During attachment Phytophthora spores secrete proteins containing thrombospondin type 1 repeats (original) (raw)

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Adhesion is crucial for the establishment of diseases caused by pathogens in both plants and animals, yet the molecular mechanisms behind the adhesion of plant pathogens, particularly oomycetes, remain poorly understood. This study successfully cloned the gene PcVsv1 from Phytophthora cinnamomi, which encodes a protein with 47 thrombospondin type 1 repeats, suggesting its role as a spore adhesin and highlighting evolutionary similarities in host attachment strategies between oomycetes and malarial parasites.

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Abstract

Adhesion is a key aspect of disease establishment in animals and plants. Adhesion anchors the parasite to the host surface and is a prerequisite for further development and host cell invasion. Although a number of adhesin molecules produced by animal pathogens have been characterised, molecular details of adhesins of plant pathogens, especially fungi, are largely restricted to general descriptions of the nature of heterogeneous secreted materials. In this paper, we report the cloning of a gene, PcVsv1, encoding a protein secreted during attachment of spores of Phytophthora, a genus of highly destructive plant pathogens. PcVsv1 contains 47 copies of the thrombospondin type 1 repeat, a motif found in adhesins of animals and malarial parasites but not in plants, green algae or true fungi. Our results suggest that PcVsv1 is a spore adhesin and highlight intriguing similarities in structural and molecular features of host attachment in oomycete and malarial parasites.

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Introduction

Adhesion has been shown to be a key step in the establishment of disease in both animals and plants (Klein 2000; Tucker and Talbot 2001). Firm attachment of pathogen cells to the surface of the potential host not only anchors them in a favourable position for host cell penetration but also is often a prerequisite for the development of specialised infection structures required for penetration (Cotter and Kavanagh 2000; Epstein and Nicholson 1997; Huynh et al. 2003; Shaw and Hoch 2001). The importance of adhesion for disease development has been demonstrated experimentally for pathogens of both animals and plants (Brandhorst et al. 1999; Gale et al. 1998; Jones and Epstein 1990; Staab et al. 1999; Stanley et al. 2002). Although a number of adhesin molecules produced by fungal pathogens infecting animals have been cloned and at least partially characterised (Boyle and Finlay 2003; Sundstrom 2002), to date, genes encoding the adhesins of fungal pathogens of plants have not been isolated. The recent cloning of a gene encoding a cellulose-binding protein from the fungus-like oomycete, Phytophthora nicotianae (Gaulin et al. 2002; Mateos et al. 1997), has given the first data on a putative hyphal adhesive, but information on adhesive proteins used by fungal or oomycete spores is still lacking.

About 20% of the world-wide expenditure on fungicides in agriculture is directed towards the control of Phytophthora and other oomycete pathogens (Schwinn and Staub 1995). Oomycetes cause some of the most devastating plant diseases known, including late blight of potato and tomato, downy mildew of grapes and lettuce, and root and stem rots of tobacco, pineapple and avocado (Erwin and Ribeiro 1996). Oomycetes have a fungus-like morphology and mode of nutrition but are phylogenetically distinct from the true fungi (Dick 1990; Govers 2001; Gunderson et al. 1987). One consequence of their phylogeny is that most fungicides are not inhibitory for oomycete species. In addition, in recent years resistance to key chemicals has developed in a number of Phytophthora species, including P. infestans and P. nicotianae (Timmer et al. 1998), and the development of new control measures is urgently needed (Fry and Goodwin 1997; Schiermeier 2001).

One of the distinguishing features of the Oomycetes is their production of motile, biflagellate zoospores. The zoospores are asexual spores produced in vast numbers under suitable conditions and, for the majority of Phytophthora species, are the main infective agent that initiates disease. Phytophthora zoospores are chemotactically attracted to favourable infection sites on potential host plants. On reaching these sites, the zoospores encyst, rapidly detaching the flagella and secreting adhesive material onto the host surface. Within 20–30 min, the encysted spores germinate and penetrate the underlying plant tissues (Hardham 2001). The adhesive material is synthesised during asexual sporulation and stored in secretory vesicles targeted to the ventral surface of the zoospores (Dearnaley et al. 1996; Hardham and Gubler 1990). During the first 2 min of plant infection, the adhesive material is secreted from the ventral vesicles and forms an adhesive pad that glues the spore to the plant surface (Gubler et al. 1989; Hardham and Gubler 1990). In this paper, we report the immunoscreening of a P. cinnamomi cDNA library with antibodies that label an approximately 220-kDa polypeptide that occurs in the ventral vesicle adhesive material, to clone the gene encoding the putative P. cinnamomi adhesive protein.

