Comprehensive Research on Strawberries

Annual Report

February 1, 1989 - January 31, 1990

Project Title:Evaluation of cDNA probes for the detection of strawberry mild _ yellow edge .

Project Leader and Cooperatin.q Personnel: Dr. Robed R. Martin, Anita Quail and Stuart MacDonald, Agriculture Canada, - 6660 NW Marine Dr., Vancouver, B.C. V6T 1X2 CANADA

Research Objectives: 1. Determine if the cDNA that we have produced from SMYEV infected Rubus is indeed that of SMYEV.

2. Prepare antiserum to the fusion protein of the coat protein of the potexvirus.

3. Test this antiserum in imunnospecific electron microscopy to determine if we can visualize rod-shaped potexvirus particles in the SMYEV infected plants.

4. Use the antiserum of the fusion protein to develop an ELISA based detection for SMYEV.

Concise General Summary: Strawberry mild yellow edge virus (SMYEV) is a major component of the _- aphid-transmitted yellows complex which can reduce strawberry yields significantly. In the current certification systems only plants at the early stages of multiplication schemes are tested for virus infection due to the tedious nature of - the graft indexing that is used for virus detection. With the development of rapid diagnostic procedures for SMYEV and other strawberry it will be possible to test a significant number of plants at all stages of propagation schemes similar to what is done for production of potato tubers. This will enable one to identify where the viruses are being reintroduced in the scheme and allow for rational management decisions to reduce the reintroduction of this virus into the plant _ production schemes. Rapid tests will also allow for epidemiological and ecological studies on these viruses to determine when and from where the viruses are coming into fruiting fields. This information will be useful in the development of spray schedules to minimize the damage done by viruses. _- Rapid virus detection tests in use today either make use of ELISA which is a serological test that requires virus specific antiserum or nucleic acid hybridizations which make use of cDNA specific for the virus being tested. It is hoped that a -- strategy developed to prepare diagnostics for SMYEV will be useable development of diagnostics for some of the other aphid-borne strawberry viruses for which there are not any diagnostics available. _ In this project we have purified disease specific double stranded RNA from SMYEV infected strawberry and Rubus plants. We have cloned this dsRNA and sequenced most of the genome of the virus. From the sequence we were able

-44- d to determine that the virus we have cloned is a potexvirs. All the previous ,_1 information on SMYEV including its mode of transmission by aphid vectors suggested that the virus would be a luteovirus. Since potexviruses are quite _1 different from luteoviruses both in mode of transmission and particle structure we carried out further experimentsto ensure that the virus we were working was not a contaminantbut actuallyinvolved in SMYE disease. ,) To this end, we contacted several people around the world that work with ;I virus diseases of small fruits and requested their isolates of SMYEV. We collected 10 isolates of SMYEV from various locations around the world (2 from California, 1 from Oregon, 1 local isolate from the Fraser Valley in British Columbia,I from Australia,2 from Germany,1 from England, 1 from Belgiumand I from Israel). Each isolate was propagatedin Fra.qariavesca Alpine seedlings and the dsRNA extracted from each isolate as well as from healthy Alpine / seedlings maintained in the same greenhouse. The dsRNA was run on a J agarose gel, transferredto nitrocellulose and probed with the with the cDNA of the potexvirus that we cloned from SMYEV infected strawberry. In these tests,

the cDNA probe hybridized to each of the isolates tested but did not give any l.Jii reaction with healthy strawberry. These results suggests that the virus we are working with is indeed a componentof the SMYE disease. From the sequencing information we were able to identify the coat protein 'il gene of the virus. We transferred this gene into an expression vector which LJ allows us to produce the virus coat protein in bacteria. This coat protein was then purified from the bacteriaand used to prepare an antiserumin rabbits. With _1 the resulting antisera (we made 2 different antisera) we were able to trap potex- J like virus particles (rod-shapedparicles) from infected strawberry plants but not from healthy plants. Early attempts to use this antiserum in ELISA to detect the virus in strawberry plants have been unsuccessful. We are currently injectingthe fusion protein into mice to make monoclonal antibodieswhich should give us a better result in ELISA tests. In addition we are transferring the coat protein of the potexvirusthat we have cloned into strawberry to determine if this will give a _ii form of virus resistance which has been reported for a number of other viruses in the past few years.

