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
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ABSTRACT |
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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.
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INTRODUCTION |
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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 vectors 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.
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METHODS |
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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 antibodyantigen complex (Harlow & Lane, 1988
). The sample was centrifuged for 25 min at 16000 g and the supernatant, which contained unbound antibody, was discarded. The antigenantibody 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 manufacturers 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 [
-32P]dATP. Southern blot hybridizations were performed in 50% formamide using Denhardts 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 manufacturers 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
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
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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Preparation of fusion-protein antisera.
Approximately 70100 µg SDS-PAGE-purified IdpA was mixed with Freunds complete adjuvant and injected into New Zealand White rabbits. Subsequent injections were at two week intervals with the same amount of immunogen mixed with Freunds 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.
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RESULTS |
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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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Identification and properties of the idpA ORF
One complete ORF of 864 nt, potentially encoding 287 aa, was identified on the cloned EcoRIHindIII fragment and designated idpA (Fig. 5). This ORF was preceded by a putative ShineDalgarno 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 3672 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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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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DISCUSSION |
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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 ShineDalgarno 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.
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ACKNOWLEDGEMENTS |
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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.
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REFERENCES |
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Berg, M. & Seemüller, E.(1999). Chromosomal organization and nucleotide sequence of the genes coding for the elongation factors G and Tu of the apple proliferation phytoplasma. Gene 226, 103-109.[Medline]
Berg, M., Davies, D. L., Clark, M. F., Vetten, H. J., Maier, G., Marcone, C. & Seemüller, E.(1999). Isolation of the gene encoding an immunodominant membrane protein of the apple proliferation phytoplasma, and expression and characterization of the gene product. Microbiology 145, 1937-1943.[Abstract]
Berg, M., Yu, J., Melcher, U. & Fletcher, J.(2000). Spiroplasma citri putative adhesin P89: development of serological and molecular markers. Phytopathology 90, S6.
Clark, M. F.(1981). Immunosorbent assays in plant pathology. Annu Rev Phytopathol 19, 83-106.
Clark, M. F., Morton, A. & Buss, S. L.(1989). Preparation of mycoplasma immunogens from plants and a comparison of polyclonal and monoclonal antibodies made against primula yellows MLO-associated antigens. Ann Appl Biol 114, 111-124.
Davies, D. L. & Clark, M. F.(1991). Production and characterization of polyclonal and monoclonal antibodies against peach yellow leafroll MLO-associated antigens. Acta Hortic 309, 275-283.
Davies, D. L., Clark, M. F. & Barbara, D. J. (1999). Cloning and sequencing of the genes determining a major membrane protein associated with the chlorante isolate of aster yellows and clover phyllody. First Internet Conference on Phytopathogenic Mollicutes. http://www.uniud.it/phytoplasma/pap/davi7140.html.
Errampalli, D. & Fletcher, J.(1993). Production of monospecific polyclonal antibodies against aster yellows mycoplasmalike organism-associated antigen. Phytopathology 83, 1279-1282.
Feinberg, A. P. & Vogelstein, B.(1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13.[Medline]
Freitag, J. H.(1964). Interaction and mutual suppression among three strains of aster yellows virus. Virology 24, 401-413.[Medline]
Gaidenko, T. A., Yang, X., Lee, Y. M. & Price, C. W.(1999). Threonine phosphorylation of modulator protein RsbR governs its ability to regulate a serine kinase in the environmental stress signaling pathway of Bacillus subtilis. J Mol Biol 288, 29-39.[Medline]
Gundersen, D. E., Lee, I. M., Rehner, S. A., Davis, R. E. & Kingsbury, D. T.(1994). Phylogeny of mycoplasmalike organisms (phytoplasmas) a basis for their classification. J Bacteriol 176, 5244-5254.[Abstract]
Gundersen, D. E., Lee, I. M., Schaff, D. A., Harrison, N. A., Chang, C. J., Davis, R. E. & Kingsbury, D. T.(1996). Genomic diversity and differentiation among phytoplasma strains in 16S rRNA groups I (aster yellows and related phytoplasmas) and Iii (X-disease and related phytoplasmas). Int J Syst Bacteriol 46, 64-75.[Abstract]
Harlow, E. & Lane, D. (1988). Antibodies: a Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
Jensen, D. D.(1957). Differential transmission of peach yellow leaf roll virus to peach and celery by the leafhopper, Collodonus montanus. Phytopathology 47, 575-578.
Jiang, Y. P., Lei, J. D. & Chen, T. A.(1988). Purification of aster yellows agent from diseased lettuce using affinity chromatography. Phytopathology 78, 828-831.
Kirkpatrick, B. C. (1986). Characterization of Western X-disease mycoplasma-like organisms. PhD thesis, University of California at Berkeley.
Kirkpatrick, B. C., Stegner, D. C., Morris, T. J. & Purcell, A. H.(1987). Cloning and detection of DNA from a nonculturable plant pathogenic mycoplasma-like organism. Science 238, 197-200.
Kirkpatrick, B. C., Fisher, G. A., Fraser, J. D. & Purcell, A. H.(1990). Epidemiological and phylogenetic studies on Western X-disease mycoplasma-like organisms. In Recent Advances in Mycoplasmology , pp. 288-296. Edited by G. Stanek, G. H. Cassell, J. G. Tully & R. F. Whitcomb. New York:Gustav Fisher Verlag.
