The Plant Journal (2012) 70, 796–808
doi: 10.1111/j.1365-313X.2012.04928.x
Putative members of the Arabidopsis Nup107-160 nuclear
pore sub-complex contribute to pathogen defense
Marcel Wiermer1,*,†, Yu Ti Cheng1,2, Julia Imkampe3, Meilan Li1,4, Dongmei Wang1,5, Volker Lipka3 and Xin Li1,6,*
Michael Smith Laboratories, Room 301, 2185 East Mall, University of British Columbia, Vancouver, BC V6T 1Z4, Canada,
2
Genetics Graduate Program, University of British Columbia, Vancouver, BC V6T 1Z3, Canada,
3
Department of Plant Cell Biology, Albrecht-von-Haller Institute for Plant Sciences, Georg August University Göttingen, Julia
Lermontowa Weg 3, 37077 Göttingen, Germany,
4
Shanxi Agricultural University, Taigu, Shanxi, China,
5
The Institute of Nuclear and Biological Technology, Xinjiang Academy of Agricultural Sciences, Urumqi, Xinjiang 830091,
China, and
6
Department of Botany, Room 3529, 6270 University Boulevard, University of British Columbia, Vancouver, BC V6T 1Z4, Canada
1
Received 22 December 2011; accepted 24 January 2012; published online 6 March 2012.
*For correspondence (e-mail [email protected] or [email protected]).
†
Present Address: Department of Plant Cell Biology, Albrecht-von-Haller Institute for Plant Sciences, Georg August University Göttingen, Julia Lermontowa Weg 3,
37077 Göttingen, Germany.
SUMMARY
In eukaryotic cells, transduction of external stimuli into the nucleus to induce transcription and export of
mRNAs for translation in the cytoplasm is mediated by nuclear pore complexes (NPCs) composed
of nucleoporin proteins (Nups). We previously reported that Arabidopsis MOS3, encoding the homolog of
vertebrate Nup96, is required for plant immunity and constitutive resistance mediated by the de-regulated Toll
interleukin 1 receptor/nucleotide-binding/leucine-rich repeat (TNL)-type R gene snc1. In vertebrates, Nup96 is
a component of the conserved Nup107-160 nuclear pore sub-complex, and implicated in immunity-related
mRNA export. Here, we used a reverse genetics approach to examine the requirement for additional subunits
of the predicted Arabidopsis Nup107-160 complex in plant immunity. We show that, among eight putative
complex members, beside MOS3, only plants with defects in Nup160 or Seh1 are impaired in basal resistance.
Constitutive resistance in the snc1 mutant and immunity mediated by TNL-type R genes also depend on
functional Nup160 and have a partial requirement for Seh1. Conversely, resistance conferred by coiled coil-type
immune receptors operates largely independently of both genes, demonstrating specific contributions to plant
defense signaling. Our functional analysis further revealed that defects in nup160 and seh1 result in nuclear
accumulation of poly(A) mRNA, and, in the case of nup160, considerable depletion of EDS1, a key positive
regulator of basal and TNL-triggered resistance. These findings suggest that Nup160 is required for nuclear
mRNA export and full expression of EDS1-conditioned resistance pathways in Arabidopsis.
Keywords: plant immunity, nucleoporins, Nup107-160 complex, mRNA export, nucleocytoplasmic trafficking,
Arabidopsis.
INTRODUCTION
In plants, a major barrier to pathogen infection is conferred
by innate immune responses of individual cells. A first line of
basal defense is mediated by cell-surface pattern recognition
receptors that sense generic pathogen-associated molecular
patterns (PAMPs); this is referred to as PAMP-triggered
immunity (Jones and Dangl, 2006; Zipfel, 2009). Although
PAMP-triggered immunity is normally sufficient to prevent
non-adapted microbes from colonizing plant tissues, hostadapted pathogens have evolved effectors that are often
796
secreted inside host cells during infection to suppress PAMPtriggered immunity, thereby allowing host invasion. Plant
cells deploy a second, largely intracellular, layer of immunity
to host-adapted pathogens that is generally conferred by
nucleotide-binding domain/leucine-rich repeat-containing
immune receptors known as resistance (R) proteins. R
proteins recognize and respond to isolate-specific microbial
effectors in a strong and robust defense response termed
effector-triggered immunity. Effector-triggered immunity
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The Plant Journal ª 2012 Blackwell Publishing Ltd
Nup107-160 complex in plant immunity 797
typically culminates in programmed cell death at attempted
infection sites as part of the hypersensitive response (HR) to
restrict pathogen growth (Greenberg and Yao, 2004; Jones
and Dangl, 2006). Pattern recognition receptor and R protein
activation triggers a major transcriptional reprogramming of
affected host cells (Tao et al., 2003; Bartsch et al., 2006) that
depends on coordinated relay of information by intracellular
signaling systems into the nucleus and nuclear export of
defense-related transcripts towards the cytoplasmic protein
synthesis machinery. Thus, communication between the
cytoplasm and the nucleus is required for both PAMP-triggered immunity and effector-triggered immunity.
The nucleo-cytoplasmic exchange of proteins and RNAs is
mediated through nuclear pore complexes (NPCs), numerous perforations in the nuclear envelope (NE) that separates
the cytoplasm and the nucleoplasm of eukaryotic cells. NPCs
are supramolecular assemblies comprising multiple copies
of approximately 30 constituent nucleoporin proteins
(Nups). Nups are modularly assembled in distinct subcomplexes and arranged radially around a central channel
that serves as the sole conduit for selective bi-directional
exchange of molecular cargoes. Translocation of proteins
typically depends on the recognition of nuclear localization/
export signal sequence motifs on the cargo by nuclear
transport receptors of the karyopherin (Kap) family that have
the capacity to transiently interact with the NPC to facilitate
nuclear import (importins) or export (exportins) (Tran and
Wente, 2006; Terry et al., 2007). General export of mRNAs
utilizes non-Kap export receptors and requires numerous
additional RNA-binding proteins and export factors that
assemble with mRNAs into export-competent messenger
ribonucleoprotein particles (Cole and Scarcelli, 2006; Köhler
and Hurt, 2007; Chinnusamy et al., 2008).
