The applicability of single and double-stranded transcript conformation polymorphism (TCP) for differentiation
among Prunus necrotic ringspot virus (PNRSV) isolates was evaluated and compared with other molecular
differentiation procedures. Single-stranded TCP was found to be suitable to differentiate closely related virus isolates.
However, due to its high variability, it was not suitable for grouping virus isolates. Double-stranded TCP, on the
other hand, enabled the division of virus isolates into major groups. Restriction fragment length polymorphism
(RFLP) of the PCR products confirmed the grouping of the virus isolates but this technique was limited in its ability
to detect a wide range of nucleotide modifications. Nucleotide sequence analysis was essential for the detection of
strain-specific sequences but did not clearly identify most other minor modifications that are necessary for virus
classification. The combination of all methods is therefore sometimes required for complete analysis

Maslenin, Ludmila

Rosner, Arie

Spiegel, Sara

Yilmaz, Mehmet Asil

Yurtmen, Melike

Firenze University Press: E-Journals

Assessing the applicability of transcript conformation polymorphism for differentiation among «Prunus nocrotic ringspot virus» isolates

Arie Rosner

Ludmila Maslenin

Mehmet Asil Yilmaz

Melike Yurtmen

Sara Spiegel

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92 Phytopathologia MediterraneaPhytopathol. Mediterr. (2002) 41, 92–98IntroductionPrunus necrotic ringspot virus (PNRSV) is amember of the genus Ilarvirus in the family Bro-moviridae with a tripartite genome. PNRSV is dis-seminated worldwide in Prunus spp. (Fulton,1983). There are numerous isolates or strains ofthe virus varying widely in their pathogenic, bio-physical and serological properties (Aebig et al.,1987). Distinguishing the various strains is, there-fore, of prime importance.Attempts to differentiate among PNRSV iso-Assessing the applicability of transcript conformationpolymorphism for differentiation amongPrunus necrotic ringspot virus isolatesMELIKE YURTMEN2, SARA SPIEGEL1, LUDMILA MASLENIN1, MEHMET ASIL YILMAZ3 and ARIE ROSNER11Virology Department, Agricultural Research Organization, The Volcani Center, Bet-Dagan 50250, Israel2Plant Protection Research Institute, Kisla Caddesi, PK 21, 01321, Adana, Turkey3The University of Cukurova, Faculty of Agriculture, Plant Protection Department, 01330,  Adana, TurkeySummary. The applicability of single and double-stranded transcript conformation polymorphism (TCP) for differ-entiation among Prunus necrotic ringspot virus (PNRSV) isolates was evaluated and compared with other moleculardifferentiation procedures. Single-stranded TCP was found to be suitable to differentiate closely related virus iso-lates. However, due to its high variability, it was not suitable for grouping virus isolates. Double-stranded TCP, on theother hand, enabled the division of virus isolates into major groups. Restriction fragment length polymorphism(RFLP) of the PCR products confirmed the grouping of the virus isolates but this technique was limited in its abilityto detect a wide range of nucleotide modifications. Nucleotide sequence analysis was essential for the detection ofstrain-specific sequences but did not clearly identify most other minor modifications that are necessary for virusclassification. The combination of all methods is therefore  sometimes required for complete analysis.Key words: PNRS, strain differentiation, TCP, RFLP.Corresponding author: A. RosnerFax: +972 3 9604180E-mail: rosnera@agri.gov.illates have been made by several research groups.Mink et al. (1987) produced polyclonal antibodiesthat distinguished several serotypes of the virus.Crosslin & Mink (1992) demonstrated variationsin the molecular weights of the coat protein of somePNRSV isolates.Recent advances in molecular biology have pro-vided new tools for the classification and identifi-cation of plant viruses. Strain-differentiation bymeans of restriction fragment length polymorphism(RFLP) analysis of PCR products has been carriedout in several laboratories (Rosner et al., 1998;Aparicio et al., 1999). Determination of the nucle-otide sequence of RNA3 for several PNRSV isolates(Guo et al., 1995, Hammond and Crosslin, 