An immunodominant membrane protein gene from the Western X-disease phytoplasma is distinct from those of other phytoplasmas

Cheryl L. Blomquist1, Dez J. Barbara2, David L. Davies3, Michael F. Clark3 and Bruce C. Kirkpatrick1

Department of Plant Pathology, University of California at Davis, Davis, CA 95616, USA1
Horticulture Research International, Wellesbourne, Warwickshire CV35 9EF, UK2
Horticulture Research International, East Malling, West Malling, Kent ME19 6BJ, UK3

Author for correspondence: Bruce C. Kirkpatrick. Tel: +1 530 752 2831. Fax: +1 530 752 5674. e-mail: bckirkpatrick{at}ucdavis.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Membrane proteins mediate several important processes, including attachment, in several Mollicute species. Phytoplasmas are non-culturable plant pathogenic mollicutes that are transmitted in a specific manner by certain phloem-feeding insect vectors. Because it is likely that phytoplasma membrane proteins are involved with some aspect of the transmission process, their identification, isolation and characterization are important first steps in understanding phytoplasma transmission. A 32 kDa immunodominant protein (IDP) from the Western X-disease (WX) phytoplasma was purified from infected plants by immunoprecipitation using monoclonal antibodies, and two peptides from a tryptic digest were sequenced. PCR primers designed from these sequences amplified a 145 bp product which hybridized with WX-related phytoplasmas in Southern blots. This PCR product was used to identify a 2·5 kbp EcoRI–HindIII fragment that was cloned and sequenced. A complete 864 bp ORF (idpA) was identified for which the putative translation product contained both of the tryptic digest peptide sequences that were used to design the PCR primers. Analysis of the predicted IdpA sequence indicated two transmembrane domains but no cleavage point. The amino acid sequence had no significant homology with other known phytoplasma IDP genes. The idpA ORF was cloned into an Escherichia coli expression vector and a fusion protein of the predicted size was identified in Western blots using a WX-specific antiserum. A rabbit polyclonal antiserum was prepared to the purified expression protein and this reacted with both the E. coli-expressed and native WX phytoplasma proteins. This newly identified WX IDP (IdpA) is distinct from other known mollicute membrane proteins.

Keywords: phytoplasma, Western X-disease, membrane protein

Abbreviations: AP, apple proliferation; AY, aster yellows; CP, clover phyllody (aster yellows clade); IDP, immunodominant protein; EcIDPS, WX IDP expressed in E. coli lacking the proposed C-terminal domain; SPWB, sweet potato witches’ broom; WX, Western X-disease.

The GenBank accession number for the sequence reported in this work is AF225904.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plant pathogenic phytoplasmas are wall-less bacteria that are members of the class Mollicutes. They have small, A+T-rich genomes. Taxonomically, they are thought to be descended from a Gram-positive clostridial ancestor (Gundersen et al., 1994 ; Lim & Sears, 1992 ; Seemüller et al., 1994 ). Phytoplasmas multiply in the phloem of their plant hosts, and the haemolymph and other tissues of their phloem-feeding insect vectors, many of which are leafhoppers (family Cicadellidae). Phytoplasmas cause hundreds of plant diseases, several of which have world-wide agricultural significance (McCoy et al., 1989 ). Despite numerous attempts, phytoplasmas have not been cultured in vitro and as a consequence there are few molecular genetic tools available to investigate how they cause disease in plants or the mechanisms involved with insect transmission.

In contrast, Mycoplasma spp., which are pathogens or symbionts of animals, insects or other hosts, can be cultured in vitro. Attachment of mycoplasmas to their host cell surfaces is the first step of a multistep disease process. This complex attachment process has been extensively studied for some mycoplasma pathogens and has been the subject of several reviews (Krause, 1996 ; Razin & Jacobs, 1992 ; Rottem & Kahane, 1993 ).