In plants, little is known about the composition of the NPC
and nuclear import and export pathways that mediate
spatial communication between the cytoplasm and nucleoplasm. The contribution of the nucleo-cytoplasmic trafficking machinery to plant immunity was first revealed in an
Arabidopsis genetic screen that aimed to identify components of auto-immune responses and related growth inhibition caused by an constitutively active variant of the
Toll interleukin 1 receptor/nucleotide-binding/leucine-rich
repeat (TNL)-type R gene snc1 (suppressor of npr1-1,
constitutive 1) (Zhang et al., 2003). Mutations in the nucleoporin genes MOS3 (MODIFIER OF SNC1, 3) and MOS7,
which encode homologs of vertebrate/Drosophila Nup96
and Nup88, respectively, and in the importin a3 homolog
MOS6 were identified as suppressors of snc1 (Palma et al.,
2005; Zhang and Li, 2005; Cheng et al., 2009). The three MOS
proteins participate in different nucleo-cytoplasmic trafficking pathways: MOS6 and MOS7 are involved in protein
import and export, respectively, and MOS3/Nup96/SAR3
is implicated in nuclear mRNA export (Palma et al.,
2005; Zhang and Li, 2005; Parry et al., 2006; Cheng et al.,
2009). Recently, the predicted RNA-binding protein MOS11
was identified as another essential component of snc1
auto-immunity, probably functioning in the same mRNA
export pathway as MOS3 (Nup96) at an early stage before
the mRNAs exit the nucleus through nuclear pores (Germain
et al., 2010).
Vertebrate Nup96 and its yeast homolog, Nup145C, are
constituents of the Nup107-160 nuclear pore sub-complex
(called the Nup84 complex in Saccharomyces cerevisiae),
that is located symmetrically on both the nuclear and
cytoplasmic sides of the nuclear pore and the largest
subunit of the NPC (Alber et al., 2007). Of the 30 bona fide
Nups so far identified in vertebrates, almost one third can
be assigned to the Nup107-160 complex, which consists of
Nup37, Nup43, Nup85, Nup96, Nup107, Nup133, Nup160,
Sec13 and Seh1. The Nup107-160/Nup84 complex is multifunctional. It mediates mRNA export in interphase and
plays roles in kinetochore functions and post-mitotic NPC
formation in the NE (Fabre et al., 1994; Vasu et al., 2001;
Harel et al., 2003; Walther et al., 2003; Zuccolo et al., 2007).
Notably, one of its members in mice, Nup96, is required
selectively for innate and adaptive immunity (Faria et al.,
2006).
The involvement of vertebrate and Arabidopsis Nup96 in
immune responses (Zhang and Li, 2005; Faria et al., 2006)
and the requirement for three additional members of
this sub-complex, Nup85, Nup133 and Seh1, for plant
responses to symbiotic microbes (Kanamori et al., 2006;
Saito et al., 2007; Groth et al., 2010) directed our attention
towards identifying additional putative Arabidopsis
Nup107-160 complex members and understanding their
possible function in response to microbial pathogen
challenge. To address this, we used a reverse genetics
approach, and analyzed available T-DNA insertion mutants
of eight putative Nup107-160 complex members predicted
in the Arabidopsis genome for defects in disease resistance. We found that two members which have not
previously been implicated in regulating plant immunity,
Nup160 and the WD40-repeat nucleoporin Seh1, are both
required for basal defense to virulent Pseudomonas syringae and contribute different activity to auto-immunity in
snc1 and TNL-conditioned resistance in response to avirulent bacterial and oomycete pathogens. In contrast, coiled
coil/nucleotide-binding/leucine-rich repeat (CNL)-mediated
immunity operates largely independently of Nup160 or
Seh1. We further establish that both the nup160 and seh1
mutants accumulate poly(A) mRNA inside the nucleus.
In addition, protein levels of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), a key positive regulator of basal and
TNL protein-mediated resistance, are strongly depleted
in the nup160 mutant. Thus, Nup160 is an important
component of EDS1-dependent resistance in Arabidopsis,
reinforcing an important role for the plant NPC in innate
immunity.
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The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 796–808
798 Marcel Wiermer et al.
RESULTS
Identification of putative Arabidopsis Nup107-160
complex members
We previously identified Arabidopsis MOS3/Nup96 from a
mutant screen for genetic suppressors of snc1 (Zhang and Li,
2005). As the MOS3 homologs in vertebrates and yeast,
Nup96 and Nup145C, respectively, are integral members of
the vertebrate Nup107-160/yeast Nup84 complex (hereafter
called the Nup107-160 complex), we wished to determine
whether additional subunits of this largest nuclear pore subcomplex also exist in plants. We performed BLAST database
searches to identify additional putative complex members in
the Arabidopsis thaliana Col-0 reference genome. The
vertebrate and yeast complexes comprise nine and seven
members, respectively (Table S1), and our analyses revealed
that eight of the nine vertebrate members have putative
homologs in Arabidopsis. These include Nup160/SAR1
(At1g33410), Nup133 (At2g05120), Nup107 (At3g14120),
Nup96/MOS3/SAR3 (At1g80680), Nup85 (At4g32910), Nup43
(At4g30840), Seh1 (At1g64350), and two genes similar to
Sec13 (At3g01340 and At2g30050) (Figure 1 and Table S1).
We conclude that the Nup107-160 complex, which appears to
be evolutionary conserved between distantly related
eukaryotes such as yeast and mammals (Belgareh et al.,
2001; Walther et al., 2003; Bai et al., 2004), also exists in a
similar form in plants, as suggested previously (Parry et al.,
2006; Tamura et al., 2010).
Nup160 and Seh1 are required for basal defense
As mutations in the Nup96 homolog MOS3 impair basal
defense against the virulent bacterial pathogen Pseudomonas syringae (Zhang and Li, 2005), we tested the putative
Figure 1. Putative Arabidopsis homologs of Nup107-160 complex members.
Schematic gene structures of predicted Arabidopsis Nup107-160 complex
members, with exons represented as black boxes and introns as solid lines.
The positions of T-DNA insertions (triangles) and mutations (asterisks) are
indicated. All T-DNA insertions are within exons except nup107-1, nup107-2
and nup43-1.
Nup107-160 complex members in a targeted reverse genetics approach for their involvement in basal resistance to
this pathogen. T-DNA insertion mutants of the putative
complex members were obtained from the Arabidopsis
Biological Resource Center (Figure 1) (Alonso et al., 2003),
and homozygous lines were isolated by PCR-based genotyping. For each gene except Nup107 (At3g14120) and
Sec13A (At2g30050) (see below), at least one line with a
T-DNA inserted within exonic sequence was obtained
(Figure 1), and disruption of functional transcripts was
confirmed by RT-PCR using cDNA-specific primers flanking
the insertions (data not shown). For Sec13A (At2g30050) no
T-DNA insertion mutant was available. Our RT-PCR analyses
of the two intronic nup107 insertion lines revealed disruption of full-length transcripts in nup107-1, suggesting loss of
Nup107 function in this mutant allele (data not shown). We
named the nup160 mutant alleles nup160-3 (SAIL_877_B01)
and nup160-4 (SALK_126801; sar1-4) because nup160-1 and
nup160-2 have previously been assigned (Dong et al., 2006).
An additional nup160 mutant allele, sar1-1 (Parry et al.,
2006), was kindly provided by Mark Estelle (Section of Cell
and Developmental Biology, University of California at San
Diego, La Jolla, CA, USA). If possible, at least two alleles for
each gene were included for the comprehensive analysis.