1995;Scott et al., 1998) enabled differentiation of sever-al PNRSV isolates.93Vol. 41, No. 2, August 2002Evaluating RNA conformation polymorphism for virus differentiationIn previous studies (Rosner et al., 1998, 1999),we differentiated PNRSV isolates by polymorphismof single and double-stranded RNA transcripts cop-ied from amplified virus-specific PCR products.This method has the advantage of not requiringprior knowledge of the nucleotide sequence of thevirus isolates. However, its unique contributionrelative to other more established molecular meth-ods has not been clearly defined.In the present study we evaluated the relevance,importance and limitations of this method and com-pared it with other molecular procedures used todifferentiate among PNRSV isolates.Materials and methodsVirus isolatesThree isolates of PNRSV served as a model sys-tem in this study. Isolate Apr152 (Apr) was fromapricot cv. Bergeron (Hungary), Ro from rose cv.Mercedes (Israel), and Pe30/4 (Pe) from peach(North Carolina, USA). These isolates were previ-ously characterized by Rosner et al. (1998) andSpiegel et al. (1999). Three unknown isolates ofPNRSV, M1, M2 and M3 originally introduced fromItaly and grafted on peach trees in different re-gions of Turkey, were also included.RNA extractionTotal RNA isolation from plant tissue (100 mgleaves) was carried out by means of the lithiummethod, avoiding phenol extraction, as detailed bySpiegel and Martin (1993).PNRSV clones, primers and RT-PCRThe forward (I) and reverse (II) primers for PCRwere as in Spiegel et al. (1999):(I) 5’-GAGCTCTGGTCCCACTCAGG-3’ (posi-tion 111-130);(II) 5’-CTGCTTGAGATCTAGAGTGA-3’ (posi-tion 701-720).The T7 promoter (5’-AATTTAATACGACGACT-CACTATA-3’) was attached to primer I or II to yieldPCR products with the promoter on its 5’ or 3’-endrespectively. The RNA of both orientations was thusobtained. A standard PCR protocol was applied. Fordetails see Rosner et al. (1998, 1999).TranscriptionA 3-µl sample of the PCR product was incubatedfor one hour at 37°C together with 4 µl rNTP mix-ture (10 mM each), 4 µl transcription buffer 5, 0.5µl RNAsin (5 U µl–1 Promega, Madison, USA), 8.5µl water (total volume 20 µl) and 1 µl T7 RNApolymerase (50 U µl–1, New England, Biolabs, USA).Preparation of double-strand transcript duplexTwo types of PCR products were made for eachvirus isolate, one with the T7 promoter on the 3’-end, the other with the promoter on the 5’-end. ThePCR products were obtained by using, for amplifi-cation, either the forward primer I with the T7promoter, or the T7 promoter attached to reverseprimer II. Copying the two types of PCR productsyielded RNA transcripts of two complementary ori-entations.Annealing of RNA transcriptsComplementary RNA transcripts were mixedand incubated for 5 min at 50°C, followed by slowcooling to room temperature prior to gel electro-phoresis.Electrophoresis of RNAOne third by volume of loading buffer (50%glycerol, 1 mM EDTA and 0.4% bromophenol blue)was added to the RNA samples and fractionationwas carried out by electrophoresis (applied volt-age 100V) in a 108 cm, 6% polyacrylamide gel(PAG) in 40 mM Tris-acetate, pH 8.0; 1 mM EDTA(TAE) buffer at 25°C in a vertical minigel appa-ratus (BioRad Laboratories Inc., Hercules, CA,USA).RFLP analysis of PCR productsAliquots (6 µl) of the amplified PCR productswere digested by the addition of 0.5 U of the ap-propriate restriction endonuclease to a recommend-ed buffer in a total volume of 10 µl. The sampleswere incubated at 37°C for 2 h. The digests werefractionated in 6% PAG by electrophoresis.Sequence analysisPCR products of the virus isolates were clonedinto pGEM-T Easy Plasmid vector (Promega) ac-cording to manufacturer’s instructions. The stand-ard forward (T7) and reverse (SP6) primers wereused for sequencing the cloned virus fragment atthe Molecular Biology Center Ltd. (MBC), Rehovot,Israel.94 Phytopathologia MediterraneaM. Yurtmen et al.ResultsThe aim of this study was to assess the suita-bility of transcript confirmation polymorphism(TCP) together with two other molecular proce-dures: RFLP and nucleotide sequencing, for differ-entiation among PNRSV isolates. Three well-char-acterized isolates, Ro, Pe and Apr (Rosner et al.,1998; Spiegel et al., 1999) and three unidentifiedones, M1, M2 and