Some Spiroplasma spp., which are helical members of the class Mollicutes, infect both plants and insects, like the phytoplasmas. The interaction between spiroplasmas, phytoplasmas and their insect vectors is highly specific (Nielson, 1979 ). For a plant pathogenic phytoplasma or spiroplasma to infect a leafhopper, several steps are involved. The mollicute must first be ingested from an infected phloem element and then attach to the vector’s gut epithelial cells. It multiplies in gut cells, crosses the intestinal wall and enters the haemolymph where it multiplies and circulates to other tissues. For transmission to a new plant host to occur, it must also penetrate and multiply in the salivary gland before finally being salivated into the plant phloem while the insect feeds. It has been clearly shown that the salivary gland is a specific barrier that a phytoplasma (Purcell et al., 1981 ) or spiroplasma (Markham & Townsend, 1979 ) must pass in order to be transmitted. Multiplication of the flavescence dorée (Fd) phytoplasma has been shown to occur in the midgut, salivary glands and haemolymph of its Euscelidius variegatus vector (Lefol et al., 1994 ). Additionally, it was shown that the Fd phytoplasma attaches specifically to the midgut and salivary glands of its insect vector in in situ assays (Lefol et al., 1993 ). Kwon et al. (1999 ) showed that Spiroplasma citri probably enters Circulifer tenellus gut epithelium by a process of receptor-mediated cell endocytosis. These authors hypothesized that specific spiroplasma membrane proteins recognize receptors on leafhopper gut epithelial cells. Two putative S. citri attachment-protein genes, P58 (Ye et al., 1997 ) and P89 (Berg et al., 2000 ) have been cloned and continue to be investigated.

Because of the important role membrane proteins play in attachment of many mollicutes to their hosts, we hypothesized that phytoplasma membrane proteins are likely candidates for mediating the specific attachment of these pathogens to their insect vectors. Both monoclonal and polyclonal antibodies have been made to several phytoplasmas, which, when used in Western blot analysis, mostly recognize only one or two abundant immunodominant proteins (IDPs). The majority of these IDPs have a molecular mass between 15 and 32 kDa (Clark et al., 1989 ; Errampalli & Fletcher, 1993 ; Jiang et al., 1988 ; Saeed et al., 1992 ; Seddas et al., 1993 ). Four IDP genes from phylogenetically diverse groups of phytoplasmas have been isolated, cloned and sequenced (Barbara et al., 1998 ; Berg et al., 1999 ; Yu et al., 1998 ). Sequence analysis showed that the IDPs possess transmembrane domains that probably localize them on or in the phytoplasma membrane.

The long-term goal of our research is to isolate and characterize phytoplasma membrane proteins that mediate insect transmission. We chose to isolate and characterize an IDP of the Western X-disease (WX) phytoplasma because (i) this product is found at reasonably high concentrations in infected celery, (ii) we have polyclonal and monoclonal antibodies to WX IDP and (iii) efficient insect vectors of the WX phytoplasma can be maintained in an insectary.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Phytoplasmas.
The WX phytoplasma (Jensen, 1957 ) was maintained in celery by serial transmission from diseased to healthy celery plants using the leafhopper vector Colladonus montanus. Vaccinium witches’ broom (kindly provided by E. Seemüller, BBA, Dosenheim, Germany), California prune X (Purcell et al., 1988 ; Kirkpatrick et al., 1990 ), a highly pathogenic strain of Western aster yellows (Freitag, 1964 ), WX (Jensen, 1957 ) and peach rosette (kindly provided by S. Scott, Clemson University, SC, USA) phytoplasma strains were maintained by graft transmission in Catharanthus roseus (Madagascar periwinkle). All healthy and diseased plants were grown in an insect-proof greenhouse.

Antisera and monoclonal antibodies.
The rabbit polyclonal antiserum against the WX phytoplasma was as described by Kirkpatrick (1986) and the 1D9 and 1F3 mouse monoclonal antibodies (both IgG1) were as described by Davies & Clark (1991) . IgG from the polyclonal antiserum was purified by Protein A affinity chromatography using low-salt conditions and eluting from the column with 0·1 M glycine, pH 3·0 (Harlow & Lane, 1988 ). Monoclonal antibodies were ammonium sulfate precipitated, the pellet dissolved in PBS and dialysed overnight against 1·5 M glycine and 3 M NaCl binding buffer. The monoclonal antibodies were purified by binding to a Protein A column and eluted from the columns with 0·1 M citric acid, pH 5·5 (Harlow & Lane, 1988 ).

