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Blackwell Science Ltd

Characterization of homologues of the apple proliferation immunodominant membrane protein gene from three related phytoplasmas

ANNE MORTON

1

DAV ID L DAV IES

2

CHERYL L BLOMQUIST

3

dagger AND DEREK J BARBARA

1

1

Sustainable Disease Resistance Horticulture Research International Wellesbourne Warwickshire CV35 9EF UK

2

Entomology and Plant Pathology Horticulture Research International East Malling West Malling Kent ME19 6BJ UK

3

Department of Plant Pathology University of California Davis CA 95616 USA

SUMMARY

Homologues of the immunodominant membrane protein genefrom apple proliferation (AP) phytoplasma have been cloned andsequenced for three further members of the AP subclade namelyEuropean stone fruit yellows peach yellow leaf roll and a Euro-pean isolate of pear decline (PD) The putative translation prod-ucts of all three were similar in size to that of AP and all hada transmembrane region towards the N-terminus and a largeC-terminal hydrophilic domain probably held on the outside of thecell membrane

in vivo

Sequence similarities for the putative pro-teins were compared with interrelationships of the phytoplasmasas measured by rRNA gene sequence similarity The proteins fromAP and PD were more similar (57 identical in the major hydro-philic domain) than those for any other pair (31ndash34) but thesetwo phytoplasmas were not more closely related by rRNA genesequences than other pairs The possibility that the relativesimilarities of these proteins is related to the host is discussedIt is suggested that the similarity of the AP and PD proteins mayreflect the fact that these two proteins have narrow plant hostranges in two closely related genera in the tribe Maloideae (fam-ily Rosaceae) whilst the other two have broader host ranges

mainly in the tribe Prunoideae

INTRODUCTION

Phytoplasmas are important plant pathogens (McCoy

et al

1989)which have been relatively little studied at the cellular and molecu-lar levels Current phytoplasma classification is based largely on16

S

rRNA gene sequences (Seemuumlller

et al

1998) and phyto-plasmas form a monophyletic clade with a number of subclades

within the Anaeroplasma division of the order Mollicutes Noneof these wall-less prokaryotes has yet been cultured

in vitro

despite their ability to grow in both plant and insect hosts Thebasis of the ability to grow

in vivo

is unknown but it has beenproposed (Barbara

et al

1998) that attachment to host cells isrequired for growth It has been found with the flavescence doreacuteephytoplasma that some component in pathogen-enrichedpreparations from infected plants binds to insect tissues as mightbe expected from this suggestion (Lefol

et al

1993) Serologicalstudies have shown that generally phytoplasmas possess singlereasonably abundant membrane proteins that are immuno-dominant (eg Clark

et al

1983 Keane

et al

1996) and locatedprimarily on the external surface of the cell (Milne

et al

1995)These proteins are candidates for involvement in hostndashpathogeninteractions Such interactions would be analogous to those ofcytadhesins in some mycoplasmas (Razin and Jacobs 1992) andof the adhesion-related protein SARP1 (P89) in

Spiroplasma citri

(Berg

et al

2001 Yu

et al

2000)Genes encoding the immunodominant membrane proteins

have been cloned from a small number of phytoplasmas namelysweet potato witchesrsquo broom (SPWB Yu

et al

1998) apple pro-liferation (AP Berg

et al

1999) Western X (WX Blomquist

et al

2001) and aster yellows (AY) and clover phyllody (CP) (Barbara

et al

2002) Although all are predicted to have large hydrophilicextracellular domains the genomic locations of the genes andthe predicted protein structures suggest that they probably rep-resent three non-homologous proteins The proteins from AY andCP have cleavable N-terminal peptide sequences and transmem-brane regions near the C-terminals (Barbara

et al

2002) Thatfrom WX has non-cleaved transmembrane regions near bothterminals (Blomquist

et al

2001) and those from AP and SPWBhave only non-cleaved C-terminal transmembrane regions (Berg

et al

1999 Yu

et al

1998) Only for the AY subclade have mem-brane protein genes been cloned from more than one phyto-plasma namely AY and CP These two proteins have similargeneral structures and clear sequence similarities in the leaderregions and in the transmembrane regions but there is very little

Correspondence

E-mail DezBarbaraHRIACUKdagger

Present address

CDFA Plant Diagnostic Center 3294 Meadowview Road SacramentoCA 95832 USA

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similarity between the major hydrophilic domains (Barbara

et al

2002) A short sequence in the AY hydrophilic domain shows aclear match to an immunoglobulin G (IgG) binding site from

Streptococcus

protein G (Barbara

et al

2002) but whether thisis related to the function of the AY protein is not known

As no other within-taxon comparison is available it is not clearwhether this high level of divergence in the hydrophilic domainsof the AY and CP proteins is related to the degree of relationshipbetween the phytoplasmas or if it may be correlated with someother property such as the host In this paper we report the clon-ing of presumed homologues of the AP immunodominant mem-brane protein (

imp

) gene from three additional phytoplasmas inthe AP subclade (Seemuumlller

et al

1998) namely European stonefruit yellows (ESFY) peach yellow leaf roll (PYLR) and pear decline(PD) These are compared with the AP gene (Berg

et al

1999)and the structurally similar and probably homologous proteinfrom SPWB a member of the peanut witchesrsquo broom subclade (Yu

et al

1998) The relative similarities between both the genes andpredicted proteins are also compared with the apparent related-ness of the phytoplasmas as determined by rRNA sequences

RESULTS AND DISCUSSION

Analysis of genes from ESFY PD and PYLR

The sequences derived from ESFY and PD each contained a singlelarge open reading frame (ORF) with the expected initiation

codon (ATG) Comparison of the PYLR sequence with those fromESFY PD and AP suggested that it had a similar ORF but that theinitiation codon was GTG a known alternative bacterial transla-tion initiation codon (Elzanowski and Ostell 2000) As expectedall three newly cloned genes had low G + C contents (247ndash264 mol similar to AP and SPWB 263 mol and 274 molrespectively) Potential ribosome binding sites and transcriptionsignals were located upstream of the ORFs (Fig 1) For compar-ative purposes schematics of the equivalent genes are shown forAP and SPWB and some features for these have been reportedpreviously (Berg

et al

1999 Yu

et al

1998) Imperfect invertedrepeats that could be arranged as hairpin structures with fewloops (that from PD shown as an example in Fig 2) were locateddownstream of the three ORFs and also that of AP (Fig 1) Theserepeats may act as transcription terminators When aligned therepeat sequences were found to be highly conserved betweenPD PYLR and AP and on the basis that they are functionally

Fig 1 Schematic representation of open reading frames (ORFs) putative translation products and other features for four phytoplasmas in the apple proliferation (AP) clade and sweet potato witchesrsquo broom (SPWB) Sequences of AP and SPWB and some features (italics) from Berg et al (1999) and Yu et al (1998) ESFY European stone fruit yellows PD pear decline PYLR peach yellow leaf roll

Fig 2 Imperfect inverted repeats in the pear decline (PD) sequence Limits of repeats were defined by comparison with European stone fruit yellows (ESFY) peach yellow leaf roll (PYLR) and apple proliferation (AP) sequences on the assumption that they are functionally equivalent The position of these repeats in the overall sequence is shown in Fig 1

Membrane protein homologues from three phytoplasmas

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equivalent the limits of the repeats were defined by sequence iden-tity between all four phytoplasmas in the AP subclade Howevervarying numbers of nucleotides closer to the stop codon could besimilarly aligned (as close as 6 bp in ESFY) The ESFY repeats wereless strongly conserved relative to the other three sequences thanthese were to each other Whilst the first section (equivalentto the first 22 nt in Fig 2) and its complement were very similarto PD the central 37 nt were less similar and gave far lowerlevels of base pairing than the equivalent sequence in the otherphytoplasmas In SPWB only relatively short repeats furtherdownstream have been identified and parts of these may form arho-independent terminator (Yu

et al

1998)

Comparisons of predicted proteins

The putative translation products of the three genes cloned herewere very similar in size and domains both to each other and tothat of AP (Berg

et al

1999) (Fig 1) The proteins are predictedto have extracellular hydrophilic domains held by a transmem-brane region close to the N-terminal with only short hydrophilicintracellular terminal domains (8ndash9 aa) None appeared to havecleavage sites associated with the transmembrane region (Nakaiand Kanehisa 1991 Nielsen

et al

1997 von Heijne 1990 computeranalysis using psort version 64) nor did any have a C-terminaltransmembrane region The large hydrophilic domains of the PDand AP putative proteins were slightly shorter (131 and 130 aarespectively) than those of ESFY and PYLR (133 and 134 aarespectively) (Fig 1) Between the four AP subclade phyto-plasmas sequence identities for the putative translation prod-ucts ranged from 36 to 61 The large C-terminal hydrophilicdomains were more different from each other than were the com-bined N-terminaltransmembrane domains which for PD andPYLR were identical The hydrophilic domain from SPWB showedonly low sequence identities with those of the four members ofthe AP subclade studied (Table 1) These differences were mir-rored at the nucleic acid level (Table 1)

Similarities in rRNA gene and membrane protein sequences

Similarities for the 16

S

rRNA sequences varied from 981 to99 between members of the AP subclade and were

c

88between them and SPWB (Table 1) For the same rRNA genesequences AY and CP were 989 identical There was no corre-lation between the 16

S

rRNA sequence similarities and the per-centage amino acid identities within the hydrophilic domain ofthe putative translation products of the four AP subclade phyto-plasma genes (Fig 3) However for AP and PD the hydrophilicdomains were clearly more similar to each other than were thoseof any other pairing suggesting a correlation with some factorother than phylogenetic relationship

Table 1 Sequence identities () for two parts of the apple proliferation (AP) imp gene homologues and their putative translation products from five phytoplasmas (below left proteins in bolditalic and nucleic acids in plain type) and sequence similarities for part of the 16S rRNA genes (above right)

ESFY PD PYLR AP SPWB

ESFY HD ndash 981 983 985 875 16S rRNA

ICTM ndash

PD HD 49 ndash 990 984 880 16S rRNA33

ICTM 88 ndash82

PYLR HD 49 54 ndash 985 877 16S rRNA32 31

ICTM 88 98 ndash82 100

APdagger HD 47 76 55 ndash 878 16S rRNA31 57 34

ICTM 82 90 88 ndash65 82 82

SPWBdagger HD 31 33 33 32 ndash 16S rRNA14 15 15 16

ICTM 38 42 41 43 ndash27 27 27 37

ESFY European stone fruit yellows PD pear decline PYLR peach yellow leaf roll SPWB sweet potato witchesrsquo broomHD major hydrophilic domain IC TM combined N-terminal hydrophilic and transmembrane domainsdaggerMembrane protein sequences for AP and SPWB from Berg et al (1999) and Yu et al (1998) respectively

Fig 3 Comparison of the interrelationships of four phytoplasmas in the apple proliferation (AP) subclade by rRNA gene sequences with the proportion () of identical amino acids for the major hydrophilic domains of the putative translation products of the AP imp gene and the homologues from the three AP subclade phytoplasmas ESFY European stone fruit yellows PD pear decline PYLR peach yellow leaf roll

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CONCLUSIONS

The three genes cloned here are almost certainly homologousto that encoding the immunodominant membrane protein fromAP (Berg

et al

1999) but there is no direct evidence for the

in vivo

products being immunodominant Indeed despite severalattempts no satisfactory antibodies (either polyclonal or mono-clonal) have been produced against PD using plant-derivedantigen (DL Davies unpublished data) It is not known whetherthis failure is due to the loss of the phytoplasma during extractionand purification or the absence of immunogen in whatever PD-derived material is present in the final preparations It is intendedto raise antisera against expressed protein from PD in order totest whether a diagnostic reagent suitable for field use can beproduced as was the case for CP (Barbara

et al

2002)Phytoplasma taxonomy is currently largely based on a single

criterion rRNA sequences although other gene sequences mayalso be used (eg

tuf

Marcone

et al

2000 Schneider

et al

1997) The relationship of the causal agents of PD and PYLR dis-eases is slightly confused In California peach orchards becomeinfected from adjacent pear orchards (Blomquist and Kirkpatrick2002) and the PD and PYLR phytoplasmas are genetically indis-tinguishable (Guerra 1997 BC Kirkpatrick unpublished dataquoted in Blomquist and Kirkpatrick 2002) Restriction fragmentlength polymorphism analysis of polymerase chain reaction(PCR)-amplified 16

S

rRNA sequences similarly failed to distin-guish European isolates of PD from Californian PYLR isolates(Kison

et al

1997) but some differences between them werefound in the actual 16

S

rRNA sequences (Seemuumlller

et al

1998)The sequences of the putative hydrophilic domains of the mem-brane protein homologues given here clearly support the differ-entiation of the European isolate of PD from the Californianisolate of PYLR studied in this paper (although the shorter intra-cellular and transmembrane regions were identical) The genesequences of further isolates of both European PD and Califor-nian PYLRPD need to be established to determine the ranges ofvariation within each group and whether these ranges overlapHowever it seems that the aetiologic agents of the Californianand European PD diseases are distinct phytoplasmas albeitclosely related by 16

S

rRNA sequences

PYLR (and by extensionCalifornian PD) has not been reported in Europe The rRNAsequences used for comparison here refer to European (specifi-cally German) isolates of AP ESFY and PD

The main purpose of cloning the homologues of the AP

imp

gene from three further members of the same subclade was toexamine whether the variation in the membrane proteins is bet-ter correlated with taxonomic position or with some other prop-erty such as the host For AY and CP there was little overallsimilarity between the two in the large extracellular hydrophilicdomain (although this region in the AY protein possibly com-prised two imperfect repeats of the CP sequence) (Barbara

et al

2002) By rRNA gene sequence comparisons the four membersof the AP subclade studied were either similarly related or slightlymore distant from each other than were AY and CP However forthe AP subclade phytoplasmas the immunodominant membraneproteins were similar in size and showed clear similarities betweenthemselves both in the relatively conserved N-terminal domainand in the large hydrophilic domain There was also similaritywith the presumably homologous protein from SPWB (Yu

et al

1998) but as expected from the distant relationship indicated byrRNA sequences the sequence of the protein from SPWB wasquite distinct from those from the AP subclade phytoplasmas

AP and PD were in the middle of the range when comparingrRNA gene sequences as an indicator of overall relatedness butthe major hydrophilic domains of the proteins from these twophytoplasmas were much more similar in sequence to each otherthan were those of any other pairing (57 identical for APPDagainst 31ndash34 for other pairings Table 1 Fig 3) Whether thisgreater similarity can be explained by interaction with and adap-tation to the host (either plant or insect) is an open question Allfour are thought to be transmitted by members of the genus