Infection of the T-DNA mutant lines with virulent Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000)
revealed that mutations in Nup160/At1g33410 and Seh1/
At1g64350 impair basal defense responses towards this
pathogen, similar to the defect observed in mos3-1
(Figure 2a). Compared to the complete breakdown of basal
resistance in the control eds1 null mutant Col eds1-2 (Bartsch et al., 2006), the nup160 and seh1 lines showed a slightly
lower level of susceptibility. Mutants in the other predicted
Nup107-160 complex members did not show altered
susceptibility upon inoculation with Pst DC3000 (Figure 2a).
We therefore focused our functional analyses on Nup160
and Seh1.
The three independent nup160 lines (Figure 1) all compromised basal resistance to Pst DC3000 to a similar degree
(Figure 2a), are slightly smaller than wild-type and showed
an early flowering phenotype (Figure 5b and Figure S1).
According to The Arabidopsis Information Resource (TAIR),
SAIL_877_B01 (nup160-3) carries a T-DNA insertion in the
12th exon of At1g33410, whereas SALK_126801 (nup160-4/
sar1-4) carries an insertion in the 17th exon (Parry et al.,
2006). The point mutation in sar1-1 introduces a premature
stop codon in the 11th exon of the gene (Parry et al., 2006)
(Figure 1).
In contrast to nup160, seh1 mutant plants are
indistinguishable from Col-0 wild-type in terms of size and
morphology (Figure S2). As only one T-DNA insertion line
for Seh1 was available (insertion in the 4th exon), we cloned
genomic Seh1/At1g64350 with 2.2 kb upstream of its translational start codon and stably expressed it in the
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The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 796–808
Nup107-160 complex in plant immunity 799
bacteria. As mutations in the other five putative Nup107-160
complex members did not compromise basal resistance
(Figure 2a), we reasoned that these members are either not
required for basal resistance to Pst DC3000, or may function
redundantly with other Nups.
Nup160 and Seh1 are required for TNL-type
R protein-mediated immunity
Figure 2. nup160 and seh1 mutants are impaired in basal defense.
(a) Five-week-old plants of the indicated genotypes were vacuum-infiltrated
with virulent Pseudomonas syringae pv. tomato (Pst) strain DC3000 at
1 · 105 colony-forming units ml)1. Bacterial growth was quantified at day 0
(d0, white bars) and day 3 (d3, black bars), and is expressed as mean values of
viable bacteria per cm2 of leaf tissue SD, using two replicate samplings for
d0 and three replicate samplings for d3.
(b) Transgenic expression of genomic At1g64350/Seh1 under the control of its
native promoter complements the enhanced disease susceptibility of seh1-1
after infection with Pst DC3000 and quantification of bacterial growth as
described in (a).
*P < 0.05 using Student’s t-test for pairwise comparison of wild-type (Col-0)
and mutants. Experiments were repeated three times with similar results.
SALK_022717 (seh1-1) mutant background to test for
complementation of the resistance defect. As shown in
Figure 2(b), genomic Seh1 fully complemented the
enhanced disease susceptibility phenotype of SALK_022717
in multiple independent stable transgenic homozygous
plants, confirming that the T-DNA integration in Seh1/
At1g64350 is responsible for compromising basal resistance
to Pst DC3000.
These data show that, in addition to MOS3/Nup96,
Nup160 and Seh1 are two additional subunits of the
predicted Nup107-160 complex in Arabidopsis required for
the plant’s response to infection by virulent Pseudomonas
Nup160 and Seh1 are equally important for basal resistance
to the invasive hemi-biotrophic pathogen Pst DC3000. This
prompted us to assess the genetic requirement for Nup160
and Seh1 in race-specific resistance mediated by various R
proteins. We found that resistance conferred by the TNL
receptor, RPS4, to Pst DC3000 expressing the effector
avrRps4 (Hinsch and Staskawicz, 1996) was compromised in
nup160 and seh1 mutants as indicated by the approximately
20-fold (seh1-1) and 40–130-fold (nup160 alleles) increase in
bacterial growth 3 days after inoculation compared to wildtype Col-0 (Figure 3a). Loss of RPS4 resistance in seh1-1
was consistently less severe in multiple independent
experiments in comparison with susceptibility of all nup160
mutants and the control mos3-1 (Figure 3a). Conversely,
resistance conferred by the CNL receptors RPM1 and RPS2
to Pst DC3000 expressing the effectors avrRpm1 and
avrRpt2, respectively (Mackey et al., 2002, 2003; Axtell and
Staskawicz, 2003), remained intact in nup160 and seh1
(Figure 3b,c). However, RPM1-mediated resistance triggered
after inoculation with Pseudomonas syringae pv. maculicola
(Psm) ES4326 expressing the effector avrB was less tight
than RPM1 resistance triggered after avrRpm1 recognition,
and slightly increased growth of Psm ES4326 (avrB) was
observed in nup160 and mos3-1 (Figures 3b and S3). This
phenotype of mos3-1 is consistent with previous data
(Zhang and Li, 2005).
We next tested the genetic requirement for Nup160 and
Seh1 in resistance to the avirulent oomycete pathogen
Hyaloperonospora arabidopsidis (Ha) isolate Emwa1, which
is recognized by the TNL protein RPP4 (van der Biezen et al.,
2002). seh1 mutants consistently prevented pathogen sporulation on leaves, as in Col-0. Staining of leaves with
lactophenol trypan blue 7 days after inoculation revealed
that seh1-1 exhibited a combination of spreading HR lesions
and occasional development of spatially restricted trailing
necrosis that typically contained pathogen infection structures before spreading across the whole leaf (Figure 3d). By
contrast, we observed a significant decrease in RPP4 function in the nup160 mutant as seen by trailing host cell
necrosis across entire leaves and the emergence of occasional sporangiophores (Figure 3d). We conclude that
Nup160 and, to a minor extent, Seh1 are essential for full
resistance conferred by TNL proteins against bacterial and
oomycete pathogens. Nup160 may also have a minor
contribution to immunity conferred by the CNL protein
RPM1 after recognition of avrB.
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800 Marcel Wiermer et al.
Figure 3. Disease resistance phenotypes of seh1
and nup160 mutants in response to avirulent
strains of Pst DC3000 and H. arabidopsidis.
(a–c) Five-week-old plants of the indicated genotypes were vacuum-infiltrated with a bacterial
suspension (1 · 105 colony-forming units ml)1)
of Pst DC3000 expressing avrRps4 (a), avrRpm1
(b) or avrRpt2 (c). Bacterial growth was quantified at d0 and d3 as described in Figure 2(a).
*P < 0.05 using Student’s t-test for pairwise
comparison of wild-type (Col-0) and mutants.
