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Keywords:

  • watermould;
  • infection;
  • protein translocation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

The fish pathogenic oomycete Saprolegnia parasitica causes the disease Saprolegniosis in salmonids and other freshwater fish, resulting in considerable economic losses in aquaculture. Very little is known about the molecular and cellular mechanisms underlying the infection process of fish pathogenic oomycetes. In order to investigate the interaction in detail, an in vitro infection assay using an Oncorhynchus mykiss (rainbow trout) cell line (RTG-2) was developed. In a zoospore/cyst cDNA library, we identified the ORF SpHtp1, which encodes a secreted protein containing an RxLR motif. Detailed expression analysis indicated that SpHtp1 is highly expressed in zoospores/cysts from S. parasitica and in the very early stages of infection on RTG-2 cells, when compared with in vitro-grown mycelium. Moreover, the protein, SpHtp1, was found to translocate into the RTG-2 trout cells, during the interaction with S. parasitica, and also when the RTG-2 cells were treated with recombinant SpHtp1 fused to a C-terminal His-tag. These findings suggest that protein translocation could play an important role in Saprolegniosis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Oomycetes contain some of the most devastating pathogens of animals and plants, causing major economic and environmental damage in natural and cultured ecosystems (Kamoun, 2003; van West, 2006; Phillips et al., 2008). One destructive oomycete pathogen of fish is Saprolegnia parasitica. It is endemic to all freshwater habitats around the world, is partly responsible for the decline in natural populations of salmonids and is a serious problem in the aquaculture industry (van West, 2006; Bruno et al., 2010). The disease symptoms include white or grey patches of filamentous mycelium on the body or the fins of freshwater fish. The cellular and molecular mechanisms underlying Saprolegnia infection have not been studied extensively (Kamoun, 2003). Instead, considerably more is known about how plant pathogenic oomycetes infect their hosts. Most oomycetes generate asexual zoospores for dispersal, which encyst and germinate when they have reached a potential host. Saprolegnia parasitica is also able to generate both primary and secondary zoospores, whereby the latter type is infectious (Phillips et al., 2008; Bruno et al., 2010). Upon finding a host, some oomycetes form a swelling at the tip of a germ tube, called an appressorium, which forms a penetration peg to enter the host cell (Grenville-Briggs et al., 2008). These appressoria-like structures have not been described so far for S. parasitica. Biotrophic and hemibiotrophic plant pathogenic oomycetes can also generate specialized hyphal branches called haustoria. These are structures that invaginate the plant cell and induce the formation of a plant-derived extrahaustorial membrane with a gel-like layer between the extrahaustorial membrane and the haustorial wall, called the extrahaustorial matrix (Bushnell, 1972; Szabo & Bushnell, 2001). Within this extrahaustorial matrix, water and nutrients are exchanged between the pathogen and the host (Voegele & Mendgen, 2003). The extracellular space is also considered important for the trafficking of secreted proteins from the pathogen, including effector proteins (Ellis et al., 2006). Effector proteins are required to establish a successful infection, but if recognized, they can also trigger a host resistance response (Birch et al., 2006, 2009; Jones & Dangl, 2006; Hogenhout et al., 2009).

Some plant and animal pathogens have evolved intriguing molecular mechanisms to inject or translocate potential effector proteins into their host cells (Coombes et al., 2004; Navarro et al., 2005; Birch et al., 2006; Jones & Dangl, 2006; Whisson et al., 2007). For example, bacterial pathogens can inject effector proteins into the host cytosol using a type-III secretion system, where these effectors can suppress basal/innate immunity, inhibit inflammatory responses, inhibit phagocytosis and induce apoptosis in macrophages (Hueck, 1998; Navarro et al., 2005; Galán & Wolf-Watz, 2006; Lewis et al., 2009). The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, is also able to translocate secreted proteins that contain a so-called PEXEL motif [amino acid motif RxLx (E, Q or D)] into the cytosol of red blood cells (Hiller et al., 2004; Marti et al., 2004; Hiss et al., 2008). During the blood stages of infection, the parasite invades mature human erythrocytes and develops within a parasitophorous vacuolar membrane. The PEXEL proteins synthesized by the parasite induce antigenic and structural changes in the cytosol and membranes of the host cell, which ultimately lead to many disease pathologies that result in death (Bhattacharjee et al., 2006).