Purification of IdpA.
Fourteen grams of healthy or WX-infected celery crown tissues were ground in MLO grinding buffer (Kirkpatrick et al., 1987 ) using a cold mortar and pestle. The extract was filtered through cheesecloth, centrifuged at 2900 g for 7 min and the pellet discarded. The supernatant was centrifuged at 19000 g for 25 min and the resulting pellet was suspended in cold phosphate-buffered saline containing 50 mM ascorbic acid (PBSa) and again centrifuged at 19000 g for 25 min. The pellet was resuspended in 0·5 ml PBSa and 200 µg WX polyclonal IgG or 100 µg each of 1D9 and 1F3 were added and the mixture incubated for 4 h at 4 °C. The two monoclonal antibodies, which we believe are specific for two different epitopes on the 32 kDa IdpA (D. L. Davies & M. F. Clark, unpublished results), were pooled to increase the avidity of the antibody–antigen complex (Harlow & Lane, 1988 ). The sample was centrifuged for 25 min at 16000 g and the supernatant, which contained unbound antibody, was discarded. The antigen–antibody complex was resuspended in 0·5 ml PBSa containing magnetic immunocapture beads coated with anti-mouse IgG1 (Dynal). After incubation for 12 h at 4 °C, the magnetic beads were collected, separated from the plant debris and washed three times in PBSa. Proteins bound to the beads were eluted by incubating at 60 °C for 10 min with 50 µl 2x SDS-PAGE sample buffer (Laemmli, 1970 ). A 30 µl sample of the eluted proteins were size fractionated by discontinuous SDS-PAGE and visualized by Coomassie blue staining.

SDS-PAGE.
Discontinuous SDS-PAGE was performed using 12% acrylamide gels (Laemmli, 1970 ). For final purification of the Escherichia coli expressed IdpA, 1·5 mm, 12% polyacrylamide single-well preparative gels were used.

Peptide sequencing.
To obtain N-terminal sequence, the SDS-PAGE-resolved proteins were transferred to a PVDF membrane (Bio-Rad) in 10 mM CAPS, pH 11. Membrane pieces containing the IDP were excised and the IDP sequenced by Edman degradation using an ABI model 477A or a 470 sequencer. For tryptic peptide digestion, the SDS-PAGE gel was stained for 3 h in Coomassie blue and destained in 50% methanol. The 32 kDa band was excised, subjected to tryptic digestion (Shevchenko et al., 1996 ) and the resulting peptideswere size fractionated using microbore HPLC (Gaidenko et al., 1999 ). The sequences of four peptides were determined as described above but using an on-line HPLC.

Degenerate PCR primers.
Degenerate PCR primers were designed from the amino acid sequence of two of the tryptic digest peptides from IdpA. Primers were designed with due regard to limited information on codon usage in phytoplasmas (Lim & Sears, 1991 ; Gundersen et al., 1996 ; Berg & Seemüller, 1999 ). WX-enriched DNA from infected celery was used as the template in a PCR reaction using conditions previously described (Smart et al., 1996 ), except that the annealing temperature was reduced to 45 °C and the extension time was increased to 3 min.

DNA preparation.
DNAs were extracted using a phytoplasma enrichment procedure (Kirkpatrick et al., 1987 ) from stems and leaves of WX-infected celery (Jensen, 1957 ) or Cat. roseus (all other phytoplasmas).

Cloning of the PCR product and Southern hybridization.
The PCR product obtained using the degenerate primers was cloned using the TA TOPO cloning kit (Invitrogen) according to the manufacturer’s instructions. Recombinant plasmids were selected by size and sequenced. The insert, released from the plasmids by digestion with EcoRI, was gel purified (Prep-A-Gene; Bio-Rad) and 50 ng insert DNA was random-primer labelled (Feinberg & Vogelstein, 1983 ) using [{alpha}-32P]dATP. Southern blot hybridizations were performed in 50% formamide using Denhardt’s buffer and standard protocols (Sambrook et al., 1989 ) with incubation overnight at 42 °C. The membranes were washed twice in 0·2xSSC containing 1 g SDS l-1 at 55 °C.

Cloning of the IDP-containing gene fragment.
Approximately 2 µg WX-infected celery DNA was digested with EcoRI/HindIII and size fractionated by electrophoresis in a 1% low-melting-point agarose gel using TAE buffer. Gel pieces containing DNA fragments between 2 and 3 kbp were excised and solubilized with ß-agarase 1 according to the manufacturer’s instructions (USB). EcoRI/HindIII-digested plasmid (pUC18) was gel purified and ligated to the size-selected WX DNA fragments using standard protocols (Sambrook et al., 1989 ). Ligated products were electroporated into E. coli DH5{alpha} cells and recombinants were selected by colony hybridization using the cloned, 32P-labelled 145 bp PCR product as a probe.

Nucleotide sequencing and sequence analysis.
DNA sequencing used standard protocols on a PE Applied Biosystems 377 DNA Sequencer with 96-well upgrades. Seqweb version 1.1 was used with the Wisconsin Package version 10 (Genetics Computer Group) for all sequence analyses except PSORT (Nakai & Kenehisa, 1991 ).