ExperimentalProceduresand Results: Virus isolates. The virus isolate used for cloning was SMYEV MY-18 (Martin & Converse, 1985) that had been grafted into Rubus.rosifolius from which the dsRNAtemplates were purified. For immunoelectronmicroscopystudies the MY- J 18 isolate, a German field isolate of SMYEV maintained in F. vesca 'Alpine' '-' indicator plants by transmissionwith Chaetosiphonfra.qaefoliiin a greenhousefor 14 years and two SMYEV isolates obtained from Dr. D. Barbara (East Mailing, [ UK) and purged of any possible nonpersistenttransmitted viruses by successive ._J aphid transmissionswere used. An isolate from Californiadesignated D-531 and dsRNAMY-18 werepurifieused fromd for SMYEVthin sectioninfectedelectronplantsmicroscopy.each of theForaboveNorthernisolatesanalysiswereof .j 1 used except only one of the isolates from East Mailing, plus an isolate from the FraserValley of British Columbia,designatedBC-1, isolate 69N from Dr. Bormans in Belgium,a second Californianisolate designated2, an isolate from R. Heath in -1 Australia,designatedQRCR, and an isolate from Dr. S. Spiegelin Israel.

Anlysis of cDNA clones. Several clones that were shown in agarose gel :-t electrophoresis to contain inserts larger than 1 kbps were mapped physically after digestion With various restriction enzymes and agarose gel electrophoresis.

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I I Overlapping clones were identified by double digestions and confirmed in Southern blots. DNA analysis and modifications were done as described in Maniatiset al. (1982)unless stated otherwise. _ Restrictionfragments of selected clones were purified after separation in 1% Iow melting point agarose gels and subcloned into the multiple cloning site of bluescript M13+ (Strategene). These plasmids were prepared for sequencing of both cDNA strands by unidirectional deletions with exonuclease III and religation - after mung bean nuclease treatment according to Henikoff (1984). DsDNA sequencing by the dideoxynucleotide chain termination method (Sanger et al., 1977) was performed using either the Klenow fragment of DNA polymerase I - (BRL) or Sequenase DNA Polymerase (USB) (Toneguzzo et al., 1988). Single strand DNA templates were prepared with the helper phage M13K07 (Pharmacia). Sequence data were collected, assembled and analyzed with an IBM-AT _ computer using the GENE,MASTERsoftware (Bio-Rad). Sequence alignmentsof the coat protein of several potexviruses was done with the progressive sequence alignmentprogramof Feng and Doolittle(1987). Constructionof the expressionplasmid. The coat proteingene was obtainedfrom cDNA clone p463 by digestionwith Rsal and ligated into the Smal site of pRIT2T. The recombinant plasmid (pCPYE12) was used to transform competent E. coli - N4830-1 cells that contained the phage lambda cl857 temperature sensitive repressor. Insertion of the desired the DNA fragment was confirmed by restriction mapping and sequence analysis. Productionof chimeric coat protein and antiserum. A N4830-1 culture containing the pCPYE12 plasmid was grown at 30C in Luria broth containing ampicillin (100ug/ml)to an OD6ooof 0.8. Fusion protein synthesis was then induced by a -- temperature shift to 42C by adding an equal volume of medium preheated to 54C. After 90 min incubationtime at 42C the cells were cooled and centrifuged at 5000 x g for 10 min. The cells were resuspendedin 1/5 volume of TST (0.05 - M Tris-CI, pH 7.4, 0.15 M NaCI, 0.05% Tween 20) and disrupted by sonication. Clarificationof the homogenatewas obtained by centrifugation at 20,000 x g for 30 min. The chimeric coat protein was then affinity purified on a rabbit globulin _ Sepharose 2B column. After several washes with TST the chimeric coat protein was eluted with 0.1 M acetic acid, lyophilized and stored in aliquots at -20C. Analysis of expression of the fusion protein and degree of purificationwas done by running an aliquot on a 12% SDS-PAGE (Laemmli, 1970) in the presence of a Iow molecular weight standards (Pharmacia) and the protein A from pRIT2T as control. A rabbit was immunized by injection of 1 mg fusion protein in the presence -- of Freunds complete adjuvant. Two booster injections were given with 0.5 mg fusion protein at 3-week intervals. Blood was collected weekly from rabbit ear veins.