Plant pathogenic oomycetes appear to have evolved a protein translocation system similar to malaria, which involves secreted proteins possessing an RxLR motif located after the signal peptide sequence (Bhattacharjee et al., 2006; Birch et al., 2006; Haldar et al., 2006; Whisson et al., 2007; Dou et al., 2008b). It was found that the RxLR motif is required for translocating these proteins into the host cells of infected plants (Whisson et al., 2007; Dou et al., 2008a). Bioinformatic analysis has shown that over 500 putative RxLR effectors are found in the potato pathogenic oomycete Phytophthora infestans, and similarly, hundreds more in other plant pathogenic oomycetes (Whisson et al., 2007; Haas et al., 2009; Tyler, 2009). It was demonstrated that the oomycete RxLR motif is functional in Plasmodium, where it can direct an RxLR–GFP fusion protein from the parasite into the host erythrocyte (Bhattacharjee et al., 2006). The PEXEL motif is also functional in P. infestans as it is able to translocate an avirulent chimaeric PEXEL-PiAvr3 protein into PiAvr3-recognizing potato plants (Grouffaud et al., 2008). Replacement of the N-terminal region of the effector protein PsAvr1b with a PEXEL motif containing leader sequences of three Plasmodium effectors resulted in the translocation of chimaeric PsAvr1b into the soybean cytoplasm (Dou et al., 2008a).

Before detailed molecular interaction studies between Saprolegnia and fish can be performed, it is essential to develop a suitable infection model. The ami-momi treatment established, which involves shaking fish in a net for approximately 2 min to remove part of the mucosal layer and subsequently challenging with Saprolegnia zoospores (Hatai & Hoshiai, 1993), is a good method to characterize the virulence of S. parasitica strains (Stueland et al., 2005). However, it is not a suitable model to study the early cellular and molecular infection mechanisms and events. In order to study these in more detail, the development of a fish cell-line infection assay is necessary. Here, we describe the identification and molecular characterization of a putative effector protein, SpHtp1, containing an RxLR motif. Microscopic studies of a Saprolegnia–fish interaction using an in vitro system involving a rainbow trout cell line showed that SpHtp1 is translocated into the fish cells, also when applied exogenously.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Culture conditions

A Saprolegnia parasitica isolate CBS223.65, obtained from the Centraal Bureau voor Schimmelcultures (CBS), the Netherlands, was grown on potato dextrose agar (Fluka) for 5 days at 18 °C, before inoculation in pea broth (125 g L−1 frozen peas, autoclaved, filtered through cheese cloth, volume adjusted to 1 L and autoclaved again) and incubation for 2 days at 24 °C. To accomplish S. parasitica sporulation, the mycelium was washed three times in sterile tap water and placed in a sterile 50 : 50 solution of demineralized water and aquarium tank water, obtained from regular fresh water aquaria. After an overnight incubation, zoospores and cysts were collected. Germinating cysts were collected after vortexing the zoospore/cyst suspension and incubation at 24 °C for 4–5 h.

Maintenance of the RTG-2 cell line

The RTG-2 cell line is a continuous cell line obtained from ATCC (ATCC CCL-55). It was derived from rainbow trout (Oncorhynchus mykiss) gonadal tissue (Wolf & Quimby, 1962) and was maintained at 24 °C in 75-cm2 cell culture flasks (Nunc) in 25 mL Leibovitz's L-15 medium (Gibco) supplemented with 10% foetal bovine serum (BioSera), 200 U mL−1 penicillin and 200 μg mL−1 streptomycin (Fisher). Flasks with confluent cell growth were inoculated weekly after splitting cells by washing three times with Hank's balanced salt solution (Gibco) at room temperature and treating the cells with 5 mL 0.5 g L−1 trypsin–EDTA (Invitrogen) until the cells were detached from the flasks. A fresh medium was added, and after gentle shaking, the cells were distributed into three to five flasks, each containing approximately 30 mL of cell suspension, or 2–4 mL was added to each well of six-well plates (Nunc), where the wells contained an autoclaved glass coverslip.

Generation and bioinformatic analysis of the expressed sequence tag (EST) library