Cacopsylla

(order Homoptera suborder Sternorrhyncha super-family Psylloidea family Psyllidae) namely AP by

C costalis

(Frisinghelli

et al

2000) (although there is an earlier report ofinefficient transmission by

Fieberiella florii

a leaf-hopper in thesuborder Auchenorrhyncha and the detection of AP in this insectby PCR Bliefernicht and Krczal 1995 Krczal

et al

1988) ESFY by

C pruni

(Carraro

et al

1998 2001 Jarausch

et al

2001) PD by

C pyricola

in North America and the UK (Davies

et al

1992Jensen

et al

1964) and probably by

C pyri

in southern Europe(Avinent

et al

1997 Giunchedi

et al

1994) and PYLR by

Cpyricola

(Blomquist and Kirkpatrick 2002) On the basis of oneshared vector PD and PYLR might be expected to be most similarHowever the relative importance of vector specificity due toadaptation to growth in particular insect species vs the possibil-ity of acquisition being governed by host choice in the insect isnot clear Similarly the degree of adaptation to the plant host isunclear Some experimental hosts are susceptible to a wide rangeof phytoplasmas All four members of the AP subclade studiedhere naturally infect mainly species in the family Rosaceae APand PD (European isolates at least) have narrow host ranges inthe closely related genera

Malus

and

Pyrus

within the tribeMaloideae whereas both ESFY and PYLR mainly infect a range ofgenera within the tribe Prunoideae It is tempting to suggest thatthe similarity in the major hydrophilic domains of the AP and PDproteins reflects interactions with two relatively similar planthosts whilst the relative dissimilarity in the other two phyto-plasma proteins in some way reflects the more diverse hostrange Additional isolates from other hosts and geographicalregions need to be tested to begin to substantiate this sugges-tion Most important would be to examine Californian isolatesof PD to test whether as seems likely from the published data

Membrane protein homologues from three phytoplasmas

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discussed above the membrane protein homologues are identicalto that already found here for PYLR (and distinct from that ofthe European isolate sequenced here) The occurrence of a singlePYLRPD-California phytoplasma in both peach and pear wouldsupport the idea that PYLR (and by extension ESFY) is able toinfect a wider range of hosts The suggestion that these mem-brane protein homologues may be involved in determining thehost range might be examined more directly by looking for inter-actions

in vitro

between membrane proteins expressed off thecloned genes and host (plant and insect) components

EXPERIMENTAL PROCEDURES

Maintenance of cultures DNA extraction and sequence analysis

Both ESFY and PD phytoplasmas were from UK sources (Daviesand Adams 2000 Davies

et al

1992) ESFY was kept in apricotwhilst PD was maintained in pear tissue culture as describedpreviously (Davies and Clark 1994) PYLR was collected fromfield-infected peach in Yuba County CA USA The tissue waslyophilized and stored at 4

deg

C until use The tissue was confirmedas infected with PYLR but not with WX phytoplasma by PCR usingprimer pairs 1PYLR and 1WX (Smart

et al

1996) DNA wasextracted from infected and equivalent uninfected material asdescribed previously (Barbara

et al

2002) All sequence assem-bly basic analysis and comparisons were performed using DNAs-tar (Lasergene) Phytoplasma interrelationships were estimatedusing 16

S

rRNA gene sequences from databases (accessionnumbers AP X68375 AY X68373 CP X83870 ESFY X68374 PDX76425 PYLR Y16394 SPWB L33770)

Cloning of PD gene

A series of primers was developed from a known sequence con-taining the AP

imp

gene (accession number AJ011678 Berg

et al

1999) When used in PCR with DNA from PD-infected plantsseveral pairs gave amplicons two of which were cloned intopMOSBlue according to the manufacturerrsquos instructions (AmershamPharmacia Biotech Little Chalfont Bucks UK) The inserts fromtwo clones of the larger amplicon (from primers 672 5

prime

-TTATT-GAAGTTTTTAGTTTGG-3

prime

and 676 5prime-TTATTTCAAATCTAAAG-CAG-3prime) were sequenced using a commercial service (SequiserveVaterstetten Germany) and conserved primers in the plasmidFrom the positions of these primers in the AP sequence theseinserts were thought likely to cover the majority of the homolo-gous gene and the upstream ORF The sequence obtained wassimilar to but distinct from that of AP The ORF thought to encodethe membrane protein homologue was incomplete and a secondround of PCR using a primer based on the PD sequence and someof the original AP primers designed from a sequence outside the

ORF was undertaken The amplicons produced from primers 6845prime-TATAGGAGTTCAATTTCATCCTG-3prime and 686 5prime-AATAAAAC-CACAGTTCAAGGTG-3prime were cloned and sequenced Based onthe new contig (1203 nt) primers (700 5prime-CTTTTTATGT-TATAATAAATGGTGTG-3prime and 701 5prime-CAAGACCTTTAACA-CATCC-3prime) were then designed to allow the amplification cloningand sequencing of the entire PD gene as a single unit These prim-ers did not yield a product with ESFY DNA extract later analysisshowed that primer 700 was in a region poorly conservedbetween the two phytoplasma species

Cloning of ESFY gene

A similar process was used to clone and sequence the ESFYgene starting with the AP-derived primers The sequence derivedfrom primers 672 and 678 5prime-AACAACTGAACCAACACC-3prime wasshorter than that from PD spanning part of the upstream ORFand intergenic region but only a small part of the desired ORFThe cloned amplicons from primer 683 5prime-GGAGAAAAAAATAAT-GGAAGCAAATCAAC-3prime with 684 and 685 5prime-CAAGACCTT-TAAGGCCACATCC-3prime were sequenced to give almost the entireORF except for that part encoding the N-terminus of the proteinwhere primer 683 overlapped with primer 678 An AP sequence-derived primer 673 5prime-GTAGAACCAAATGATAAAG-3prime was usedwith 696 5prime-GCCAAAAACTCATAGACCAAGC-3prime designed fromthe ESFY sequence to complete the ORF Finally primers 724 5prime-TAATCAGTGTATTAAATTAAC-3prime and 725 5prime-CTTTGTTTAAAAA-TTTTATTA-3prime were designed from the contig to enable the ampli-fication cloning and sequencing of the entire ORF as one amplicon

Cloning of PYLR gene

Primer 700 (PD derived) with both 701 and 684 (PD and APderived respectively) yielded amplicons from extracts of PYLR-infected plants which when cloned and sequenced were bothfound to contain an entire ORF From this initial PYLR sequencefurther primers 729 5prime-GTTACAAATATTTACTAGGGGTAG-3prime and730 5prime-CAAGACCTTTAAGACCGCATCC-3prime were designed and usedto amplify clone and sequence the entire ORF as a single unit

The details of these approaches meant that for the PD andESFY sequence of the imp gene homologue the intergenic regionand part of the upstream ORF were obtained However for PYLRonly the protein ORF and part of the intergenic region were sequenced

The GENBANK accession numbers for the sequences reported inthis paper are ESFY AF400587 PD AF400588 PYLR AF400589

ACKNOWLEDGEMENTS

This work was funded by the Biotechnology and Biological Sci-ences Research Council through its competitive strategic grant toHorticulture Research International

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REFERENCES

Avinent L Llaacutecer G Almacellas J and Toraacute R (1997) Pear decline inSpain Plant Pathol 46 694ndash698

Barbara DJ Davis DL and Clark MF (1998) Cloning and sequencingof a major membrane protein from chlorante (AY) phytoplasma In Proceed-ings of the 12th International Organisation of Mycoplasmology SydneyAustralia International Organisation of Mycoplasmology p 183

Barbara DJ Morton A Clark MF and Davis DL (2002) Immuno-dominant membrane proteins from two phytoplasmas in the asteryellows clade (chlorante aster yellows and clover phyllody) are highlydivergent in the major hydrophilic region Microbiology 148 157ndash167

Berg M Davis DL Clark MF Vetten HJ Maier G Marcone Cand Seemuumlller E (1999) Isolation of the gene encoding an immuno-dominant membrane protein of the apple proliferation phytoplasma andexpression and characterisation of the gene product Microbiology 1451937ndash1945

Berg M Melcher U and Fletcher J (2001) Characterization of aSpiroplasma citri adhesion related protein SARP1 which contains adomain of a novel family designated sarpin Gene 275 57ndash64

Bliefernicht K and Krczal G (1995) Epidemiological studies on appleproliferation disease in southern Germany Acta Hortic 386 444ndash447

Blomquist CL Barbara DJ Davies DL Clark MF andKirkpatrick BC (2001) Cloning and characterization of a major mem-brane protein of the X-disease phytoplasma Microbiology 147 571ndash580

Blomquist CL and Kirkpatrick BC (2002) Identification of phyto-plasma strains and insect vectors of peach yellow leaf roll disease inCalifornia Plant Dis 86 759ndash763

Carraro L Loi N and Ermacora P (2001) Transmission characteristicsof the European stone fruit yellows phytoplasma and its vector Cacops-ylla pruni Eur J Plant Pathol 107 695ndash700

Carraro L Osler R Loi N Ermacora P and Refatti E (1998) Trans-mission of European stone fruit yellows phytoplasma by Cacopsyllapruni J Plant Pathol 80 233ndash239

Clark MF Barbara DJ and Davies DL (1983) Production and char-acteristics of antisera to Spiroplasma citri and clover phyllody-associatedantigens derived from plants Ann Appl Biol 103 251ndash259

Davies DL and Adams AN (2000) European stone fruit yellows phyto-plasma associated with a decline disease of apricot in southern EnglandPlant Pathol 49 635ndash639

Davies DL and Clark MF (1994) Maintenance of mycoplasma-likeorganisms occurring in pyrus species by micropropagation and their elim-ination by tetracycline therapy Plant Pathol 43 819ndash823

Davies DL Guise CM Clark MF and Adams AN (1992) Parryrsquosdisease of pears is similar to pear decline and is associated withmycoplasma-like organisms transmitted by Cacopsylla pyricola PlantPathol 41 195ndash203

Elzanowski A and Ostell J (2000) The genetic codes lthttpwwwncbinlmnihgovhtbin-post Taxonomygt

Frisinghelli C Delaiti L Grando MS Forti D and Vindimian ME(2000) Cacopsylla costalis (Flor 1861) as a vector of apple proliferationin Trentino J Phytopathol 148 425ndash431

Giunchedi L Poggi Pollini C Biondi S and Babini AR (1994) PCRdetection of MLOs in quick decline-affected pear trees in Italy Ann ApplBiol 124 399ndash403

Guerra LJ (1997) Biological and molecular characterization of phyto-plasmas infecting fruit and nut trees in California PhD Thesis Davis CAUniversity of California

von Heijne G (1990) The signal peptide J Membr Biol 115 195ndash201

Jarausch W Danet JL Labonne G Dosba D Broquaire JMSaillard C and Garnier M (2001) Mapping the spread of apricotchlorotic leaf roll (ACLR) in southern France and implication of Cacopsyllapruni as a vector of European stone fruit yellows (ESFY) phytoplasmasPlant Pathol 50 782ndash790

Jensen DD Griggs WH Gonzales CQ and Schneider H (1964)Pear decline virus transmission by pear psylla Phytopathology 541346ndash1351

Keane G Edwards E and Clark MF (1996) Differentiation of group16Sr-IB aster yellows phytoplasmas with monoclonal antibodies Diag-nostics Crop Prod BCPC Symp Series 65 263ndash268

Kison H Kirkpatrick BC and Seemuumlller E (1997) Genetic compar-ison of the peach yellow leaf roll agent with European fruit tree phyto-plasmas of the apple proliferation group Plant Pathol 46 538ndash544

Krczal G Krczal H and Kunze L (1988) Fieberiella florii (Stal) a vectorof the apple proliferation agent Acta Hortic 235 99ndash107

Lefol C Caudwell A Herminier JL and Larrue J (1993) Attachmentof the flavescence doreacutee pathogen (MLO) to leafhopper vectors and otherinsects Ann Appl Biol 123 611ndash622

Marcone C Lee I-M Davis RE Ragozzino A and Seemuumlller E(2000) Classification of aster yellows-group phytoplasmas based oncombined analyses of rRNA and tuf gene sequences Int J Syst EvolMicrobiol 50 1703ndash1713

McCoy RE Caudwell A Chang CJ Chen TA Chiykowski LNCousin MT Dale JL de Leeuw GTN Golino DA Hackett KJKirkpatrick BC Marwitz R Petzold H Sinha RC Sugiura MWhitcomb RF Yang IL Zhu BM and Seemuumlller E (1989) Plantdiseases associated with mycoplasma-like organisms In The Mycoplas-mas Vol V (Whitcomb RF and Tully JG eds) pp 546ndash623 San DiegoAcademic Press

Milne RG Ramassso E Lenzi R Masenga V Sarindu N andClark MF (1995) Pre- and post-embedding immunogold labelling andelectron microscopy in plant host tissues of three antigenically unrelatedMLOs primula yellows tomato big bud and bermudagrass white leafEur J Plant Pathol 101 57ndash67

Nakai K and Kanehisa M (1991) Expert systems for predicting proteinlocalization sites in Gram-negative bacteria Proteins Struct FunctGenet 11 95ndash110

Nielsen H Engelbrecht J Brunak S and von Heijne G (1997) Iden-tification of prokaryotic and eukaryotic signal peptides and prediction oftheir cleavage sites Protein Eng 10 1ndash6

Razin S and Jacobs E (1992) Mycoplasma adhesion J Gen Microbiol138 407ndash422

Schneider B Gibb KS and Seemuumlller E (1997) Sequence and RFLPanalysis of the elongation factor Tu gene used in differentiation and clas-sification of phytoplasmas Microbiology 143 3381ndash3389

Seemuumlller E Marcone C Lauer U Ragozzino A and Goumlschl M(1998) Current status of molecular classification of the phytoplasmasJ Plant Pathol 80 3ndash26

Smart CD Schneider B Blomquist CL Guerra L Harrison NAAhrens U Lorenz KH Seemuumlller E and Kirkpatrick BC (1996)Phytoplasma-specific PCR primers based on sequences of the 16Sminus23SrRNA spacer region Appl Environ Microbiol 62 2988ndash2993

Yu J Wayadande AC and Fletcher J (2000) Spiroplasma citri surfaceprotein P89 implicated in adhesion to cells of the vector Circulifer tenellusPhytopathology 90 716ndash722

Yu Y-L Yeh K-W and Lin CP (1998) An antigenic protein gene of aphytoplasma associated with sweet potato witchesrsquo broom Microbio-logy 144 1257ndash1262

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et al

MOLECULAR PLANT PATHOLOGY

(2003)

