Download Active uptake of cyst nematode parasitism proteins into the plant cell

International Journal for Parasitology 37 (2007) 1269–1279
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Active uptake of cyst nematode parasitism proteins
into the plant cell nucleus
Axel A. Elling a, Eric L. Davis b, Richard S. Hussey c, Thomas J. Baum
a
a,*
Interdepartmental Genetics Program and Department of Plant Pathology, Iowa State University, Ames, IA 50011, USA
b
Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695, USA
c
Department of Plant Pathology, University of Georgia, Athens, GA 30602, USA
Received 30 January 2007; received in revised form 11 March 2007; accepted 13 March 2007
Abstract
Cyst nematodes produce parasitism proteins that contain putative nuclear localisation signals (NLSs) and, therefore, are predicted to
be imported into the nucleus of the host plant cell. The in planta localisation patterns of eight soybean cyst nematode (Heterodera glycines) parasitism proteins with putative NLSs were determined by producing these proteins as translational fusions with the GFP and
GUS reporter proteins. Two parasitism proteins were found to be imported into the nuclei of onion epidermal cells as well as Arabidopsis
protoplasts. One of these two parasitism proteins was further transported into the nucleoli. Mutations introduced into the NLS domains
of these two proteins abolished nuclear import and caused a cytoplasmic accumulation. Furthermore, we observed active nuclear uptake
for three additional parasitism proteins, however, only when these proteins were synthesised as truncated forms. Two of these proteins
were further transported into nucleoli. We hypothesise that nuclear uptake and nucleolar localisation are important mechanisms for
H. glycines to modulate the nuclear biology of parasitised cells of its host plant.
2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
Keywords: Heterodera glycines; NLS; Nucleus; Secretion; Plant-parasitic nematode
1. Introduction
Cyst nematodes are important obligate biotrophic plant
parasites affecting crops worldwide. The soybean cyst nematode Heterodera glycines causes estimated annual damage
of almost US $800 million to soybean production in the
USA alone (Wrather et al., 2001). The parasitic behaviour
of this nematode species begins with infective second-stage
juveniles (J2) that penetrate into host roots and migrate
intracellulary through the root cortex until they reach the
vicinity of the vascular tissue. There, they become sedentary and initiate the formation of a feeding site, the syncytium (Endo, 1964). Both the migration process through
root tissue as well as the induction and maintenance of
the syncytium are mediated by the secreted products of
*
Corresponding author. Tel.: +1 515 294 2398; fax: +1 515 294 9420.
E-mail address: [email protected] (T.J. Baum).
parasitism genes (i.e., parasitism proteins), which encompass a variety of cell wall-degrading enzymes as well as proteins that are thought to alter the normal host plant
physiology after they have been secreted into the apoplast
or into the cytoplasm of plant cells by the nematode (Davis
et al., 2000, 2004; Gao et al., 2003; Baum et al., 2007). Most
cyst nematode parasitism proteins have an N-terminal signal peptide for secretion and are produced in three secretory gland cells that are associated with the nematode
oesophagus, the so-called oesophageal glands. Two of
these gland cells are the subventral glands, which are predominantly active in early stages of parasitism, and one
is the dorsal gland, which enlarges and becomes more
active as parasitism progresses. Parasitism proteins are
injected by the nematode into host tissues or cells through
a stylet, a protrusible hollow mouth spear that is connected
to the gland cells via the oesophagus (Hussey, 1989; Davis
et al., 2000; Vanholme et al., 2004; Lilley et al., 2005).
0020-7519/$30.00 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijpara.2007.03.012
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A.A. Elling et al. / International Journal for Parasitology 37 (2007) 1269–1279
The cyst nematode feeding site, the syncytium, is a multinucleate and physiologically active aggregation of fused
root cells that exclusively provides the nematode with
nourishment during its sedentary life stages (Gheysen and
Fenoll, 2002; Jasmer et al., 2003). While the exact molecular mechanisms that lead to the differentiation of this nematode-induced structure are still unknown, interference
with the normal nuclear biology of the host cell might play
an important role (Goverse et al., 2000; Davis et al., 2004;
Tytgat et al., 2004; Baum et al., 2007). Cyst nematodes
change the normal fate of parasitised cells that are destined
to develop into a syncytium by activating the cell cycle
(Goverse et al., 2000). In brief, the cell cycle consists of
the mitotic (M) phase, in which the nuclear division takes
place, and the interphase, which can be divided into the
first gap (G1), DNA synthesis (S) and second gap (G2)
phases. Previous studies showed that the M phase of the
cell cycle takes place in plant cells adjacent to the syncytium, but not in the syncytium itself (Endo, 1964) and that
the size of nuclei in syncytia increases due to DNA endoreduplication (Endo, 1971). Even though no mitotic activity
could be found in syncytia, an upregulation of transcriptional activity for certain mitotic cyclins that are markers
for the G2 phase could be detected during syncytium formation (Niebel et al., 1996; de Almeida-Engler et al.,
1999). In other words, the cell cycle in syncytia progresses
until G2, and cyst nematodes cause repeated cycles of
DNA endoreduplication (G1, S, G2) while shunting the
M phase (Niebel et al., 1996). These observations suggest
that DNA endoreduplication and interference with the
normal nuclear biology are crucial for successful syncytium
establishment. Cell cycle changes were also observed in
mammalian muscle cells infected by the intracellular nematode parasite Trichinella spiralis (Jasmer et al., 2003).