RNA was isolated from the preinfection stages of S. parasitica strain CBS223.65, including zoospores, cysts and germinating cysts, at Vertis Biotechnology AG (Germany), using a Trizol-based extraction. From total RNA, polyA+ was prepared and cDNA was synthesized according to the Vertis Biotechnology AG standard protocol for full-length enriched cDNA using an oligo(dT)-NotI primer for first-strand synthesis. Before cloning, the cDNA was amplified with 13 cycles of PCR. For directional cloning, cDNA was subjected to a limited exonuclease treatment to generate EcoRI overhangs at the 5′ end, and was subsequently digested with NotI. Size-fractioned cDNA fractions >0.5 kb were ligated into EcoRI and NotI digested pcDNA3.1 (Invitrogen) and subsequently transformed via electroporation into T1 phage-resistant TransforMax™ EC100™-T1R electrocompetent cells (Epicentre Biotechnologies). The transformants were stored in 15% v/v glycerol at −80 °C. End-sequencing was performed on plasmid DNA isolated from 1000 clones of the cDNA library by a single pass sequence from the 5′ end with a primer specific for the pcDNA3.1 vector by GATC Biotech (Cambridge, UK) using an ABI3730 system. The EST sequences were trimmed to remove vector sequence and validated using seqclean (http://compbio.dfci.harvard.edu/tgi/software/), and subsequently, contigs were assembled using cap3 (http://pbil.univ-lyon1.fr/cap3.php). Screening for secreted proteins was performed by signalp (http://www.cbs.dtu.dk/services/SignalP/) analysis using both hidden markov models and neural networks programs, and subsequently, the sequences were screened by word searches for the presence of an RxLR motif. blastp analyses were performed at the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and the search for specific domains was performed using interproscan (http://www.ebi.ac.uk/Tools/InterProScan/). Alignments were generated using clustalx 1.81 or clustalw (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The sequence of the SpHtp1 has been deposited in GenBank under accession number GU345745.

Time-course experiment and gene expression analysis

RTG-2 cells were grown as a confluent monolayer in 75 cm2 in cell culture flasks (Nunc) and challenged with 5 × 104 zoospore/cysts at 24 °C. At several time points, media were discarded, except for time point 0, and 5 mL of Qiazol (Qiagen) or Trizol reagent (Invitrogen) was added to each flask. Cells were scraped loose with a cell scraper (Fisher) and the suspension was aliquoted as 1 mL portions into 2-mL screw-cap tubes containing 10–35 glass beads of 1 mm diameter (Biospec). Samples were frozen immediately in liquid N2. Frozen cells were homogenized in a Fastprep machine (ThermoSavant) and shaken several times at speed 5.0 for 45 s until defrosted and homogenized. RNA was isolated with Trizol (Invitrogen) according to the manufacturer's protocol, modified with an extra 1 : 1 (v/v) phenol : chloroform extraction after first chloroform addition. A similar approach was used for RNA isolation from zoospores/cysts and germinated cysts. RNA was isolated from mycelium and sporulating mycelium using the Qiagen RNeasy kit, according to the manufacturer's protocol for filamentous fungi. RNA was treated with Turbo DNA-free DNase (Ambion) according to the manufacturer's protocol and checked for genomic DNA contamination by PCR with the primers used for quantitative RT-qPCR (Q-PCR). The concentration and purity of RNA were determined spectrophotometrically with Nanodrop at 260 and 260/280 nm ratios, respectively. Samples with a 260/280 nm ratio lower than 1.7 were discarded. Subsequent cDNA synthesis was performed using a First strand cDNA synthesis kit (GE Healthcare) with 3–5 μg of RNA per 33-μL sample using the pd(N)6 random hexamers according to the manufacturer's protocol. Transcript levels of SpHtp1 were analysed with a LightCycler® 480 (Roche), using the LightCycler® 480 SYBR Green I Master mix (Roche), with 1 μL of cDNA in a total of 10 μL and according to the manufacturer's protocol. The reaction was performed with an initial incubation at 95 °C for 5 min, followed by 45 cycles of 95 °C for 10 s, 58 °C for 10 s and 72 °C for 5 s, respectively. A dissociation curve as described in the LightCycler® 480 SYBR Green I Master mix (Roche) was performed to check the specificity of the primers. The amplicon length and optimized concentrations of the primers were 104 bp and 250 nM for SpHtp1, respectively, and 129 bp and 400 nM for SpTub-b, respectively. To correct for differences in the template concentration, several reference genes suggested by Yan & Liou (2006) were tested initially (Supporting Information, Fig. S4) and, based on the lowest variation in transcript levels between the life stages, the SpTubB gene coding for β-tubulin was used as a reference gene. The primers used for the Q-PCR were as follows: for SpHtp1 5′-CGTCATCATCGGAGAAATCC-3′ (forward) and 5′-CGCTTTGTTCAAGTTGTTCC-3′ (reverse); for SpTub-b 5′-AGGAGATGTTCAAGCGCGTC-3′ (forward) and 5′-GATCGTTCATGTTGGACTCGGC-3′ (reverse). For analysis, a standard curve of a pool of the cDNA of all samples was included to normalize the transcript levels. Subsequent analysis was performed with lightcycler® 480 software release 1.5.0 (Roche), using the second derivative maximum method, which calculates and includes PCR efficiency according to Pfaffl (2001). Q-PCR analysis was performed with three technical replicates of four independent RNA isolations (biological replicates). Statistically significant differences were determined by anova (P<0.05), followed by the Bonferroni post hoc multiple comparison.