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(2 ) 109ndash114 copy 2003 BLACKWELL PUBL ISH ING LTD

similarity between the major hydrophilic domains (Barbara

et al

2002) A short sequence in the AY hydrophilic domain shows aclear match to an immunoglobulin G (IgG) binding site from

Streptococcus

protein G (Barbara

et al

2002) but whether thisis related to the function of the AY protein is not known

As no other within-taxon comparison is available it is not clearwhether this high level of divergence in the hydrophilic domainsof the AY and CP proteins is related to the degree of relationshipbetween the phytoplasmas or if it may be correlated with someother property such as the host In this paper we report the clon-ing of presumed homologues of the AP immunodominant mem-brane protein (

imp

) gene from three additional phytoplasmas inthe AP subclade (Seemuumlller

et al

1998) namely European stonefruit yellows (ESFY) peach yellow leaf roll (PYLR) and pear decline(PD) These are compared with the AP gene (Berg

et al

1999)and the structurally similar and probably homologous proteinfrom SPWB a member of the peanut witchesrsquo broom subclade (Yu

et al

1998) The relative similarities between both the genes andpredicted proteins are also compared with the apparent related-ness of the phytoplasmas as determined by rRNA sequences

RESULTS AND DISCUSSION

Analysis of genes from ESFY PD and PYLR

The sequences derived from ESFY and PD each contained a singlelarge open reading frame (ORF) with the expected initiation

codon (ATG) Comparison of the PYLR sequence with those fromESFY PD and AP suggested that it had a similar ORF but that theinitiation codon was GTG a known alternative bacterial transla-tion initiation codon (Elzanowski and Ostell 2000) As expectedall three newly cloned genes had low G + C contents (247ndash264 mol similar to AP and SPWB 263 mol and 274 molrespectively) Potential ribosome binding sites and transcriptionsignals were located upstream of the ORFs (Fig 1) For compar-ative purposes schematics of the equivalent genes are shown forAP and SPWB and some features for these have been reportedpreviously (Berg

et al

1999 Yu

et al

1998) Imperfect invertedrepeats that could be arranged as hairpin structures with fewloops (that from PD shown as an example in Fig 2) were locateddownstream of the three ORFs and also that of AP (Fig 1) Theserepeats may act as transcription terminators When aligned therepeat sequences were found to be highly conserved betweenPD PYLR and AP and on the basis that they are functionally

Fig 1 Schematic representation of open reading frames (ORFs) putative translation products and other features for four phytoplasmas in the apple proliferation (AP) clade and sweet potato witchesrsquo broom (SPWB) Sequences of AP and SPWB and some features (italics) from Berg et al (1999) and Yu et al (1998) ESFY European stone fruit yellows PD pear decline PYLR peach yellow leaf roll

Fig 2 Imperfect inverted repeats in the pear decline (PD) sequence Limits of repeats were defined by comparison with European stone fruit yellows (ESFY) peach yellow leaf roll (PYLR) and apple proliferation (AP) sequences on the assumption that they are functionally equivalent The position of these repeats in the overall sequence is shown in Fig 1

Membrane protein homologues from three phytoplasmas

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equivalent the limits of the repeats were defined by sequence iden-tity between all four phytoplasmas in the AP subclade Howevervarying numbers of nucleotides closer to the stop codon could besimilarly aligned (as close as 6 bp in ESFY) The ESFY repeats wereless strongly conserved relative to the other three sequences thanthese were to each other Whilst the first section (equivalentto the first 22 nt in Fig 2) and its complement were very similarto PD the central 37 nt were less similar and gave far lowerlevels of base pairing than the equivalent sequence in the otherphytoplasmas In SPWB only relatively short repeats furtherdownstream have been identified and parts of these may form arho-independent terminator (Yu

et al

1998)

Comparisons of predicted proteins

The putative translation products of the three genes cloned herewere very similar in size and domains both to each other and tothat of AP (Berg

et al

1999) (Fig 1) The proteins are predictedto have extracellular hydrophilic domains held by a transmem-brane region close to the N-terminal with only short hydrophilicintracellular terminal domains (8ndash9 aa) None appeared to havecleavage sites associated with the transmembrane region (Nakaiand Kanehisa 1991 Nielsen

et al

1997 von Heijne 1990 computeranalysis using psort version 64) nor did any have a C-terminaltransmembrane region The large hydrophilic domains of the PDand AP putative proteins were slightly shorter (131 and 130 aarespectively) than those of ESFY and PYLR (133 and 134 aarespectively) (Fig 1) Between the four AP subclade phyto-plasmas sequence identities for the putative translation prod-ucts ranged from 36 to 61 The large C-terminal hydrophilicdomains were more different from each other than were the com-bined N-terminaltransmembrane domains which for PD andPYLR were identical The hydrophilic domain from SPWB showedonly low sequence identities with those of the four members ofthe AP subclade studied (Table 1) These differences were mir-rored at the nucleic acid level (Table 1)

Similarities in rRNA gene and membrane protein sequences

Similarities for the 16

S

rRNA sequences varied from 981 to99 between members of the AP subclade and were

c

88between them and SPWB (Table 1) For the same rRNA genesequences AY and CP were 989 identical There was no corre-lation between the 16

S

rRNA sequence similarities and the per-centage amino acid identities within the hydrophilic domain ofthe putative translation products of the four AP subclade phyto-plasma genes (Fig 3) However for AP and PD the hydrophilicdomains were clearly more similar to each other than were thoseof any other pairing suggesting a correlation with some factorother than phylogenetic relationship

Table 1 Sequence identities () for two parts of the apple proliferation (AP) imp gene homologues and their putative translation products from five phytoplasmas (below left proteins in bolditalic and nucleic acids in plain type) and sequence similarities for part of the 16S rRNA genes (above right)

ESFY PD PYLR AP SPWB

ESFY HD ndash 981 983 985 875 16S rRNA

ICTM ndash

PD HD 49 ndash 990 984 880 16S rRNA33

ICTM 88 ndash82

PYLR HD 49 54 ndash 985 877 16S rRNA32 31

ICTM 88 98 ndash82 100

APdagger HD 47 76 55 ndash 878 16S rRNA31 57 34

ICTM 82 90 88 ndash65 82 82

SPWBdagger HD 31 33 33 32 ndash 16S rRNA14 15 15 16

ICTM 38 42 41 43 ndash27 27 27 37

ESFY European stone fruit yellows PD pear decline PYLR peach yellow leaf roll SPWB sweet potato witchesrsquo broomHD major hydrophilic domain IC TM combined N-terminal hydrophilic and transmembrane domainsdaggerMembrane protein sequences for AP and SPWB from Berg et al (1999) and Yu et al (1998) respectively

Fig 3 Comparison of the interrelationships of four phytoplasmas in the apple proliferation (AP) subclade by rRNA gene sequences with the proportion () of identical amino acids for the major hydrophilic domains of the putative translation products of the AP imp gene and the homologues from the three AP subclade phytoplasmas ESFY European stone fruit yellows PD pear decline PYLR peach yellow leaf roll

112

A MORTON

et al

MOLECULAR PLANT PATHOLOGY

(2003)

4

(2 ) 109ndash114 copy 2003 BLACKWELL PUBL ISH ING LTD

CONCLUSIONS

The three genes cloned here are almost certainly homologousto that encoding the immunodominant membrane protein fromAP (Berg

et al

1999) but there is no direct evidence for the

in vivo

products being immunodominant Indeed despite severalattempts no satisfactory antibodies (either polyclonal or mono-clonal) have been produced against PD using plant-derivedantigen (DL Davies unpublished data) It is not known whetherthis failure is due to the loss of the phytoplasma during extractionand purification or the absence of immunogen in whatever PD-derived material is present in the final preparations It is intendedto raise antisera against expressed protein from PD in order totest whether a diagnostic reagent suitable for field use can beproduced as was the case for CP (Barbara

et al

2002)Phytoplasma taxonomy is currently largely based on a single

criterion rRNA sequences although other gene sequences mayalso be used (eg

tuf

Marcone

et al

2000 Schneider

et al

1997) The relationship of the causal agents of PD and PYLR dis-eases is slightly confused In California peach orchards becomeinfected from adjacent pear orchards (Blomquist and Kirkpatrick2002) and the PD and PYLR phytoplasmas are genetically indis-tinguishable (Guerra 1997 BC Kirkpatrick unpublished dataquoted in Blomquist and Kirkpatrick 2002) Restriction fragmentlength polymorphism analysis of polymerase chain reaction(PCR)-amplified 16

S

rRNA sequences similarly failed to distin-guish European isolates of PD from Californian PYLR isolates(Kison

et al

1997) but some differences between them werefound in the actual 16

S

rRNA sequences (Seemuumlller

et al

1998)The sequences of the putative hydrophilic domains of the mem-brane protein homologues given here clearly support the differ-entiation of the European isolate of PD from the Californianisolate of PYLR studied in this paper (although the shorter intra-cellular and transmembrane regions were identical) The genesequences of further isolates of both European PD and Califor-nian PYLRPD need to be established to determine the ranges ofvariation within each group and whether these ranges overlapHowever it seems that the aetiologic agents of the Californianand European PD diseases are distinct phytoplasmas albeitclosely related by 16

S

rRNA sequences

PYLR (and by extensionCalifornian PD) has not been reported in Europe The rRNAsequences used for comparison here refer to European (specifi-cally German) isolates of AP ESFY and PD

The main purpose of cloning the homologues of the AP

imp

gene from three further members of the same subclade was toexamine whether the variation in the membrane proteins is bet-ter correlated with taxonomic position or with some other prop-erty such as the host For AY and CP there was little overallsimilarity between the two in the large extracellular hydrophilicdomain (although this region in the AY protein possibly com-prised two imperfect repeats of the CP sequence) (Barbara

et al

2002) By rRNA gene sequence comparisons the four membersof the AP subclade studied were either similarly related or slightlymore distant from each other than were AY and CP However forthe AP subclade phytoplasmas the immunodominant membraneproteins were similar in size and showed clear similarities betweenthemselves both in the relatively conserved N-terminal domainand in the large hydrophilic domain There was also similaritywith the presumably homologous protein from SPWB (Yu

et al

1998) but as expected from the distant relationship indicated byrRNA sequences the sequence of the protein from SPWB wasquite distinct from those from the AP subclade phytoplasmas

AP and PD were in the middle of the range when comparingrRNA gene sequences as an indicator of overall relatedness butthe major hydrophilic domains of the proteins from these twophytoplasmas were much more similar in sequence to each otherthan were those of any other pairing (57 identical for APPDagainst 31ndash34 for other pairings Table 1 Fig 3) Whether thisgreater similarity can be explained by interaction with and adap-tation to the host (either plant or insect) is an open question Allfour are thought to be transmitted by members of the genus

Cacopsylla

(order Homoptera suborder Sternorrhyncha super-family Psylloidea family Psyllidae) namely AP by

C costalis

(Frisinghelli

et al

2000) (although there is an earlier report ofinefficient transmission by

Fieberiella florii

a leaf-hopper in thesuborder Auchenorrhyncha and the detection of AP in this insectby PCR Bliefernicht and Krczal 1995 Krczal

et al

1988) ESFY by

C pruni

(Carraro

et al

1998 2001 Jarausch

et al

2001) PD by

C pyricola

in North America and the UK (Davies

et al

1992Jensen

et al

1964) and probably by

C pyri

in southern Europe(Avinent

et al

1997 Giunchedi

et al

1994) and PYLR by

Cpyricola

(Blomquist and Kirkpatrick 2002) On the basis of oneshared vector PD and PYLR might be expected to be most similarHowever the relative importance of vector specificity due toadaptation to growth in particular insect species vs the possibil-ity of acquisition being governed by host choice in the insect isnot clear Similarly the degree of adaptation to the plant host isunclear Some experimental hosts are susceptible to a wide rangeof phytoplasmas All four members of the AP subclade studiedhere naturally infect mainly species in the family Rosaceae APand PD (European isolates at least) have narrow host ranges inthe closely related genera

Malus

and

Pyrus

within the tribeMaloideae whereas both ESFY and PYLR mainly infect a range ofgenera within the tribe Prunoideae It is tempting to suggest thatthe similarity in the major hydrophilic domains of the AP and PDproteins reflects interactions with two relatively similar planthosts whilst the relative dissimilarity in the other two phyto-plasma proteins in some way reflects the more diverse hostrange Additional isolates from other hosts and geographicalregions need to be tested to begin to substantiate this sugges-tion Most important would be to examine Californian isolatesof PD to test whether as seems likely from the published data

Membrane protein homologues from three phytoplasmas

113

copy 2003 BLACKWELL PUBL ISH ING LTD

MOLECULAR PLANT PATHOLOGY

(2003)

4

(2 ) 109ndash114

discussed above the membrane protein homologues are identicalto that already found here for PYLR (and distinct from that ofthe European isolate sequenced here) The occurrence of a singlePYLRPD-California phytoplasma in both peach and pear wouldsupport the idea that PYLR (and by extension ESFY) is able toinfect a wider range of hosts The suggestion that these mem-brane protein homologues may be involved in determining thehost range might be examined more directly by looking for inter-actions

in vitro

between membrane proteins expressed off thecloned genes and host (plant and insect) components

EXPERIMENTAL PROCEDURES

Maintenance of cultures DNA extraction and sequence analysis

Both ESFY and PD phytoplasmas were from UK sources (Daviesand Adams 2000 Davies

et al

1992) ESFY was kept in apricotwhilst PD was maintained in pear tissue culture as describedpreviously (Davies and Clark 1994) PYLR was collected fromfield-infected peach in Yuba County CA USA The tissue waslyophilized and stored at 4

deg

C until use The tissue was confirmedas infected with PYLR but not with WX phytoplasma by PCR usingprimer pairs 1PYLR and 1WX (Smart

et al

1996) DNA wasextracted from infected and equivalent uninfected material asdescribed previously (Barbara

et al

2002) All sequence assem-bly basic analysis and comparisons were performed using DNAs-tar (Lasergene) Phytoplasma interrelationships were estimatedusing 16

S

rRNA gene sequences from databases (accessionnumbers AP X68375 AY X68373 CP X83870 ESFY X68374 PDX76425 PYLR Y16394 SPWB L33770)