(d) Infection phenotypes of leaves inoculated
with H. arabidopsidis Emwa1, which is recognized by RPP4 in Col-0. Leaves of the indicated
genotypes were stained with lactophenol trypan
blue 7 days after inoculation to visualize pathogen hyphae and plant cell death. HR, hypersensitive response; TN, trailing necrosis; h, hyphae.
All experiments were repeated at least three
times with similar results.
snc1 auto-immunity is suppressed to different levels
by nup160 and seh1
Mutations in MOS3/Nup96 suppress the auto-immune phenotypes of the auto-activated TNL variant, snc1, suggesting
that this nucleoporin contributes to snc1-mediated resistance (Zhang and Li, 2005). To determine whether Nup160
and Seh1 are also involved in snc1-mediated auto-immunity, we crossed snc1 with nup160 and seh1 to obtain snc1
nup160 and snc1 seh1 double mutants, respectively. snc1
plants are stunted and have curly leaves, but snc1 nup160
plants no longer exhibit snc1-like morphology and resemble
the nup160 mutant, which is slightly smaller than wild-type
(Figure 4a). Mutations in Nup160 further suppress constitutive expression of the pathogenesis-related (PR) genes PR-1
and PR-2 in the snc1 mutant background as determined by
semi-quantitative RT-PCR (Figure 4c). The snc1 single
mutant exhibits enhanced resistance to virulent Pseudomonas syringae (Li et al., 2001; Zhang et al., 2003). To test
whether enhanced disease resistance is impaired in the snc1
nup160 double mutant, we infected plants with Pst DC3000.
As shown in Figure 4(d), resistance to Pst DC3000 was
completely suppressed when Nup160 function was disabled
in the snc1 mutant background, with an approximately
10-fold higher titer of Pst DC3000 compared with Col-0. In
addition to increased resistance, the snc1 mutant accumulates high levels of the defense hormone salicylic acid (SA).
HPLC analyses of SA extracts revealed a marked reduction of
endogenous total SA levels in the snc1 nup160 mutant to
levels comparable with those of Col-0 (Figure 4e). Together,
our results show that nup160 fully suppresses snc1-related
auto-immune phenotypes, consistent with a function for
Nup160 in TNL-mediated immunity (Figure 3a,d).
The rather complete suppression of snc1 by nup160 is in
contrast to the partial suppression observed when combining snc1 with seh1-1. snc1 seh1-1 double mutants retain the
typical stunted morphology and curly leaves of snc1, but are
significantly bigger than snc1 plants (Figure 4a,b). The
elevated PR1 and PR2 gene expression (Figure 4c) and
accumulation of SA (Figure 4e) in snc1 seh1-1 resemble the
snc1 single mutant. The enhanced disease resistance of snc1
to Pst DC3000 is only partially suppressed by seh1, with
bacterial titers in the snc1 seh1-1 double mutant being
intermediate between those in snc1 and Col-0 plants
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The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 796–808
Nup107-160 complex in plant immunity 801
Figure 4. snc1 auto-immune responses are suppressed to different levels by nup160 and seh1.
(a) Morphology of 4-week-old plants of the
indicated genotypes grown on soil under a 8 h
light regime. Scale bar = 1 cm.
(b) Mean fresh weight (FW) SD of 4-week-old
snc1 and snc1 seh1-1 plants calculated from the
aerial tissue of 40 plants per genotype. P < 0.001
using Student’s t-test for comparison of genotypes.
(c) RT-PCR analysis for PR-1 and PR-2 on RNA
extracted from 4-week-old plants of the indicated
genotypes. Tubulin expression was used as
control.
(d) Five-week-old plants of the indicated genotypes were vacuum-infiltrated with Pst DC3000 at
1 · 105 colony-forming units ml)1. Bacterial
growth was quantified at d0 and d3 as described
in Figure 2(a). *P < 0.05 using Student’s t-test for
pairwise comparison of the indicated mutants
(horizontal bars).
(e) Total SA levels in leaves of 4-week-old plants.
Values are means SD (n = 4 replicates).
*P < 0.001 using Student’s t-test for pairwise
comparison of snc1 and double mutants.
(Figure 4d). In summary, the partial suppression of snc1
auto-immune responses and related growth inhibition by
seh1-1 are consistent with the data that the seh1-1 mutation
partially impairs the function of the TNL receptors RPS4 and
RPP4 (Figure 3a,d).
Defects in both Nup160 and Seh1 do not further impair
basal defense responses
An important function of both Nup160 and Seh1 is to
maintain the basal resistance layer to virulent Pst DC3000
(Figure 2a). To test the relationship between the two genes
within this resistance pathway, we generated nup160 seh1
double mutants and infected them with Pst DC3000. Bacterial growth in nup160-4 seh1-1 was not further increased
compared with the single mutants (Figure 5a). The susceptibility of other double mutant combinations between
nup160 and nup43-2, nup107-1 or nup133-1 was also similar
to that of the nup160 single mutant (Figure 5a). Whereas
nup160 seh1, nup160 nup43 and nup160 nup107 double
mutants are similar to the nup160 mutant in terms of morphology and the timing of floral transition, nup160-4
nup133-1 plants show compound developmental defects.
Although nup133-1 mutants are indistinguishable from
wild-type, the nup160 early-flowering phenotype is further
exacerbated in the nup160-4 nup133-1 double mutant and
accompanied by a further reduction in rosette size
(Figure 5b). These plants produce siliques that are significantly decreased in size and contain few or no seeds (inset in
Figure 5b). An even more severe phenotype was reported
previously for nup160 mos3 double mutants that exhibit
seedling lethality (Parry et al., 2006). Our epistasis analyses
suggest that Nup160 and Seh1 function within the same
pathway that confers basal resistance to virulent Pseudomonas bacteria. Because nup160-4 nup133-1 double
mutants show more severe developmental defects than
nup160, our results indicate a partially redundant function of
Nup133 and Nup160, and suggest that loss of both subunits
results in a further decrease in NPC function that is important for several aspects of plant development.
Seh1 is a nuclear and cytoplasmic protein
In vivo localization of translational Nup160 and Nup96/
MOS3 fusions with green fluorescent protein (GFP) to the
nuclear rim has previously been demonstrated in transient
expression assays and roots of stable transgenic plants
(Zhang and Li, 2005; Dong et al., 2006). Seh1 is a WD40repeat protein that localizes to multiple subcellular locations
in yeast, including the NPC, the nucleoplasm and the cytoplasm (Rout et al., 2000; Bai et al., 2004; Matsuyama et al.,
2006; Alber et al., 2007), suggesting that part of its cellular
pool is not permanently associated with the NPC. In transiently transfected Arabidopsis protoplasts, Seh1 has been
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The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 796–808
802 Marcel Wiermer et al.
Figure 5. Basal resistance and developmental phenotypes of nucleoporin
single and double mutants.