Identification, cloning, overexpression and protein purification of SpHtp1

A 1406-bp fragment containing SpHtp1 and including flanking regions was amplified from genomic DNA by the primers 5′-GTTTGAATGGAGCAGCGTGCT-3′ (forward) and 5′-TACGATGAATTCTAATCGAATGTCGGGACGACCTGG-3′ (reverse) and subsequently sequenced. The obtained sequence was analysed for the start and the stop codon and the oomycete promoter region. For overexpression, a fragment of SpHtp1 was amplified, encoding for amino acids (aa) 24-198 lacking the putative N-terminal signal peptide and the C-terminal stop codon. The fragment was amplified by PCR from mycelial cDNA using KOD-Hot start DNA polymerase (Novagen) at an annealing temperature of 55 °C and in the presence of 3% DMSO. The primers used were 5′-GGGCGCATATGCGCATTCACCACCCGTTGACC-3′ (SpHtp124-198 forward) and 5′-CCGGGAATTCGGATCGAATGTCGGGACG-3′ (SpHtp124-198 reverse). The forward primer contained an NdeI and the reverse primer contained an EcoRI restriction site. The blunt end PCR-product was cloned into pETblue-2 (Novagen) and, after NdeI and EcoRI digestion and gel purification, cloned into the NdeI- and EcoRI-digested vector pET21b (Novagen) in frame with the (His)6 tag. The resulting plasmid SpHtp124-198-(His)6 was checked by sequencing and transformed into Rosetta gami B Escherichia coli cells (DE3, pLys; Novagen). SpHtp124-198-(His)6-overexpressing cells were grown in Luria–Bertani media to an OD600 nm of 0.6–0.8 and induced with 1 mM IPTG for 6 h at 37 °C. Cells were centrifuged and the pellet was resuspended in 40 mL of 50 mM sodium phosphate (pH 7.1) and incubated with 250 U of benzonase (Sigma-Aldrich), two dissolved tablets of protease inhibitor (Roche) and 0.1 g lysozyme (Fluka). After a 30-min incubation on ice, the solution was French-pressed and diluted 1 : 5 in 25 mM sodium phosphate buffer (pH 7.0) before the soluble fraction was separated from the nonsoluble via centrifugation at 48 000 g for 1 h. The supernatant was applied to a Fractogel-EMD-SO3-column (Merck, 2 cm diameter × 15 cm) and washed with 10 volumes of 25 mM sodium phosphate buffer (pH 7.0) containing 25 mM potassium chloride. The flow-through was applied to a QAE-Sephadex column (GE Healthcare, 2 cm diameter × 15 cm). After washing the column with 10 volumes 25 mM sodium phosphate buffer (pH 7.0) containing 25 mM KCl, the bound proteins were eluted with a gradient from 0 to 1.5 M KCl in 25 mM Tris/HCl (pH 7.5) and the fractions were checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The fractions containing the SpHtp124-198-(His)6 recombinant protein were pooled and applied to an Ni-NTA Agarose column (Invitrogen, 1 cm diameter × 10 cm). The column was washed with 100 mL of 25 mM Tris/HCl containing 30 mM imidazol (pH 7.5) and proteins were eluted with 25 mM Tris/HCl containing 300 mM imidazol adjusted to pH 7.1, and checked by SDS-PAGE. The purified protein was concentrated using Vivaspin 6 centricons (MWCO 5000), dialysed three times against 3 L 25 mM sodium phosphate buffer (pH 7.0) and checked by Coomassie staining on SDS-PAGE. The protein was further characterized by circular-dichroism (CD) spectroscopy to investigate its secondary structure. CD-spectra were recorded on a Jasco J710 spectrometer using 5-μM protein in a 1 mm cell in 50 mM potassium phosphate buffer (pH 7.2) (Fig. S6). SDS-PAGE was essentially performed according to the manufacturer's instructions (Invitrogen). Gradient 4–12% Bis-Tris NuPage gels were used with NuPage MES-SDS running buffer (Invitrogen). Protein samples were dissolved in Laemmli SDS buffer (Invitrogen) containing 8M urea and 2%β-mercaptoethanol.