Cloning of PD gene

A series of primers was developed from a known sequence con-taining the AP

imp

gene (accession number AJ011678 Berg

et al

1999) When used in PCR with DNA from PD-infected plantsseveral pairs gave amplicons two of which were cloned intopMOSBlue according to the manufacturerrsquos instructions (AmershamPharmacia Biotech Little Chalfont Bucks UK) The inserts fromtwo clones of the larger amplicon (from primers 672 5

prime

-TTATT-GAAGTTTTTAGTTTGG-3

prime

and 676 5prime-TTATTTCAAATCTAAAG-CAG-3prime) were sequenced using a commercial service (SequiserveVaterstetten Germany) and conserved primers in the plasmidFrom the positions of these primers in the AP sequence theseinserts were thought likely to cover the majority of the homolo-gous gene and the upstream ORF The sequence obtained wassimilar to but distinct from that of AP The ORF thought to encodethe membrane protein homologue was incomplete and a secondround of PCR using a primer based on the PD sequence and someof the original AP primers designed from a sequence outside the

ORF was undertaken The amplicons produced from primers 6845prime-TATAGGAGTTCAATTTCATCCTG-3prime and 686 5prime-AATAAAAC-CACAGTTCAAGGTG-3prime were cloned and sequenced Based onthe new contig (1203 nt) primers (700 5prime-CTTTTTATGT-TATAATAAATGGTGTG-3prime and 701 5prime-CAAGACCTTTAACA-CATCC-3prime) were then designed to allow the amplification cloningand sequencing of the entire PD gene as a single unit These prim-ers did not yield a product with ESFY DNA extract later analysisshowed that primer 700 was in a region poorly conservedbetween the two phytoplasma species

Cloning of ESFY gene

A similar process was used to clone and sequence the ESFYgene starting with the AP-derived primers The sequence derivedfrom primers 672 and 678 5prime-AACAACTGAACCAACACC-3prime wasshorter than that from PD spanning part of the upstream ORFand intergenic region but only a small part of the desired ORFThe cloned amplicons from primer 683 5prime-GGAGAAAAAAATAAT-GGAAGCAAATCAAC-3prime with 684 and 685 5prime-CAAGACCTT-TAAGGCCACATCC-3prime were sequenced to give almost the entireORF except for that part encoding the N-terminus of the proteinwhere primer 683 overlapped with primer 678 An AP sequence-derived primer 673 5prime-GTAGAACCAAATGATAAAG-3prime was usedwith 696 5prime-GCCAAAAACTCATAGACCAAGC-3prime designed fromthe ESFY sequence to complete the ORF Finally primers 724 5prime-TAATCAGTGTATTAAATTAAC-3prime and 725 5prime-CTTTGTTTAAAAA-TTTTATTA-3prime were designed from the contig to enable the ampli-fication cloning and sequencing of the entire ORF as one amplicon

Cloning of PYLR gene

Primer 700 (PD derived) with both 701 and 684 (PD and APderived respectively) yielded amplicons from extracts of PYLR-infected plants which when cloned and sequenced were bothfound to contain an entire ORF From this initial PYLR sequencefurther primers 729 5prime-GTTACAAATATTTACTAGGGGTAG-3prime and730 5prime-CAAGACCTTTAAGACCGCATCC-3prime were designed and usedto amplify clone and sequence the entire ORF as a single unit

The details of these approaches meant that for the PD andESFY sequence of the imp gene homologue the intergenic regionand part of the upstream ORF were obtained However for PYLRonly the protein ORF and part of the intergenic region were sequenced

The GENBANK accession numbers for the sequences reported inthis paper are ESFY AF400587 PD AF400588 PYLR AF400589

ACKNOWLEDGEMENTS

This work was funded by the Biotechnology and Biological Sci-ences Research Council through its competitive strategic grant toHorticulture Research International

114 A MORTON et al

MOLECULAR PLANT PATHOLOGY (2003) 4 (2 ) 109ndash114 copy 2003 BLACKWELL PUBL ISH ING LTD

REFERENCES

Avinent L Llaacutecer G Almacellas J and Toraacute R (1997) Pear decline inSpain Plant Pathol 46 694ndash698

Barbara DJ Davis DL and Clark MF (1998) Cloning and sequencingof a major membrane protein from chlorante (AY) phytoplasma In Proceed-ings of the 12th International Organisation of Mycoplasmology SydneyAustralia International Organisation of Mycoplasmology p 183

Barbara DJ Morton A Clark MF and Davis DL (2002) Immuno-dominant membrane proteins from two phytoplasmas in the asteryellows clade (chlorante aster yellows and clover phyllody) are highlydivergent in the major hydrophilic region Microbiology 148 157ndash167

Berg M Davis DL Clark MF Vetten HJ Maier G Marcone Cand Seemuumlller E (1999) Isolation of the gene encoding an immuno-dominant membrane protein of the apple proliferation phytoplasma andexpression and characterisation of the gene product Microbiology 1451937ndash1945

Berg M Melcher U and Fletcher J (2001) Characterization of aSpiroplasma citri adhesion related protein SARP1 which contains adomain of a novel family designated sarpin Gene 275 57ndash64

Bliefernicht K and Krczal G (1995) Epidemiological studies on appleproliferation disease in southern Germany Acta Hortic 386 444ndash447

Blomquist CL Barbara DJ Davies DL Clark MF andKirkpatrick BC (2001) Cloning and characterization of a major mem-brane protein of the X-disease phytoplasma Microbiology 147 571ndash580

Blomquist CL and Kirkpatrick BC (2002) Identification of phyto-plasma strains and insect vectors of peach yellow leaf roll disease inCalifornia Plant Dis 86 759ndash763

Carraro L Loi N and Ermacora P (2001) Transmission characteristicsof the European stone fruit yellows phytoplasma and its vector Cacops-ylla pruni Eur J Plant Pathol 107 695ndash700

Carraro L Osler R Loi N Ermacora P and Refatti E (1998) Trans-mission of European stone fruit yellows phytoplasma by Cacopsyllapruni J Plant Pathol 80 233ndash239

Clark MF Barbara DJ and Davies DL (1983) Production and char-acteristics of antisera to Spiroplasma citri and clover phyllody-associatedantigens derived from plants Ann Appl Biol 103 251ndash259

Davies DL and Adams AN (2000) European stone fruit yellows phyto-plasma associated with a decline disease of apricot in southern EnglandPlant Pathol 49 635ndash639

Davies DL and Clark MF (1994) Maintenance of mycoplasma-likeorganisms occurring in pyrus species by micropropagation and their elim-ination by tetracycline therapy Plant Pathol 43 819ndash823

Davies DL Guise CM Clark MF and Adams AN (1992) Parryrsquosdisease of pears is similar to pear decline and is associated withmycoplasma-like organisms transmitted by Cacopsylla pyricola PlantPathol 41 195ndash203

Elzanowski A and Ostell J (2000) The genetic codes lthttpwwwncbinlmnihgovhtbin-post Taxonomygt

Frisinghelli C Delaiti L Grando MS Forti D and Vindimian ME(2000) Cacopsylla costalis (Flor 1861) as a vector of apple proliferationin Trentino J Phytopathol 148 425ndash431

Giunchedi L Poggi Pollini C Biondi S and Babini AR (1994) PCRdetection of MLOs in quick decline-affected pear trees in Italy Ann ApplBiol 124 399ndash403

Guerra LJ (1997) Biological and molecular characterization of phyto-plasmas infecting fruit and nut trees in California PhD Thesis Davis CAUniversity of California

von Heijne G (1990) The signal peptide J Membr Biol 115 195ndash201

Jarausch W Danet JL Labonne G Dosba D Broquaire JMSaillard C and Garnier M (2001) Mapping the spread of apricotchlorotic leaf roll (ACLR) in southern France and implication of Cacopsyllapruni as a vector of European stone fruit yellows (ESFY) phytoplasmasPlant Pathol 50 782ndash790

Jensen DD Griggs WH Gonzales CQ and Schneider H (1964)Pear decline virus transmission by pear psylla Phytopathology 541346ndash1351

Keane G Edwards E and Clark MF (1996) Differentiation of group16Sr-IB aster yellows phytoplasmas with monoclonal antibodies Diag-nostics Crop Prod BCPC Symp Series 65 263ndash268

Kison H Kirkpatrick BC and Seemuumlller E (1997) Genetic compar-ison of the peach yellow leaf roll agent with European fruit tree phyto-plasmas of the apple proliferation group Plant Pathol 46 538ndash544

Krczal G Krczal H and Kunze L (1988) Fieberiella florii (Stal) a vectorof the apple proliferation agent Acta Hortic 235 99ndash107

Lefol C Caudwell A Herminier JL and Larrue J (1993) Attachmentof the flavescence doreacutee pathogen (MLO) to leafhopper vectors and otherinsects Ann Appl Biol 123 611ndash622

Marcone C Lee I-M Davis RE Ragozzino A and Seemuumlller E(2000) Classification of aster yellows-group phytoplasmas based oncombined analyses of rRNA and tuf gene sequences Int J Syst EvolMicrobiol 50 1703ndash1713

McCoy RE Caudwell A Chang CJ Chen TA Chiykowski LNCousin MT Dale JL de Leeuw GTN Golino DA Hackett KJKirkpatrick BC Marwitz R Petzold H Sinha RC Sugiura MWhitcomb RF Yang IL Zhu BM and Seemuumlller E (1989) Plantdiseases associated with mycoplasma-like organisms In The Mycoplas-mas Vol V (Whitcomb RF and Tully JG eds) pp 546ndash623 San DiegoAcademic Press

Milne RG Ramassso E Lenzi R Masenga V Sarindu N andClark MF (1995) Pre- and post-embedding immunogold labelling andelectron microscopy in plant host tissues of three antigenically unrelatedMLOs primula yellows tomato big bud and bermudagrass white leafEur J Plant Pathol 101 57ndash67

Nakai K and Kanehisa M (1991) Expert systems for predicting proteinlocalization sites in Gram-negative bacteria Proteins Struct FunctGenet 11 95ndash110

Nielsen H Engelbrecht J Brunak S and von Heijne G (1997) Iden-tification of prokaryotic and eukaryotic signal peptides and prediction oftheir cleavage sites Protein Eng 10 1ndash6

Razin S and Jacobs E (1992) Mycoplasma adhesion J Gen Microbiol138 407ndash422

Schneider B Gibb KS and Seemuumlller E (1997) Sequence and RFLPanalysis of the elongation factor Tu gene used in differentiation and clas-sification of phytoplasmas Microbiology 143 3381ndash3389

Seemuumlller E Marcone C Lauer U Ragozzino A and Goumlschl M(1998) Current status of molecular classification of the phytoplasmasJ Plant Pathol 80 3ndash26

Smart CD Schneider B Blomquist CL Guerra L Harrison NAAhrens U Lorenz KH Seemuumlller E and Kirkpatrick BC (1996)Phytoplasma-specific PCR primers based on sequences of the 16Sminus23SrRNA spacer region Appl Environ Microbiol 62 2988ndash2993

Yu J Wayadande AC and Fletcher J (2000) Spiroplasma citri surfaceprotein P89 implicated in adhesion to cells of the vector Circulifer tenellusPhytopathology 90 716ndash722

Yu Y-L Yeh K-W and Lin CP (1998) An antigenic protein gene of aphytoplasma associated with sweet potato witchesrsquo broom Microbio-logy 144 1257ndash1262

Membrane protein homologues from three phytoplasmas

111

copy 2003 BLACKWELL PUBL ISH ING LTD

MOLECULAR PLANT PATHOLOGY

(2003)

4

(2 ) 109ndash114

equivalent the limits of the repeats were defined by sequence iden-tity between all four phytoplasmas in the AP subclade Howevervarying numbers of nucleotides closer to the stop codon could besimilarly aligned (as close as 6 bp in ESFY) The ESFY repeats wereless strongly conserved relative to the other three sequences thanthese were to each other Whilst the first section (equivalentto the first 22 nt in Fig 2) and its complement were very similarto PD the central 37 nt were less similar and gave far lowerlevels of base pairing than the equivalent sequence in the otherphytoplasmas In SPWB only relatively short repeats furtherdownstream have been identified and parts of these may form arho-independent terminator (Yu

et al

1998)

Comparisons of predicted proteins

The putative translation products of the three genes cloned herewere very similar in size and domains both to each other and tothat of AP (Berg

et al

1999) (Fig 1) The proteins are predictedto have extracellular hydrophilic domains held by a transmem-brane region close to the N-terminal with only short hydrophilicintracellular terminal domains (8ndash9 aa) None appeared to havecleavage sites associated with the transmembrane region (Nakaiand Kanehisa 1991 Nielsen

et al

1997 von Heijne 1990 computeranalysis using psort version 64) nor did any have a C-terminaltransmembrane region The large hydrophilic domains of the PDand AP putative proteins were slightly shorter (131 and 130 aarespectively) than those of ESFY and PYLR (133 and 134 aarespectively) (Fig 1) Between the four AP subclade phyto-plasmas sequence identities for the putative translation prod-ucts ranged from 36 to 61 The large C-terminal hydrophilicdomains were more different from each other than were the com-bined N-terminaltransmembrane domains which for PD andPYLR were identical The hydrophilic domain from SPWB showedonly low sequence identities with those of the four members ofthe AP subclade studied (Table 1) These differences were mir-rored at the nucleic acid level (Table 1)

Similarities in rRNA gene and membrane protein sequences

Similarities for the 16

S

rRNA sequences varied from 981 to99 between members of the AP subclade and were

c

88between them and SPWB (Table 1) For the same rRNA genesequences AY and CP were 989 identical There was no corre-lation between the 16

S

rRNA sequence similarities and the per-centage amino acid identities within the hydrophilic domain ofthe putative translation products of the four AP subclade phyto-plasma genes (Fig 3) However for AP and PD the hydrophilicdomains were clearly more similar to each other than were thoseof any other pairing suggesting a correlation with some factorother than phylogenetic relationship

Table 1 Sequence identities () for two parts of the apple proliferation (AP) imp gene homologues and their putative translation products from five phytoplasmas (below left proteins in bolditalic and nucleic acids in plain type) and sequence similarities for part of the 16S rRNA genes (above right)

ESFY PD PYLR AP SPWB

ESFY HD ndash 981 983 985 875 16S rRNA

ICTM ndash

PD HD 49 ndash 990 984 880 16S rRNA33

ICTM 88 ndash82

PYLR HD 49 54 ndash 985 877 16S rRNA32 31

ICTM 88 98 ndash82 100

APdagger HD 47 76 55 ndash 878 16S rRNA31 57 34

ICTM 82 90 88 ndash65 82 82

SPWBdagger HD 31 33 33 32 ndash 16S rRNA14 15 15 16

ICTM 38 42 41 43 ndash27 27 27 37

ESFY European stone fruit yellows PD pear decline PYLR peach yellow leaf roll SPWB sweet potato witchesrsquo broomHD major hydrophilic domain IC TM combined N-terminal hydrophilic and transmembrane domainsdaggerMembrane protein sequences for AP and SPWB from Berg et al (1999) and Yu et al (1998) respectively