M3, were chosen for that pur-pose. These isolates were initially characterized bythe more established methods of RFLP and nucle-otide sequencing.RFLP analysis of PCR productsA 600 bp fragment originating in the coat pro-tein gene of PNRSV was amplified by PCR andanalyzed by restriction cleavage (Fig. 1). Three ofthe virus isolates, M1, M2 and Pe, yielded simi-lar cleavage patterns using RsaI, Asp718 andHpaII. The digest patterns of M3 and Ro weredifferent. The RsaI cleavage of Apr differed fromall the others, while its Asp718 and HpaII cleav-age patterns were as in M1, M2 and Pe. The posi-tion of the restriction sites along the amplifiedfragment of each virus isolate is shown in Fig. 2.On the basis of RFLP analysis, the virus isolateswere divided into three main groups: (i) M1, M2and Pe, (ii) M3 and Ro and (iii) Apr. We were un-able, however, to distinguish individual membersof the group by this method.Nucleotide sequence analysisIn order to differentiate further between the close-ly related isolates, the nucleotide sequence of the PCRproducts was determined (Fig. 3). An additional re-peated six-nucleotides-long sequence was found inthe amplified products of M3 and Ro at positions 151–156 of RNA 3. This addition resulted in two corre-sponding extra amino acids arginine (N) and aspar-agine (R) in the putative polypeptide of the virus coatprotein (Fig. 4). These isolates were, therefore, as-signed to group PV32, which was previously describedby Aparicio et al. (1999). Positive identification of themembers belonging to that group can be made onlyby nucleotide sequence analysis. These six repeatedextra nucleotides are clearly identified as strain-spe-cific; however, other minor nucleotide modificationswere not so clearly defined. Therefore, the differenti-ation among individual isolates cannot be made solelyon the basis of sequence data. TCP was used in anattempt to secure better differentiation.Transcript conformation polymorphismSingle-strand transcripts of M1 and M2 (whichhave 99% similarity) (Table 1) copied from the 3’-end slightly differed in their electrophoretic mo-bility (Fig. 5A). The mobility of the Pe isolate wasthe same as that of M2, and both had 98% similar-ity (Table 1). The M3 and Ro transcripts both con-tain the six extra nucleotides (Fig. 3) and have thesame restriction patterns (Fig. 1), and were clear-Fig. 1. Differentiation among Prunus necrotic ringspot virus isolates by restriction endonuclease cleavage of PCRproducts. PCR products amplified from total RNA extracted from isolates M1, M2, Pe, M3, Ro and Apr were cleavedwith RsaI (A), HpaII (B) and Asp718 (C). Digests were fractionated in 6% PAG and stained with ethidium bromide.M, DNA size marker III (Boehringer, Mannheim, Germany).(A) RsaI (B) HpaII (C) Asp71895Vol. 41, No. 2, August 2002Evaluating RNA conformation polymorphism for virus differentiationM1      GAGCTCTGGTCCCACTCAGGGCTCAACAGAGGGCTGCGAATAACCC......GAATAGAA  164M2      ----------------------------------------------......--------Pe      ----------------------------------------------......--------M3      ----------------------------a-----------------gaatag------g-Ro      -----a----------------------a-------------g---gaatag--------Apr     at-----------------a--------a-----------------......--------M1      ACCCGAATAGGGCTTCGAGTGGTACCGGACCAGTGGTCCGACCACAACCGGTCGTGAAGA  224M2      ------------------------------------------------------------Pe      ----------------------gta-c-ga------------------------------M3      ------------t----g-c----ta-----t-c---------g----------------Ro      ------------t------c----ta-----t-c---------g----------------Apr     -----gtg----t-----a--t-gta-----t-ca--------ca----a----------Fig. 2. Restriction map of the PCR products of Prunus necrotic ringspot virus isolates; M1, M2, Pe, M3, Ro and Apr.No. nt: number of nucleotides.Fig. 3. Nucleotide sequence comparison. Nucleotide (nt) sequences of PCR products derived from the six Prunusnecrotic ringspot virus isolates were aligned. Identical nucleotides are marked by a dash, modified ones by smallletters, deletions by dots, and  bold letters mark duplicated nucleotides.Fig. 4. Amino acid alignment. Multiple alignment of the putative coat protein sequences of the Prunus necroticringspot virus isolates M1, M2, M3, Pe, Ro and Apr translated from the nucleotide sequences shown in Fig. 3.M1      ALVPLRAQQRAANNP..NRNPNRASSGTGPVVRPQPVVKTTWTVRGPNVPPRIPKGFVAHNH  84M2      ---------------..