The nucleus is compartmentalised from the cytoplasm
by the nuclear membrane, but large protein complexes
that span this membrane, so-called nuclear pore complexes (NPCs), provide an entry gate for larger molecules
like proteins and nucleic acids (Görlich and Kutay, 1999;
Stoffler et al., 1999; Lim and Fahrenkrog, 2006). Proteins
that are larger than the NPC diffusion barrier of about
40 kDa require hours to diffuse passively through the
NPC, while active import of proteins with a nuclear
localisation signal (NLS) is much more efficient (Merkle,
2001, 2004). The first NLS motif was discovered in the
simian virus 40 (SV40) large T-antigen and consists of
a short stretch of basic amino acids, mostly lysine residues (Görlich et al., 1994). NLSs with similarity to this
type of motif are called SV40-like or monopartite. A second class of NLS is called bipartite and is characterised
by two short stretches of basic residues, which are separated by a short spacer (Görlich et al., 1995). Both types
of NLSs are known from animal as well as plant proteins. The NLS-dependent nuclear import of cargo proteins involves cytoplasmic and nuclear receptors that
recognise the NLS motif (Görlich et al., 1996; Meier,
2005).
In an earlier study, Gao et al. (2003) found that a group
of H. glycines parasitism proteins contain putative NLS
motifs and proposed that these proteins are targeted to
the plant cell nucleus after secretion by the cyst nematode
into the host cell cytoplasm. Cyst nematode parasitism proteins that contain a functional NLS could be hypothesised
to play regulatory roles for processes in the host nucleus
that are required for successful parasitism, like modifying
host gene expression and/or cell cycle regulation. In this
report, we were able to demonstrate the in planta localisation patterns of seven H. glycines parasitism proteins that
are synthesised in the dorsal gland as well as one parasitism
protein produced in the subventral glands. All proteins
characterised here are predicted to contain SV40 or bipartite forms of NLSs, as well as signal peptides. Five of these
parasitism proteins are of unknown function and show no
similarities to any known proteins.
2. Materials and methods
2.1. Sequence analysis and manipulation
A subset of eight parasitism genes (Table 1) from a
H. glycines oesophageal gland cell cDNA library (Gao
et al., 2003) was chosen for analyses here based upon predicted subcellular localisation and putative NLS domains
of the translated sequence using PSORT II (Nakai and
Horton, 1999) and WoLF PSORT (Horton, P., Park, K.J., Obayashi, T., Nakai, K. 2006. Protein subcellular localization prediction with WoLF PSORT. Proc. Asian Pacific
Bioinformatics Conf., pp. 39–48, Taipeh). Putative protein
domains and families were identified using InterProScan
(Zdobnov and Apweiler, 2001). BLASTP (Altschul et al.,
1990) was used to identify similar sequences meeting the
following cutoff criteria: e-value < 1e-05, bit score > 50.
SignalP 3.0 (Bendtsen et al., 2004) was used to detect signal
peptides in the translated parasitism gene sequences.
Full-length sequences without the signal peptide-encoding region were amplified from the gland cell cDNA clones
in pGEM-TEasy (Promega, Madison, WI) (Gao et al.,
2003) and HindIII and SalI restriction sites (for 5D06 HindIII and BstZI; for 10A06 XbaI and SalI) as well as a start
codon were added by PCR for subcloning into the respective sites in pRJG23 (Grebenok et al., 1997). The primers
used for generating mutated and truncated sequences are
listed in Table 2. To mutate the bipartite NLS domain in
6E07, two overlapping PCR fragments were generated.
The first fragment was amplified with 6E07-5 0 and
6E07mut-1 and the second fragment with 6E07mut-2 and
6E07-3 0 . A third PCR fused both 6E07 fragments using
primers 6E07-5 0 and 6E07-3 0 . Subcloning of all nematode
cDNA regions generated in-frame translational fusions
between the nematode sequence of interest and green fluorescent protein (GFP) and b-glucuronidase (GUS) reporter
gene coding sequences. All constructs were confirmed by
DNA sequencing. Plasmids were transformed into
Escherichia coli DH10b by electroporation and DNA was
A.A. Elling et al. / International Journal for Parasitology 37 (2007) 1269–1279
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Table 1
Overview of Heterodera glycines parasitism protein characteristics
Protein
Accession
no.