Materials and methods

Oomycete cultures

P. cinnamomi (H1000, ATCC 200982) and P. nicotianae (H1111, ATCC MYA 141) were grown on V8 nutrient medium as previously described (Hardham et al. 1991; Mitchell and Hardham 1999). Asexual sporulation of P. cinnamomi was induced by washing mycelia in nutrient-free mineral salts solution. Asexual sporulation of P. nicotianae occurred during extended incubation in liquid V8 nutrient medium. Zoospores were produced from Plasmopara viticula, P. halstedii and Albugo sp. inoculated onto host leaves in a growth cabinet.

Nucleic acid isolation and analysis

A randomly primed cDNA library constructed in λgt11(Marshall et al. 2001) was screened by standard protocols with a mixture of four purified monoclonal antibodies (Pn3F4, Pn8G8, Pn17E7, Pn19F2) directed towards the Phytophthora ventral vesicle antigen (Robold and Hardham 2004). Antibodies were purified on a HiTrap G column (Pharmacia) after partial purification by 45% ammonium sulphate precipitation, solubilisation and dialysis and were used at a concentration of 20 μg/ml. A cDNA clone containing a fragment of the gene encoding the ventral vesicle protein, designated PcVsv1, was isolated. A P. cinnamomi genomic library constructed in EMBL3 (Weerakoon et al. 1998) was screened under high stringency conditions (Marshall et al. 2001) with a radiolabelled DNA probe containing the insert of the PcVsv1 cDNA. DNA fragments resulting from a restriction digest of the positive genomic clone with the enzymes _Sal_I, _Sac_I and _Xho_I recognised by the PcVsv1 cDNA were subcloned into the bacterial vector pBluescript. Genomic DNA isolation and Southern blotting were as described by Marshall et al. (2001) and plasmid DNA was extracted using a commercial kit (Qiagen).

DNA sequencing and sequence analysis

DNA sequencing was done using an Applied Biosystems automated fluorescent DNA sequencer. DNA and protein sequence searches were conducted against the non-redundant, expressed sequence tag (EST) and selected genome sequence databases using BLAST programs through the websites of the National Centre for Biological Information (http://www.ncbi.nih.gov/BLAST), the Phytophthora Functional Genomics Database (http://www.pfgd.org/) and the Joint Genome Initiative (http://genome.jgi-psf.org/physo00/physo00.info.html). The DNA sequence was searched for introns using the software program FGENESH (http://www.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind). The molecular weight and theoretical isoelectric point were calculated with Protparam on the ExPasy server (http://kr.expasy.org/tools/protparam.html). The inferred amino acid sequence was searched for a signal peptide using SignalP software (http://www.cbs.dtu.dk/services/SignalP/).

Gene expression analysis

Ribonucleic acid dot-blots were conducted as described by Marshall et al. (2001). The dot-blots were hybridised at high stringency, using a radiolabelled probe made from the insert of the PcVsv1 cDNA. The hybridisation intensity of six replicate spots was measured using Metamorph software (Universal Imaging Corp., West Chester, Pa). For reverse transcriptase PCR, total RNA was isolated from hyphal material (vegetative or 4 h after induction of sporulation) using Trizol reagent (GibcoBRL), contaminating genomic DNA was removed with RQ1 RNase-free DNase (Promega) and the RNA was reverse-transcribed using SuperScript II RNase H− reverse transcriptase (Invitrogen), 5 μg of RNA and 2 pmol of the gene-specific reverse primer 5′-gcactcacttggcaatcac-3′ in a final volume of 12 μl. Two microlitres of the reverse-transcribed RNA reaction were added to 5 μl of 2× PCR mix (Promega) and 0.2 μmol each of forward primer 5′-cagcggtcaacaatggag-3′ and reverse primer in a final volume of 10 μl and 35 PCR cycles were carried out. Both primers annealled to the coding region of the PcVsv1 gene. All reagents and enzymes were used as recommended by the manufacturer.

