Esfandiari, Neda

Hohn, Thomas

Kohi-Habibi, Mina

Pooggin, Mikhail

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Complete genome sequence of an Iranian isolate of Potato virus Xfrom the legume plant Pisum sativumNeda Esfandiari Æ Mina Kohi-Habibi ÆThomas Hohn Æ Mikhail M. PoogginPublished online: 31 May 2009 Springer Science+Business Media, LLC 2009The natural host range of Potato virus X (PVX) has so farbeen restricted to the families Amaranthaceae, Cruciferae,and Solanaceae. Here we provide evidence that in IranPVX also occurs on pea (Pisum sativum), a legume plant.To understand the molecular basis for this expansion of thevirus host range, we sequenced a complete RNA genome ofthe Iranian isolate of PVX (PVX-Iran) and compared itwith the genomes of 14 known PVX isolates. The PVX-Iran nucleotide sequence shares 95.4–96.7% identity withthe Eurasian isolates and 77.2–78.0% with the SouthAmerican ones and, like all other isolates, codes for RNA-dependent RNA-polymerase (RdRP), triple gene blockproteins (TGBp) 1, 2, and 3, and coat protein (CP). At theamino acid level, PVX-Iran is most closely related to theKorean isolate (PVX-KR) sharing 98.1–99.2% identity and98.6–100% similarity. Further inspection of PVX-Iranrevealed several unique amino acid substitutions that donot occur in any other isolate, most notably A542 in RdRP,T10 in TGBp3, and T24 in CP, which individually or incombination could have contributed to breaking pearesistance to PVX.Potato virus X (PVX) is the type member of genusPotexvirus in the family Flexiviridae. Its capped andpolyadenylated, single-stranded RNA(?) genome, 6432–6435 nt in length [excluding the poly(A) tail], which isencapsidated in flexuous filamentous virions, contains fiveconserved ORFs. ORF1 codes for RNA-dependent RNApolymerase (RdRP) of 166 KDa, involved in genomereplication and production of three subgenomic RNAs. Theoverlapping ORFs 2, 3, and 4 code for triple gene blockproteins (TGBp) of 25, 12, and 8 kDa, respectively, allrequired for cell-to-cell movement. In addition, TGBp1 is asuppressor of plant defense based on RNA silencing [1].ORF 5 encodes the coat protein (CP) of 27 KDa, which inaddition to building the virions and aiding to cell-to-cellmovement can elicit hypersensitive response in plantscarrying specific resistance genes (for recent review onPVX, see [14]).The natural host range of PVX has so far included plantspecies of the families Amaranthaceae, Cruciferae, andSolanaceae. Using serological methods, electron micros-copy, and RT-PCR we detected PVX on pea (Pisum sati-vum; the family Leguminosae or Fabaceae) in Iran [4, 5];unpublished data). Among other viruses identified onsymptomatic pea plants in several commercial fields of theTeheran province during 2002 and 2004, the incidence ofPVX systemic infection was the highest (67%). The PVXsymptoms observed in naturally-infected pea plants inclu-ded stunting and wilting as well as premature death ofyoung seedlings. To our knowledge, this is the first case ofnatural occurrence of PVX on pea, though PVX wasreported, following experimental inoculation, to causesystemic infection on crimsom clover (Trifolium incarna-tum) but not other legume plants tested [15] and localnecrotic lesions on some cultivars of pea [3]. The latterfinding suggests that pea and other legumes carry gene(s)conferring resistance to PVX, which in some cases triggerhypersensitive response. As a first step to understanding theElectronic supplementary material The online version of thisarticle (doi:10.1007/s11262-009-0371-0) contains supplementarymaterial, which is available to authorized users.N. Esfandiari  T. Hohn  M. M. Pooggin (&)Institute of Botany, University of Basel, Schoenbeinstrasse 6,Basel 4058, Switzerlande-mail: mikhail.pooggin@unibas.ch; pooggin@yahoo.comN. Esfandiari  