Best BLASTP matcha
4E02
5D06
5D08
6E07
8H07
AAO33473.1
AAN32891.1
AAO33475.1
AAO33476.1
AAP30763.1b
Novel
VmcA lipoprotein (Mycoplasma capricolum)
Novel
Novel
S phase kinase-associated protein 1A (Danio rerio)
10A06
AAP30834.1
SKP1-like protein ASK10 (Arabidopsis thaliana)
S phase kinase-associated protein 1A (Ictalurus
punctatus)
RING-H2 zinc finger protein-like (Oryza sativa)
10A07
13A06
AAP30760.1
AAP30759.1
Novel
Novel
a
b
BLASTP
score/evalue
52.4/4e-05
94.7/6e-18
Length
(amino
acids)
Signal
peptide
InterProScan match
92
489
136
214
398
1-27
1-19
1-18
1-23
1-17
No
No
No
No
IPR001232 (SKP1
components)
308
1-17
278
222
1-15
1-17
IPR001841 (Zinc finger,
RING)
IPR005819 (Histone H5)
No
94.7/6e-18
94.7/6e-18
53.9/8e-06
Best descriptor other than H. glycines secretory protein hits.
Gene had multiple BLASTP hits with same score and e-value.
Table 2
Primers used to generate mutated and truncated forms of Heterodera glycines genes
Primer
Sequence
4E02-5 0
4E02-mut-3 0
6E07-5 0
6E07mut-1
6E07mut-2
6E07-3 0
5D06R-5 0
5D06R-3 0
5D08R-5 0
5D08R-3 0
8H07R-5 0
8H07R-3 0
10A06R-5 0
10A06R-3 0
10A07R-5 0
10A07R-3 0
13A06R-5 0
13A06R-3 0
5 0 -CCC AAG CTT ACA TGG AAG AGG GAG GGC GAG TGA AGC-3 0
5 0 -CAT GTC GAC ATA TGT TTG GGC GCC GCC CCG CAA CAT GCC CAC ACG TAA TTT TTG TCG CAA C-3 0
5 0 -CCC AAG CTT ACA TGT CAA AAG TAG TCA AAA AAG ACA ATA AA-3 0
5 0 -GCC GAT TTA CCT TTT TTT GTT GGC GCT GCA TTT GCA ACT GCA ATG CCT TTG GTT TC-3 0
5 0 -GAA ACC AAA GGC ATT GCA GTT GCA AAT GCA GCG CCA ACA AAA AAA GGT AAA TCG GC-3 0
5 0 -CAT GTC GAC ATT TGC CCC GAC TCT CCT CTC TCA TA-3 0
5 0 -CCC AAG CTT ACA TGC CAA TAA TTG AAA AAT ATG TTG ATG AA-3 0
5 0 -CAT GTC GAC ATG ATC TTC ACC GTT TGA TTA GGA TTT TTC-3 0
5 0 -CCC AAG CTT ACA TGA AAG CGC CCT CTG GCG AAA GT-3 0
5 0 -CAT GTC GAC ATC CAT CCT CCG ACG TAT CCG C-3 0
5 0 -CCC AAG CTT ACA TGA GCG ATT TTG GCC TAA ACT TAG C-3 0
5 0 -CAT GTC GAC ATA GCT GTG TTC ATA ACG CTT ATT-3 0
5 0 -CCC AAG CTT ACA TGA AGT TGA AAA GCG ATT TTG GCC TA-3 0
5 0 -CAT GTC GAC ATA GCT TCA GAT GCC GAG TCC T-3 0
5 0 -CCC AAG CTT ACA TGG CAT CGC CAA AAG GAG GCA-3 0
5 0 -CAT GTC GAC ATT GCG AGT TTT TTG ACT GTC TTA GGC-3 0
5 0 -CCC AAG CTT ACA TGG TTA GTA AAA AAG ATA ACA AAT TGA AA-3 0
5 0 -CAT GTC GAC ATG ACT TTC TTG GCT GTT TTT G-3 0
recovered using a QiaFilter maxiprep kit (Qiagen, Valencia, CA).
2.2. Expression in onion epidermal cells
The inner epidermal layers of white onions were peeled
off and placed on modified Murashige and Skoog (MS)
media [(per L: 4.3 g MS salt, 10 mg myoinositol, 180 mg
KH2PO4, 30 g sucrose, 2.5 mg amphotericin, pH 5.7,
0.6% agar) (Varagona et al., 1992; modified)]. Gold particles (1.6 lm, 1.5 mg) (Biorad, Carlsbad, CA) were coated
with 3 lg plasmid DNA using standard procedures. Onion
cells were bombarded at 1100 psi and 8 cm distance using a
Biolistic Particle Delivery System PDS-1000/He (E.I. du
Pont de Nemours & Co., Wilmington, DE) and incubated
at 25 C in darkness for about 24 h. Tissue was stained for
GUS activity [per 50 ml: 5 mg X-Gluc salt (RPI, Mt. Pros-
pect, IL), 10 ml dimethyl sulfoxide (DMSO), 5 ml KPO4
pH 7.0, 2 drops Triton X-100] for 3–4 h at 37 C. All transformations were performed at least four times
independently.