Sequence data

GenBank accession number for nucleotide sequence data is AY973234.

Antibody production, immunofluorescence microscopy and immunoblotting

Six Balbc mice were inoculated with a synthetic peptide with the sequence SRGASAEGDTFWGPLGGTSAYGTSAYGTS conjugated to keyhole limpet haemocyanin (KLH). The peptide corresponded to the N-terminus of the mature PcVsv1 protein inferred from the PcVsv1 gene sequence. Mouse sera and monoclonal antibodies were used in immunofluorescence and immunoblotting assays as described by Cope et al. (1996). In brief, for immunofluorescence microscopy, Phytophthora zoospores were fixed in 4% formaldehyde in 50 mM Pipes buffer, pH 7.0, and air-dried onto multi-well slides. After rehydration in phosphate-buffered saline, the cells were incubated in 15 μl of primary antibody for 1 h at 37°C, rinsed and incubated in 15 μl of sheep anti-mouse immunoglobulin conjugated to fluorescein isothiocyanate for 1 h at 37°C. After rinsing, the slides were mounted in a glycerol-based mounting medium and viewed using epifluorescence and a Zeiss Axioplan microscope. For immunoblotting, Phytophthora zoospores were freeze-dried and proteins solubilised in 8 M urea. The preparations were homogenised and centrifuged at 13,000 g for 3 min and then the supernatant was loaded onto polyacrylamide gels and immunoblotted. After blocking in 5% skim milk powder, membranes were incubated for 1 h at room temperature in primary antibody, rinsed and incubated in secondary antibody (sheep anti-mouse conjugated to alkaline phosphatase) for 1 h. For immunofluorescence and immunoblotting assays, in negative controls the primary antibody was omitted before incubation in secondary antibody.

Results

Identification of a cDNA clone encoding the ventral vesicle protein

Vsv-1 is a monoclonal antibody that was raised against a ventral vesicle protein in P. cinnamomi (Hardham and Gubler 1990). In immunofluorescence assays, Vsv-1 labels vesicles predominantly distributed along the ventral surface in all Phytophthora species tested (Hardham et al. 1994; Robold and Hardham 2004), in Pythium aphanidermatum and P. butleri (Cope et al. 1996), in the downy mildew pathogens Plasmopara viticola and P. halstedii and in a species of the white rust, Albugo (Fig. 1). Immunogold labelling with Vsv-1 shows antibody binding to small vesicles near the ventral surface (Fig. 1g). Vsv-1 antibody reacts with a polypeptide of molecular mass approximately 220 kDa in immunoblots of Phytophthora cinnamomi proteins, but does not give a positive reaction in immunoblots with proteins from other Phytophthora or other oomycete species (Robold and Hardham 2004). Screening of two cDNA libraries constructed from mRNA isolated from P. cinnamomi hyphae 4 h after the induction of sporulation with Vsv-1 did not yield any positive clones. However, five monoclonal antibodies that were raised against P. nicotianae zoospore components also labelled the zoospore ventral vesicles in immunofluorescence assays of P. nicotianae (Fig. 1d) and P. cinnamomi (Fig. 1e) and cross-reacted with the ventral vesicle protein from P. nicotianae and P. cinnamomi in immunoblots (Robold and Hardham 2004). These antibodies, like Vsv-1, also labelled the contents of the ventral vesicles after their secretion onto the cyst surface during zoospore encystment (Fig. 1e). In immunocytochemical and immunoblotting experiments, negative controls (in which the primary antibody was omitted and only the secondary antibody was used) gave no labelling.