Subcloning of idpA into an expression vector.
idpA lacking the C-terminal hydrophobic region was PCR amplified using primers wxES and wxB, and the full length gene using primers wxEL and wxB (Table 1). PCR was performed in 100 µl reaction mixture containing 200 ng target plasmid ES-4 or EL-6 (see Results), 4 mM MgCl2, 2·5 U Pfu polymerase (Stratagene), 0·15 mM dNTPs (Pharmacia) in 1xPfu buffer. Thermocycling parameters were 94 °C for 5 min, followed by 25 cycles of 94 °C for 1 min, 45 °C for 1 min and 72 °C for 3 min, followed by a final 8 min extension at 72 °C. The PCR products were purified (QIAquick PCR purification kit; Qiagen), digested with BamHI/EcoRI and small fragments removed by ethanol precipitation. The expression vector pRSETB (Invitrogen) was digested with BamHI, EcoRI and PvuII (the latter to prevent religation of the vector). Both the full-length and truncated IDP genes were ligated to pRSETB and this was electroporated into E. coli DH5{alpha} cells using standard protocols (Sambrook et al., 1989 ). Transformants were selected on 2xYT medium (Rojas et al., 1997 ) containing 100 µg ampicillin ml-1, and recombinants were selected by size and sequenced.


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Table 1. Sequence of PCR primers and peptides used in this study

 
Expression and purification of an IdpA fusion protein.
The selected plasmids, pRSET-WXES or pRSET-WXEL, were transformed into E. coli JM109 D3 cells by electroporation according to manufacturer’s protocols (Bio-Rad) and grown overnight on 2xYT medium containing 100 µg ampicillin ml-1. A single colony of JM109 D3(pRSET-WXES) was selected and grown overnight at 37 °C with shaking at 250 r.p.m. in 250 ml 2xYT containing 100 µg ampicillin ml-1. Induction of expression and purification of the fusion proteins was done as described by Rojas et al. (1997) , except that after induction the culture was incubated for 5 h before harvesting and lysing the cells. Because SDS-PAGE analysis showed that the expressed protein was not present in inclusion bodies, the cell membrane-containing fraction (Rojas et al., 1997 ) was extracted directly with 0·1 M NaHCO3, pH 9, 1 g SDS l-1. The solubilized protein lysate was purified by nickel column chromatography (Rojas et al., 1997 ) and 10 µl of each 0·5 ml fraction was checked by SDS-PAGE. The fusion-protein-containing fractions were further purified by preparative SDS-PAGE. The edges of the gel were stained, the location of the fusion protein in the unstained gel determined and the band excised. The excised gel strips were lyophilized and ground to a fine powder before injecting into rabbits.

Preparation of fusion-protein antisera.
Approximately 70–100 µg SDS-PAGE-purified IdpA was mixed with Freund’s complete adjuvant and injected into New Zealand White rabbits. Subsequent injections were at two week intervals with the same amount of immunogen mixed with Freund’s incomplete adjuvant. Sera were collected at two week intervals after the second injection and tested by double-antibody sandwich ELISA. The serum with the highest titre was used for the Western blot analyses.

Western blot and ELISA analysis of IdpA fusion proteins.
Proteins from healthy and WX-infected celery, and fusion-protein-containing E. coli were separated by SDS-PAGE and transferred onto nitrocellulose in Towbin transfer buffer (Sambrook et al., 1989 ) at 100 V for 1 h. After blocking with powdered non-fat dry milk in PBS at a concentration of 10 g l-1, the membrane was incubated with either the anti-IDP antiserum (1:500) or the monoclonal antibodies 1D9 and 1F3 (10 µg ml-1) for 2 h at room temperature. Alkaline phosphatase-labelled goat anti-rabbit antibodies (1:500) (Sigma) or alkaline phosphatase-labelled goat anti-mouse antibodies (1:3000) (Bio-Rad) were added and the membrane incubated at room temperature for 1·5 h. Bound enzyme was detected using NBT/BCIP (Bio-Rad) as the substrate.

A double-antibody sandwich ELISA was performed as described by Clark (1981) with 1D9 and 1F3, each at 10 µg ml-1, as the trapping antibodies. The anti-IDP antiserum was diluted 1:2700 in PBS. The reporter antibody was an alkaline phosphatase-labelled goat anti-rabbit diluted 1:5000 (Sigma) and p-nitrophenyl phosphate was used as the substrate.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Western blot analysis using antiserum against plant-derived antigen
Two predominant proteins of 32 kDa and 20 kDa were detected using the anti-WX polyclonal antibody in Western blots of WX-infected celery proteins (Fig. 1). No proteins of these molecular masses were detected in healthy celery.