2.3. Expression in Arabidopsis protoplasts
Arabidopsis suspension cells were maintained at room
temperature in 250 ml flasks on an orbital shaker at
125 rpm in modified Linsmaier and Skoog (LS) media
[per L: 4.3 g LS salts (Caisson Laboratories, Rexburg,
ID), 20 g sucrose, 50 ll kinetin (1 mg/ml stock), 1 ml 1naphthaleneacetic acid (NAA) (1 mg/ml stock), 590 mg 2(N-morpholino)ethanesulfonic acid (MES), pH 5.7] and
subcultured weekly. Five-day-old cells were harvested by
centrifugation (20g) to generate and transform protoplasts
(Sheen, J., 2002. A transient expression assay using
1272
A.A. Elling et al. / International Journal for Parasitology 37 (2007) 1269–1279
Arabidopsis mesophyll protoplasts. http://genetics.mgh.
harvard.edu/sheenweb/). In brief, suspension cells were
digested for about 3.5 h in 40 ml enzyme solution [0.125 g
Onozuka RS cellulose (Yakult Honsha, Tokyo, Japan),
0.063 g macerozyme R-10 (Yakult Honsha, Tokyo, Japan),
20 ml artificial salt water (ASW) pH 6.0 (311 mM NaCl,
18.8 mM MgSO4, 6.8 mM CaCl2, 10 mM MES, 6.9 mM
KCl), 20 ml of 0.6 M mannitol] in darkness at room temperature at about 40 rpm. The suspension was passed
through a 75 lm cell strainer and cells were collected by
centrifugation for 5 min at 20g. Protoplasts were washed
twice in 10 ml W5 (0.4 M mannitol, 70 mM CaCl2, 5 mM
MES pH 5.7) and collected by centrifugation for 5 min at
20g at 4 C. After the second wash, cells were resuspended
in 2 ml chilled MMg (0.4 M mannitol, 15 mM MgCl2,
5 mM MES, pH 5.7). One hundred microlitres of protoplasts were gently mixed with 30 lg plasmid DNA, 20 lg salmon sperm carrier DNA and 400 ll polyethylene glycol
(PEG) solution [40% (w/v) PEG 4000, 0.4 M mannitol,
1 M CaCl2] and incubated on ice for 20 min. Cells were
transferred to 5 ml W5 0 (0.4 M mannitol, 125 mM CaCl2,
5 mM KCl, 5 mM glucose, 1.5 mM MES, pH 5.7) and centrifuged at 20g for about 10 min. After removal of the
supernatant, the protoplasts were gently resuspended in
1.5 ml modified LS media [per L: 4.3 g LS salts (Caisson
Laboratories, Rexburg, ID), 20 g sucrose, 50 ll kinetin
(1 mg/ml stock), 1 ml NAA (1 mg/ml stock), 590 mg
MES, 0.4 M mannitol, pH 5.7] and incubated in darkness
at room temperature for 16–24 h on an orbital shaker
(40 rpm). All transformations were performed at least four
times independently.
2.4. Microscopy
Onion and Arabidopsis cells were observed for GUS or
GFP activity using a Zeiss Axiovert 100 microscope (Zeiss,
Jena, Germany). GFP expression was monitored with a
Piston GFP filter set (Chroma, Rockingham, VT). Pictures
were taken at 20· (onion) or 63· (Arabidopsis) with a Zeiss
Axiocam MRc5 digital camera and processed with Zeiss
Axiovision software (Zeiss, Jena, Germany) and Adobe
Photoshop.
3. Results
3.1. Sequence analysis of parasitism proteins with NLS
domains
Similarity searches to known proteins using the
BLASTP algorithm (Altschul et al., 1990) found strong
similarities for only parasitism genes 8H07 and 10A06
(Table 1). The predicted 8H07 gene product matches S
phase kinase-associated protein 1 (SKP1) proteins of various species and the predicted 10A06 protein shows similarity to a RING-H2 zinc finger protein from rice. BLASTP
searches revealed a somewhat weaker match for 5D06 to
a variable Mycoplasma capricolum subspecies capricolum
protein A (VmcA) lipoprotein. To complement these
BLAST search results, we used InterProScan (Zdobnov
and Apweiler, 2001) analyses, which detected known protein domains for 8H07, 10A06 and 10A07. InterProScan
identified multiple motifs for SKP1 components in 8H07,
and zinc finger and RING domains in 10A06, which confirmed the BLASTP matches for these proteins. Even
though 10A07 did not have any significant BLASTP hits,
the InterProScan analysis identified histone H5 domains
in this protein.
Amino acid sequence alignments revealed that 6E07 and
13A06 are 94% identical, but, as shown in Fig. 1, show
strong differences in their N- and C-termini. Similarly,
8H07 and 10A06 amino acid sequences are near identical
for about the first fourth of the sequence while the remaining sequences of these proteins are very different. Furthermore, the putative NLS domains of 6E07 and 13A06 are
identical, but are different between 8H07 and 10A06
(Fig. 1).