M. Kohi-HabibiDepartment of Plant Protection, Faculty of HorticulturalSciences and Plant Protection Agriculture, The Universityof Tehran, Karaj, Iran123Virus Genes (2009) 39:141–145DOI 10.1007/s11262-009-0371-0Table1Percentageofnucleotideidentityandaminoacididentity/similarityofPVX-IrantootherPVXisolatesPVXisolateAccessionno.Wholegenome6435nt50 UTR84ntRdRP4371nt1457aaTGB1681nt227aaTGB2348nt116aaTGB3213nt71aaCP714nt238aa30 UTR82ntPVX-KRKoreaAF37378296.796.496.398.1/99.297.299.6/100.098.099.1/100.097.298.6/98.697.299.2/99.2100.0PVX-TaiwanChinaAF27273696.697.696.298.4/99.196.898.2/99.697.798.3/99.197.795.8/97.297.699.2/99.2100.0PVX-X3EuropeD0034496.5100.096.197.9/98.996.998.7/99.197.799.1/100.097.297.2/98.697.299.2/99.2100.0PVX-FX21ChinaEF42357296.496.496.298.1/98.896.098.2/99.196.399.1/100.097.797.2/97.297.199.2/99.2100.0PVX-MoscowRussiaM72416M37458,X0519896.297.695.998.2/99.096.898.2/99.696.699.1/100.097.797.2/97.296.197.5/99.2100.0PVX-OSJapanAB05671896.297.695.698.3/99.196.597.8/99.198.099.1/100.098.698.6/98.697.398.7/98.7100.0PVX-UK3UKM9551696.096.495.697.9/98.795.997.8/98.297.799.1/100.095.898.6/98.697.298.7/98.7100.0PVX-BSJapanAB05671995.898.895.397.9/98.996.298.2/99.196.697.4/100.098.197.2/97.295.997.5/97.9100.0PVX-Roth1UKAF11119395.6100.095.397.7/98.695.098.7/100.097.799.1/100.096.798.6/98.695.998.3/98.798.6PVX-TulaRussiaEU02121595.495.295.398.1/99.095.698.2/100.097.199.1/100.097.298.6/98.695.097.9/98.7100.0PVX-HBSouthAmericaX7221478.088.177.590.0/94.577.389.9/94.382.188.8/94.871.970.8/79.278.788.2/93.395.8PVX-HBhSouthAmericaZ2325677.988.177.490.0/94.577.088.1/93.082.689.7/95.771.469.4/79.279.289.5/94.595.8PVX-CPSouthAmericaX5580277.388.176.689.3/93.877.789.9/93.079.188.8/94.872.771.8/81.779.689.1/94.195.8PVX-CP4SouthAmericaAF17225977.288.176.689.9/94.277.689.0/92.579.689.7/94.870.670.4/80.378.989.9/95.095.8142 Virus Genes (2009) 39:141–145123molecular basis for breaking pea resistance to PVX, wesequenced a complete genome of the Iranian isolate ofPVX (PVX-Iran) and performed its phylogenetic analysis.The complete genomes of fourteen PVX isolates havebeen reported (Table 1; [6–11, 13, 16]. To sequence acomplete RNA genome of PVX-Iran we designed RT-PCRprimers (Table S1), which would anneal to the regionsconserved in the sequenced PVX isolates.PVX-Iran was purified after three successive singlelesion transfers on Gompherena globosa and one systemicinfection on Nicotiana glutinosa and used for the hostrange studies with various plant species [4, 5] includingNicotiana benthamiana. A back passage of the virus isolateto pea resulted in leaf wilting followed by death of infectedplants, indicating that the pea-infecting phenotype wasstable over the passages. Total RNA was isolated fromsystemically-infected leaves of N. benthamiana 14 dayspost-inoculation using an RNeasy plant mini kit (Qiagen)and transcribed by SuperScript III reverse transcriptase(Invitrogen) with the RT primer (Table S1) complementaryFig. 1 A Neighbor Joining treeshowing the genetic distancesbetween the nucleotidesequences of 15 isolates ofPVX. Analysis was performedwith PAUP 4.0 (PhylogeneticAnalysis Using Parsimony,Version 4; Sinauer Associates,Sunderland, Massachusetts).The tree was rooted using fourSouth American isolates (HB,HBh, CP and CP4) as anoutgroup. The bootstrap valuesare indicated. The cutoff forcollapsing the branches was60%Virus Genes (2009) 39:141–145 143123to the absolutely conserved PVX 30-terminal sequencepreceding poly(A). The resulting viral cDNA was used forconsecutive PCR reactions with Vent polymerase (BioLabs)to amplify eight overlapping fragments, *0.8–1.2 kbp inlength, covering the entire genome of PVX-Iran. Each PCRproduct was gel-purified and sequenced in both orientationsusing the corresponding forward and reverse PCR primers(Table