(a) Defects in both Nup160 and Seh1 do not further impair basal defense
compared to the respective single mutants. Plants were infected with virulent
Pst DC3000 (1 · 105 colony-forming units ml)1), and bacterial growth was
quantified at d0 and d3 as described in Figure 2(a). *P < 0.05 using Student’s
t-test for pairwise comparison of wild-type (Col-0) and mutants. The experiment was repeated three times with similar results.
(b) Developmental defects of nup160 are exacerbated by nup133-1. Fourweek-old plants representative of the indicated single and double mutants
grown on soil under a 16 h light regime are shown. nup160-4 nup133-1
double mutants produce siliques that are significantly reduced in size and
contain few or no seeds (inset). Scale bars = 1 cm.
Figure 6. Seh1–CFP localizes to the nucleus and the cytoplasm.
(a) Transgenic expression of genomic At1g64350/Seh1 fused to cyan fluorescent protein (CFP) (gSeh1::CFP) under the control of its native promoter
(promSeh1) or the double 35S promoter (prom35SS) complements the
enhanced disease susceptibility of seh1-1 after infection with Pst DC3000 at
1 · 105 colony-forming units ml)1. Bacterial growth was quantified at d0 and
d3 as described in Figure 2(a). *P < 0.05 using Student’s t-test for pairwise
comparison of wild-type (Col-0) and mutant.
(b) Confocal laser scanning microscope images of Seh1–CFP fluorescence in
4-week-old leaves of stable transgenic plants expressing Seh1–CFP under the
control of the double 35S promoter. N, nucleus; CS, cytoplasmic strands; NL,
nucleolus.
reported to localize to the nucleus, the pre-vacuolar compartment and the Golgi complex, as determined by immunostaining of affinity-tagged Seh1 protein in fixed protoplasts
(Lee et al., 2006). To investigate the subcellular localization
of Seh1 in living Arabidopsis cells, we fused cyan fluorescent protein (CFP) to the C-terminus of Seh1 and expressed
the fusion protein in the seh1-1 mutant background under
the control of endogenous 2.2 kb sequence upstream of the
Seh1 translation initiation codon or a CaMV double 35S
promoter. Western blot analyses on leaf total protein
extracts using an antibody recognizing CFP revealed
expression of the Seh1–CFP fusion protein and the absence
of detectable degradation products or free CFP (data not
shown). To test whether the Seh1–CFP fusion protein is
functional, multiple independent homozygous lines carrying
a single insertion of the transgene were infected with viru-
lent Pst DC3000. As shown in Figure 6(a), the Seh1–CFP
fusion protein complemented the enhanced disease susceptibility phenotype of seh1-1 towards Pst DC3000 when
expressed under the control of either the native promoter or
the double 35S promoter, suggesting that the Seh1–CFP
fusion protein is functional and correctly localized in
Arabidopsis cells. The intracellular localization of stably
expressed Seh1–CFP was examined in Arabidopsis leaves
by confocal laser scanning microscopy (CLSM). We were
unable to detect Seh1–CFP fluorescence in transgenic
plants expressing the fusion protein under the control of
the endogenous Seh1 promoter, probably due to low
expression levels. In contrast, strong CFP fluorescence was
observed inside the nuclei and cytoplasm of multiple independent transgenic plants expressing a translational Seh1
fusion to CFP under control of the double 35S promoter
ª 2012 The Authors
The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 796–808
Nup107-160 complex in plant immunity 803
(Figure 6b). Localization of Seh1–CFP to the nucleus and
cytoplasm is in contrast to the localization of Nup160 and
MOS3/Nup96 to the NE (Zhang and Li, 2005; Dong et al.,
2006), and suggests that Arabidopsis Seh1 shows dynamic
association with the Nup107-160 complex. However, we
cannot exclude the possibility that stable association of a
proportion of Seh1–CFP with the NPC and thus concentration of CFP fluorescence at the nuclear rim of leaf cells is
masked due to over-expression of the fusion protein by the
double 35S promoter.
The nup160 and seh1 mutants are impaired in nuclear
mRNA export and EDS1 protein accumulation
In vertebrate and yeast cells, the Nup107-160 complex has
an important function in mRNA export during interphase,
and defects in or depletion of individual members of this
complex result in nuclear mRNA accumulation (Fabre et al.,
1994; Vasu et al., 2001). In Arabidopsis, loss-of-function
nup160/sar1 and mos3/sar3/nup96 mutant plants are also
defective in nuclear mRNA export (Dong et al., 2006; Parry
et al., 2006). As Seh1 is a predicted member of the Nup160/
SAR1- and MOS3/SAR3/Nup96-containing Arabidopsis
Nup107-160 nuclear pore sub-complex, and all three genes
are required for disease resistance, we tested whether Seh1
is also essential for export of mRNAs from the nucleus to the
cytoplasm. To localize poly(A) mRNAs in leaves of 7-day-old
wild-type and seh1-1 seedlings, we performed whole mount
in situ hybridization using a 5¢-Alexa488-labeled oligo(dT)45
probe. CLSM revealed strong fluorescence inside nuclei of
the seh1 mutant that was similar to the extent of fluorescence observed in nup160-3 and mos3-1 using identical
microscope settings for image acquisition (Figure 7a) (Dong
et al., 2006; Parry et al., 2006). In contrast, wild-type Col-0
showed only weak nuclear fluorescence (Figure 7a). These
results indicate that seh1-1 mutant plants accumulate mRNA
inside the nucleus, and that the functions of Nup160/SAR1,
Nup96/MOS3/SAR3 and Seh1 are required for proper export
of mRNAs from the nucleus to the cytoplasm.
In vertebrates, Nup96 plays an important role in immunity,
where it is required for selective nuclear mRNA export of key
interferon c-regulated genes in response to viral infection
(Faria et al., 2006). Depletion of Nup96 results in nuclear
retention of these specific classes of mRNAs, and probably
contributes to the lower expression level of interferon
c-regulated proteins. We therefore reasoned that the defects
of the nup160 and seh1 mutants in basal and TNL-triggered
immunity may, at least in part, be caused by reduced
accumulation of regulators of these resistance pathways. To
test this, we generated total protein extracts from leaves of
both mutants and wild-type plants. Our immunoblot
analyses revealed that total amounts of EDS1, an essential
regulator of basal resistance and TNL receptor-mediated
Figure 7. Poly(A) RNA accumulation in nuclei of nup160 and seh1 mutants, and selective depletion of EDS1 protein in nup160.
(a) Leaves from 7-day-old plate-grown seedlings of the indicated genotypes were fixed and probed with Alexa488-tagged oligo(dT)45 oligonucleotides. The green
color in confocal images corresponds to the location of poly(A) mRNA, which accumulates to higher levels in nuclei of seh1-1, nup160-3 and mos3-1 cells compared
to wild-type. Arrows indicate nuclei.