PSORT II (Nakai and Horton, 1999) was used to determine the location of the putative NLS motifs within the
protein sequence and to classify the NLS motifs into
SV40 and bipartite-like groups (Table 3). These analyses
were conducted using the parasitism protein sequences
without the predicted N-terminal signal peptide sequence
since signal peptides are cleaved off in the endoplasmic
reticulum (ER) of the secretory cell and only the cleaved
protein actually is secreted. 5D06, 6E07, 10A07 and
13A06 contain putative bipartite NLS domains as well as
SV40-like NLS motifs that are predicted to overlap with
the bipartite domains. 4E02, 5D08, 8H07 and 10A06 are
predicted to have only SV40-like NLSs (Table 3). According to PSORT II, all proteins had the highest probability to
be localised in the nucleus compared with other possible
subcellular destinations. To obtain more robust predictions
about subcellular localisation we complemented the
PSORT II analyses by WoLF PSORT (Horton, P., Park,
K.-J., Obayashi, T., Nakai, K. 2006. Protein subcellular
localization prediction with WoLF PSORT. Proc. Asian
Pacific Bioinformatics Conf., pp. 39–48, Taipeh) for all
eight proteins under study without their signal peptides.
Using a plant setting in the WoLF PSORT software,
6E07, 10A06, 10A07 and 13A06 were predicted to accumulate most likely in nuclei while 5D08 was predicted to be
imported with equal likelihood into nuclei or mitochondria. The remaining proteins were predicted by WoLF
PSORT to accumulate in other compartments (Table 3).
3.2. Localisation of fusion proteins in plant cells
To test the predicted nuclear localisation of these eight
H. glycines parasitism proteins, we created double CaMV
35S promoter-driven gene cassettes that translationally
fused parasitism gene cDNA sequences minus signal peptide-coding sequences with the GFP and GUS reporter
protein coding sequences (Grebenok et al., 1997). These
constructs were expressed in onion epidermal cells and
A.A. Elling et al. / International Journal for Parasitology 37 (2007) 1269–1279
1273
Fig. 1. Sequence alignment of Heterodera glycines parasitism proteins 6E07 and 13A06 (A) and 8H07 and 10A06 (B), respectively. Conserved residues are
shown in grey. Signal peptides are underlined and nuclear localisation signal regions are bold.
Arabidopsis protoplasts as described in Materials and
methods. All experiments also contained cytoplasmic and
nuclear control gene constructs (Grebenok et al., 1997) as
reference treatments (data not shown), which allowed
unequivocal recognition of nuclear and nucleolar protein
accumulation in these experiments.
Even though all eight parasitism proteins studied here
were predicted to have NLS motifs, only reporter fusions
of the full-length proteins 4E02 and 6E07 (minus signal
peptide-coding sequence) showed nuclear localisation in
onion and Arabidopsis cells. The 4E02 fusion product accumulated strongly in the nucleus and to a considerably lesser
degree in the cytoplasm of both plant species used here
(Fig. 2). Similarly, the 6E07 fusion product showed strong
nuclear localisation and weak cytoplasmic accumulation.
In addition, 6E07 displayed strong nucleolar localisation
in Arabidopsis protoplasts with a weaker accumulation in
the remaining regions of the nucleus (Fig. 2). Reporter
fusions of the full-length proteins 5D06, 5D08, 8H07,
10A06 and 10A07 (minus signal peptide) accumulated only
in the cytoplasm when expressed in both onion epidermal
cells and Arabidopsis protoplasts (Fig. 2). Interestingly,
for 5D06 we only saw very few transformed cells compared
with the controls and the other constructs. During our
analyses of parasitism gene 13A06 we could not detect
any reporter gene activity in either plant species even after
multiple transformation events using different independent
gene constructs that all were verified by nucleotide
sequencing.
To test whether the predicted NLS motifs of the 4E02
and 6E07 proteins were in fact responsible for the observed
nuclear uptake, we exchanged selected lysine residues of
these NLS motifs with alanine residues by PCR-directed
mutagenesis. In these efforts we converted the SV40-like
NLS of the 4E02 product from 88KKPK91 to 88AAPK91.