S1). The assembled genome sequence of PVX-Iran(GenBank accession no. FJ461343) was complete withexception of the 50- and the 30-terminal sequences of 24 and28 nts, respectively (corresponding to the terminal PCRprimers; Table S1), in which some nucleotide substitu-tion(s) could not be excluded. The comparison of PVX-Iranwith the 14 other PVX isolates at the nucleotide and aminoacid levels were done by the Needleman–Wunsch globalpair-wise alignment and the CLUSTAL W multiple align-ment programs (http://bioweb2.pasteur.fr/intro-en.html)and the results are shown in Table 1 and SupplementaryData. A Neighbor Joining tree showing the genetic dis-tances between the 15 isolates is shown in Fig. 1.Phylogenetically, PVX-Iran falls within the Eurasian(EA) group of PVX isolates and shows 95.4–96.7% iden-tity at the whole genome nucleotide level to 10 othermembers of the group described previously [11, 16]. It ismore distantly related to 4 isolates of the South American(SA) group (72.2–78.0% identity) (Table 1 and Fig. 1).PVX-Iran contains five conserved ORFs for RdRP, TGBp1,TGBp2, TGBp3, and CP, which are identical in positionand length to those of all the other EA isolates. At theamino acid level, PVX-Iran is most closely related to theKorean isolate (PVX-KR) showing 98.1–99.2% identityand 98.6–100% similarity and least related to the PVX-CP4isolate from the SA group showing 70.4–89.9% identityand 80.3–95.0% similarity, depending on the protein(Table 1). Inspection of the multiple protein alignments(Supplementary Data) revealed several unique amino acidsubstitutions present in PVX-Iran but not in any other EAor SA isolate. These include E134 (D in other EA isolates;H in SA isolates), A490 (V/I/G in EA; P in SA), A542 (E/Din EA; T in SA), A613 (V in all others), I651 (M in allothers) and V811 (I in all others) in RdRP; D177 (E in allothers) in TGBp1; T10 (I in all others) in TGBp3; and T24(V in PVX-OS; A in all others) in CP. In TGBp2 no uniqueamino acid was found, except that PVX-Iran and PVX-Tula share H98, while other EA isolates have Y and SAisolates have C at this position. Based on amino acidsimilarity scores, the RdRP A490, the TGBp3 T10 and theCP T24 are most dissimilar at the corresponding positions.The PVX coat protein is one of the principle determinantsof the outcome of interactions between a virus isolate andpotato cultivars carrying distinct resistance genes, leadingto resistance or systemic infection [2, 9, 12]. Our furthercomparison of the CP amino acid sequences of PVX-Iranand 36 other isolates found in the Genbank [16] confirmedthat T24 is a unique signature of the PVX-Iran coat protein(Supplementary Data).We propose that the amino acid substitutions in CP and/or other viral proteins could have contributed to breakingpea resistance to PVX. However, we cannot exclude thatchanges at the nucleotide level, especially in untranslatedregions, could have also played a role. Thus, future studiesshould address which of the changes in the PVX genomeare responsible the expansion of its host range to a legumeplant.Acknowledgments We thank Dirk Redecker for help with thephylogenetic analysis. The work was supported by Swiss NationalScience Foundation.References1. E.H. Bayne, D.V. Rakitina, S.Y. Morozov, D.C. Baulcombe,Cell-to-cell movement of potato potex virus X is dependent onsuppression of RNA silencing. Plant J. 44, 471–482 (2005). doi:10.1111/j.1365-313X.2005.02539.x2. A. Bendahmane, K. 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Complete genome sequence of an Iranian isolate of Potato virus X from the legume plant Pisum sativum

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Complete genome sequence of an Iranian isolate of Potato virus X from the legume plant Pisum sativum

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