(b) EDS1 protein is selectively depleted in nup160 mutants. Immunoblot analyses of proteins recognized by the indicated antibodies in total protein extracts derived
from 4-week-old leaf tissues of the indicated genotypes. Equal loading was monitored by staining the membrane with Ponceau S. Numbers above the blot indicate
band intensities relative to the EDS1 signal in Col-0, as quantified using IMAGEJ software, http://rsb.info.nih.gov/ij/. Experiments were repeated twice with similar
results.
(c) EDS1 transcript abundance in nup160 is reduced in both the cytoplasm and the nucleus. Quantitative RT-PCR analysis for EDS1A using RNA extracted from
subcellular fractions of 4-week-old leaf tissues of wild-type (Col-0) and nup160-3. Bars represent mean EDS1A expression values SD, normalized to the expression
of Ubiquitin5 (UBQ5). Prior to RNA extraction, efficient subcellular fractionation was monitored by immunoblot analysis using anti-PEPC as a cytosolic marker and
anti-histone H3 as a nuclear marker.
ª 2012 The Authors
The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 796–808
804 Marcel Wiermer et al.
immunity (Parker et al., 1996; Aarts et al., 1998), are strongly
reduced in nup160 mutant lines (to approximately 25%
of the wild-type level) but less severely affected in the
seh1-1 mutant (to approximately 70% of the wild-type level)
(Figure 7b). EDS1 protein accumulation was unchanged in
control nup85-1 plants that did not show enhanced susceptibility to Pst DC3000 (Figures 2a and S4). Significantly,
the mutations did not obviously alter total levels of extractable PEPC, HSC70, TGA2, VDAC1 and histone H3 (Figure 7b),
suggesting a preferential depletion of EDS1 in the nup160
mutant.
We next examined whether selectively impaired export of
EDS1 transcripts from nuclei of the nup160 mutant may
account for the strong depletion of EDS1 protein in nup160.
Unexpectedly, our quantitative RT-PCR analysis of fractionated cytoplasmic and nuclear RNA did not reveal a preferential accumulation of EDS1 transcripts in nup160 nuclei.
Instead, we observed that EDS1 mRNA levels were reduced
in both the cytoplasmic and nuclear fraction of the nup160
mutant compared to wild-type levels (Figure 7c). We quantified EDS1 transcripts from total cellular RNA by quantitative RT-PCR to confirm a slight but significant reduction of
EDS1 mRNA expression in nup160 (Figure S5). The reduction in overall EDS1 expression in nup160 (to approximately
75% of wild-type levels) was less severe than the decrease in
EDS1 protein abundance (to approximately 25% of wild-type
levels) (Figures 7b and S5). Our data suggest that both
reduced nuclear mRNA export activity (Figure 7a) and
overall reduced EDS1 expression (Figures 7c and S5) in
nup160 affect EDS1-conditioned basal and TNL-type R
protein-mediated resistance.
DISCUSSION
Transport of proteins and RNAs across the NE is fundamental for eukaryotic cellular communication and function,
providing important means to control cell homeostasis,
gene expression and signaling networks (Kaffman and
O’Shea, 1999; Orphanides and Reinberg, 2002). Despite the
pivotal role NPCs play in selectively regulating the bi-directional flow of information between the cytoplasm and the
nucleoplasm, our knowledge of plant NPC composition and
the function of its subunits is limited.
In this study, we used a reverse genetics approach to
identify putative subunits of the Arabidopsis Nup107-160
nuclear pore sub-complex that are required for basal
defense signaling. Of eight complex members predicted in
the Col-0 reference genome (Figure 1 and Table S1),
defense-related functions were demonstrated for two:
Nup160 and Seh1. A third complex member, Nup96/MOS3,
has previously been identified based on its requirement for
constitutive resistance caused by the auto-activated TNL R
gene, snc1 (Zhang and Li, 2005). Our pathology assays
revealed that mutants of Nup160 and Seh1 show altered
defense, not only in basal resistance (Figure 2) but also in
TNL-type R protein-mediated immunity (Figure 3a,d).
Although both genes are equally required for basal defense
to virulent Pseudomonas bacteria, Nup160 appears to play a
more prominent role in TNL receptor-triggered immunity to
avirulent pathogens than Seh1 (Figure 3a,d). This is also
reflected by the fact that the seh1-1 mutation only partially
suppresses the constitutive resistance and growth inhibition
conditioned by snc1 (Figure 4a,b,d). As SA accumulation
and PR-1/PR-2 expression in snc1 seh1-1 were not significantly different from snc1 (Figure 4c,e), part of the nuclear
and/or cytoplasmic activity of Seh1 may contribute to the
SA-independent branch of plant defense responses activated in snc1 (Zhang et al., 2003). Significantly, growth of
Pst DC3000 expressing the effectors avrRpm1 (recognized by
the CNL-type R protein RPM1) or avrRpt2 (recognized by the
CNL protein RPS2) was largely unaffected in nup160 and
seh1 mutants (Figure 3b,c), demonstrating pathway specificity of these two components in plant defense signaling.
Because resistance to Psm ES4326 expressing avrB was
slightly relaxed in nup160 (Figure S3), we cannot exclude
the possibility of a minor contribution of Nup160 to RPM1triggered immunity after recognition of avrB, which may be
less efficient than recognition of avrRpm1. The lack of
obvious developmental defects in the seh1-1 mutant (Figures S1 and S2) and the rather mild pleiotropic defects in
nup160 (Figures 5b and S1) further indicate a selective
involvement of these two nuclear pore components in
regulating responses to microbial pathogens in Arabidopsis.
Faria et al. (2006) previously reported that mice with
reduced levels of the Nup107-160 complex member Nup96
are impaired in immunity due to specifically reduced export
and thus translation of mRNAs encoding key immune
regulators. The authors concluded that the reduced protein
accumulation of immune regulators probably accounts for
the immunity defects of Nup96-depleted mice (Faria et al.,
2006). Because nup160 plants are selectively impaired in
basal and TNL-type R protein-mediated resistance and
accumulation of EDS1, a key positive regulator of both
defense pathways (Figures 3 and 7b) (Feys et al., 2005;
Wiermer et al., 2005), we assessed whether nup160 specifically affects EDS1 mRNA export from the nucleus. We did
not observe a preferential retention of EDS1 mRNA in nuclei
of the nup160 mutant, which is inconsistent with a primary
role of Nup160 in mediating selectively the nuclear export of
transcripts encoding EDS1. Our quantitative RT-PCR analysis of fractionated RNA does not exclude the possibility that
EDS1 is among the transcripts retained inside nuclei of
nup160. Instead, our data suggest a more general mRNA
export defect in which the mRNAs of the housekeeping
genes Ubiquitin5 (UBQ5) (Figure 7c) or Protein Phosphatase
2A (PP2A) (data not shown) used for data normalization
show the same subcellular distribution pattern. The
observed accumulation of poly(A) mRNA inside mutant
nuclei would thus reflect a broad range of transcripts.