Similarly, we converted the bipartite NLS motif of the
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A.A. Elling et al. / International Journal for Parasitology 37 (2007) 1269–1279
Table 3
Predicted nuclear localisation signal (NLS) domains and localisation of Heterodera glycines parasitism proteins
Protein Predicted NLSa
4E02
88
KKPK91
5D06
40
43
NLS type PSORT IIb
SV40
WoLF PSORTc
Nuclear, 52.2% 10 mitochondria 4 nucleus
RKKP
SV40
KKSRQLFAECMQKILRK421 Bipartite
Nuclear, 69.6% 10 cytoplasm
4 nucleus
RRKR83
PSGERRK82
SV40
SV40
Nuclear, 34.8% 4 nucleus
4 mitochondria 3 extracellular 2 chloroplasts
PTKKGKS89
PGKDKKS113
70
KKETKGIKVKNAKPTKK86
SV40
SV40
Bipartite
Nuclear, 69.6% 13 nucleus
1 mitochondria
94
PVPKGRR100
PKGRRGK102
SV40
SV40
Nuclear, 69.6% 7 cytoplasm
5 nucleus
PVPKGKK100
PKGKKVE102
SV40
SV40
Nuclear, 82.6% 13 nucleus
1 cytoskeleton
KPKK65
PAKKGKA82
59
KKLKPKKDAKGIKAKKA75
64
KKDAKGIKAKKAKPAKK80
SV40
SV40
Bipartite
Bipartite
Nuclear, 78.3% 12 nucleus
2 mitochondria
77
SV40
SV40
Bipartite
Nuclear, 65.2% 14 nucleus
405
5D08
80
76
6E07
83
107
8H07
96
10A06
94
96
10A07
62
76
13A06
PTKKGKS83
PGKDKKS107
64
KKETKGIKVKNAKPTKK80
101
a
b
c
1 cytoplasm
1 chloroplasts 1 mitochondria
NLS domain as predicted by PSORT II.
PSORT II prediction with likelihood of subcellular localisation.
Number of nearest neighbors for subcellular compartments as predicted by WoLF PSORT.
6E07 product from 70KKETKGIKVKNAKPTKK86 to
70
KKETKGIAVANAAPTKK86.
These
mutations
(4E02M, 6E07M) prevented transport of the respective
proteins into plant nuclei (Fig. 3), which confirmed the
veracity of the NLS prediction.
To analyse those parasitism proteins that were not
imported into nuclei (i.e., 5D06, 5D08, 8H07, 10A06,
10A07) or that were not detected at all (13A06), we tested
whether shorter protein fragments containing the predicted
NLSs are imported into nuclei. For this purpose, we
expressed 105 to 165 nucleotides of coding sequences that
included the predicted NLS to generate appropriate fusion
products (labeled ‘‘R’’, preceded by the gene name) as
described in Materials and methods and conducted the
plant transformation assays as before (Fig. 3). We found
that 5D06R, 5D08R and 8H07R did not show a different
localisation pattern than their longer forms and remained
localised to the cytoplasm. However, 10A06R showed
strong reporter gene activity in the nucleus with some
minor accumulation in the cytoplasm in both onion and
Arabidopsis cells. This observation is a strikingly different
localisation result than was obtained with the full-length
10A06, which could only be detected in the cytoplasm.
Most interestingly, while full-length 10A07 protein fusions
were restricted to the cytoplasm and 13A06 protein fusions
did not show any detectable reporter protein activity, both
10A07R and 13A06R products localised strongly to the
nucleus in onion epidermal cells when stained for GUS
activity and led to strong fluorescence of the nucleolus
and a weaker signal in the remaining regions of the nucleus
when GFP-fusions were observed in Arabidopsis protoplasts. Both fusion proteins remained largely excluded from
the cytoplasm.
In summary, we have shown here that out of eight
H. glycines parasitism proteins with predicted NLSs, two
(4E02, 6E07) are imported into plant nuclei when expressed
as full-length proteins without signal peptides and that
6E07 fusion products further target the nucleolus upon
nuclear import in planta. Second, mutation analyses confirmed the prediction of NLSs for 4E02 and 6E07. Third,
shorter protein regions that included the NLS motifs led
to nuclear import of three additional parasitism proteins
(10A06R, 10A07R, 13A06R), with 10A07R and 13A06R
fusion products accumulating primarily in the nucleolus.
Other than 10A06, which is similar to a rice RING-H2 zinc
finger protein, none of the other four parasitism proteins
that are actively imported into plant nuclei either as fulllength protein or as truncated variant show similarities to
any proteins known to date.
4. Discussion
In this project we performed experiments to test the
PSORT II predictions that eight parasitism proteins of
the soybean cyst nematode contained NLSs for import into
host cell nuclei. For this purpose, translational fusions were
created between the respective parasitism gene cDNAs
without the signal peptide-encoding region and the GFP
and GUS genes. These constructs were expressed in onion
epidermal cells as well as in Arabidopsis protoplasts, two
A.A. Elling et al. / International Journal for Parasitology 37 (2007) 1269–1279
1275
Fig. 2. Transient expression of Heterodera glycines parasitism protein genes without signal peptide-coding sequence fused to GFP and GUS reporter
proteins. Histochemical staining for GUS activity in onion cells (A). Fluorescence microscopy for Arabidopsis protoplasts showing GFP (B), brightfield
(C) and overlay of GFP and brightfield photographs (D). Scale bars: 100 lm (black) for panel A and 25 lm (white) for panels B, C, D.
experimental systems that have been used successfully for
characterisation of nuclear import mechanisms in the past
(Varagona et al., 1992; Hwang and Sheen, 2001; Tzfira and
Citovsky, 2001) and that allow the easy recognition of
nuclei and nucleoli using conventional light and fluorescence microscopy. The use of fusions to a tandem construct
of GFP and GUS has been proven useful to prevent passive diffusion of low molecular weight proteins into the
nucleus (Grebenok et al., 1997). Furthermore, this
approach gives more flexibility in the choice of detection
assays in cases where one of the reporter proteins is less
readily detected in planta.