Electron microscopy. For immunosorbentelectron microscopy (ISEM) the crude antiserumwas diluted 1:1000 in 0.1 M Na/K-phosphate,pH 7.0. Pioloform-carbon coated nickel grids were floated for 5 min on diluted antiserum, rinsed with 2 mi dilution buffer and transferred to leaf samples homogenized in 0.1 M Na/K- phosphate, pH 7.0, containing 2% PVP 10,000, 0.2% sodium sulfite and 0.05% sodium azide and incubatedovernight at 20C. Decorationof particleswas done - with antiserum diluted 1:5 using 15-30 min incubation times. Before staining with 5 drops of 2% uranyl acetate, grids were rinsed with 4 mi of H20. Excess liquid

-46- 'i ,J was carefully removedwith filter paper, and grids examined using a Zeiss EM 10 C electron microscope. For ultrathin sections, samples were taken from veins of SMYEV infected R. rosifolius (isolate MY-18) and F. vesca 'AlPine' (isolate D-531) leaves. 'l'issu_ was fixed in 4% glutaraldehyde buffered in 0.1 M cacodylate, pH 7.0 (CB), postfixed in 1% osmium tetroxide in CB, dehydrated a in series of ethanol and :t propyleneoxide mixtures,and embeddedin epon. Thin sections were cut with a diamond knife, stained with urany! acetate and lead citrate, and examinedwith a Hitachielectronmicroscope. ,I Northern Analysis. DsRNAs were electrophoresed on 1% agarose gels and electroblotted onto Nytran (Schleicher and Schuell). Oligolabelled probes were prepared as per manufacturersdirections (BRL). Hybridizations,washings and ;I autoradiographywere done as describedin Maniatiset al. (1982). _,J

RESULTSAnalysis and sequencin.q. Mapping of selectedclones by restrictionfragment J analysis revealed a physical map of about 6.0 kb in length (Fig. 1). This indicated that about 97% of the genomic dsRNA, estimated from denaturing methylmercurichydroxide agarose gels to be 6.2 kb in size, was represented in J the cloned cDNA. CDNA clones p309 and p463 hybridized to Northerns of dsRNAs purified from MY-18 infected R. rosifoliuS and dsRNA from an Israeli isoiate of SMYEV. il All sequence data presented in Fig. 2 were deduced from both strands of cDNA clone p463. A terminal poly(A) segment of 13 bases was detected at the 3'end of this clone and presumed to be the 3'end of the viral genome. Clone ri p441 contained a poly(A) Of 41 residues and was found to be identical with 13463 J to the Apal site at position 714; The sequence of 854 nucleotides (Fig. 2)

representsposition 727anwiopenth anreadingUAA terminationframe (ORF)codon,fromencodingthe first aAUGproteincodonof Mrending25,714at !_ (26K). A second AUG codon in the same frame appears at position 58. If this AUG is the functional start codon the ORF would translate to a polypeptide of Mr 23,810 (24K). Located internal to this ORF is a second ORF between positions 295 and 614 encoding a protein of Mr 11,216 (11K). The 3'non-coding region .J consists of 128 nucleotides. Computer assisted progressive sequence alignments of the 26K polypeptide ,t revealed strong amino acid homology with the coat proteins of six potexviruses, (PVX; Huismannet al., 1988), white clover mosaic (WCIMV; Forster et al., 1988; Harbison et al., 1988), papaya mosaic (PMV; Short et al., , I 1986), potato acuba mosaic (PAMV; Bundin et al., 1986), clover yellow mosaic (CYMV; Abouhaidar and Lai, 1989) and mosaic (NMV; Zuidema et al., 1989) viruses. Percent homologybetween the viruses is shown in Table 1. No homologywas found with the 11K proteinto the 10K protein of NMV and the 7K l protein of WCIMV (Zuidema et al., 1989) both of which are located internal to their respective coat proteins.

Purification of pCPYE12 fusion protein. To raise an antiserum against the j proposed potexvirus, a gene fusion consisting of the affinity tail of the protein A gene from Staphylococcus aureus and the potexvirus coat protein gene was c_ constructed. The expressed fusion protein was purified on affinity columns and _j separated with SDS-PAGE. A protein of approximatelyMr 52,000, of which Mr 25,714 (26K) representsthe coat protein gene product was used to immunize a :j -47- ,J r i rabbit. The resulting polyclonal antiserum was utilized for the immunoelectron microscopical (IEM) studies described below.