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The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 796–808
Nup107-160 complex in plant immunity 805
However, the finding that the nup160 and seh1 mutants are
selectively impaired in basal and TNL-triggered immunity
(Figure 3a,d) and EDS1 protein accumulation (Figure 7b) is
remarkable and inconsistent with a major inhibition of bulk
mRNA export, as this would have a more significant impact
on CNL-triggered immunity as well and probably result in
catastrophic phenotypes and/or death. Our data therefore
suggest that the mRNA export defect of nup160 and seh1 is
rather mild, possibly because these mutants (like several
other tested Nup107-160 complex mutants) may produce
truncated proteins and thus may not be functionally null
(Figure S6). This suggests that EDS1-dependent resistance
pathways are particularly prone to disturbances in mRNA
export. Loss of Seh1 function might have a minor effect on
EDS1 protein abundance (Figure 7b) and TNL-triggered
immunity (Figures 3a,d and 4) due to partial compensation
by Nup160 or other Nups.
Notably, our analyses also revealed a slight reduction of
total EDS1 transcript in the nup160 mutant (Figure S5),
suggesting that Nup160 may also contribute to transcriptional activation mechanisms. An intriguing possibility is
that Nup160 itself promotes EDS1 expression. Studies in
yeast demonstrated that members of the Nup84 complex
(which is equivalent to the vertebrate Nup107-160 complex)
are capable of activating transcription in vivo, and, importantly, do so in the normal context of the NPC by tethering
target genes to the inner side of the nuclear pore, thereby
coupling transcription with efficient mRNA export (Menon
et al., 2005). Another recent study revealed a positive role for
the Nup84 complex in RNA polymerase II-mediated transcription elongation that is functionally coupled to its role in
mRNA export as part of an intact NPC (Tous et al., 2011).
Transcriptional elongation probably contributes to the control of transcript accumulation and thus transcriptional
efficiency (Akhtar and Gasser, 2007). Therefore, it is also
possible that Nup160 may fine-tune EDS1 transcript accumulation at the level of elongation.
Alternatively, Nup160 might contribute to the nuclear
accumulation of transcriptional regulators required for full
expression of EDS1 and/or regulate nuclear translocation
rates of immune regulatory proteins essential for basal and
TNL-mediated resistance. Consistent with this idea, Parry
et al. (2006) observed reduced nuclear accumulation of the
transcriptional repressor IAA17 in the nup160/sar1 mutant,
providing a possible explanation for its altered auxin
sensitivity. In contrast, nuclear import of the cold response
regulator ICE1 is unchanged in nup160 plants, which are
also defective in tolerance to cold stress (Dong et al., 2006),
suggesting a different extent to which reduced nuclear
accumulation of protein regulators contributes to the
observed defects in nup160. In this context, we tested
whether mutations in Nup160 affect the proper coordination
of EDS1 nuclear and cytoplasmic pools across the NE which
appears to be essential for full immunity (Garcia et al.,
2010). We observed that the level of EDS1 protein was
reduced proportionally in both the cytoplasm and nucleus
of nup160 plants (Figure S7). Although this may be due
to impaired EDS1 expression and mRNA export in the
nup160 mutant, we cannot exclude the existence of additional processes that influence EDS1 protein stability.
A post-transcriptional effect was observed in Arabidopsis
mos7/nup88 mutant plants, in which reduced nuclear
retention of EDS1 apparently influences EDS1 protein
stability, and reduced EDS1 amounts become equilibrated
between cellular compartments over time (Cheng et al.,
2009). Despite this reduction in EDS1 protein accumulation,
mos7-1 plants show normal EDS1 transcript levels, and are,
like their nup88/mbo counterpart in Drosophila, unlikely to
accumulate mRNA inside the nucleus (Roth et al., 2003;
Cheng et al., 2009). Also distinct from nup160 and seh1,
mos7-1 mutants are strongly affected in CNL receptormediated resistance, implying selective contributions of
individual Nups to the regulation of plant immune responses by nucleo-cytoplasmic transport.
EXPERIMENTAL PROCEDURES
Plant growth, mutant isolation and pathology assays
Plants were grown in soil at 22C under a 10 h (for pathology
assays) or 16 h light regime. The sar1-1 (Parry et al., 2006) and
mos3-1 (Zhang and Li, 2005) mutants have been described previously. T-DNA insertion mutants were obtained from the Arabidopsis
Biological Resource Center, and genotyped by PCR using insertionflanking primers. Plant cell death and H. arabidopsidis infection
structures were visualized at 7 days post-inoculation under a light
microscope after staining leaves with lactophenol trypan blue (Aarts
et al., 1998). Virulent and avirulent Pst DC3000 strains were as
described previously (Aarts et al., 1998). For bacterial growth
assays, suspensions of 1 · 105 colony-forming units ml)1 5 mM
MgCl2 with 0.001% v/v Silwet L-77, http://www.lehleseeds.com/cgibin/hazel.cgi?action=DETAIL&item=85 were vacuum-infiltrated into
leaves of 5-week-old plants, bacterial titers were determined after
1 h (d0) and 3 days (d3), and colony numbers were compared
between lines using a two-tailed Student’s t-test.
Protein analyses
Total and nuclear or nuclei-depleted protein extracts for protein
gel-blot analysis were prepared from 4-week-old leaf material as
described previously (Feys et al., 2005). Antibodies used for
immunoblot analyses were as described previously: anti-EDS1,
anti-histone H3 and anti-HSC70 (Feys et al., 2005), anti-PEPC (Noël
et al., 2007) and anti-TGA2 (Cheng et al., 2009). Anti-VDAC1
(At3g01280) was purchased from Agrisera, http://www.agrisera.
com/en/info/home.html.
Nuclear/cytoplasmic fractionation, RNA extraction and
gene expression analyses
Nuclear and cytosolic fractions for RNA extraction were obtained
using a protocol modified from that described by Park et al. (2005).