Immunolocalization of SpHtp1

A polyclonal SpHtp1 antiserum was raised in rabbits against a peptide consisting of the aa 93-107 of SpHtp1 (TKDKTTPMKNALFK) (Sigma-Genosys), and specificity was tested on purified SpHtp124-198-(His)6 using Western blot analysis. Purified SpHtp124-198-(His)6 and a protein extract of Saprolegnia-infected RTG-2 cells were run on an SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was incubated overnight at 4 °C in phosphate-buffered saline+0.2% Tween-20 (PBS-T) and 5% skimmed milk powder. After washing the membrane several times in PBS-T, it was incubated for 1 h with preimmune or final bleed antibody, diluted 1 : 400 in PBS. Membranes were washed several times in PBS-T and incubated for 1 h in secondary horse-radish peroxidase-conjugated anti-rabbit antibody (Sigma-Aldrich, No. A0545), diluted 1 : 16000 in PBS-T. After several washes, membranes were developed using Pierce ECL Western Blotting Substrate (Thermo Scientific), according to the manufacturer's protocol. Membranes were exposed to a Kodak BioMax XAR film (Amersham Biosciences). RTG-2 cells were grown as a confluent monolayer onto coverslips in six-well plates and challenged as described above. The infected monolayers were washed carefully three times with PBS, before fixation in 4% paraformaldehyde/PBS for 1 h at room temperature. Samples were permeabilized with 0.1% Triton-X 100 for 15 min and incubated in the presence of either 1 : 400 diluted preimmune or final bleed SpHtp1 antisera at 37 °C for 1 h. Subsequently, the samples were washed three times with PBS and incubated with the secondary antibody (goat-anti-rabbit Alexa Fluor 488 conjugate; Invitrogen, No. A31627) according to the manufacturer's protocol. After washing three times with PBS, the immunostained coverslips were viewed using a Zeiss LSM 510 Meta confocal microscope with a Plan Apochromat × 63/1.0 water-dipping objective lens.

For the uptake experiment of recombinant SpHtp1 proteins, RTG-2 cells were washed three times with HBSS before a 20–30-min incubation with 20 μM recombinant SpHtp124-198(His)6 protein in L-15 medium containing 10% FCS. After washing cells three times with PBS, they were fixed as described above. Fixed cells were washed three times with PBS, permeabilized for 15 min with PBS containing 0.1% Triton-X 100 and washed again three times before incubation with the primary penta-His antibody at 37 °C for 1 h (Qiagen, No. 34660; titre 1 : 300). Subsequently, the samples were washed three times with PBS, and incubated at 37 °C for 1 h with the secondary antibody [fluorescein isothiocyanate (FITC) 488-conjugated goat-anti-mouse immunoglobulin G; Jackson ImmunoResearch] according to the manufacturer's protocol. The immunostained coverslips were washed again three times with PBS and mounted onto microscope slides. Microscopy was carried out using a Zeiss LSM 510 META confocal microscope (Fluor 488/FITC: excitation is 488 nm, filter settings BP 505-530; detector gain 750. Iodide: excitation is 633 nm, filter settings LP 650; detector gain 540, all 1 μm slices).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Identification of SpHtp1 and expression analysis

In order to identify and investigate genes of S. parasitica that are expressed in the preinfection and early infection stages, we set up a cDNA library from RNA isolated from zoospores, cysts and germinated cysts of S. parasitica and generated ESTs. End-sequencing of the cloned cDNA library and subsequent preliminary bioinformatic analysis resulted in the identification of a putative secreted protein with an RxLR motif located within the first 40 aa after the predicted signal peptide cleavage site. The ORF, SpHtp1 (S. parasitica host targeting protein 1), encodes a putative protein, SpHtp1, of 198 aa, of which the first 23 aa encode a signal peptide (Fig. 1a). The RxLR motif is located 22 aa downstream of the predicted signal peptide cleavage site, which is comparable to all known and characterized oomycete RxLR effector proteins (Fig. 1b, Fig. S1). Genome sequencing confirmed that the ORF is present in the genome and revealed an intron of 55 nt long, ranging from 74 nt up to 129 nt. Also, the oomycete conserved sequence motif in the promoter region of SpHtp1 was identified 35 nt upstream of the start codon (Pieterse et al., 1994; McCleod et al., 2004) (Fig. S2).

image

Figure 1.  (a) Protein sequence of SpHtp1. Signal peptide according to signalp is underlined; the RxLR motif is boxed; the xPTx region according to interproscan is indicated in bold; the region for polyclonal antibody antiserum is indicated in grey. (b) Alignment of the N-terminus of a subset of oomycete RxLR effector proteins and a PEXEL protein from malaria. The amino acids encoding for the signal peptide are underlined, the RxLR motif is boxed and the EER motif is indicated in grey. ‘*’ indicates that the residues or nucleotides in that column are identical in all sequences in the alignment; ‘:’ indicates that conserved substitutions have been observed, according to the physicochemical properties as described for clustalw; ‘.’ indicates that semi-conserved substitutions are observed. Sequences used are PsAvr1b, 3-9fHa of Phytophthora sojae, accession numbers AAM20934 and AY183415, respectively; Atr1 and Atr13 of Hyaloperonospora parasitica, accession numbers AY842877 and AY785306B, respectively; Avr3a, Avr4 and Avr-blb1 of Phytophthora infestans, accession numbers AJ893357, EF672355 and L23939, respectively; KAHRP of Plasmodium falciparum, accession number CAA68268 and SpHtp1 of Saprolegnia parasitica, accession number GU345745. (c) Transcript levels of SpHtp1 in various life stages of S. parasitica and after challenge of fibroblast cells of the rainbow trout cell line RTG-2 with S. parasitica zoospores/cysts. Transcript levels are relative to the transcript levels of SpHtp1 in mycelium and normalized against the reference gene SpTub-b encoding for tubulin. Error bars correspond to four biological replicates. M, mycelium; SM, sporulating mycelium; Z/C, zoospores/cysts; C/GC, cysts/germinating cysts; T0, T4, T8, T16, T24, time point 0, 4, 8, 16, 24 h after inoculation of S. parasitica cysts on the RTG-2 cell line. Asterisks indicate statistically significant (P<0.05) differences relative to mycelium.

