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International Journal for Parasitology 37 (2007) 1269–1279 www.elsevier.com/locate/ijpara 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 1270 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 1271 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 1274 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 1276 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 1278 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. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. 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