Fig 3 Comparison of the interrelationships of four phytoplasmas in the apple proliferation (AP) subclade by rRNA gene sequences with the proportion () of identical amino acids for the major hydrophilic domains of the putative translation products of the AP imp gene and the homologues from the three AP subclade phytoplasmas ESFY European stone fruit yellows PD pear decline PYLR peach yellow leaf roll

112

A MORTON

et al

MOLECULAR PLANT PATHOLOGY

(2003)

4

(2 ) 109ndash114 copy 2003 BLACKWELL PUBL ISH ING LTD

CONCLUSIONS

The three genes cloned here are almost certainly homologousto that encoding the immunodominant membrane protein fromAP (Berg

et al

1999) but there is no direct evidence for the

in vivo

products being immunodominant Indeed despite severalattempts no satisfactory antibodies (either polyclonal or mono-clonal) have been produced against PD using plant-derivedantigen (DL Davies unpublished data) It is not known whetherthis failure is due to the loss of the phytoplasma during extractionand purification or the absence of immunogen in whatever PD-derived material is present in the final preparations It is intendedto raise antisera against expressed protein from PD in order totest whether a diagnostic reagent suitable for field use can beproduced as was the case for CP (Barbara

et al

2002)Phytoplasma taxonomy is currently largely based on a single

criterion rRNA sequences although other gene sequences mayalso be used (eg

tuf

Marcone

et al

2000 Schneider

et al

1997) The relationship of the causal agents of PD and PYLR dis-eases is slightly confused In California peach orchards becomeinfected from adjacent pear orchards (Blomquist and Kirkpatrick2002) and the PD and PYLR phytoplasmas are genetically indis-tinguishable (Guerra 1997 BC Kirkpatrick unpublished dataquoted in Blomquist and Kirkpatrick 2002) Restriction fragmentlength polymorphism analysis of polymerase chain reaction(PCR)-amplified 16

S

rRNA sequences similarly failed to distin-guish European isolates of PD from Californian PYLR isolates(Kison

et al

1997) but some differences between them werefound in the actual 16

S

rRNA sequences (Seemuumlller

et al

1998)The sequences of the putative hydrophilic domains of the mem-brane protein homologues given here clearly support the differ-entiation of the European isolate of PD from the Californianisolate of PYLR studied in this paper (although the shorter intra-cellular and transmembrane regions were identical) The genesequences of further isolates of both European PD and Califor-nian PYLRPD need to be established to determine the ranges ofvariation within each group and whether these ranges overlapHowever it seems that the aetiologic agents of the Californianand European PD diseases are distinct phytoplasmas albeitclosely related by 16

S

rRNA sequences

PYLR (and by extensionCalifornian PD) has not been reported in Europe The rRNAsequences used for comparison here refer to European (specifi-cally German) isolates of AP ESFY and PD

The main purpose of cloning the homologues of the AP

imp

gene from three further members of the same subclade was toexamine whether the variation in the membrane proteins is bet-ter correlated with taxonomic position or with some other prop-erty such as the host For AY and CP there was little overallsimilarity between the two in the large extracellular hydrophilicdomain (although this region in the AY protein possibly com-prised two imperfect repeats of the CP sequence) (Barbara

et al

2002) By rRNA gene sequence comparisons the four membersof the AP subclade studied were either similarly related or slightlymore distant from each other than were AY and CP However forthe AP subclade phytoplasmas the immunodominant membraneproteins were similar in size and showed clear similarities betweenthemselves both in the relatively conserved N-terminal domainand in the large hydrophilic domain There was also similaritywith the presumably homologous protein from SPWB (Yu

et al

1998) but as expected from the distant relationship indicated byrRNA sequences the sequence of the protein from SPWB wasquite distinct from those from the AP subclade phytoplasmas

AP and PD were in the middle of the range when comparingrRNA gene sequences as an indicator of overall relatedness butthe major hydrophilic domains of the proteins from these twophytoplasmas were much more similar in sequence to each otherthan were those of any other pairing (57 identical for APPDagainst 31ndash34 for other pairings Table 1 Fig 3) Whether thisgreater similarity can be explained by interaction with and adap-tation to the host (either plant or insect) is an open question Allfour are thought to be transmitted by members of the genus

Cacopsylla

(order Homoptera suborder Sternorrhyncha super-family Psylloidea family Psyllidae) namely AP by

C costalis

(Frisinghelli

et al

2000) (although there is an earlier report ofinefficient transmission by

Fieberiella florii

a leaf-hopper in thesuborder Auchenorrhyncha and the detection of AP in this insectby PCR Bliefernicht and Krczal 1995 Krczal

et al

1988) ESFY by

C pruni

(Carraro

et al

1998 2001 Jarausch

et al

2001) PD by

C pyricola

in North America and the UK (Davies

et al

1992Jensen

et al

1964) and probably by

C pyri

in southern Europe(Avinent

et al

1997 Giunchedi

et al

1994) and PYLR by

Cpyricola

(Blomquist and Kirkpatrick 2002) On the basis of oneshared vector PD and PYLR might be expected to be most similarHowever the relative importance of vector specificity due toadaptation to growth in particular insect species vs the possibil-ity of acquisition being governed by host choice in the insect isnot clear Similarly the degree of adaptation to the plant host isunclear Some experimental hosts are susceptible to a wide rangeof phytoplasmas All four members of the AP subclade studiedhere naturally infect mainly species in the family Rosaceae APand PD (European isolates at least) have narrow host ranges inthe closely related genera

Malus

and

Pyrus

within the tribeMaloideae whereas both ESFY and PYLR mainly infect a range ofgenera within the tribe Prunoideae It is tempting to suggest thatthe similarity in the major hydrophilic domains of the AP and PDproteins reflects interactions with two relatively similar planthosts whilst the relative dissimilarity in the other two phyto-plasma proteins in some way reflects the more diverse hostrange Additional isolates from other hosts and geographicalregions need to be tested to begin to substantiate this sugges-tion Most important would be to examine Californian isolatesof PD to test whether as seems likely from the published data

Membrane protein homologues from three phytoplasmas

113

copy 2003 BLACKWELL PUBL ISH ING LTD

MOLECULAR PLANT PATHOLOGY

(2003)

4

(2 ) 109ndash114

discussed above the membrane protein homologues are identicalto that already found here for PYLR (and distinct from that ofthe European isolate sequenced here) The occurrence of a singlePYLRPD-California phytoplasma in both peach and pear wouldsupport the idea that PYLR (and by extension ESFY) is able toinfect a wider range of hosts The suggestion that these mem-brane protein homologues may be involved in determining thehost range might be examined more directly by looking for inter-actions

in vitro

between membrane proteins expressed off thecloned genes and host (plant and insect) components

EXPERIMENTAL PROCEDURES

Maintenance of cultures DNA extraction and sequence analysis

Both ESFY and PD phytoplasmas were from UK sources (Daviesand Adams 2000 Davies

et al

1992) ESFY was kept in apricotwhilst PD was maintained in pear tissue culture as describedpreviously (Davies and Clark 1994) PYLR was collected fromfield-infected peach in Yuba County CA USA The tissue waslyophilized and stored at 4

deg

C until use The tissue was confirmedas infected with PYLR but not with WX phytoplasma by PCR usingprimer pairs 1PYLR and 1WX (Smart

et al

1996) DNA wasextracted from infected and equivalent uninfected material asdescribed previously (Barbara

et al

2002) All sequence assem-bly basic analysis and comparisons were performed using DNAs-tar (Lasergene) Phytoplasma interrelationships were estimatedusing 16

S

rRNA gene sequences from databases (accessionnumbers AP X68375 AY X68373 CP X83870 ESFY X68374 PDX76425 PYLR Y16394 SPWB L33770)

Cloning of PD gene

A series of primers was developed from a known sequence con-taining the AP

imp

gene (accession number AJ011678 Berg

et al

1999) When used in PCR with DNA from PD-infected plantsseveral pairs gave amplicons two of which were cloned intopMOSBlue according to the manufacturerrsquos instructions (AmershamPharmacia Biotech Little Chalfont Bucks UK) The inserts fromtwo clones of the larger amplicon (from primers 672 5

prime

-TTATT-GAAGTTTTTAGTTTGG-3

prime

and 676 5prime-TTATTTCAAATCTAAAG-CAG-3prime) were sequenced using a commercial service (SequiserveVaterstetten Germany) and conserved primers in the plasmidFrom the positions of these primers in the AP sequence theseinserts were thought likely to cover the majority of the homolo-gous gene and the upstream ORF The sequence obtained wassimilar to but distinct from that of AP The ORF thought to encodethe membrane protein homologue was incomplete and a secondround of PCR using a primer based on the PD sequence and someof the original AP primers designed from a sequence outside the

ORF was undertaken The amplicons produced from primers 6845prime-TATAGGAGTTCAATTTCATCCTG-3prime and 686 5prime-AATAAAAC-CACAGTTCAAGGTG-3prime were cloned and sequenced Based onthe new contig (1203 nt) primers (700 5prime-CTTTTTATGT-TATAATAAATGGTGTG-3prime and 701 5prime-CAAGACCTTTAACA-CATCC-3prime) were then designed to allow the amplification cloningand sequencing of the entire PD gene as a single unit These prim-ers did not yield a product with ESFY DNA extract later analysisshowed that primer 700 was in a region poorly conservedbetween the two phytoplasma species

Cloning of ESFY gene

A similar process was used to clone and sequence the ESFYgene starting with the AP-derived primers The sequence derivedfrom primers 672 and 678 5prime-AACAACTGAACCAACACC-3prime wasshorter than that from PD spanning part of the upstream ORFand intergenic region but only a small part of the desired ORFThe cloned amplicons from primer 683 5prime-GGAGAAAAAAATAAT-GGAAGCAAATCAAC-3prime with 684 and 685 5prime-CAAGACCTT-TAAGGCCACATCC-3prime were sequenced to give almost the entireORF except for that part encoding the N-terminus of the proteinwhere primer 683 overlapped with primer 678 An AP sequence-derived primer 673 5prime-GTAGAACCAAATGATAAAG-3prime was usedwith 696 5prime-GCCAAAAACTCATAGACCAAGC-3prime designed fromthe ESFY sequence to complete the ORF Finally primers 724 5prime-TAATCAGTGTATTAAATTAAC-3prime and 725 5prime-CTTTGTTTAAAAA-TTTTATTA-3prime were designed from the contig to enable the ampli-fication cloning and sequencing of the entire ORF as one amplicon

Cloning of PYLR gene

Primer 700 (PD derived) with both 701 and 684 (PD and APderived respectively) yielded amplicons from extracts of PYLR-infected plants which when cloned and sequenced were bothfound to contain an entire ORF From this initial PYLR sequencefurther primers 729 5prime-GTTACAAATATTTACTAGGGGTAG-3prime and730 5prime-CAAGACCTTTAAGACCGCATCC-3prime were designed and usedto amplify clone and sequence the entire ORF as a single unit

The details of these approaches meant that for the PD andESFY sequence of the imp gene homologue the intergenic regionand part of the upstream ORF were obtained However for PYLRonly the protein ORF and part of the intergenic region were sequenced

The GENBANK accession numbers for the sequences reported inthis paper are ESFY AF400587 PD AF400588 PYLR AF400589

ACKNOWLEDGEMENTS

This work was funded by the Biotechnology and Biological Sci-ences Research Council through its competitive strategic grant toHorticulture Research International

114 A MORTON et al

MOLECULAR PLANT PATHOLOGY (2003) 4 (2 ) 109ndash114 copy 2003 BLACKWELL PUBL ISH ING LTD

REFERENCES

Avinent L Llaacutecer G Almacellas J and Toraacute R (1997) Pear decline inSpain Plant Pathol 46 694ndash698

Barbara DJ Davis DL and Clark MF (1998) Cloning and sequencingof a major membrane protein from chlorante (AY) phytoplasma In Proceed-ings of the 12th International Organisation of Mycoplasmology SydneyAustralia International Organisation of Mycoplasmology p 183

Barbara DJ Morton A Clark MF and Davis DL (2002) Immuno-dominant membrane proteins from two phytoplasmas in the asteryellows clade (chlorante aster yellows and clover phyllody) are highlydivergent in the major hydrophilic region Microbiology 148 157ndash167

Berg M Davis DL Clark MF Vetten HJ Maier G Marcone Cand Seemuumlller E (1999) Isolation of the gene encoding an immuno-dominant membrane protein of the apple proliferation phytoplasma andexpression and characterisation of the gene product Microbiology 1451937ndash1945

Berg M Melcher U and Fletcher J (2001) Characterization of aSpiroplasma citri adhesion related protein SARP1 which contains adomain of a novel family designated sarpin Gene 275 57ndash64

Bliefernicht K and Krczal G (1995) Epidemiological studies on appleproliferation disease in southern Germany Acta Hortic 386 444ndash447

Blomquist CL Barbara DJ Davies DL Clark MF andKirkpatrick BC (2001) Cloning and characterization of a major mem-brane protein of the X-disease phytoplasma Microbiology 147 571ndash580

Blomquist CL and Kirkpatrick BC (2002) Identification of phyto-plasma strains and insect vectors of peach yellow leaf roll disease inCalifornia Plant Dis 86 759ndash763

Carraro L Loi N and Ermacora P (2001) Transmission characteristicsof the European stone fruit yellows phytoplasma and its vector Cacops-ylla pruni Eur J Plant Pathol 107 695ndash700

Carraro L Osler R Loi N Ermacora P and Refatti E (1998) Trans-mission of European stone fruit yellows phytoplasma by Cacopsyllapruni J Plant Pathol 80 233ndash239

Clark MF Barbara DJ and Davies DL (1983) Production and char-acteristics of antisera to Spiroplasma citri and clover phyllody-associatedantigens derived from plants Ann Appl Biol 103 251ndash259

Davies DL and Adams AN (2000) European stone fruit yellows phyto-plasma associated with a decline disease of apricot in southern EnglandPlant Pathol 49 635ndash639

Davies DL and Clark MF (1994) Maintenance of mycoplasma-likeorganisms occurring in pyrus species by micropropagation and their elim-ination by tetracycline therapy Plant Pathol 43 819ndash823

Davies DL Guise CM Clark MF and Adams AN (1992) Parryrsquosdisease of pears is similar to pear decline and is associated withmycoplasma-like organisms transmitted by Cacopsylla pyricola PlantPathol 41 195ndash203

Elzanowski A and Ostell J (2000) The genetic codes lthttpwwwncbinlmnihgovhtbin-post Taxonomygt