---------------------------------------------Pe      ---------------..----------yrt----------i---------------------M3      ---------------nr------v-g-i--a-------------------------y-----Ro      -m-----------s-nr------v---i--a-------------------------y-----Apr     ---------------..----v-v-nvv--a---k-------------------------s-96 Phytopathologia MediterraneaM. Yurtmen et al.ly distinguished by electrophoretic migration ingel. Single-stranded-TCP thus enabled the differ-entiation of the isolates, which had been impossi-ble with either RFLP or sequence analysis alone.The transcripts initiated from the 5’-end, on theother hand, though they differentiated the Apr iso-late from all the others, failed to differentiate be-tween M1, Ro, M3 and Pe (Fig. 5B). These resultsNucleotideSequenceIdentity(%)M2 Pe M3 Ro AprM1 99 98 95 96 84M2 98 94 95 84Pe 93 94 83M3 98 85Ro86Table 1. Percentage of nucleotide sequence identity. The nucleotide sequence (about 600 base pairs long) of theamplified PCR products derived from the Prunus necrotic ringspot virus isolates M1,  M2, M3, Pe, Ro and Apr werecompared and % similarity between them was determined.Fig. 5. Single-stranded Transcript Conformation Polymorphism (TCP) of Prunus necrotic ringspot virus (PNRSV).RNA transcripts copied from the 3’(A) or the 5’(B)-end were made from PCR products of six PNRSV isolates, M1,M2, Pe, M3, Ro and Apr, and analyzed by PAGE. (C) Differentiation of isolates by ds-TCP. Transcript of isolate M1at one orientation (transcription with the T7 promoter on the 3’-end of the PCR products) was used as a reference.It was separately annealed to opposite oriented transcripts (copied from a T7 promoter on the 5’-end) of isolates M1,M2, Pe, M3, Ro and Apr to form RNA duplexes which were analyzed by PAGE.further demonstrate the unpredictable nature ofTCP.Electrophoretic migration of double-strandedRNA made by annealing a reference transcript ofone of the isolates (M1) to complementary tran-scripts of the other isolates (Fig. 5C) resulted inM1, Pe and M2 duplexes which migrated for aboutthe same distance and the M3 and Ro transcripts97Vol. 41, No. 2, August 2002Evaluating RNA conformation polymorphism for virus differentiationwhich also migrated an equal distance. These twotypes of isolates each belonged to a specific virusgroup (Fig. 1 and Table 1). The duplex of Apr dif-fered and belonged to a separate group. Hence ds-TCP was useful in the initial sorting of virus iso-lates into groups. However, members within eachgroup (M1, M2 and Pe; and M3 and Ro) could notbe distinguished from one another by the electro-phoresis of transcript duplexes.DiscussionThe applicability of TCP in combination withother molecular methods for differentiation amongvirus strains was evaluated. All the isolates wereinitially characterized by the more-establishedRFLP and nucleotide sequencing methods. RFLPof PCR products was found to be convenient as ameans of dividing virus isolates into groups at aninitial stage (Fig. 1), provided the strain-specificrestriction sites were well characterized. Thismethod however, was limited in its ability to de-tect most other nucleotide variations. It identifiedonly nucleotide transitions found within the shortrecognition sequence of a particular restrictionendonuclease (e.g. differentiation among M2 andM3 in Fig. 1 and 2) but did not detect most othermodifications residing outside that sequence (seedigest of M1, M2 and Pe in Fig. 1)Nucleotide sequence analysis is essential todetect well-defined strain-specific repeated se-quences in RNA3, as shown with M3 and Ro (Fig.2). It should be noted that the addition of theseextra six nucleotides, which was repeated in M3and Ro, was correlated with the absence of theAsp718 site and with a shift in the position of theHpaII site (Fig. 1 and 2). These restriction sitesmay serve as convenient molecular markers, avoid-ing sequence analysis. However, most minor nu-cleotide modifications were not well characterized,and therefore were not sufficiently reliable to dif-ferentiate the virus isolates. TCP is generally be-lieved to be affected by multiple