Fig. 1

Fig. 1

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Immunofluorescence (a–f; bar 5 μm) and immunogold (g; bar 0.5 μm) labelling of the ventral vesicle protein in zoospores and in the secreted adhesive pad on the surface of a cyst. a Vsv-1 antigen in P. cinnamomi ventral vesicles labelled with Vsv-1. b Vsv-1 antigen in Plasmopara viticola ventral vesicles labelled with Vsv-1. c Vsv-1 antigen in Albugo sp. ventral vesicles labelled with Vsv-1. d Labelling of ventral vesicles in Phytophthora nicotianae by Pn19F2. e Zoospore ventral vesicles (left) and surface of young cyst (right) of P. cinnamomi labelled with Pn19F2. f Labelling of P. cinnamomi ventral vesicles by polyclonal antibodies raised against KLH-PcVsv1 N-terminal peptide. g Labelling of ventral vesicles in P. cinnamomi zoospore with Vsv-1-Au15. h Diagram showing ventral vesicles aligned along the ventral groove of a Phytophthora zoospore

Four of the five antibodies that cross-reacted with P. cinnamomi (Pn3F4, Pn8G8, Pn17E7, Pn19F2) were purified, mixed together and used to screen a randomly primed λgt11 cDNA library constructed from mRNA isolated from P. cinnamomi hyphae 4 h after the induction of sporulation. This immunoscreening led to the identification and purification of a phage colony that reacted strongly with the antibodies. PCR amplification of the cDNA insert yielded a DNA fragment of approximately 550 bp. Sequencing of the cDNA clone showed that an open reading frame extended in both directions from the clone, indicating that the insert was not from the C- or N-terminus of the gene. The gene was designated PcVsv1 (P. c nnamomi ventral surface vesicle protein

Identification and sequence analysis of a genomic clone encoding the PcVsv1 protein

The cDNA was used to probe a P. cinnamomi genomic library constructed in EMBL3 in order to obtain the full sequence of the PcVsv1 gene. Two genomic clones hybridising to the probe were identified and purified to homogeneity. DNA from one of the genomic clones was isolated and subjected to restriction analysis. Restriction with _Sal_I showed the presence of a fragment of approximately 3.5 kb that was recognised by the cDNA probe on a Southern blot of the genomic clone DNA and P. cinnamomi genomic DNA (Fig. 2). The cDNA also hybridised weakly with a lower band in the genomic DNA, indicating either that there are two copies of the PcVsv1 gene in P. cinnamomi or that the probe is detecting the second allele in the diploid P. cinnamomi genome.

Fig. 2

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Southern blots of Phytophthora DNA after _Sal_1 restriction. a PcVsv1 genomic clone hybridised with the PcVsv1 cDNA probe. b P. cinnamomi (left), P. nicotianae (centre) and P. infestans (right) genomic DNA hybridised with the PcVsv1 cDNA probe. Sizes (right) are indicated in kilobases

DNA of the genomic clone was digested with _Sal_I, _Xho_I or _Sac_I and fragments subcloned into pBluescript II. The fragments overlapped extensively and were used to obtain the full PcVsv1 gene sequence by primer walking. The PcVsv1 gene consists of an open reading frame of 7,356 nucleotides, contains no introns and encodes an inferred protein of 2,452 amino acids with a molecular mass of 262 kDa and a pI of 5.52. The inferred protein sequence begins with a 22-amino-acid N-terminal signal peptide directing insertion into the endoplasmic reticulum, predicted according to the method of Nielson et al. (1997). Cleavage of the signal peptide yields a mature protein with a bipartite N-terminal domain consisting of an initial sequence of 17 amino acids and a subsequent domain of 43 amino acids containing six copies of a motif with the sequence GTSAY (Fig. 3a). The PcVsv1 protein ends with a 59-amino-acid C-terminal region. There is no transmembrane domain present and none of the N- or C-terminal sequences show homology to protein sequences from other organisms in the publicly available databases. In between the terminal regions, the PcVsv1 protein consists of 47 copies of a domain approximately 50 amino acids in length that shows homology to thrombospondin type 1 repeats (TSR1s) found in a large number of adhesive extracellular matrix proteins in animals (Adams and Tucker 2000) and secreted adhesins in apicomplexan parasites (Tomley and Soldati 2001). Alignment of the TSR1 domains from PcVsv1 with those in adhesins from Caenorhabditis elegans and the apicomplexan Cryptosporidium parvum reveals that the TSR1s in the P. cinnamomi protein contain nine of the 11 highly conserved residues in the typical TSR1 module (Fig. 3b). BLAST searches show that one or other of the two cysteine residues missing from the P. cinnamomi TSR1s is also absent from some TSR1 modules in proteins from other organisms. The PcVsv1 cDNA, which was recognised by the P. nicotianae monoclonal antibodies, spans TSR1 modules 8–10 (Fig. 3c).