Frisinghelli C Delaiti L Grando MS Forti D and Vindimian ME(2000) Cacopsylla costalis (Flor 1861) as a vector of apple proliferationin Trentino J Phytopathol 148 425ndash431

Giunchedi L Poggi Pollini C Biondi S and Babini AR (1994) PCRdetection of MLOs in quick decline-affected pear trees in Italy Ann ApplBiol 124 399ndash403

Guerra LJ (1997) Biological and molecular characterization of phyto-plasmas infecting fruit and nut trees in California PhD Thesis Davis CAUniversity of California

von Heijne G (1990) The signal peptide J Membr Biol 115 195ndash201

Jarausch W Danet JL Labonne G Dosba D Broquaire JMSaillard C and Garnier M (2001) Mapping the spread of apricotchlorotic leaf roll (ACLR) in southern France and implication of Cacopsyllapruni as a vector of European stone fruit yellows (ESFY) phytoplasmasPlant Pathol 50 782ndash790

Jensen DD Griggs WH Gonzales CQ and Schneider H (1964)Pear decline virus transmission by pear psylla Phytopathology 541346ndash1351

Keane G Edwards E and Clark MF (1996) Differentiation of group16Sr-IB aster yellows phytoplasmas with monoclonal antibodies Diag-nostics Crop Prod BCPC Symp Series 65 263ndash268

Kison H Kirkpatrick BC and Seemuumlller E (1997) Genetic compar-ison of the peach yellow leaf roll agent with European fruit tree phyto-plasmas of the apple proliferation group Plant Pathol 46 538ndash544

Krczal G Krczal H and Kunze L (1988) Fieberiella florii (Stal) a vectorof the apple proliferation agent Acta Hortic 235 99ndash107

Lefol C Caudwell A Herminier JL and Larrue J (1993) Attachmentof the flavescence doreacutee pathogen (MLO) to leafhopper vectors and otherinsects Ann Appl Biol 123 611ndash622

Marcone C Lee I-M Davis RE Ragozzino A and Seemuumlller E(2000) Classification of aster yellows-group phytoplasmas based oncombined analyses of rRNA and tuf gene sequences Int J Syst EvolMicrobiol 50 1703ndash1713

McCoy RE Caudwell A Chang CJ Chen TA Chiykowski LNCousin MT Dale JL de Leeuw GTN Golino DA Hackett KJKirkpatrick BC Marwitz R Petzold H Sinha RC Sugiura MWhitcomb RF Yang IL Zhu BM and Seemuumlller E (1989) Plantdiseases associated with mycoplasma-like organisms In The Mycoplas-mas Vol V (Whitcomb RF and Tully JG eds) pp 546ndash623 San DiegoAcademic Press

Milne RG Ramassso E Lenzi R Masenga V Sarindu N andClark MF (1995) Pre- and post-embedding immunogold labelling andelectron microscopy in plant host tissues of three antigenically unrelatedMLOs primula yellows tomato big bud and bermudagrass white leafEur J Plant Pathol 101 57ndash67

Nakai K and Kanehisa M (1991) Expert systems for predicting proteinlocalization sites in Gram-negative bacteria Proteins Struct FunctGenet 11 95ndash110

Nielsen H Engelbrecht J Brunak S and von Heijne G (1997) Iden-tification of prokaryotic and eukaryotic signal peptides and prediction oftheir cleavage sites Protein Eng 10 1ndash6

Razin S and Jacobs E (1992) Mycoplasma adhesion J Gen Microbiol138 407ndash422

Schneider B Gibb KS and Seemuumlller E (1997) Sequence and RFLPanalysis of the elongation factor Tu gene used in differentiation and clas-sification of phytoplasmas Microbiology 143 3381ndash3389

Seemuumlller E Marcone C Lauer U Ragozzino A and Goumlschl M(1998) Current status of molecular classification of the phytoplasmasJ Plant Pathol 80 3ndash26

Smart CD Schneider B Blomquist CL Guerra L Harrison NAAhrens U Lorenz KH Seemuumlller E and Kirkpatrick BC (1996)Phytoplasma-specific PCR primers based on sequences of the 16Sminus23SrRNA spacer region Appl Environ Microbiol 62 2988ndash2993

Yu J Wayadande AC and Fletcher J (2000) Spiroplasma citri surfaceprotein P89 implicated in adhesion to cells of the vector Circulifer tenellusPhytopathology 90 716ndash722

Yu Y-L Yeh K-W and Lin CP (1998) An antigenic protein gene of aphytoplasma associated with sweet potato witchesrsquo broom Microbio-logy 144 1257ndash1262

112

A MORTON

et al

MOLECULAR PLANT PATHOLOGY

(2003)

4

(2 ) 109ndash114 copy 2003 BLACKWELL PUBL ISH ING LTD

CONCLUSIONS

The three genes cloned here are almost certainly homologousto that encoding the immunodominant membrane protein fromAP (Berg

et al

1999) but there is no direct evidence for the

in vivo

products being immunodominant Indeed despite severalattempts no satisfactory antibodies (either polyclonal or mono-clonal) have been produced against PD using plant-derivedantigen (DL Davies unpublished data) It is not known whetherthis failure is due to the loss of the phytoplasma during extractionand purification or the absence of immunogen in whatever PD-derived material is present in the final preparations It is intendedto raise antisera against expressed protein from PD in order totest whether a diagnostic reagent suitable for field use can beproduced as was the case for CP (Barbara

et al

2002)Phytoplasma taxonomy is currently largely based on a single

criterion rRNA sequences although other gene sequences mayalso be used (eg

tuf

Marcone

et al

2000 Schneider

et al

1997) The relationship of the causal agents of PD and PYLR dis-eases is slightly confused In California peach orchards becomeinfected from adjacent pear orchards (Blomquist and Kirkpatrick2002) and the PD and PYLR phytoplasmas are genetically indis-tinguishable (Guerra 1997 BC Kirkpatrick unpublished dataquoted in Blomquist and Kirkpatrick 2002) Restriction fragmentlength polymorphism analysis of polymerase chain reaction(PCR)-amplified 16

S

rRNA sequences similarly failed to distin-guish European isolates of PD from Californian PYLR isolates(Kison

et al

1997) but some differences between them werefound in the actual 16

S

rRNA sequences (Seemuumlller

et al

1998)The sequences of the putative hydrophilic domains of the mem-brane protein homologues given here clearly support the differ-entiation of the European isolate of PD from the Californianisolate of PYLR studied in this paper (although the shorter intra-cellular and transmembrane regions were identical) The genesequences of further isolates of both European PD and Califor-nian PYLRPD need to be established to determine the ranges ofvariation within each group and whether these ranges overlapHowever it seems that the aetiologic agents of the Californianand European PD diseases are distinct phytoplasmas albeitclosely related by 16

S

rRNA sequences

PYLR (and by extensionCalifornian PD) has not been reported in Europe The rRNAsequences used for comparison here refer to European (specifi-cally German) isolates of AP ESFY and PD

The main purpose of cloning the homologues of the AP

imp

gene from three further members of the same subclade was toexamine whether the variation in the membrane proteins is bet-ter correlated with taxonomic position or with some other prop-erty such as the host For AY and CP there was little overallsimilarity between the two in the large extracellular hydrophilicdomain (although this region in the AY protein possibly com-prised two imperfect repeats of the CP sequence) (Barbara

et al

2002) By rRNA gene sequence comparisons the four membersof the AP subclade studied were either similarly related or slightlymore distant from each other than were AY and CP However forthe AP subclade phytoplasmas the immunodominant membraneproteins were similar in size and showed clear similarities betweenthemselves both in the relatively conserved N-terminal domainand in the large hydrophilic domain There was also similaritywith the presumably homologous protein from SPWB (Yu

et al

1998) but as expected from the distant relationship indicated byrRNA sequences the sequence of the protein from SPWB wasquite distinct from those from the AP subclade phytoplasmas

AP and PD were in the middle of the range when comparingrRNA gene sequences as an indicator of overall relatedness butthe major hydrophilic domains of the proteins from these twophytoplasmas were much more similar in sequence to each otherthan were those of any other pairing (57 identical for APPDagainst 31ndash34 for other pairings Table 1 Fig 3) Whether thisgreater similarity can be explained by interaction with and adap-tation to the host (either plant or insect) is an open question Allfour are thought to be transmitted by members of the genus

Cacopsylla

(order Homoptera suborder Sternorrhyncha super-family Psylloidea family Psyllidae) namely AP by

C costalis

(Frisinghelli

et al

2000) (although there is an earlier report ofinefficient transmission by

Fieberiella florii

a leaf-hopper in thesuborder Auchenorrhyncha and the detection of AP in this insectby PCR Bliefernicht and Krczal 1995 Krczal

et al

1988) ESFY by

C pruni

(Carraro

et al

1998 2001 Jarausch

et al

2001) PD by

C pyricola

in North America and the UK (Davies

et al

1992Jensen

et al

1964) and probably by

C pyri

in southern Europe(Avinent

et al

1997 Giunchedi

et al

1994) and PYLR by

Cpyricola

(Blomquist and Kirkpatrick 2002) On the basis of oneshared vector PD and PYLR might be expected to be most similarHowever the relative importance of vector specificity due toadaptation to growth in particular insect species vs the possibil-ity of acquisition being governed by host choice in the insect isnot clear Similarly the degree of adaptation to the plant host isunclear Some experimental hosts are susceptible to a wide rangeof phytoplasmas All four members of the AP subclade studiedhere naturally infect mainly species in the family Rosaceae APand PD (European isolates at least) have narrow host ranges inthe closely related genera

Malus

and

Pyrus

within the tribeMaloideae whereas both ESFY and PYLR mainly infect a range ofgenera within the tribe Prunoideae It is tempting to suggest thatthe similarity in the major hydrophilic domains of the AP and PDproteins reflects interactions with two relatively similar planthosts whilst the relative dissimilarity in the other two phyto-plasma proteins in some way reflects the more diverse hostrange Additional isolates from other hosts and geographicalregions need to be tested to begin to substantiate this sugges-tion Most important would be to examine Californian isolatesof PD to test whether as seems likely from the published data

Membrane protein homologues from three phytoplasmas

113

copy 2003 BLACKWELL PUBL ISH ING LTD

MOLECULAR PLANT PATHOLOGY

(2003)

4

(2 ) 109ndash114

discussed above the membrane protein homologues are identicalto that already found here for PYLR (and distinct from that ofthe European isolate sequenced here) The occurrence of a singlePYLRPD-California phytoplasma in both peach and pear wouldsupport the idea that PYLR (and by extension ESFY) is able toinfect a wider range of hosts The suggestion that these mem-brane protein homologues may be involved in determining thehost range might be examined more directly by looking for inter-actions

in vitro

between membrane proteins expressed off thecloned genes and host (plant and insect) components

EXPERIMENTAL PROCEDURES

Maintenance of cultures DNA extraction and sequence analysis

Both ESFY and PD phytoplasmas were from UK sources (Daviesand Adams 2000 Davies

et al

1992) ESFY was kept in apricotwhilst PD was maintained in pear tissue culture as describedpreviously (Davies and Clark 1994) PYLR was collected fromfield-infected peach in Yuba County CA USA The tissue waslyophilized and stored at 4

deg

C until use The tissue was confirmedas infected with PYLR but not with WX phytoplasma by PCR usingprimer pairs 1PYLR and 1WX (Smart

et al

1996) DNA wasextracted from infected and equivalent uninfected material asdescribed previously (Barbara

et al

2002) All sequence assem-bly basic analysis and comparisons were performed using DNAs-tar (Lasergene) Phytoplasma interrelationships were estimatedusing 16

S

rRNA gene sequences from databases (accessionnumbers AP X68375 AY X68373 CP X83870 ESFY X68374 PDX76425 PYLR Y16394 SPWB L33770)

Cloning of PD gene

A series of primers was developed from a known sequence con-taining the AP

imp

gene (accession number AJ011678 Berg

et al

1999) When used in PCR with DNA from PD-infected plantsseveral pairs gave amplicons two of which were cloned intopMOSBlue according to the manufacturerrsquos instructions (AmershamPharmacia Biotech Little Chalfont Bucks UK) The inserts fromtwo clones of the larger amplicon (from primers 672 5

prime

-TTATT-GAAGTTTTTAGTTTGG-3

prime

and 676 5prime-TTATTTCAAATCTAAAG-CAG-3prime) were sequenced using a commercial service (SequiserveVaterstetten Germany) and conserved primers in the plasmidFrom the positions of these primers in the AP sequence theseinserts were thought likely to cover the majority of the homolo-gous gene and the upstream ORF The sequence obtained wassimilar to but distinct from that of AP The ORF thought to encodethe membrane protein homologue was incomplete and a secondround of PCR using a primer based on the PD sequence and someof the original AP primers designed from a sequence outside the

ORF was undertaken The amplicons produced from primers 6845prime-TATAGGAGTTCAATTTCATCCTG-3prime and 686 5prime-AATAAAAC-CACAGTTCAAGGTG-3prime were cloned and sequenced Based onthe new contig (1203 nt) primers (700 5prime-CTTTTTATGT-TATAATAAATGGTGTG-3prime and 701 5prime-CAAGACCTTTAACA-CATCC-3prime) were then designed to allow the amplification cloningand sequencing of the entire PD gene as a single unit These prim-ers did not yield a product with ESFY DNA extract later analysisshowed that primer 700 was in a region poorly conservedbetween the two phytoplasma species

Cloning of ESFY gene

A similar process was used to clone and sequence the ESFYgene starting with the AP-derived primers The sequence derivedfrom primers 672 and 678 5prime-AACAACTGAACCAACACC-3prime wasshorter than that from PD spanning part of the upstream ORFand intergenic region but only a small part of the desired ORFThe cloned amplicons from primer 683 5prime-GGAGAAAAAAATAAT-GGAAGCAAATCAAC-3prime with 684 and 685 5prime-CAAGACCTT-TAAGGCCACATCC-3prime were sequenced to give almost the entireORF except for that part encoding the N-terminus of the proteinwhere primer 683 overlapped with primer 678 An AP sequence-derived primer 673 5prime-GTAGAACCAAATGATAAAG-3prime was usedwith 696 5prime-GCCAAAAACTCATAGACCAAGC-3prime designed fromthe ESFY sequence to complete the ORF Finally primers 724 5prime-TAATCAGTGTATTAAATTAAC-3prime and 725 5prime-CTTTGTTTAAAAA-TTTTATTA-3prime were designed from the contig to enable the ampli-fication cloning and sequencing of the entire ORF as one amplicon