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Fig. 1. Western blot of extracts from WX-infected and healthy plants using a polyclonal antiserum raised against plant-derived WX phytoplasma antigen. Arrows (with approximate molecular masses) indicate the two major antigenic WX proteins. Lanes: 1, WX-infected celery; 2, healthy celery.

 
Immunoprecipitation and sequencing of the 32 kDa WX IDP
Because purification by immunoprecipitation using the WX polyclonal antibody gave insufficient yields of the IDPs for sequencing (results not shown), the two monoclonal antibodies to the WX phytoplasma, 1D9 and 1F3, were used. SDS-PAGE profiles of proteins that were eluted from the immunocapture magnetic beads are shown in Fig. 2. The two most abundant, non-immunoglobulin proteins that were isolated corresponded to the same molecular masses, 32 and 20 kDa (Fig. 1). Although some healthy celery proteins bound non-specifically to the magnetic beads, including one which co-migrated with the 32 kDa protein (lane H, Fig. 2), none of these proteins reacted with the WX rabbit polyclonal antiserum in Western blots (data not shown). As expected, mouse IgG1 proteins were also present in the WX-infected celery protein magnetic bead eluant (lane WX, Fig. 2). This experiment was repeated twice with similar results obtained each time.



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Fig. 2. Immunocapture purification of WX proteins using WX monoclonal antibodies, 1F3 and 1D9. Lanes: S, molecular mass standards; IgG, Protein A purified 1F3 IgG (10 µg); H, healthy celery plant proteins; WX, WX-infected celery plant proteins. Arrows indicate the two major WX-derived antigens. Note that a small amount of another healthy celery protein of the same apparent molecular mass co-migrated with the 32 kDa WX-derived protein from infected celery.

 
After transfer to a PVDF membrane, N-terminal sequencing of the 32 kDa protein was attempted twice and failed both times, possibly due to a blocked N terminus. The 32 kDa protein was digested with trypsin and the resulting peptides were size separated by HPLC. Two of the HPLC-purified digested peptides were N-terminally sequenced (Table 1). The 20 kDa protein was not analysed further.

Degenerate PCR primers for amplifying the gene encoding the 32 kDa WX IDP
Degenerate primers wxp1 and wxp6 were designed using the amino acid sequence data from the tryptic peptides (Table 1). Using wxp1 and wxp6 as PCR primers, a WX-specific 145 bp product was amplified from WX-infected celery (Fig. 3). No 145 bp PCR product was amplified from healthy celery although a 300 bp product was detected routinely (Fig. 3).



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Fig. 3. PCR amplification products from WX-infected celery leaf DNA using degenerate primers based on the WX 32 kDa protein sequence. Lanes: 1, healthy celery DNA; 2, WX-infected celery DNA. Arrows indicate the 145 bp WX-derived band and the 300 bp band routinely amplified from healthy plant extracts.

 
Cloning and specificity of the 145 bp PCR product
To confirm that the 145 bp PCR product was phytoplasma-specific, it was cloned, sequenced and used as a hybridization probe against restriction endonuclease-digested DNAs from different phytoplasmas. The 32P-labelled 145 bp insert hybridized with a 5·5 kbp EcoRI fragment from WX, peach rosette and prune X strains, but did not hybridize to DNA from vaccinium witches’ broom, healthy celery (Fig. 4), aster yellows, elm yellows or healthy Cat. roseus (not shown).



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Fig. 4. Southern blot of EcoRI-digested DNAs from healthy and phytoplasma-infected plants using the cloned 145 bp WX-specific PCR fragment as a probe. Lane: 1, peach rosette; 2, prune X; 3, WX; 4, vaccinium witches’ broom; 5, healthy celery.

 
Cloning of the gene for the 32 kDa WX IDP
Because of the low G+C content of the WX phytoplasma genome, many restriction enzymes with sites in the multiple cloning sites of standard vectors will not digest phytoplasma DNA, or will do so only infrequently. To maximize the chances of generating fragments that were of a suitable size for cloning and likely to contain the idpA gene, several pairwise combinations of restriction endonucleases were tested. A 2·5 kbp EcoRI–HindIII WX DNA fragment hybridized with the 145 bp PCR product (data not shown). DNA fragments (2–3 kbp) of EcoRI/HindIII-digested WX DNA were purified from agarose gels and ligated to EcoRI/HindIII-digested pUC18. Recombinants were selected by colony and Southern blot hybridization using the 32P-labelled, 145 bp cloned PCR product. One of the positive hybridization recombinants, WX389, was selected and fully sequenced on both strands.