Fig. 3

Fig. 3

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Sequence analysis of the PcVsv1 gene. a Domain structure of PcVsv1. b Alignment of predicted amino acid sequences of TSR1 domains of Caenorhabditis elegans (Ce, NM_077715), Cryptosporidium parvum (Cp, AF017267) and P. cinnamomi (Pc) proteins. Conserved amino acids according to Adams and Tucker (2000) are boxed. c The amino acid sequence of the PcVsv1 cDNA. The cDNA spans TSR1 modules 8–10 in the PcVsv1 protein sequence

Verification that PcVsv1 encodes the Vsv-1 antigen stored in the ventral vesicle

A peptide 29 amino acids in length and corresponding to the N-terminal sequence of the predicted mature PcVsv1 protein was synthesised and used to immunise six mice. Immunofluorescence assays (Fig. 1f) and immunoblotting (Fig. 4a) revealed that the resultant polyclonal antisera labelled the ventral vesicles in P. cinnamomi zoospores and the 220-kDa Vsv-1 band on immunoblots. Negative controls in which the primary antibody was omitted gave no labelling and antisera raised against peptides not found in PcVsv1 did not label the ventral vesicles (data not shown). This evidence confirms that the cloned gene, PcVsv1, encodes the Vsv-1 antigen stored in the zoospore ventral vesicles.

Fig. 4

Fig. 4

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PcVsv1 identification and expression. a Immunoblot of P. cinnamomi proteins labelled with Vsv-1 (left lane) and polyclonal antiserum against the KLH-PcVsv1 N-terminal peptide (right lane). Arrow 200-kDa marker. b Immunoblot of P. cinnamomi proteins isolated 0–10 h after induction of sporulation and labelled with Vsv-1. Arrow 200-kDa marker. c Reverse transcriptase (RT)-PCR using RNA from P. cinnamomi hyphae grown vegetatively (lanes 1–3) or harvested 4 h after induction of sporulation (lanes 6–8). RNA in lanes 1, 3, 6 and 8 was treated with DNase. RNA was omitted from samples in lanes 2 and 7 and RT omitted from samples in lanes 3 and 8 before conducting the entire RT-PCR process. Lane 4 Nucleic acids omitted and only the PCR step carried out. Lane 5 Genomic DNA of P. cinnamomi used as template and subjected to PCR only. A band at around 0.5 kb indicates the presence of the 584-nucleotide sequence in the genomic DNA. A band of the same size is present in the sporulation-induced sample (lane 6), but not in the vegetative hyphae sample (lane 1). Numbers (left) indicate size in kilobases. d Intensity of hybridisation of PcVsv1 cDNA probe with dot-blots of P. cinnamomi RNA isolated 0–24 h after induction of sporulation

Expression of the PcVsv1 gene during asexual sporulation

Immunoblots of P. cinnamomi proteins isolated from mycelia at various times after the induction of sporulation show that the Vsv-1 antigen becomes detectable about 6 h after the induction of sporulation (Fig. 4b), consistent with the appearance of ventral vesicles at this time (Dearnaley et al. 1996). RNA isolated from P. cinnamomi hyphae before and 4 h after the induction of sporulation and used as a template for reverse transcriptase PCR showed that PcVsv1 transcripts were present in induced hyphae but absent from vegetative hyphae (Fig. 4c). Hybridisation of the PcVsv1 cDNA probe with dot-blots containing RNA isolated from hyphae 0–24 h after the induction of sporulation was weak but quantitation showed a rapid increase in levels of the PcVsv1 transcript after induction, followed by a gradual decrease (Fig. 4d). Thus, the up-regulation of PcVsv1 gene expression precedes the appearance of the Vsv-1 antigen in the ventral vesicles. The maintenance of relatively high levels of PcVsv1 transcript is consistent with the continued synthesis of Vsv-1 antigen and production of ventral vesicles during progressive sporulation over this time-frame.