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blastp analysis of SpHtp1 yielded sequence similarity only to regions of low complexity in other proteins such as the elicitin 6 precursor proteins of Phytophthora medicaginis, Phytophthora ramorum and Phytophthora sojae (E-values 9e-19, 2e-16, 3e-16 and 52%, 68% and 51% identity, respectively). Elicitins are structurally related secreted proteins that induce a hypersensitive response in plants (Ponchet et al., 1999), although this has not been demonstrated for the elicitin 6 precursor proteins identified by our blast analysis. However, SpHtp1 aligns only with the C-terminal region of the elicitin 6 precursor proteins identified by the blast analysis, containing an xPTx repeat region, and not with INF1, which is the highly expressed elicitin in mycelium stages from P. infestans (Kamoun et al., 1997) (Fig. S3). Moreover, blast of SpHtp1 against INF1 results in an E-value of 8.7. interpro analysis shows that the xPTx repeat region is observed in a variety of proteins; however, it is not known whether they are homologous to each other and no specific function of this repeat region has been identified so far.

In vitro transcript analysis showed that SpHtp1 is expressed in all life stages of S. parasitica, but compared with the transcript levels in mycelia, SpHtp1 transcripts are more abundant in zoospores/cysts and germinating cysts when normalized to transcript levels of the reference gene SpTub-b (Fig. 1c). In the RTG-2 model system, it was observed that SpHtp1 transcript levels were very high at time point 0, representing the addition of the zoospores/cysts as an inoculum source. A decrease over time was observed, representing germination and subsequent mycelial growth (Fig. 1c). Similar results were obtained when other reference genes were used. However, the SpTub-b transcript levels showed the lowest variation between the life stages (Fig. S4). These results indicate that SpHtp1 is predominantly expressed in the preinfection stages and in the very early stages of infection.

SpHtp1 is translocated into fish cells during the interaction

To investigate whether SpHtp1 is secreted and where the protein is located during the infection of S. parasitica on RTG-2 cells, a final bleed polyclonal antiserum was generated that was directed against a peptide of the SpHtp1 sequence (Fig. 1a). Western blot analysis showed that the antiserum recognized SpHtp1 synthesized in E. coli and a protein band of the same size in a protein fraction isolated from infected RTG-2 cells (Fig. S5). Several other bands in the protein samples isolated from uninfected and infected RTG-2 cells were recognized by the final bleed polyclonal antiserum, but these were also detected with the preimmune antiserum. Subsequent fluorescent immunolocalization studies on S. parasitica-infected RTG-2 cells resulted in SpHtp1 detection inside fish cells, surrounding the host nucleus, that are in close contact with the S. parasitica hyphae (Figs 2 and 3). This localization pattern was neither observed in infected RTG-2 cells treated with only preimmune antiserum nor in uninfected RTG-2 cells treated with preimmune or the final bleed polyclonal antiserum (Fig. 2), thereby demonstrating that the immunolocalization pattern in the infected cells of RTG-2 is only derived from translocated SpHtp1. Z-scans of fish cells that are in contact with hyphae from S. parasitica show that SpHtp1 is translocated inside the trout cells (Fig. 3).

image

Figure 2.  Immunolocalization of SpHtp1 on rainbow trout cell line RTG-2 challenged with Saprolegnia parasitica. Fixed cells were incubated with either preimmune or the final bleed SpHtp1 antiserum and the secondary antibody goat-anti-rabbit (Alexa Fluor 488). On the left bright field, a phase-contrast image (DIC) is shown and subsequently the fluorescent images for the secondary antibody Alexa Fluor 488, for the nuclear stain TO-PRO-3 iodide and the merged image. At the bottom, close-up pictures are shown of S. parasitica-challenged RTG-2 cells treated with final bleed antiserum and secondary antibody. Fish cells in which the SpHtp1 protein is detected with the SpHtp1 antiserum are indicated with an arrow. Saprolegnia parasitica hyphae are also indicated (h).