Particle morpholo.qy and immunoelectron microscopy ('IEM). Filamentous particles were trapped from sap extracts of SMYE- infected plants on grids coated with the antiserum to the fusion protein. Mostly scattered single particles were observed, clusters of particles seldom occured. On uncoated grids particles could be observed infrequently. On antiserum-coated grids at least a 100-fold increase in particle number could be obtained, compared to normal-serum-coated grids. The particle numbers trapped from different extracts varied considerably. Thus a -- difference in particle concentrations between the isolates analyzed could not be detected. No virus particles could be detected in healthy control plants of F. vesca or R. rosifolius. The filamentous particles showed a modal length of 482 - nm and a diameter of 13 nm. In decoration tests the particles showed a strong reaction with the antiserum to the fusion protein. In further decoration tests 27 antisera to definitive or tentative potexviruses were tested for serological reactions with the filamentous particles from the German isolate of SMYEV (Table 2). Eight of these antisera gave weak reactions. Essentially the same result was obtained with the filamentous particles of isolate MY-18. IEM investigations with antisera to beet western yellows (BWYV) and potato - leafroll viruses (PLRV) were conducted to test for the presence of luteovirus particles anticipating a possible heterologous reactivity of these antisera as reported for an antiserum to BWYV by Spiegel et al. (1986). Whereas luteovirus- -- like particles were reported to be associated with the MY-18 isolate (Martin & Converse, 1985) no such particles could be observed in our experiments.

_ Thin section electron microscopy. In order to investigate the location of the potexvirus particles ultrathin sections were taken from leaf tissues of R. rosifolius infected with the MY-18 isolate and F. vesca 'Alpine' infected with D-531 isolate of SMYEV. Bundles of filamentous particles, which are believed to represent the potexvirus particles, were observed in phloem parenchyma cells with both isolates.

- Northern Analysis. On nondenaturing agarose gels the dsRNAs purified from 9 different isolates of SMYEV all comigrated with the dsRNAs obtained from R. rosifolius infected with the MY-18 isolate of SMYEV, whereas we did not see any dsRNA in purifications from healthy strawberry. In Northerns, oligolabelled MY-18 cDNA probes hybridized to the dsRNAs of each of the 9 SMYEV isolates tested but not to nucleic acids from healthy strawberry.

CONCLUSIONS

- The nucleotide sequence of the 3'end of cDNA clone p463 has been determined. Interviral homology of the 26K ORF with published plant viral sequences indicates that the coat protein of an unknown potexvirus from _ strawberries was identified. A 3' poly(A) tail has been observed as is present in other potexviruses: PMV (Short et al., 1986); (CYMV) (Abouhaidar, 1988); PVX (Huismann et al, 1988; Morozov et al., 1987); PAMV (Bundin et al., 1986); WCIMV (Harbison et al., 1988) and NMV (Zuidema et al., 1989) but is not known from luteoviruses (Mayo et al., 1982; Veldt et al., 1988; Miller et al., 1988). The dsRNA was shown to be associated with SMYE disease of different sources

-48- (Spiegel, 1987) and was cloned from the MY-18 isolate of SMYEV. Northern J hybridizations of the dsRNAs from nine isolates from diverse geographicalareas along with lack of hybridization to dsRNA extracted from healthy strawberry I eliminates the possibility of a contaminationby a latent potexvirus present in R. : rosifolius or strawberryindicator plants. Since the MY-18 isolate of SMYEV was isolated by aphids, purged of the possible Presenceof semi-persistentviruses by successive 2h feedings (Martin & Converse, 1985) and aphid transmissionused for maintenance,the potexvirusor its RNA genome is presumably aphid transmissible. This may be by the mechanism of heterologousencapsidationwith a luteovirus as has been reported J for several luteoviruses, ie. carrot red leaf virus and carrot mottle (Waterhouse and Murant, 1983), beet westem yellows virus and lettuce speckles (Falk et al., 1979) or by some other as yet unknown mechanism. Whether or not this is true 1 has to be demonstrated by identifying and differentiating both proposed viruses, _J the luteovirus and the potexvirusassociatedwith SMYE disease. The capsid protein is either 26K or 24K in size depending on the functional AUG codon. A second AUG was also found at the N-terminus of PMV (Short et .J al., 1986; Abouhaidar, 1988) but the first one was thought to be not functional. The significance of the 11K protein located within the coat protein remains unknown. A protein similar in size and location occurs in NMV (Zuidema et al., _ 1989) and WCIMV,reported by Zuidemaet al. (1989); In order to demonstratethe presence of this proposed potexvirus in SMYE affected plants, antiserum prepared to a chimeric coat protein was used to trap t and decorate filamentous particles from infected plants. The homologous ,.J antiserumgave a strong reaction in decoration experimentswhile only eight of 27 different potexvirus antisera tested showed weak reactions possibly indicating r 1 distant serological relatedness. These results demonstrate that the structural J confirmationof the fusion protein resembles epitopes of the native target particles and that this potexvirusis likely a new member,of the potexvirus group. While the preparationof structural or non-structuralviral fusion proteins using a variety of systems has becomea widely utilized technique(Mackenzie& Tremaine,1986; J Forstova et al., 1989; Gombart et al., 1989; Ray et al., 1989) no reports were available about the preparation of antisera to plant viruses by immunizationwith i in-vitro expressedcoat protein. Furthermorethe results of this paper indicate that ,.J cloning of dsRNA, identification of coat protein genes and their expression by fusion protein systems is a suitable technique for the preparation of antisera for I recalcitrant viruses where virions can not be purified in sufficient purity or J concentrations for antiserum production. From results reported here the causal agent responsible for SMYE disease may be either the proposed luteovirus (Converse et al., 1987) or the potexvirus described here or a combination of the two. Our results show the presence of potexvirusparticles in several geographicallydiverse isolates of SMYEV. Isolates MY-18 was obtained originally and propagatd by aphid transmission for several I years in strawberry (Marrtin and Converse, 1985) and the German source of ,J SMYEV was propagated by aphid transmission over a period of 14 years (H. Krczal, pers. comm.). Although this suggests the potexvirus is aphid 1 transmissiblethe possibility of some helper mechanism has not been ruled out. ,.J Generallypotexvirusesare not suspectedto be transmitted by aphids (Koenig and Lesemann, 1978). However,a !ow proportion of aphid transmissionwas reported