Identification and properties of the idpA ORF
One complete ORF of 864 nt, potentially encoding 287 aa, was identified on the cloned EcoRI–HindIII fragment and designated idpA (Fig. 5). This ORF was preceded by a putative Shine–Dalgarno sequence (5'-AAAGGA-3') and putative -10 RNA polymerase binding site (Fig. 5). No putative -35 RNA polymerase binding site was identified. The ORF began with an ATG start codon and terminated with two TAA stop codons. There was an inverted repeat, which may signal rho-independent termination, located 36–72 bp downstream of the beginning of the TAA stop codon. As expected, the ORF sequence had a low G+C content (26·6 mol%) with a corresponding bias to use A+T-rich codons. The idpA ORF corresponded to a protein of 32769 Da with a pI of 10·01. The most abundant amino acid was lysine at 13·9 mol% followed by isoleucine (12·2 mol%), threonine (11·2 mol%) and leucine (9·4 mol%). There was one tryptophan present and it was coded by TGG. There were no cysteines. When the putative translation product of the ORF was compared with the sequences of the two tryptic peptides (shown in Table 1) obtained from the immunocapture-purified IdpA, each peptide matched exactly a portion of the translated idpA ORF.



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Fig. 5. Schematic representation of the ORFs and proposed ribosome-binding sites on insert DNA in WX389. S-D, putative Shine–Dalgarno sequence; -10, putative -10 promoter region; IDP, immunodominant protein gene; ORF 2, putative DNA polymerase III partial gene.

 
PSORT (Nakai & Kenehisa, 1991 ) predicted two transmembrane domains at aa 9–29 and 251–281. The first of these was predicted to be a non-cleavable, N-terminal signal sequence. The 221 aa domain between the two transmembrane regions was predicted to be hydrophilic and relatively neutral (predicted pI 7·36). These data suggest that IdpA is a membrane protein with the hydrophilic central domain held on the outside of the cell surface with only short terminal regions within the cell. Database BLAST searches with both the ORF nucleic acid sequence and the predicted translation product showed no significant similarities to any other entries or to any of the phytoplasma IDPs that have been previously characterized (Table 2).


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Table 2. Comparison of IdpA with other mollicute membrane proteins

 
WX dnaX ORF
A 606 bp partial ORF was located on the opposite strand 536 bp from the start of the IDP gene (ORF2, Fig. 5). No other potential ORFs were identified on either strand in any reading frame. Database searches using BLASTP suggested that this ORF encodes a DNA polymerase III. Using GAP (Wisconsin GCG) to align the sequences, the partial WX ORF (dnaX) was shown to share 51% amino acid sequence identity with the tau and gamma subunits of DNA polymerase III from Bacillus subtilis and 46% amino acid sequence identity with the DNA polymerase III from Mycoplasma genitalium and Mycoplasma pneumoniae. The accession numbers for the DNA sequences encoding these B. subtilis, M. genitalium and M. pneumoniae proteins are S13786, D64246 and S73550 respectively. There was a putative Shine–Dalgarno sequence of 5'-AAAGGA-3' located 11 bases upstream and a putative -10 RNA promoter sequence of 5'-TATAAT-3' located 40 bp upstream from the ATG start codon of WX dnaX. No clear -35 sequence was identified for dnaX.

Expression of the gene encoding IDP in E. coli
Primers wxEL and wxB (Table 1) were used to amplify the entire idpA ORF and the primers wxES and wxB (Table 1) were used to amplify the IDP gene ORF without the putative C-terminal transmembrane domain, using the 2·5 kb WX389 clone as a template. The wxES and wxEL primers introduced an EcoRI site on the C-terminal end of the amplicon and the wxB primer a BamHI site on the N-terminal end. These primers were designed to allow cloning in-frame with the vector His-tag site. The PCR products obtained from both sets of primers (wxEL/wxB; wxES/wxB) were digested with EcoRI/BamHI and ligated into the fusion-protein expression vector pRSETB. Recombinants were selected by size of the cloned insert and the insert sequenced. The selected clones were designated pRSETWXEL (full-length IDP) and pRSETWXES (truncated IDP), and following induction with IPTG, produced fusion proteins of the expected sizes. Both fusion proteins reacted with the WX polyclonal antiserum in Western blot analysis (data not shown). The smaller fusion protein from pRSETWXES, which lacked the proposed C-terminal transmembrane domain, was designated EcIDPS (E. coli expressed IDP, short form) and was chosen as the immunogen for rabbits because its protein yield was greater than that obtained from the full-length fusion protein produced from pRSETWXEL. The monoclonal antibodies 1F3 and 1D9 reacted with the EcIDPs and the 32 kDa IdpA from celery in Western blots (Fig. 6). Neither monoclonal antibody reacted with the 20 kDa protein from WX-infected celery in Western blots, but could immunocapture this protein in its native form from solution (Fig. 2). No reaction occurred with the monoclonal antibodies used in Western blots against healthy celery proteins or E. coli proteins from JM109 cells containing vector alone (Fig. 6).