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image

Figure 3.  Immunolocalization of SpHtp1 on rainbow trout cell line RTG-2 challenged with Saprolegnia parasitica. Fixed cells were incubated with either preimmune or the final bleed SpHtp1 antiserum and the secondary antibody goat-anti-rabbit (Alexa Fluor 488). Z-stack images of merged channels (Fluor 488 and TO-PRO-3 iodide, left) and an overlay with DIC (right) are shown. One hypha (h) of S. parasitica is indicated. Confocal Z-stack images were acquired comprising sequential optical XY sections taken at 1.2-μm Z-intervals.

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To investigate whether the uptake of SpHtp1 can be caused by physical disruption of the membranes by Saprolegnia, a His-tagged SpHtp1 fusion protein without the putative signal peptide, SpHtp124-198-His, was synthesized in E. coli, purified and characterized (Fig. S6). Treatment with the final bleed SpHtp1 antibody in combination with the secondary antibody Fluor 488 showed no fluorescence in or on RTG-2 cells. When the RTG-2 cells were treated with SpHtp124-198-His, no fluorescence was detected when the preimmune antiserum was used in combination with the secondary antibody Fluor 488, also showing that the treatment of SpHtp1-His did not affect the fish cells (Fig. 4). However, SpHtp124-198-His and final bleed SpHtp1 antibody-treated RTG-2 cells showed SpHtp124-198-His localization on the surface of the fish cells and also inside the fish cells, surrounding the nucleus (Fig. 4). Furthermore, when the fish cells were incubated with SpHtp124-198-His and only labelled with the primary or the secondary antibody, no fluorescence was observed inside or outside the cells. Identical results were observed when an anti-His antibody was used for the immunolocalization studies (Fig. S7).

image

Figure 4.  Uptake of exogenous SpHtp1 in fibroblast cells of the rainbow trout cell line RTG-2. The fish cells were untreated, or treated with 20 μM His-tagged SpHtp124-198 and subsequently treated with either preimmune or final bleed SpHtp1 antiserum and the secondary antibody goat-anti-rabbit (Alexa Fluor 488). On the left bright field, a phase-contrast image (DIC) is shown and subsequently the fluorescent images for the secondary antibody Fluor 488, for the nuclear stain TO-PRO-3 iodide and the merged image.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Setting up a model infection system without having to sacrifice animals has many obvious advantages as it helps to fulfil the ultimate goal of the three Rs, whereby the aim is to reduce, refine and replace animals in experimental research. Here, we have shown that the trout RTG-2 cell line represents an excellent in vitro system for studying the very early interactions between fish cells and S. parasitica, and that it can also be used to study the molecular mechanism of infection.

Analysis of ESTs from zoospores and germinated cysts resulted in the identification of a putative RxLR effector protein, demonstrating that these types of proteins are possibly not only present in plant pathogenic oomycetes but also in animal pathogenic oomycetes. SpHtp1 is expressed in the preinfection and the very early infection stages of S. parasitica, as are many RxLR effector genes from P. sojae and P. infestans (Whisson et al., 2007; Dong et al., 2009). Analysis of the protein sequence revealed that SpHtp1 lacks the ‘so-called’ EER motif, which is found closely behind the RxLR motif in about 500 putative P. infestans RxLR effector proteins (Whisson et al., 2007) (Fig. 1a and b). The EER motif in the PiAvr3a and PsAvr1b proteins seems to be required for effector translocation of P. infestans and P. sojae, respectively (Whisson et al., 2007; Dou et al., 2008a). However, another intracellularly recognized RxLR effector protein, Atr13 of Hyaloperonospora arabidopsidis, lacks the EER motif (Allen et al., 2004), suggesting that the presence of an EER motif is not always essential for the translocation of every RxLR effector into host cells, or for inducing a hypersensitive response. Indeed, several studies have shown that specific domains and amino acid residues present in the C-terminal region of the effector proteins are involved in recognition by the host and not the RxLR–EER sequence (Bos et al., 2006; Kamoun, 2006; Dou et al., 2008b). Therefore, it is possible that the EER motif or the amino acids in the region located after the RxLR motif in Atr13 and SpHtp1 play an important role in the folding of these proteins and thereby targeting specific recognition sites in the host. Related to this, positive selection was found to have acted mostly on the C-terminal region of RxLR proteins, which is consistent with the view that the N-terminal RxLR–EER region functions as a translocation signal and that it is not required for effector activity (Morgan & Kamoun, 2007; Win et al., 2007). An exception to the recognition of the C-terminal part of oomycete effectors by cognate resistance genes in plants comes from the recently published P. sojae PsAvr4/6 effector, where the RxLR–EER region is recognized by Rps4 from soybean (Dou et al., 2010).