PcVsv1 homologues in other Phytophthora species

Southern blot analysis was used to investigate the occurrence of PcVsv1 homologues in other Phytophthora species. In both P. nicotianae and P. infestans, a single band was detected after hybridisation with the PcVsv1 cDNA probe (Fig. 2b), indicating that a copy of the PcVsv1 gene is present in the genomes of these species. TBLASTX searches of the recently released P. sojae and P. ramorum genomes (http://genome.jgi-psf.org/physo00.info.html) also detected homologous sequences in these two Phytophthora species. The PcVsv1 cDNA probe did not hybridise to DNA from species of Pythium or Saprolegnia (data not shown), suggesting that conservation at the nucleotide level is apparently not sufficient to allow hybridisation to a putative PcVsv1 homologue outside the Phytophthora genus, even though immunolabelling indicates that such homologues do occur. TBLASTX searches against GenBank and a number of complete fungal genomes, including those of Magnaporthe grisea, Ustilago maydis and Aspergillus nidulans, did not reveal any sequences in true fungi that were homologous to PcVsv1.

Discussion

There is strong evidence that the PcVsv1 protein is a component of the Phytophthora spore adhesive material. Immunolabelling shows that the spatial and temporal aspects of secretion of PcVsv1 (the Vsv-1 antigen) from Phytophthora spores are consistent with a role for PcVsv1 in the adhesion of spores to an adjacent surface (Hardham and Gubler 1990). The zoospores adopt a specific orientation prior to encystment, such that the ventral surface faces the host and the contents of the ventral vesicles are secreted to form a pad of adhesive between the pathogen spore and the plant. Assays that measure the adhesiveness of the spores show that the spores become sticky about 2 min after the induction of encystment, concomitant with the timing of secretion of the ventral vesicle contents. Although the ventral vesicles may also contain other components involved in spore attachment, cloning and sequencing of the PcVsv1 gene, as reported in the present study, reveals that PcVsv1 contains multiple copies of a domain known to participate in cell attachment in animal cells and in malarial parasites (Adams and Tucker 2000; Deng et al. 2002; Tomley and Soldati 2001; Witcombe et al. 2003), namely the TSR1. Taken together, these data indicate that the PcVsv1 protein is likely to be an adhesin that plays an important role in the adhesion of Phytophthora spores to the underlying surface.

The difference in the molecular masses of PcVsv1 predicted from the inferred PcVsv1 sequence (260 kDa) and that measured in immunoblots (220 kDa) could be due either to an undetected intron(s) in the PcVsv1 genomic sequence or to inaccuracies in molecular mass estimation from polyacrylamide gels, which occur especially for large proteins. We consider the latter to be the more likely reason.