nucleotide modifi-cations within the RNA molecule (Sarkar et al.,1992; Rosner et al., 1998). In this report we havedemonstrated that ss-TCP distinguishes closelyrelated virus isolates (as judged by the degree ofsequence similarity among them, see % similarityin Table 1). Transcripts of M1 and M2 were distin-guished by PAGE as were M3 and Ro (Fig. 5A)which otherwise could not be distinguished by ei-ther RFLP (Fig. 1) or sequence analysis (Fig. 3).M1 and Pe seemed to be identical by all tested cri-teria. However, single-stranded-TCP is an empiri-cal procedure that does not follow strict rules andis almost totally unpredictable in nature. There-fore, it cannot be used to group virus isolates. In-stead, this could be achieved by applying ds-TCP,as shown in Fig. 5C. Migration of RNA duplexes ingel was correlated with their classification ingroups by RFLP or nucleotide sequence analysis.It is concluded that virus strain differentiationcannot solely rely on any one of the methods de-scribed above, but rather needs a combination ofall three, each complementing the other at a dif-ferent level of analysis. Double-stranded-TCP orRFLP could be used for the initial division of virusisolates into major groups. Nucleotide sequenceanalysis is essential in assigning virus isolates con-taining strain-specific sequences, and ss-TCP helpsin distinguishing between closely related virus iso-lates.AcknowledgementsThis project was supported by an Israeli For-eign Ministry grant (MASHAV) to M. Yurtmen, andit was carried out as part of her Ph.D. thesis.Literature citedAebig J.A., R.L. Jordan and H.T. Hsu, 1987. Immunochem-ical and biological properties of a mouse monoclonalantibody reactive to prunus necrotic ringspot ilarvirus.Intervirology 28, 57–64.Aparicio F., A. Myrta, B. Di Terlizzi and V. Pallas, 1999.Molecular variability among isolates of prunus necrot-ic ringspot virus from different Prunus spp. Phytopa-thology 89, 991–999.Crosslin J.M. and G.I. Mink, 1992. Biophysical differencesamong prunus necrotic ringspot ilarviruses. Phytopa-thology 82, 200–206.Fulton R.W., 1983. Ilarvirus group. CMI/AAB Descriptionof Plant Viruses No. 260 Dundee, UK.Guo D., E. Maiss, G. Adam and R. Casper, 1995. Prunusnecrotic ringspot ilarvirus: Nucleotide sequence ofRNA3 and the relationship to other ilarviruses basedon coat protein comparison. Journal of General Virolo-gy 76, 1073–1079.Hammond R.W. and J.M. Crosslin, 1995. The completenucleotide sequence of RNA3 of a peach isolate of pru-nus necrotic ringspot virus. Virology 208, 349–353.Mink G.I., W.E. Howell, A. Cole and S. Regev, 1987. Three98 Phytopathologia MediterraneaM. Yurtmen et al.Accepted for publication: May 14, 2002serotypes of prunus necrotic ringspot virus isolated fromrugose mosaic-diseased sweet cherry trees in Washing-ton. Plant Disease 71, 91–93.Rosner A., L. Maslenin and S. Spiegel, 1998. Differentia-tion among isolates of prunus necrotic ringspot virusby transcript conformation polymorphism. Journal ofVirological Methods 74, 109–115.Rosner A., L. Maslenin and S. Spiegel, 1999.Double-strand-ed conformation polymorphism of heterologous RNAtranscripts and its use for virus strain differentiation.Plant Pathology 48, 235–239.Sarkar G., H.S. Yoon, S. Sommer, 1992. Screening for mu-tations by RNA single-strand conformation polymor-phism (rSSCP): comparison with DNA-SSCP. NucleicAcids Research 20, 871–878.Scott S.W., M.T. Zimmerman, X. Ge and D.J. MacKenzie,1998. The coat protein and putative movement proteinsof isolates of prunus necrotic ringspot virus from differ-ent host species and geographic origins are extensivelyconserved. European Journal of Plant Pathology 104,155–161.Spiegel S. and R.R. Martin, 1993. Improved detection ofpotato leafroll virus in dormant potato tubers and mi-crotubers by the polymerase chain reaction and ELI-SA. Annals of Applied Biology 122, 493–500.Spiegel S., Y. Tam, L. Maslenin, M. Kolber, M. Nemeth andA. Rosner, 1999. Typing prunus necrotic ringspot virusisolates by serology and restriction endonuclease anal-ysis of PCR products. Annals of Applied Biology 135,395–400.

Assessing the applicability of transcript conformation polymorphism for differentiation among «Prunus nocrotic ringspot virus» isolates

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