Transcript analysis showed that SpHtp1 is mainly expressed in the zoospores/cysts and in the onset of the challenge of the RTG-2 cell line, corresponding to the zoospore/cysts stage. Transcript analysis of 38 predicted RxLR–EER effectors from P. infestans showed various expression patterns: ‘predominantly upregulated in preinfection only; predominantly in preinfection and biotrophy; preinfection and throughout infection; biotrophy only’ (Whisson et al., 2007). Also, seven genes encoding RxLR proteins lacking the EER motif conformed to the profiles seen for the RxLR–EER effectors (Whisson et al., 2007). Although no time point 0 was included in their analysis and it is therefore not possible to compare that time point with our results, the expression profile of SpHtp1 would fit best in the group of preinfection only.

Translocated SpHtp1 was detected inside the host cells, using an antiserum directed against a peptide of SpHtp1 (Figs 2 and S5), which has not been shown for any other oomycete RxLR effector thus far. In a P. infestans transformant expressing recombinant Avr3a-mRFP, the RxLR protein localizes in the haustoria and the extrahaustorial matrix of infected potato leaves. However, translocation inside the infected plant cells was only observed in a recombinant Avr3a-GUS-expressing transformant (Whisson et al., 2007). Moreover, substitution of the RxLR and EER residues with alanine abolished Avr3a-GUS translocation inside the plant cells (Whisson et al., 2007). Here, we show that recombinant and exogenously applied SpHtp1 is also taken up into the fish cells (Fig. 4). Furthermore, Dou et al. (2008a) showed that recombinant PsAvr1b from P. sojae can also be translocated into nonhost onion cells, suggesting that the RxLR translocation mechanism is used by both plant and animal pathogenic oomycetes.

In summary, we have isolated a putative RxLR effector protein from an animal pathogenic oomycete, which is translocated inside fish cells. This detection inside the fish cells is not due to the physical disruption of S. parasitica. When the fish cells were treated with recombinant SpHtp1, translocation without the presence of the pathogen is observed. These results suggest that S. parasitica may have a biotrophic stage in the infection process, which is similar to what has been found in biotrophic and hemibiotrophic plant pathogenic oomycetes. Consequently, it is conceivable that the pathogen has an early infection stage, whereby it does not kill the host cells, but instead, host cells are kept alive in order to enhance its own growth. It is interesting to note that the cells in which SpHtp1 has been translocated, in the presence of S. parasitica (Fig. 2), seem to be somewhat smaller than the surrounding fish cells. It could be that the cells are in fact not smaller, but that the focal plane is not showing the true size of the cells. Alternatively, Saprolegnia is absorbing nutrients from the cells, which results in smaller fish cells. At present, we do not know how many RxLR effectors or whether other RxLR-like effectors are produced by S. parasitica during an infection. However, the completion of the genome sequence in the near future (at The Broad Institute with Nusbaum, van West, Haas, Russ, Dieguez-Uribeondo and Tyler) will enable a more in-depth analysis of the number of putative RxLR proteins produced by S. parasitica.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Our work was supported by the BBSRC (I.d.B., K.L.M., A.J.P., S.W., C.J.S., P.v.W.), the University of Aberdeen (E.J.R., V.L.A., C.J.S., P.v.W.) and The Royal Society (P.v.W.). We would like to acknowledge the Broad Institute (Carsten Russ, Rays Jiang, Brian Haas and Chad Nusbaum), Brett Tyler (VBI) and P.v.W. for early release of draft supercontigs of the genome sequence of isolate CBS233.65, which helped us choose the best control gene for the Q-PCR experiments and helped resolve the promoter sequence of SpHtp1.

Authors' contribution

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

I.d.B., K.L.M., A.J.P., E.J.R. and S.W. contributed equally to this work.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Fig. S1. Alignment of the full sequences of a subset of oomycete RxLR effector proteins.

Fig. S2. Alignment of the upstream region of SpHtp1 with the conserved motif in the oomycete core promoter sequence and genome sequence of SpHtp1.

Fig. S3. Alignment of SpHtp1 with elicitin-like precursor proteins obtained by blastp analysis of SpHtp1 against nonredundant protein database in NCBI.

Fig. S4. Primer sequences used for quantitative real time RT-PCR and reference gene analysis.

Fig. S5. SpHtp1 is detected during infection.

Fig. S6. Biochemical characterization of SpHtp124-198(His)6.

Fig. S7. Uptake of SpHtp1 in fibroblast cells of the rainbow trout cell-line RTG-2.

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