
Nimchuk et al. (2000
For tobacco and Arabidopsis, 15 leaf discs of each sample were extracted in
350 µL of buffer and were fractionated as described
(Nimchuk et al., 2000 Chemical crosslinking with DTSSP (Pierce, Rockford, IL) was performed by adding the indicated amount of the crosslinker to microsomes prepared in phosphate-buffered saline (PBS). Reactions were incubated at room temperature for 30 min before SDS-PAGE and western-blot analysis.
Two-phase partitioning experiments were performed as described by Larsson
et al. (1987 For immunodetection, blots were probed with a monoclonal antibody to the HA epitope (Roche Applied Science, Mannheim, Germany) at a dilution of 1:1,000 or a monoclonal antibody to the c-myc epitope (Sigma) at a dilution of 1:500. The mouse monoclonal anti-BiP antibody (Stressgen Biotechnologies, Victoria, British Columbia, Canada) was used at 2 µg mL–1 dilution, and the rabbit polyclonal anti-AHA2 (gift from Michael Palmgren's laboratory) was used at 1: 3,000 dilution. Peroxidase-conjugated anti-mouse immunoglobulin (Ig) G at 1:10,000 dilution (Sigma) or anti-rabbit IgG at 1:3,000 dilution (Bio-Rad, Munich) was used as a secondary antibody.
Microsomes prepared from plants expressing NHL3-HA were treated with denaturation buffer and PNGase F from the deglycosylation kit (Roche Applied Science, Mannheim, Germany) for 30 min at 37°C. The proteins were subjected to SDS-PAGE and western blotting with the anti-HA antibody. To strip subclasses of proteins from membranes, aliquots of microsomal fractions were incubated in NaCl or NaCO3 solutions at a final concentration of 0.1 M, or in the presence of 1% (v/v) Triton X-100 for 1 h at 4°C. After incubation, samples were centrifuged (100,000g for 1.5 h), the supernatant was retained, and the pellet was resuspended in Tris-EDTA, pH 8.0. The proteins were subjected to SDS-PAGE and western blotting with the anti-HA antibody.
Three-week-old Arabidopsis plants were sprayed with 20 µM
DEX. After 24 h, small pieces of leaves were fixed with 4% (w/v)
paraformaldehyde/0.1% (v/v) Triton X-100 in PBS (135 mM NaCl, 3
mM KCl, 1.5 mM KH2PO4, and 8
mM Na2HPO4), dehydrated by a graded series of
ethanol, and embedded in polyethylene glycol for fluorescence microscopy as
described (Hause et al.,
1996 For electron microscopy, ethanol of dehydrated specimens was substituted by LR-White (Polysciences, Warrington, PA). Immunolabeling of ultrathin sections was carried out with the mouse monoclonal antibody to the HA epitope (diluted 1:500 in PBS containing 1% [w/v] acetylated BSA and 0.1% [v/v] Tween 20) and a goat anti-mouse IgG conjugated with 10 nm colloidal gold (Sigma). After immunolabeling, sections were poststained with uranyl acetate and lead citrate. Sections were visualized with a electron microscope (TEM 900; Ziess).
We thank Guido van den Ackerveken (Utrecht University, Utrecht, The Netherlands) for performing the resistance test with Peronospora parasitica, and Michael Palmgren for the gift of anti-AHA2 antibodies (Royal Veterinary and Agricultural University, Copenhagen, Denmark). Many thanks also to Rebecca Boston (North Carolina State University, Raleigh, NC) and Estelle Hrabak (University of New Hampshire, Durham, NH) for advice on antibodies against membrane markers. We also thank Thomas Lahaye, Norbert Nass (Martin-Luther University, Halle/Saale, Germany), and Boris Szurek (Unité de Recherche en Génomique Végétale, Evry, France) for critical comments on the manuscript. Received January 14, 2003; returned for revision March 12, 2003; accepted May 14, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.020438.
1 This work was supported in part by "Fonds der chemischen
Industrie." * Corresponding author; e-mail jlee{at}ipb-halle.de; fax 49–345–5582–1409.
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TOP
ABSTRACT
RESULTS
-1–3-linked core Fuc
residues (Tarentino and Plummer,
1987
-glucosaminyl) asparagine amidase and
endo-