Comparison of PcVsv1 with TSR1-containing proteins in animals and apicomplexans

In animals, proteins that contain TSR1 modules include thrombospondins, F-spondins, SCO-spondin and members of the semaphorin 5 family (Adams and Tucker 2000; Tucker 2004). Many of the TSR1-containing proteins are components of the extracellular matrix, are often expressed in the developing nervous system and have roles that include cell guidance, attachment and aggregation. In apicomplexan parasites, including species of Plasmodium, Cryptosporidium and Eimeria, the TSR1-containing proteins are stored in and secreted from apical microneme vesicles during the early stages of host infection and mediate attachment to the host cell (Naitza et al. 1998; Soldati et al. 2001; Tomley and Soldati 2001). The TSR1 is just one of a number of repeated sequence modules in the animal and parasitic adhesive proteins; and it has been recognised as arising before the evolutionary separation of nematodes and chordates (Adams and Tucker 2000). It contains a number of highly conserved amino acid residues, including W8, S9, W11, C14, C18, R25, R27, C29, C41, C51 and C56 (Adams and Tucker 2000). These conserved amino acids are key components in three regions of the TSR1 that have been shown to bind a variety of glycoconjugates, including heparin and fibronectin (Guo et al. 1992; Sipes et al. 1993), and to function in cell adhesion. These regions are the WSXW and CSVTCG motifs (amino acids 14–19) and the basic residues that lie downstream of the CSVTCG motif (including R25 and R27). Comparison of the TSR1s in Phytophthora cinnamomi PcVsv1 reveals that PcVsv1 contains the WSXW motif (with variants) and the basic region (including R25 and R27) in all 47 TSR1s, but only four of the TSR1s contain the sequence CXXXCG, as a version of the CSVTCG motif. All 47 PcVsv1 TSR1 modules contain W8, while in ten modules W11 is replaced by tyrosine and in five copies W11 is replaced by phenylalanine. Of the six conserved cysteine residues, all of the TSR1 modules in PcVsv1 lack C29 and all but four lack C18. However, as illustrated in Fig. 3b, not all TSR1s in animal proteins contain all six cysteine residues and C18 and C29 appear to be generally less highly conserved than the other cysteines.

Phylogenetic distribution of PcVsv1 homologues

Southern blotting and BLAST searches demonstrate the presence of PcVsv1 homologues in P. nicotianae, P. infestans, P. sojae and P. ramorum. An EST with homology to a thrombospondin-related adhesive has also been reported from P. sojae (Qutob et al. 2000). Immunolabelling with Vsv-1 monoclonal antibody indicates that homologues of the PcVsv1 protein also occur in at least three other oomycete genera, namely Pythium (Cope et al. 1996), Plasmopara and Albugo, suggesting that the Vsv protein may be used in spore attachment throughout the Oomycetes.

Outside the Oomycetes, the closest homologue of PcVsv1 in GenBank is currently a TSR1-containing protein from the apicomplexan malarial parasite, C. parvum (AA039046; Deng et al. 2002). This homology between Phytophthora and apicomplexan adhesives draws attention to other similarities, both molecular and structural, between Phytophthora zoospores and apicomplexan zoites. Our understanding of protist phylogeny has developed extensively in recent years and the novel assemblages that have emerged through a redefinition of the boundaries between the major eukaryotic groups include the stramenopiles (incorporating the Oomycetes) and the alveolates (incorporating the Apicomplexa). These groups are characterised by the possession of tripartite tubular hairs and flattened membranous alveoli, respectively (Patterson and Sogin 1992; Van de Peer et al. 1996). Connections between these two groups based on traditional ultrastructural analyses have been limited (e.g. possession of tubular cristae in mitochondria; Patterson and Sogin 1992). However, the recognition of phylogenetic affinities has been strengthened by comparisons of rRNA sequence data (Van de Peer et al. 1996; Van de Peer and De Wachter 1997). The new information on the Phytophthora TSR1-containing putative adhesive protein spotlights common features in infection strategies of these two parasitic groups. In both, onset of infection is marked by rapid, regulated secretion of adhesive-containing vesicles from a localised region of the pathogen aligned to face the host (Hardham and Gubler 1990; Joiner and Roos 2002). In Phytophthora, adhesive-containing ventral vesicles are confined to the ventral surface (Hardham and Gubler 1990). In apicomplexans, adhesin-containing micronemes are part of the apical complex that gives rise to the name of the group (Tomley and Soldati 2001). Remarkably, the cortical region at which secretion occurs is, in both cases, free of an underlying system of flattened membranes—the taxon-defining alveoli in the apicomplexans (typically called the inner membrane complex) and the zoospore peripheral cisternae in Phytophthora and related Oomycetes.