Krause, D. C.(1996). Mycoplasma pneumoniae cytadherence unravelling the tie that binds. Mol Microbiol 20, 247-253.[Medline]
Kwon, M. O., Wayadande, A. C. & Fletcher, J.(1999). Spiroplasma citri movement into the intestines and salivary glands of its leafhopper vector, Circulifer tenellus. Phytopathology 89, 1144-1151.
Laemmli, U. K.(1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Lefol, C., Caudwell, A., Lherminier, J. & Larrue, J.(1993). Attachment of the flavescence doree pathogen (MLO) to leafhopper vectors and other insects. Ann Appl Biol 123, 611-622.
Lefol, C., Lherminier, J., Boudon-Padieu, E., Larrue, J., Louis, C. & Caudwell, A.(1994). Propagation of flavescence doree MLO (mycoplasma-like organism) in the leafhopper vector Euscelidius variegatus Kbm. J Invertebr Pathol 63, 285-293.
Lim, P. O. & Sears, B. B.(1991). DNA sequence of the ribosomal protein genes rp12 and rps19 from a plant-pathogenic mycoplasma-like organism. FEMS Microbiol Lett 84, 71-74.
Lim, P. O. & Sears, B. B.(1992). Evolutionary relationships of a plant-pathogenic mycoplasma-like organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences. J Bacteriol 174, 2606-2611.[Abstract]
McCoy, R. E., Caudwell, A., Chang, C. J. & 16 other authors (1989). Plant diseases associated with mycoplasma-like organisms. In The Mycoplasmas, pp. 545640. Edited by R. F. Whitcomb & J. G. Tully. New York: Academic Press.
Markham, P. J. & Townsend, R.(1979). Experimental vectors of spiroplasmas. In Leafhopper Vectors and Plant Disease Agents , pp. 413-445. Edited by K. Maramorosch & K. F. Harris. New York:Academic Press.
Milne, R. G., Masenga, V., Lenzi, R., Ramasso, E. & Sarindu, N.(1991). Gold immunolabeling and electron microscopy of mycoplasma-like organisms in plant tissues using pre-embedding and post-embedding techniques. Phytoparasitica 19, 263.
Nakai, K. & Kenehisa, M.(1991). Expert system for predicting protein localization sites in Gram-negative bacteria. Proteins 11, 95-110.[Medline]
Nielson, M. W.(1979). Taxonomic relationships of leafhopper vectors of plant pathogens. In Leafhopper Vectors and Plant Disease Agents , pp. 3-27. Edited by K. Maramorosch & K. F. Harris. New York:Academic Press.
Purcell, A. H., Richardson, J. & Finlay, A.(1981). Multiplication of the agent of X-disease in a non-vector leafhopper Macrosteles fascifrons. Ann Appl Biol 99, 283-289.
Purcell, A. H., Suslow, K. G. & Kirkpatrick, B. C.(1988). Vector transmission of X-disease mycoplasma-like organisms from California. In Stone Fruit Tree Decline, Fourth Workshop Proceedings , pp. 60. Edited by M. V. McKenry. Parlier, CA:US Department of Agriculture.
Razin, S. & Jacobs, E.(1992). Mycoplasma adhesion. J Gen Microbiol 138, 407-422.[Medline]
Rojas, M. R., Zerbini, F. M., Allison, R. F., Gilbertson, R. L. & Lucas, W. J.(1997). Capsid protein and helper component proteinase function as potyvirus cell-to-cell movement proteins. Virology 237, 283-295.[Medline]
Rottem, S. & Kahane, I. (1993). Mycoplasma Cell Membranes. New York: Plenum.
Saeed, E., Rage, P. & Cousin, M. T.(1992). Determination of the antigenic protein size associated with Faba bean phyllody MLO by using (SDS-PAGE) electrophoresis and immunotransfer. J Phytopathol 136, 1-8.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
Seddas, A., Meignoz, R., Daire, X., Boudon-Padieu, E. & Caudwell, A.(1993). Purification of grapevine flavescence doree MLO (mycoplasma-like organism) by immunoaffinity. Curr Microbiol 27, 229-236.
Seemüller, E., Schneider, B., Mäurer, R. & 8 other authors (1994). Phylogenetic classification of phytopathogenic mollicutes by sequence analysis of 16S ribosomal DNA. Int J Syst Bacteriol 44, 440446.[Abstract]
Shevchenko, A., Wilm, M., Vorm, O. & Mann, M.(1996). Mass spectrometic sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68, 850-858.[Medline]
Smart, C. D., Schneider, B., Blomquist, C. L., Guerra, L. J., Harrison, N. A., Ahrens, U., Lorenz, K. H., Seemüller, E. & Kirkpatrick, B. C.(1996). Phytoplasma-specific PCR primers based on sequences of the 16S23S rRNA spacer region. Appl Environ Microbiol 62, 2988-2993.[Abstract]
Ye, F. C., Melcher, U. & Fletcher, J.(1997). Molecular characterization of a gene encoding a membrane protein of Spiroplasma citri. Gene 189, 95-100.[Medline]
Yu, Y.-L., Yeh, K.-W. & Lin, C.-P.(1998). An antigenic protein gene of a phytoplasma associated with sweet potato witches broom. Microbiology 144, 1257-1262.[Abstract]
Received 27 July 2000;
revised 2 November 2000;
accepted 15 November 2000.