Signal peptide-containing proteins are imported cotranslationally into the ER, which is the starting point of
the secretory pathway and also the site where cleavage of
the signal peptide occurs. Since all eight parasitism proteins
under study by definition encompass a predicted signal
peptide in addition to a NLS, the NLS cannot function
inside the nematode gland cell but has to play its role inside
plant cells once the protein has been secreted by the nematode into the host cell cytoplasm. Because of this mechanism, we expressed all parasitism genes without their
respective signal peptide-encoding sequences in the nuclear
localisation assays.
The products of the 4E02 and 6E07 parasitism genes,
when expressed as full-length proteins, were clearly
imported into the nucleus. Interestingly, 6E07 was further
targeted to the nucleolus in Arabidopsis protoplasts. In
our experiments, very few onion cells expressed GFP. Consequently, we stained for GUS activity in onion cells and
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A.A. Elling et al. / International Journal for Parasitology 37 (2007) 1269–1279
Fig. 3. Transient expression of mutated (M) and partial (R) Heterodera glycines parasitism protein genes without signal peptide-coding sequence fused to
GFP and GUS reporter proteins. Histochemical staining for GUS activity in onion cells (A). Fluorescence microscopy for Arabidopsis protoplasts showing
GFP (B), brightfield (C) and overlay of GFP and brightfield photographs (D). Scale bars: 100 lm (black) for panel A and 25 lm (white) for panels
B, C, D.
relied on fluorescent microscopy to detect GFP in Arabidopsis protoplasts. This means that the seeming disparity
between nuclear and nucleolar localisation in onion and
Arabidopsis cells for 6E07 can be explained by diffusion
of the GUS stain from the nucleoli into the remaining
regions of the nucleus. The nucleolus is not enclosed by a
membrane, but rather an open and dynamic structure consisting mostly of protein components and rRNA (Raška
et al., 2006). Therefore, the nucleolus is unable to retain
stains like the product of GUS reactions which does not
specifically bind to nucleolar components. GFP fluorescence, however, is an intrinsic property of the GFP protein
which does not diffuse in our Arabidopsis assays. Since
there is no known nucleolus localisation signal and the
nucleolus is not compartmentalised by a membrane but is
a dynamic and open structure, it is assumed that nucleolar
proteins are targeted to their destination by interacting
with other macromolecules that are already present in the
nucleolus (Raška et al., 2006) and that they are retained
by a recently discovered GTP-driven cycle (Tsai and
McKay, 2005). Neither parasitism protein 4E02 nor 6E07
had any significant BLAST matches, so that their putative
functions remain elusive.
Kemen et al. (2005) found that the NLS-containing Rust
Transferred Protein 1 (RTP1p) from the plant-pathogenic
fungus Uromyces fabae rarely displayed reporter protein
activity when fused to GFP and expressed in plant cells.
However, when using a truncated form of RTP1p that
A.A. Elling et al. / International Journal for Parasitology 37 (2007) 1269–1279
included the predicted NLS motif, strong nuclear GFP
fluorescence was obtained. Since we were only able to
observe very few plant cells that displayed GUS or GFP
activity for the fusion product of 5D06 and could not find
any reporter gene activity for the product of 13A06, we
performed similar experiments to determine whether a
shorter parasitism gene fragment containing the predicted
NLS domain would result in nuclear import for the products of the 5D06 and 13A06 genes. Similarly, the four genes
that produced only cytoplasmic accumulation (5D08,
8H07, 10A06, 10A07) also were subjected to such assays.
The truncated NLS-containing parasitism protein versions
10A06R, 10A07R and 13A06R were imported into nuclei
in both plant cell types and 10A07R and 13A06R were further targeted to the nucleolus in Arabidopsis cells while
their full-length protein counterparts remained in the cytoplasm (10A06, 10A07) or did not show any detectable
reporter protein activity at all (13A06). Again, the discrepancy between translocation into the nucleoli in Arabidopsis
and nuclear import in onion cells for 10A07R and 13A06R
can be explained by the fact that we relied on GFP detection in Arabidopsis protoplasts and used a histochemical
GUS stain in onion cells as explained above.