for white clover (Goth, 1962). Additionallythe aphidtransmission of b-.ii2 potato aucuba mosaic virus is known but is dependent on the presence of a helper virus of the potato virus Y group (Kassanis & Govier, 1971). Since ] -,t9- !-] luteoviruses have the ability for heterologous encapsidation of several plant viral RNA genomes (Falk et al., 1979; Waterhouse & Murant, 1983) the potexvirus - might be transmitted with the help of the SMYE associated luteovirus reported previously (Yoshikawa et al., 1984; Martin & Converse, 1985; and Spiegel et al., 1986). A name for this virus is not being proposed until its role, if any, in the _ SMYE disease is determined.

Fig. 1. Restriction endonuclease map of cDNA clones prepared from dsRNA associated with the MY-18 isolate of SMYEV. Agarose gel electrophoresis based size estimation of inserts revealed a genomic length of 6.0 kb. Abbreviations of enzymes are as follows: (A) Apal, (B) BamHI, (E) EcoRI, (H) Hindlll, (Hi) Hincll, (K) Kpnl, (P) Pstl, (S) Smal, (Sc) Sac!, (X) Xbal. A(13) and A(41) indicate the _ poly(A) residues found in the respective cDNA clones.

SMYEV POTEXVIRUS MAP 5' 3' 0 1 2 3 4 5 6 Kb I I I I I I I

XS Hi E ScB PHSc P398 !l II II ffr,It'l

P441 A(41}

-50- J Table 1. Per cent sequence identity between the coat protein sequences of SMYEV and six other potexviruses. PMV CYMV PVX WCLMV NMV PAMV SMYEV ;-J PMV 100.00 CYMV 41.35 100.00 ! PVX 39.42 31.43100.00 _ d WCLM 31.28 33.15 38.83 100.00 NMV 27.27 29.52 32.59 50.00 100.00 PAMV 27.80 27.67 27.71 42.47 43.05 100.00 J SMYE 31.37 28,43 29.69 30.27 25.11 23.18 100.00 ] J

] U

i _J'r

U

-51- I Table 2. Decoration tests with fusion protein antiserum and 27 antisera to other -- potexviruses using the German source of the SMYE-associated potexvirus' in extracts of Fra.qaria vesca 'Alpine'.

- Antiserum_ Decoration intensity3

SMYE-associated potex +++ Argent. plantago Boussingaultia mosaic + CactusX - Cassava common mosaic (Brasil) - Cassava common mosaic (Columbia) + Cassavacommonmosaic(WF) ChicoryX + Cloveryellowmosaic Cymbidium mosaic _ Daphne X - Hevea + Hydrangearingspot LilyX Lychnispotex - Narcissusmosaic Papayamosaiv + PepinoX - PlantagoX Plantagoseveremosaic + Potatoaucubamosaic PotatoX PotexSieg + Tamus potex -- TulipX Viola mottle White clover mosaic _ Wineberry latent +

1 virus was trapped with homologous antiserum at 1:1000 dilution 2 antiserum dilution was 1:50 for homologous, and 1:5 for all heterologous combinations 3 (-) = no decoration, (+) = weak decoration, (+++) = strong decoration

Publications or Reports: This work has been submitted to the Journal of General virology for publication. Also, the early part of the work was reported at .-.- the 5th International Symposium Small Fruit Viruses, in Thessaloniki, Greece in June of 1988. The proceedings of this meeting were published in Acta Horticulturae.

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