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Fig. 6. Western blot of EcIDPS, healthy and WX-infected celery proteins probed with the monoclonal antibodies 1F3 (lanes 1–4) and 1D9 (lanes 5–8). Lanes: 1 and 5, proteins purified from E. coli containing pRSET only; 2 and 6, proteins purified from E. coli containing pRSETWXES; 3 and 7, healthy celery; 4 and 8, WX-infected celery. Note that neither 1F3 nor 1D9 reacted with the 20 kDa protein by Western blotting (compare with Fig. 2).

 
The polyclonal antiserum produced from the purified EcIDPS fusion protein reacted with the expressed EcIDPS protein and the native WX phytoplasma protein in double-antibody sandwich ELISA. ELISA A405 readings were 1·04±0·16 (±standard deviation; n=3) from WX-infected periwinkle and 0·05±0·01 (n=3) from healthy periwinkle. The antiserum prepared against EcIDPS also reacted with EcIDPS and IdpA proteins from plants in Western blots (Fig. 7). Only minor background reactions occurred with preimmune serum or EcIDPS antiserum used in Western blots against healthy celery proteins or E. coli proteins from JM109 cells containing vector alone (Fig. 7).



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Fig. 7. Western blot of healthy and WX-infected celery proteins probed with polyclonal antiserum prepared against the WX fusion protein, EcIDPS. Lanes: 1, WX-infected celery; 2, healthy celery; 3, proteins purified from E. coli containing pRSET only; 4, proteins purified from E. coli containing pRSETWXES. Lanes 5–8 are the same protein preparations probed with pre-immune antiserum. Note that the strong positive band in lane 4 representing the expressed IDP has a larger apparent molecular mass than that of the plant-derived antigen in lane 1 due to the presence of a short tag (33 amino acids; 3·8 kDa) sequence from pRSET as a fusion protein with IdpA.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Several phytoplasma IDPs have been identified using monoclonal and polyclonal antibodies (Clark et al., 1989 ; Errampalli & Fletcher, 1993 ; Jiang et al., 1988 ; Saeed et al., 1992 ; Seddas et al., 1993 ). Monoclonal and polyclonal antibodies raised to phytoplasmas have generally been found to be quite specific. They react with the phytoplasma strain they were made from and occasionally with other members of the same phylogenetic clade, but not with phytoplasmas in other clades. The monoclonal antibodies used in this study, 1D9 and 1F3, reacted by double-antibody sandwich ELISA with peach rosette and all strains of WX tested, but not with vaccinium witches’ broom (data not shown). This is interesting because vaccinium witches’ broom is phylogenetically very similar to WX and peach rosette based on 16S rRNA gene sequence (Seemüller et al., 1994 ) and our Western blot results suggest much more rapid divergence in the IDP genes of these phytoplasmas than the rRNA genes. Hybridization analyses using the 145 bp idpA-derived PCR product as the probe agreed with these serological results as it hybridized to DNA from all members of the WX clade tested except vaccinium witches’ broom. At least in this region, the vaccinium witches’ broom IDP gene is clearly different from the WX and peach rosette IDP genes.

Although Western blot analyses have identified IDPs of numerous phytoplasmas, the corresponding genes from only five phytoplasmas have been cloned and sequenced (Barbara et al., 1998 ; Berg et al., 1999 ; Davies et al., 1999 ; Yu et al., 1998 ). These are the IDPs from the WX, apple proliferation (AP), sweet potato witches’ broom (SPWB), the chlorante strain of aster yellows (AY) and clover phyllody (CP) phytoplasmas (the last two are from the aster yellows phylogenetic clade). There are some similarities between some of these phytoplasma IDPs, but also many differences in both DNA and amino acid sequence, as well as their predicted secondary structures.

Structural properties of the WX IDP compared with other phytoplasma proteins
All of the IDP genes isolated from phytoplasmas have a high lysine content ranging from 11·0 mol% for CP to 14·6 mol% in the AY IDP. In all five cases, tryptophan was encoded by TGG rather than the unusual TGA codon, normally a termination signal, which occurs in Mycoplasma spp., Spiroplasma spp. and some other mollicutes. This was expected as the TGG codon is known to be used for tryptophan in other phytoplasma proteins such as the ribosomal proteins (Gundersen et al., 1996 ; Lim & Sears, 1991 ), fus and tuf genes (Berg & Seemüller, 1999 ). Like P1 in M. pneumoniae, the WX, AP, CP and SPWB IDPs contain no cysteines, but the AY IDP has two cysteines (Barbara et al., 1998 ; Davies et al., 1999 ).