The cause for differential accumulation of the truncated
proteins 10A06R, 10A07R and 13A06R and the failure of
nuclear import for both the full-length and truncated versions of 5D06, 5D08 and 8H07 is unresolved. It is possible
that the PSORT II predictions about putative NLS
domains and subcellular localisation for these proteins
are erratic. WoLF PSORT predicts that 5D08 is equally
likely to be imported into nuclei and mitochondria, while
it predicts 5D06 and 8H07 to be retained in the cytoplasm.
On the other hand, 4E02, which we showed to be clearly
imported into nuclei of both plant species tested here,
was strongly predicted to be imported into mitochondria
rather than nuclei by WoLF PSORT. While we have demonstrated that shorter H. glycines parasitism protein
regions that include the putative NLS domain are imported
into plant nuclei we can only speculate whether nuclear
uptake events of these proteins also take place during the
natural infection process. Furthermore, it is possible that
N-terminal fusions of the H. glycines proteins to GFP
and GUS as opposed to the C-terminal fusions created here
would result in a protein conformation that allows nuclear
uptake of the full-length proteins.
Even though parasitism proteins 5D06, 5D08 and 8H07
were not imported into nuclei in our studies and 10A06,
10A07 and 13A06 only as truncated versions, they might
still play an important role as cytoplasmic effectors in a
nematode-infected plant cell. Interestingly, H. glycines possesses signal peptide-bearing SKP1 (8H07) and RING
(10A06) variants that have more similarity with plant proteins than with homologues in the fully sequenced Caenorhabditis elegans and Caenorhabditis briggsae genomes.
Similarly, 5D06, with a weak similarity to a mycoplasma
VmcA lipoprotein that is thought to generate surface variation essential for host adaptations (Wise et al., 2006),
1277
might play an equally important role in the cytoplasm of
plant cells parasitised by the soybean cyst nematode. Further studies are needed to identify a potential role during
the host–parasite interaction for these proteins.
Although alignments revealed 94% identity for the
amino acid sequences of 6E07 and 13A06, we observed that
only 6E07 was translocated to nuclei (and nucleoli) and
that 13A06 did not show any reporter protein activity when
expressed as full-length protein. A truncated 13A06 version, however, yielded the same localisation pattern as
the full-length 6E07 protein. This is interesting as it possibly suggests that the sequence differences in 13A06 lead to
its rapid degradation in planta. Similarly, the first fourth of
the amino acid sequences of 8H07 and 10A06 is almost
identical while the remaining parts of the proteins are very
different. Since we could not find any known domains for
the identical regions of 8H07 and 10A06, we can only speculate as to the functions of this part of the proteins.
Few studies have been done on nuclear import of nematode secretory proteins. In addition to our comprehensive
analysis of NLS-containing H. glycines parasitism proteins
to date, a Heterodera schachtii ubiquitin extension protein
was reported to be imported into plant nucleoli (Tytgat
et al., 2004) and weak nuclear accumulation was obtained
for two Meloidogyne incognita 14-3-3 protein homologues
(Jaubert et al., 2004). Furthermore, secreted antigens of
the animal-parasitic nematode T. spiralis were detected in
muscle cell nuclei and are believed to be involved in cell
cycle changes in infected cells (Vassilatis et al., 1992; Yao
and Jasmer, 1997; Jasmer et al., 2003).
On the other hand, nuclear uptake of NLS-containing
proteins in other plant–pathogen systems is well-studied,
e.g., the Xanthomonas campestris pathovar vesicatoria
avirulence protein AvrBs3 (Van den Ackerveken et al.,
1996; Szurek et al., 2001), the VirE2 protein (and Transfer
(T) DNA) of Agrobacterium (Gelvin, 2000; Citovsky et al.,
2004) as well as nucleic acid and protein components of a
wide variety of viruses (Whittaker and Helenius, 1996)
are all translocated into plant nuclei. However, none of
these proteins have any similarity to H. glycines parasitism
proteins with NLSs.
In summary, we could observe active nuclear uptake of
two full-length (4E02, 6E07) H. glycines parasitism proteins and of three (10A06R, 10A07R, 13A06R) truncated
proteins in both onion and Arabidopsis. Three of these proteins (6E07, 10A07R, 13A06R) were further targeted to the
nucleolus. The demonstration here of active NLS domains
in parasitism proteins secreted by H. glycines into host
plant cells suggests the potential for direct regulatory activity of effector molecules within the host nucleus to promote
successful parasitism.
Acknowledgements
This is a Journal Paper of the Iowa Agriculture and
Home Economics Station, Ames, Iowa, Project No. 3608,
and supported by Hatch Act and State of Iowa funds. This
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A.A. Elling et al. / International Journal for Parasitology 37 (2007) 1269–1279
study was funded by a grant from the United Soybean
Board to T.J.Baum, E.L.Davis and R.S.Hussey. A.A.Elling was in part supported by a Storkan-Hanes-McCaslin
Research Foundation fellowship. We are grateful to David
Galbraith for the kind gift of plasmids pRJG23 and
pRJG32 and to Daniel Voytas for an Arabidopsis suspension cell line. We also thank Tom Maier for technical
support.
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