Microneme proteins characterised to date have 1–16 copies of the TSR1 domain and other motifs associated with cell–cell or cell–matrix adhesion (Deng et al. 2002; Tomley and Soldati 2001; Witcombe et al. 2003). PcVsv1 lacks other known adhesive motifs but, with 47 copies, it has the largest number of TSR1 domains reported to date. Recent studies of microneme contents revealed the presence of auxiliary proteins, including chaperones (Brydges et al. 2000), escorters (Meissner et al. 2002; Reiss et al. 2001) and proteases (Opitz et al. 2002). Future investigations of the protein complement of Phytophthora ventral vesicles may reveal the presence of similar processing enzymes, characterisation of which will help elucidate the molecular mechanism underlying PcVsv1 function. Analysis of these secreted proteins may also yield the first information on targeting motifs for oomycete proteins.

Fungal and oomycete adhesives

Fungal pathogens of animals and plants are likely to use a spectrum of adhesive molecules to attach to their hosts. Some molecules (e.g. hydrophobins; Tucker and Talbot 2001) mediate non-specific binding to the adjacent substratum, such as the hydrophobic interactions displayed by a range of fungi and Oomycetes (Apoga et al. 2001; Doyle 2000; Gubler et al. 1989; Mercure et al. 1994; Wright et al. 2002b). Other adhesive molecules bind to specific ligands associated with the host surface. A number of the genes cloned from pathogenic yeasts, for example, recognise and bind to RGD-containing proteins on the animal cell surface (Hostetter 2000). There is some evidence that RGD-binding adhesive molecules also exist in fungal phytopathogens (Corrêa et al. 1996), but as yet genes encoding these or any other fungal adhesive molecules have not been cloned. Indeed, our understanding of the adhesives of phytopathogenic fungi remains limited largely to observations of the site of storage of adhesive material in conidia, the timing of adhesive secretion and the glycoprotein nature of some adhesives (Hamer et al. 1988; Hughes et al. 1999; Kwon and Epstein 1993; Tucker and Talbot 2001; Wright et al. 2002a). It is clear that the extracellular matrix material secreted by fungal conidia contains many different components, as exemplified by surface iodination experiments in Bipolaris sorokiniana which revealed about 40 labelled proteins (Apoga et al. 2001) and by the demonstration of esterases that digest cutin and alter the properties of the host surface (Deising et al. 1992; Gevens et al. 2001). This heterogeneity hampers the identification of fungal adhesins.

A gene encoding a 34-kDa glycoprotein, CBEL, that is expressed in hyphae and binds to cellulose fibres and cellulose in plant cell walls, has been cloned from P. nicotianae (Mateos et al. 1997) but silencing this gene does not affect pathogenicity (Gaulin et al. 2002) and its role in pathogen attachment to host plants remains to be determined. PcVsv1 is one component of the adhesive material stored in and secreted from Phytophthora ventral vesicles during spore attachment and the presence of multiple copies of the TSR1 adhesive motif suggest that PcVsv1 is a Phytophthora spore adhesin. As such, PcVsv1 would be the first gene encoding a spore adhesive to be cloned from a fungal or oomycete plant pathogen. In future studies, confirmation of the role of PcVsv1 in spore attachment and its ligand specificity may be achieved through biochemical studies that demonstrate the specific inhibition of adhesion by PcVsv1-directed antibodies, peptides or other ligands or through transformation and a gene-silencing approach. A better understanding of the molecular basis of adhesion in Phytophthora may provide a foundation for the development of much-needed novel controls of Phytophthora diseases.

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Acknowledgements

We thank V. Maclean and J. Elliott for excellent technical assistance, L. Blackman for Fig. 4a, F. Gubler for Fig. 1g and L. Lange for the production of Plasmopara zoospores.

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  1. Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University, Canberra, ACT 2601, Australia
    Andrea V. Robold & Adrienne R. Hardham

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  1. Andrea V. Robold
  2. Adrienne R. Hardham

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Correspondence toAdrienne R. Hardham.

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Communicated by U. Kück

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Robold, A.V., Hardham, A.R. During attachment Phytophthora spores secrete proteins containing thrombospondin type 1 repeats.Curr Genet 47, 307–315 (2005). https://doi.org/10.1007/s00294-004-0559-8

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