Cloning of PYLR gene

Primer 700 (PD derived) with both 701 and 684 (PD and APderived respectively) yielded amplicons from extracts of PYLR-infected plants which when cloned and sequenced were bothfound to contain an entire ORF From this initial PYLR sequencefurther primers 729 5prime-GTTACAAATATTTACTAGGGGTAG-3prime and730 5prime-CAAGACCTTTAAGACCGCATCC-3prime were designed and usedto amplify clone and sequence the entire ORF as a single unit

The details of these approaches meant that for the PD andESFY sequence of the imp gene homologue the intergenic regionand part of the upstream ORF were obtained However for PYLRonly the protein ORF and part of the intergenic region were sequenced

The GENBANK accession numbers for the sequences reported inthis paper are ESFY AF400587 PD AF400588 PYLR AF400589

ACKNOWLEDGEMENTS

This work was funded by the Biotechnology and Biological Sci-ences Research Council through its competitive strategic grant toHorticulture Research International

114 A MORTON et al

MOLECULAR PLANT PATHOLOGY (2003) 4 (2 ) 109ndash114 copy 2003 BLACKWELL PUBL ISH ING LTD

REFERENCES

Avinent L Llaacutecer G Almacellas J and Toraacute R (1997) Pear decline inSpain Plant Pathol 46 694ndash698

Barbara DJ Davis DL and Clark MF (1998) Cloning and sequencingof a major membrane protein from chlorante (AY) phytoplasma In Proceed-ings of the 12th International Organisation of Mycoplasmology SydneyAustralia International Organisation of Mycoplasmology p 183

Barbara DJ Morton A Clark MF and Davis DL (2002) Immuno-dominant membrane proteins from two phytoplasmas in the asteryellows clade (chlorante aster yellows and clover phyllody) are highlydivergent in the major hydrophilic region Microbiology 148 157ndash167

Berg M Davis DL Clark MF Vetten HJ Maier G Marcone Cand Seemuumlller E (1999) Isolation of the gene encoding an immuno-dominant membrane protein of the apple proliferation phytoplasma andexpression and characterisation of the gene product Microbiology 1451937ndash1945

Berg M Melcher U and Fletcher J (2001) Characterization of aSpiroplasma citri adhesion related protein SARP1 which contains adomain of a novel family designated sarpin Gene 275 57ndash64

Bliefernicht K and Krczal G (1995) Epidemiological studies on appleproliferation disease in southern Germany Acta Hortic 386 444ndash447

Blomquist CL Barbara DJ Davies DL Clark MF andKirkpatrick BC (2001) Cloning and characterization of a major mem-brane protein of the X-disease phytoplasma Microbiology 147 571ndash580

Blomquist CL and Kirkpatrick BC (2002) Identification of phyto-plasma strains and insect vectors of peach yellow leaf roll disease inCalifornia Plant Dis 86 759ndash763

Carraro L Loi N and Ermacora P (2001) Transmission characteristicsof the European stone fruit yellows phytoplasma and its vector Cacops-ylla pruni Eur J Plant Pathol 107 695ndash700

Carraro L Osler R Loi N Ermacora P and Refatti E (1998) Trans-mission of European stone fruit yellows phytoplasma by Cacopsyllapruni J Plant Pathol 80 233ndash239

Clark MF Barbara DJ and Davies DL (1983) Production and char-acteristics of antisera to Spiroplasma citri and clover phyllody-associatedantigens derived from plants Ann Appl Biol 103 251ndash259

Davies DL and Adams AN (2000) European stone fruit yellows phyto-plasma associated with a decline disease of apricot in southern EnglandPlant Pathol 49 635ndash639

Davies DL and Clark MF (1994) Maintenance of mycoplasma-likeorganisms occurring in pyrus species by micropropagation and their elim-ination by tetracycline therapy Plant Pathol 43 819ndash823

Davies DL Guise CM Clark MF and Adams AN (1992) Parryrsquosdisease of pears is similar to pear decline and is associated withmycoplasma-like organisms transmitted by Cacopsylla pyricola PlantPathol 41 195ndash203

Elzanowski A and Ostell J (2000) The genetic codes lthttpwwwncbinlmnihgovhtbin-post Taxonomygt

Frisinghelli C Delaiti L Grando MS Forti D and Vindimian ME(2000) Cacopsylla costalis (Flor 1861) as a vector of apple proliferationin Trentino J Phytopathol 148 425ndash431

Giunchedi L Poggi Pollini C Biondi S and Babini AR (1994) PCRdetection of MLOs in quick decline-affected pear trees in Italy Ann ApplBiol 124 399ndash403

Guerra LJ (1997) Biological and molecular characterization of phyto-plasmas infecting fruit and nut trees in California PhD Thesis Davis CAUniversity of California

von Heijne G (1990) The signal peptide J Membr Biol 115 195ndash201

Jarausch W Danet JL Labonne G Dosba D Broquaire JMSaillard C and Garnier M (2001) Mapping the spread of apricotchlorotic leaf roll (ACLR) in southern France and implication of Cacopsyllapruni as a vector of European stone fruit yellows (ESFY) phytoplasmasPlant Pathol 50 782ndash790

Jensen DD Griggs WH Gonzales CQ and Schneider H (1964)Pear decline virus transmission by pear psylla Phytopathology 541346ndash1351

Keane G Edwards E and Clark MF (1996) Differentiation of group16Sr-IB aster yellows phytoplasmas with monoclonal antibodies Diag-nostics Crop Prod BCPC Symp Series 65 263ndash268

Kison H Kirkpatrick BC and Seemuumlller E (1997) Genetic compar-ison of the peach yellow leaf roll agent with European fruit tree phyto-plasmas of the apple proliferation group Plant Pathol 46 538ndash544

Krczal G Krczal H and Kunze L (1988) Fieberiella florii (Stal) a vectorof the apple proliferation agent Acta Hortic 235 99ndash107

Lefol C Caudwell A Herminier JL and Larrue J (1993) Attachmentof the flavescence doreacutee pathogen (MLO) to leafhopper vectors and otherinsects Ann Appl Biol 123 611ndash622

Marcone C Lee I-M Davis RE Ragozzino A and Seemuumlller E(2000) Classification of aster yellows-group phytoplasmas based oncombined analyses of rRNA and tuf gene sequences Int J Syst EvolMicrobiol 50 1703ndash1713

McCoy RE Caudwell A Chang CJ Chen TA Chiykowski LNCousin MT Dale JL de Leeuw GTN Golino DA Hackett KJKirkpatrick BC Marwitz R Petzold H Sinha RC Sugiura MWhitcomb RF Yang IL Zhu BM and Seemuumlller E (1989) Plantdiseases associated with mycoplasma-like organisms In The Mycoplas-mas Vol V (Whitcomb RF and Tully JG eds) pp 546ndash623 San DiegoAcademic Press

Milne RG Ramassso E Lenzi R Masenga V Sarindu N andClark MF (1995) Pre- and post-embedding immunogold labelling andelectron microscopy in plant host tissues of three antigenically unrelatedMLOs primula yellows tomato big bud and bermudagrass white leafEur J Plant Pathol 101 57ndash67

Nakai K and Kanehisa M (1991) Expert systems for predicting proteinlocalization sites in Gram-negative bacteria Proteins Struct FunctGenet 11 95ndash110

Nielsen H Engelbrecht J Brunak S and von Heijne G (1997) Iden-tification of prokaryotic and eukaryotic signal peptides and prediction oftheir cleavage sites Protein Eng 10 1ndash6

Razin S and Jacobs E (1992) Mycoplasma adhesion J Gen Microbiol138 407ndash422

Schneider B Gibb KS and Seemuumlller E (1997) Sequence and RFLPanalysis of the elongation factor Tu gene used in differentiation and clas-sification of phytoplasmas Microbiology 143 3381ndash3389

Seemuumlller E Marcone C Lauer U Ragozzino A and Goumlschl M(1998) Current status of molecular classification of the phytoplasmasJ Plant Pathol 80 3ndash26

Smart CD Schneider B Blomquist CL Guerra L Harrison NAAhrens U Lorenz KH Seemuumlller E and Kirkpatrick BC (1996)Phytoplasma-specific PCR primers based on sequences of the 16Sminus23SrRNA spacer region Appl Environ Microbiol 62 2988ndash2993

Yu J Wayadande AC and Fletcher J (2000) Spiroplasma citri surfaceprotein P89 implicated in adhesion to cells of the vector Circulifer tenellusPhytopathology 90 716ndash722

Yu Y-L Yeh K-W and Lin CP (1998) An antigenic protein gene of aphytoplasma associated with sweet potato witchesrsquo broom Microbio-logy 144 1257ndash1262

Membrane protein homologues from three phytoplasmas

113

copy 2003 BLACKWELL PUBL ISH ING LTD

MOLECULAR PLANT PATHOLOGY

(2003)

4

(2 ) 109ndash114

discussed above the membrane protein homologues are identicalto that already found here for PYLR (and distinct from that ofthe European isolate sequenced here) The occurrence of a singlePYLRPD-California phytoplasma in both peach and pear wouldsupport the idea that PYLR (and by extension ESFY) is able toinfect a wider range of hosts The suggestion that these mem-brane protein homologues may be involved in determining thehost range might be examined more directly by looking for inter-actions

in vitro

between membrane proteins expressed off thecloned genes and host (plant and insect) components

EXPERIMENTAL PROCEDURES

Maintenance of cultures DNA extraction and sequence analysis

Both ESFY and PD phytoplasmas were from UK sources (Daviesand Adams 2000 Davies

et al

1992) ESFY was kept in apricotwhilst PD was maintained in pear tissue culture as describedpreviously (Davies and Clark 1994) PYLR was collected fromfield-infected peach in Yuba County CA USA The tissue waslyophilized and stored at 4

deg

C until use The tissue was confirmedas infected with PYLR but not with WX phytoplasma by PCR usingprimer pairs 1PYLR and 1WX (Smart

et al

1996) DNA wasextracted from infected and equivalent uninfected material asdescribed previously (Barbara

et al

2002) All sequence assem-bly basic analysis and comparisons were performed using DNAs-tar (Lasergene) Phytoplasma interrelationships were estimatedusing 16

S

rRNA gene sequences from databases (accessionnumbers AP X68375 AY X68373 CP X83870 ESFY X68374 PDX76425 PYLR Y16394 SPWB L33770)

Cloning of PD gene

A series of primers was developed from a known sequence con-taining the AP

imp

gene (accession number AJ011678 Berg

et al

1999) When used in PCR with DNA from PD-infected plantsseveral pairs gave amplicons two of which were cloned intopMOSBlue according to the manufacturerrsquos instructions (AmershamPharmacia Biotech Little Chalfont Bucks UK) The inserts fromtwo clones of the larger amplicon (from primers 672 5

prime

-TTATT-GAAGTTTTTAGTTTGG-3

prime

and 676 5prime-TTATTTCAAATCTAAAG-CAG-3prime) were sequenced using a commercial service (SequiserveVaterstetten Germany) and conserved primers in the plasmidFrom the positions of these primers in the AP sequence theseinserts were thought likely to cover the majority of the homolo-gous gene and the upstream ORF The sequence obtained wassimilar to but distinct from that of AP The ORF thought to encodethe membrane protein homologue was incomplete and a secondround of PCR using a primer based on the PD sequence and someof the original AP primers designed from a sequence outside the

ORF was undertaken The amplicons produced from primers 6845prime-TATAGGAGTTCAATTTCATCCTG-3prime and 686 5prime-AATAAAAC-CACAGTTCAAGGTG-3prime were cloned and sequenced Based onthe new contig (1203 nt) primers (700 5prime-CTTTTTATGT-TATAATAAATGGTGTG-3prime and 701 5prime-CAAGACCTTTAACA-CATCC-3prime) were then designed to allow the amplification cloningand sequencing of the entire PD gene as a single unit These prim-ers did not yield a product with ESFY DNA extract later analysisshowed that primer 700 was in a region poorly conservedbetween the two phytoplasma species

Cloning of ESFY gene

A similar process was used to clone and sequence the ESFYgene starting with the AP-derived primers The sequence derivedfrom primers 672 and 678 5prime-AACAACTGAACCAACACC-3prime wasshorter than that from PD spanning part of the upstream ORFand intergenic region but only a small part of the desired ORFThe cloned amplicons from primer 683 5prime-GGAGAAAAAAATAAT-GGAAGCAAATCAAC-3prime with 684 and 685 5prime-CAAGACCTT-TAAGGCCACATCC-3prime were sequenced to give almost the entireORF except for that part encoding the N-terminus of the proteinwhere primer 683 overlapped with primer 678 An AP sequence-derived primer 673 5prime-GTAGAACCAAATGATAAAG-3prime was usedwith 696 5prime-GCCAAAAACTCATAGACCAAGC-3prime designed fromthe ESFY sequence to complete the ORF Finally primers 724 5prime-TAATCAGTGTATTAAATTAAC-3prime and 725 5prime-CTTTGTTTAAAAA-TTTTATTA-3prime were designed from the contig to enable the ampli-fication cloning and sequencing of the entire ORF as one amplicon

Cloning of PYLR gene

Primer 700 (PD derived) with both 701 and 684 (PD and APderived respectively) yielded amplicons from extracts of PYLR-infected plants which when cloned and sequenced were bothfound to contain an entire ORF From this initial PYLR sequencefurther primers 729 5prime-GTTACAAATATTTACTAGGGGTAG-3prime and730 5prime-CAAGACCTTTAAGACCGCATCC-3prime were designed and usedto amplify clone and sequence the entire ORF as a single unit

The details of these approaches meant that for the PD andESFY sequence of the imp gene homologue the intergenic regionand part of the upstream ORF were obtained However for PYLRonly the protein ORF and part of the intergenic region were sequenced

The GENBANK accession numbers for the sequences reported inthis paper are ESFY AF400587 PD AF400588 PYLR AF400589

ACKNOWLEDGEMENTS

This work was funded by the Biotechnology and Biological Sci-ences Research Council through its competitive strategic grant toHorticulture Research International

114 A MORTON et al

MOLECULAR PLANT PATHOLOGY (2003) 4 (2 ) 109ndash114 copy 2003 BLACKWELL PUBL ISH ING LTD

REFERENCES

Avinent L Llaacutecer G Almacellas J and Toraacute R (1997) Pear decline inSpain Plant Pathol 46 694ndash698

Barbara DJ Davis DL and Clark MF (1998) Cloning and sequencingof a major membrane protein from chlorante (AY) phytoplasma In Proceed-ings of the 12th International Organisation of Mycoplasmology SydneyAustralia International Organisation of Mycoplasmology p 183