Leaf material (2 g) from 4-week-old soil-grown plants was ground in
liquid nitrogen and mixed with 2 ml cell-wall disruption buffer
[10 mM Tris pH 7.5, 10 mM NaCl, 10 mM MgCl2, 10 mM b-mercaptoethanol, 10% v/v glycerol, 100 U ml)1 Ribolock (Fermentas, http://
ª 2012 The Authors
The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 796–808
806 Marcel Wiermer et al.
www.fermentas.com)]. The homogenate was spun through a 95 lm
nylon mesh, and the flow-through was centrifuged at 2500 g and
4C for 10 min to pellet the nuclei. The supernatant was re-centrifuged at 13 000 g and 4C for 15 min, and the supernatant of this
second centrifugation was saved as the cytoplasmic fraction. The
nuclear pellet from the first centrifugation was carefully resuspended in nuclei washing buffer (10 mM Tris pH 7.5, 10 mM NaCl,
10 mM MgCl2, 10 mM b-mercaptoethanol, 1 M hexylene glycol, 0.5%
Triton X-100), and centrifuged at 1500 g for 10 min at 4C. The
supernatant was discarded, and washing and centrifugation of
nuclear pellets was repeated five or six times. Each nuclear pellet
was resuspended in 75 ll cell-wall disruption buffer, and nuclear
fractions of each genotype were pooled for RNA extraction. As
quality controls for the fractionation, immunoblot analyses were
performed using the nuclear marker protein histone H3 and the
cytoplasmic marker protein PEPC. Total RNA was isolated from
cytoplasmic and nuclear fractions or directly from 4-week-old soilgrown plants using TRIzol (Invitrogen, http://www.invitrogen.com/).
A 1.5 lg aliquot of RNA was reverse-transcribed using RevertAid H
Minus M-MuLV reverse transcriptase (Fermentas) and 0.5 lg
oligo(dT)18V primer at 42C in a 20 ll reaction volume. Aliquots of
reverse transcription reaction products were used for semi-quantitative PCR (0.5 ll) or quantitative PCR (1 ll of a 1:7.5 dilution).
Quantitative RT-PCRs were performed using a CFX96 real-time PCR
detection system (Bio-Rad, http://www.bio-rad.com/) using SsoFast
EvaGreen Supermix (Bio-Rad). Ubiquitin5 (UBQ5, At3g62250) and
Protein Phosphatase 2A (PP2A, At1g13320) transcript levels were
used for data normalization. Relative transcript levels were calculated using CFX Manager software (version 2.1) and the non-linear
regression method. All primers are listed in Table S2.
DNA constructs, transgenic plants and protein
localization studies
The construct used to complement the seh1-1 mutation was generated by PCR amplification of a Col-0 genomic fragment containing
the Seh1/At1g64350 coding region and 2.2 kb upstream of the ATG
start codon as well as 2.2 kb downstream of the stop codon. Primers
Seh1.F.BamHI
(5¢-CGCGGATCCGACTTGTATATACCGTCTCG-3¢)
and Seh1.R.EcoRI (5¢-CCGGAATTCGTCGCAGACTGAGATTCTTG-3¢)
were used for PCR, and the BamHI/EcoRI-digested fragment was
cloned into pGreenII0229 (Hellens et al., 2000) for complementation
analysis. The construct was introduced into Agrobacterium tumefaciens strain GV3101 (pMP90) harboring the helper plasmid
pSOUP (Hellens et al., 2000), and transformed into SALK_022717
using the floral-dip method (Clough and Bent, 1998). To generate
plants expressing Seh1–CFP under the control of the 35S promoter
or the native Seh1 promoter, a genomic fragment spanning the fulllength Col-0 Seh1 gene without the stop codon was PCR-amplified
either with 2.2 kb upstream of the translation initiation codon
(promSeh1-gSeh1) or without the promoter sequence (gSeh1),
using the primers gAt1g64350.D-TOPO.F (5¢-CACCATGGCGAAA
TCAATGGCGACG-3¢) and Seh1R.GW (5¢-GGAGGGAACTGGTT
CAAGCG-3¢) for gSeh1, and Seh1.F.GW (5¢-CACCGACTTGTATAT
ACCGTCTCG-3¢) and Seh1.R.GW (5¢-GGAGGGAACTGGTTCAAGC
G-3¢) for promSeh1-gSeh1. Both fragments were cloned into
pENTR/D-TOPO (Invitrogen) and sequenced. Gateway LR reactions
were performed between the pENTR/D-TOPO clones and the binary
destination vectors pXCG-CFP and pXCSG-CFP (Feys et al., 2005) to
generate the expression constructs pXCG-promSeh1::gSeh1::CFP
and pXCSG-prom35SS::gSeh1::CFP, respectively. Constructs were
transferred to A. tumefaciens GV3101 (pMP90RK) and transformed
into SALK_022717 as described above. CLSM was performed on a
Leica SP5-DM6000 (http://www.leica.com/) using an excitation
wavelength of 458 nm.
SA measurements
Endogenous SA levels were determined as described previously (Li
et al., 1999).
Whole-mount in situ localization of mRNA
Poly(A) RNA in situ hybridizations were performed on 7-day-old
seedlings grown on half-strength MS medium as previously
described (Gong et al., 2005; Germain et al., 2010) using a
5¢-Alexa488-labeled oligo(dT)45 probe. Leaves were observed using a
Nikon PCM-2000 confocal laser scanning microscope (http://www.
nikon.com/) equipped with a 488 nm argon excitation laser. All
images were taken using a 40· objective at equivalent laser intensity.
ACKNOWLEDGEMENTS
We thank the Arabidopsis Biological Resource Center for providing
T-DNA insertion mutants, Mark Estelle (Section of Cell and Developmental Biology, University of California at San Diego, La Jolla,
CA, USA) for sar1-1 seeds, Jane Parker (Department of PlantMicrobe Interactions, Max Planck Institute for Plant Breeding
Research, Cologne, Germany) for Col eds1-2 seeds and purified
EDS1 antisera, and Rene Fuchs for help with CLSM. We are grateful
for financial support to M.W. by a research fellowship from the
Alexander von Humboldt Foundation and the Deutsche Forschungsgemeinschaft. We acknowledge a Natural Sciences and
Engineering Research Council of Canada postgraduate fellowship
to Y.T.C., and thank the Natural Sciences and Engineering Research
Council of Canada (Discovery Grant program) for funding to X.L.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Phenotypes of 4-week-old Col-0 wild-type and mutant
plants grown on soil under a 16 h light regime.
Figure S2. Morphology of 5-week-old Col-0 wild-type and seh1-1
(SALK_022717) plants grown on soil under a 10 h light regime.
Figure S3. Disease resistance phenotypes of seh1 and nup160
mutants in response to Psm ES4326 expressing avrB.
Figure S4. EDS1 protein accumulation in wild-type and nup85-1.
Figure S5. EDS1 expression is affected in nup160 mutants.
Figure S6. RT-PCR analyses of nucleoporin transcripts expressed 5¢
and 3¢ of the indicated mutations and in wild-type plants.
Figure S7. EDS1 protein abundance in subcellular fractions of wildtype and nup160-3 mutant plants.
Table S1. Mammalian, S. cerevisiae and putative A. thaliana homologs of Nup107-160 complex members.
Table S2. Primers used for semi-quantitative and quantitative
RT-PCR.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
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