The hydrophobicity profiles of the AP and SPWB IDPs are similar, having a hydrophobic transmembrane region near the N terminus, with the remainder of the protein being relatively hydrophilic. The hydrophobicity profiles of CP, AY and WX IDPs were similar to each other but different from those of AP and SPWB. In these three IDPs there were two hydrophobic regions, one near the C terminus and one near the N terminus, while the central domain was largely hydrophilic. Both the AP and SPWB proteins seem to lack a cleavable N-terminal signal peptide and contain one hydrophobic sequence. Berg et al. (1999) suggested that these proteins are anchored in the phytoplasma cell membrane by the N-terminal transmembrane region with the remaining, more hydrophilic, part of the protein outside the cell. The CP and AY proteins are not predicted to be lipoproteins, and seem to possess cleavable N-terminal signal sequences and hydrophobic C-terminal anchors, suggesting a final orientation of the C terminus anchored in the membrane, the central hydrophilic domain outside the cell membrane and the N-terminal signal sequence removed during export (Barbara et al., 1998 ; Davies et al., 1999) . IdpA appears to have an non-cleavable N-terminal signal sequence, which suggests that both the N- and C-terminal transmembrane regions are anchored in the cell membrane, probably with the central hydrophilic domain on the outer cell surface and the two short regions at the extreme ends of the protein inside the cell.

Sequence similarity of idpA with other phytoplasma IDP genes
When the idpA sequence was compared to the four other phytoplasma IDP sequences, no significant amino acid similarity was found (Table 2). However, similarities have been found between other phytoplasma IDPs. The chlorante strain of AY and the closely related CP phytoplasma have significant similarity near the N and C termini, but not in the hydrophilic central domain (Barbara et al., 1998 ). The molecular masses of the IDPs vary from 32·8 kDa for WX to 17·6 kDa for CP and their predicted pIs from 8·9 to 10·3 (Table 2). Although the WX and the AY IDPs share similar hydrophobicity profiles, there are significant differences between these genes. Not only are the primary amino acid sequences quite different, but the genes appear to be in different locations in the genomes. A chaperonin homologue is located directly upstream from the coding sequence of the AY IDP (Davies et al., 1999 ; D. J. Barbara, A. Morton, M. F. Clark & D. L. Davies, unpublished) whereas immediately upstream from idpA there is a putative DNA polymerase III gene and no chaperonin. Using the Coilscan program to identify possible coiled-coil structures, four coiled-coil sequences were predicted in IdpA, but no coiled-coil regions were found in any of the other phytoplasma IDPs. BLAST searches indicated homology between IdpA and a large number of proteins which possess coiled-coil regions. In all cases, the homology was only between the coiled-coil regions in the proteins, not other regions with different predicted secondary structure. Further study is necessary to understand the significance of the coiled-coil regions in IdpA.

All of the IDP genes characterized to date have putative Shine–Dalgarno ribosome-binding sites. As with idpA, Yu et al. (1998) found an inverted repeat downstream of the TAA stop codon for the SPWB IDP gene.

Membrane proteins play an important role in the attachment of many mycoplasma pathogens to their hosts. Although the target host cells are different, it is likely that phytoplasmas also possess cell-surface proteins which mediate a highly specific interaction between pathogen and host cells. The IDPs that are present on the surface of the phytoplasma are good candidates for interacting with insect host cells because they are apparently abundant, and both sequence analysis and electron microscopy studies (Milne et al., 1991 ) strongly suggest they are localized on the outside of the phytoplasma plasmalemma. Studies are now under way to investigate the possible role of the WX 32 kDa IDP in phytoplasma/host interactions.


   ACKNOWLEDGEMENTS
 
We would like to thank Alexander Purcell and Stuart Saunders for providing the Western X-disease infected celery, Erich Seemüller for the vaccinium witches’ broom and S. Scott for the peach rosette isolates used in these studies.

This work was funded in part by the California Cling Peach Advisory Board and the California Integrated Pest Management program. D.J.B., D.L.D. and M.F.C. were funded by the Biotechnology and Biological Sciences Research Council.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 27 July 2000; revised 2 November 2000; accepted 15 November 2000.