Barbara DJ Morton A Clark MF and Davis DL (2002) Immuno-dominant membrane proteins from two phytoplasmas in the asteryellows clade (chlorante aster yellows and clover phyllody) are highlydivergent in the major hydrophilic region Microbiology 148 157ndash167

Berg M Davis DL Clark MF Vetten HJ Maier G Marcone Cand Seemuumlller E (1999) Isolation of the gene encoding an immuno-dominant membrane protein of the apple proliferation phytoplasma andexpression and characterisation of the gene product Microbiology 1451937ndash1945

Berg M Melcher U and Fletcher J (2001) Characterization of aSpiroplasma citri adhesion related protein SARP1 which contains adomain of a novel family designated sarpin Gene 275 57ndash64

Bliefernicht K and Krczal G (1995) Epidemiological studies on appleproliferation disease in southern Germany Acta Hortic 386 444ndash447

Blomquist CL Barbara DJ Davies DL Clark MF andKirkpatrick BC (2001) Cloning and characterization of a major mem-brane protein of the X-disease phytoplasma Microbiology 147 571ndash580

Blomquist CL and Kirkpatrick BC (2002) Identification of phyto-plasma strains and insect vectors of peach yellow leaf roll disease inCalifornia Plant Dis 86 759ndash763

Carraro L Loi N and Ermacora P (2001) Transmission characteristicsof the European stone fruit yellows phytoplasma and its vector Cacops-ylla pruni Eur J Plant Pathol 107 695ndash700

Carraro L Osler R Loi N Ermacora P and Refatti E (1998) Trans-mission of European stone fruit yellows phytoplasma by Cacopsyllapruni J Plant Pathol 80 233ndash239

Clark MF Barbara DJ and Davies DL (1983) Production and char-acteristics of antisera to Spiroplasma citri and clover phyllody-associatedantigens derived from plants Ann Appl Biol 103 251ndash259

Davies DL and Adams AN (2000) European stone fruit yellows phyto-plasma associated with a decline disease of apricot in southern EnglandPlant Pathol 49 635ndash639

Davies DL and Clark MF (1994) Maintenance of mycoplasma-likeorganisms occurring in pyrus species by micropropagation and their elim-ination by tetracycline therapy Plant Pathol 43 819ndash823

Davies DL Guise CM Clark MF and Adams AN (1992) Parryrsquosdisease of pears is similar to pear decline and is associated withmycoplasma-like organisms transmitted by Cacopsylla pyricola PlantPathol 41 195ndash203

Elzanowski A and Ostell J (2000) The genetic codes lthttpwwwncbinlmnihgovhtbin-post Taxonomygt

Frisinghelli C Delaiti L Grando MS Forti D and Vindimian ME(2000) Cacopsylla costalis (Flor 1861) as a vector of apple proliferationin Trentino J Phytopathol 148 425ndash431

Giunchedi L Poggi Pollini C Biondi S and Babini AR (1994) PCRdetection of MLOs in quick decline-affected pear trees in Italy Ann ApplBiol 124 399ndash403

Guerra LJ (1997) Biological and molecular characterization of phyto-plasmas infecting fruit and nut trees in California PhD Thesis Davis CAUniversity of California

von Heijne G (1990) The signal peptide J Membr Biol 115 195ndash201

Jarausch W Danet JL Labonne G Dosba D Broquaire JMSaillard C and Garnier M (2001) Mapping the spread of apricotchlorotic leaf roll (ACLR) in southern France and implication of Cacopsyllapruni as a vector of European stone fruit yellows (ESFY) phytoplasmasPlant Pathol 50 782ndash790

Jensen DD Griggs WH Gonzales CQ and Schneider H (1964)Pear decline virus transmission by pear psylla Phytopathology 541346ndash1351

Keane G Edwards E and Clark MF (1996) Differentiation of group16Sr-IB aster yellows phytoplasmas with monoclonal antibodies Diag-nostics Crop Prod BCPC Symp Series 65 263ndash268

Kison H Kirkpatrick BC and Seemuumlller E (1997) Genetic compar-ison of the peach yellow leaf roll agent with European fruit tree phyto-plasmas of the apple proliferation group Plant Pathol 46 538ndash544

Krczal G Krczal H and Kunze L (1988) Fieberiella florii (Stal) a vectorof the apple proliferation agent Acta Hortic 235 99ndash107

Lefol C Caudwell A Herminier JL and Larrue J (1993) Attachmentof the flavescence doreacutee pathogen (MLO) to leafhopper vectors and otherinsects Ann Appl Biol 123 611ndash622

Marcone C Lee I-M Davis RE Ragozzino A and Seemuumlller E(2000) Classification of aster yellows-group phytoplasmas based oncombined analyses of rRNA and tuf gene sequences Int J Syst EvolMicrobiol 50 1703ndash1713

McCoy RE Caudwell A Chang CJ Chen TA Chiykowski LNCousin MT Dale JL de Leeuw GTN Golino DA Hackett KJKirkpatrick BC Marwitz R Petzold H Sinha RC Sugiura MWhitcomb RF Yang IL Zhu BM and Seemuumlller E (1989) Plantdiseases associated with mycoplasma-like organisms In The Mycoplas-mas Vol V (Whitcomb RF and Tully JG eds) pp 546ndash623 San DiegoAcademic Press

Milne RG Ramassso E Lenzi R Masenga V Sarindu N andClark MF (1995) Pre- and post-embedding immunogold labelling andelectron microscopy in plant host tissues of three antigenically unrelatedMLOs primula yellows tomato big bud and bermudagrass white leafEur J Plant Pathol 101 57ndash67

Nakai K and Kanehisa M (1991) Expert systems for predicting proteinlocalization sites in Gram-negative bacteria Proteins Struct FunctGenet 11 95ndash110

Nielsen H Engelbrecht J Brunak S and von Heijne G (1997) Iden-tification of prokaryotic and eukaryotic signal peptides and prediction oftheir cleavage sites Protein Eng 10 1ndash6

Razin S and Jacobs E (1992) Mycoplasma adhesion J Gen Microbiol138 407ndash422

Schneider B Gibb KS and Seemuumlller E (1997) Sequence and RFLPanalysis of the elongation factor Tu gene used in differentiation and clas-sification of phytoplasmas Microbiology 143 3381ndash3389

Seemuumlller E Marcone C Lauer U Ragozzino A and Goumlschl M(1998) Current status of molecular classification of the phytoplasmasJ Plant Pathol 80 3ndash26

Smart CD Schneider B Blomquist CL Guerra L Harrison NAAhrens U Lorenz KH Seemuumlller E and Kirkpatrick BC (1996)Phytoplasma-specific PCR primers based on sequences of the 16Sminus23SrRNA spacer region Appl Environ Microbiol 62 2988ndash2993

Yu J Wayadande AC and Fletcher J (2000) Spiroplasma citri surfaceprotein P89 implicated in adhesion to cells of the vector Circulifer tenellusPhytopathology 90 716ndash722

Yu Y-L Yeh K-W and Lin CP (1998) An antigenic protein gene of aphytoplasma associated with sweet potato witchesrsquo broom Microbio-logy 144 1257ndash1262

114 A MORTON et al

MOLECULAR PLANT PATHOLOGY (2003) 4 (2 ) 109ndash114 copy 2003 BLACKWELL PUBL ISH ING LTD

REFERENCES

Avinent L Llaacutecer G Almacellas J and Toraacute R (1997) Pear decline inSpain Plant Pathol 46 694ndash698

Barbara DJ Davis DL and Clark MF (1998) Cloning and sequencingof a major membrane protein from chlorante (AY) phytoplasma In Proceed-ings of the 12th International Organisation of Mycoplasmology SydneyAustralia International Organisation of Mycoplasmology p 183

Barbara DJ Morton A Clark MF and Davis DL (2002) Immuno-dominant membrane proteins from two phytoplasmas in the asteryellows clade (chlorante aster yellows and clover phyllody) are highlydivergent in the major hydrophilic region Microbiology 148 157ndash167

Berg M Davis DL Clark MF Vetten HJ Maier G Marcone Cand Seemuumlller E (1999) Isolation of the gene encoding an immuno-dominant membrane protein of the apple proliferation phytoplasma andexpression and characterisation of the gene product Microbiology 1451937ndash1945

Berg M Melcher U and Fletcher J (2001) Characterization of aSpiroplasma citri adhesion related protein SARP1 which contains adomain of a novel family designated sarpin Gene 275 57ndash64

Bliefernicht K and Krczal G (1995) Epidemiological studies on appleproliferation disease in southern Germany Acta Hortic 386 444ndash447

Blomquist CL Barbara DJ Davies DL Clark MF andKirkpatrick BC (2001) Cloning and characterization of a major mem-brane protein of the X-disease phytoplasma Microbiology 147 571ndash580

Blomquist CL and Kirkpatrick BC (2002) Identification of phyto-plasma strains and insect vectors of peach yellow leaf roll disease inCalifornia Plant Dis 86 759ndash763

Carraro L Loi N and Ermacora P (2001) Transmission characteristicsof the European stone fruit yellows phytoplasma and its vector Cacops-ylla pruni Eur J Plant Pathol 107 695ndash700

Carraro L Osler R Loi N Ermacora P and Refatti E (1998) Trans-mission of European stone fruit yellows phytoplasma by Cacopsyllapruni J Plant Pathol 80 233ndash239

Clark MF Barbara DJ and Davies DL (1983) Production and char-acteristics of antisera to Spiroplasma citri and clover phyllody-associatedantigens derived from plants Ann Appl Biol 103 251ndash259

Davies DL and Adams AN (2000) European stone fruit yellows phyto-plasma associated with a decline disease of apricot in southern EnglandPlant Pathol 49 635ndash639

Davies DL and Clark MF (1994) Maintenance of mycoplasma-likeorganisms occurring in pyrus species by micropropagation and their elim-ination by tetracycline therapy Plant Pathol 43 819ndash823

Davies DL Guise CM Clark MF and Adams AN (1992) Parryrsquosdisease of pears is similar to pear decline and is associated withmycoplasma-like organisms transmitted by Cacopsylla pyricola PlantPathol 41 195ndash203

Elzanowski A and Ostell J (2000) The genetic codes lthttpwwwncbinlmnihgovhtbin-post Taxonomygt

Frisinghelli C Delaiti L Grando MS Forti D and Vindimian ME(2000) Cacopsylla costalis (Flor 1861) as a vector of apple proliferationin Trentino J Phytopathol 148 425ndash431

Giunchedi L Poggi Pollini C Biondi S and Babini AR (1994) PCRdetection of MLOs in quick decline-affected pear trees in Italy Ann ApplBiol 124 399ndash403

Guerra LJ (1997) Biological and molecular characterization of phyto-plasmas infecting fruit and nut trees in California PhD Thesis Davis CAUniversity of California

von Heijne G (1990) The signal peptide J Membr Biol 115 195ndash201

Jarausch W Danet JL Labonne G Dosba D Broquaire JMSaillard C and Garnier M (2001) Mapping the spread of apricotchlorotic leaf roll (ACLR) in southern France and implication of Cacopsyllapruni as a vector of European stone fruit yellows (ESFY) phytoplasmasPlant Pathol 50 782ndash790

Jensen DD Griggs WH Gonzales CQ and Schneider H (1964)Pear decline virus transmission by pear psylla Phytopathology 541346ndash1351

Keane G Edwards E and Clark MF (1996) Differentiation of group16Sr-IB aster yellows phytoplasmas with monoclonal antibodies Diag-nostics Crop Prod BCPC Symp Series 65 263ndash268

Kison H Kirkpatrick BC and Seemuumlller E (1997) Genetic compar-ison of the peach yellow leaf roll agent with European fruit tree phyto-plasmas of the apple proliferation group Plant Pathol 46 538ndash544

Krczal G Krczal H and Kunze L (1988) Fieberiella florii (Stal) a vectorof the apple proliferation agent Acta Hortic 235 99ndash107

Lefol C Caudwell A Herminier JL and Larrue J (1993) Attachmentof the flavescence doreacutee pathogen (MLO) to leafhopper vectors and otherinsects Ann Appl Biol 123 611ndash622

Marcone C Lee I-M Davis RE Ragozzino A and Seemuumlller E(2000) Classification of aster yellows-group phytoplasmas based oncombined analyses of rRNA and tuf gene sequences Int J Syst EvolMicrobiol 50 1703ndash1713

McCoy RE Caudwell A Chang CJ Chen TA Chiykowski LNCousin MT Dale JL de Leeuw GTN Golino DA Hackett KJKirkpatrick BC Marwitz R Petzold H Sinha RC Sugiura MWhitcomb RF Yang IL Zhu BM and Seemuumlller E (1989) Plantdiseases associated with mycoplasma-like organisms In The Mycoplas-mas Vol V (Whitcomb RF and Tully JG eds) pp 546ndash623 San DiegoAcademic Press

Milne RG Ramassso E Lenzi R Masenga V Sarindu N andClark MF (1995) Pre- and post-embedding immunogold labelling andelectron microscopy in plant host tissues of three antigenically unrelatedMLOs primula yellows tomato big bud and bermudagrass white leafEur J Plant Pathol 101 57ndash67

Nakai K and Kanehisa M (1991) Expert systems for predicting proteinlocalization sites in Gram-negative bacteria Proteins Struct FunctGenet 11 95ndash110

Nielsen H Engelbrecht J Brunak S and von Heijne G (1997) Iden-tification of prokaryotic and eukaryotic signal peptides and prediction oftheir cleavage sites Protein Eng 10 1ndash6

Razin S and Jacobs E (1992) Mycoplasma adhesion J Gen Microbiol138 407ndash422

Schneider B Gibb KS and Seemuumlller E (1997) Sequence and RFLPanalysis of the elongation factor Tu gene used in differentiation and clas-sification of phytoplasmas Microbiology 143 3381ndash3389

Seemuumlller E Marcone C Lauer U Ragozzino A and Goumlschl M(1998) Current status of molecular classification of the phytoplasmasJ Plant Pathol 80 3ndash26

Smart CD Schneider B Blomquist CL Guerra L Harrison NAAhrens U Lorenz KH Seemuumlller E and Kirkpatrick BC (1996)Phytoplasma-specific PCR primers based on sequences of the 16Sminus23SrRNA spacer region Appl Environ Microbiol 62 2988ndash2993

Yu J Wayadande AC and Fletcher J (2000) Spiroplasma citri surfaceprotein P89 implicated in adhesion to cells of the vector Circulifer tenellusPhytopathology 90 716ndash722

Yu Y-L Yeh K-W and Lin CP (1998) An antigenic protein gene of aphytoplasma associated with sweet potato witchesrsquo broom Microbio-logy 144 1257ndash1262