Chili leaf curl betasatellite is associated with a distinct recombinant begomovirus, Pepper leaf curl Lahore virus, in Capsicum in Pakistan

Virus Research 149 (2010) 109–114

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Short communication

Chili leaf curl betasatellite is associated with a distinct recombinant begomovirus, Pepper leaf curl Lahore virus, in Capsicum in Pakistan Muhammad Tahir a,∗ , Muhammad Saleem Haider a , Rob W. Briddon b a b

School of Biological Sciences, University of the Punjab, New Campus, Lahore, Pakistan National Institute for Biotechnology and Genetic Engineering, Jhang Road, Faisalabad, Pakistan

a r t i c l e

i n f o

Article history: Received 28 January 2009 Received in revised form 18 November 2009 Accepted 30 December 2009 Available online 15 January 2010 Keywords: Begomovirus Betasatellite Recombinant Pepper

a b s t r a c t Capsium spp. are an important vegetable crop cultivated through Pakistan. Leaf curl disease is the major disease of Capsicum spp. in Pakistan caused by viruses. The disease has previously been shown to be associated with begomoviruses and betasatellites. We have cloned and sequenced a begomovirus and its associated betasatellite from Capsicum originating from central Pakistan. The begomovirus isolated was distinct from all previously characterised viruses and we propose the name Pepper leaf curl Lahore virus (PepLCLV) for this new species. Comparison of the sequence of PepLCLV with previously characterised begomoviruses shows it likely to have resulted from recombination between Papaya leaf curl virus and Chili leaf curl virus (ChiLCV), two species that have previously been identified in Pakistan. The betasatellite associated with PepLCLV in Capsicum was identified as Chili leaf curl betasatellite (ChLCB). This is the first identification of a cognate begomovirus for ChLCB infecting Capsicum, although this betasatellite has been shown in association with ChiLCV infecting potato in Pakistan. PepLCLV is one of an increasing number of monopartite begomoviruses shown to be associated with a betasatellite and one of the numerous species that affect Capsicum. In view of their only having been identified in Pakistan, PepLCLV and ChLCB likely represent a geographically distinct, Capsicum adapted, begomovirus–betasatellite complex. © 2010 Published by Elsevier B.V.

Capsicum spp. are a globally important commodity used as a vegetable, spice, medicinal herb, and ornamental plant. In Pakistan two species, Capsicum annum and Capsicum frutescens are known, although most of the cultivated varieties are C. annum (chili peppers) or C. annum var. grossum (sweet/bell peppers). Across Asia diseases caused by viruses are a major limitation to Capsicum production (Ali, 2006) and prime among these on the subcontinent is leaf curl, a disease associated with geminiviruses. Geminiviruses (family Geminiviridae) are circular, single-stranded DNA viruses with characteristic twinned quasiicosahedral particles. The viruses in this family are divided across four genera that are distinguished on the basis of host range, insect vector and genome arrangement (Stanley et al., 2005). The most important geminiviruses, and by far the most numerous, are the members of the genus Begomovirus. Begomoviruses are transmitted exclusively by the whitefly Bemisia tabaci and in the New World they have bipartite genomes (components known as DNA A and DNA B). In the Old World both bipartite and monopartite begomoviruses have been identified (Stanley et al., 2005). The component of bipartite begomoviruses known as DNA A encodes all factors required for virus replication, over-

∗ Corresponding author. Tel.: +92 322 4415661; fax: +92 42 9230980. E-mail address: [email protected] (M. Tahir). 0168-1702/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.virusres.2009.12.007

coming host defences, insect transmission and control of gene expression, while DNA B component encodes factors required for inter- and intra-cellular movement in host plants (Rojas et al., 2005). Begomoviruses that lack a DNA B component are more numerous than their bipartite cousins in the Old World. The majority of these “monopartite” begomoviruses are instead associated with a newly identified class of single-stranded DNA satellites termed betasatellites. Betasatellite molecules are about half the size of the helper begomoviruses (∼1350 bp; Saunders et al., 2000; Briddon et al., 2003). They are involved in symptom induction, host range determination and overcoming host defences (reviewed by Briddon and Stanley, 2006). Leaf samples of chili pepper (CAL) and sweet pepper (CAGL) with leaf curl symptoms were collected in areas around Lahore, Pakistan, in 2004 and a sweet pepper (CAGS) with leaf curl symptoms was collected from Sahiwal in 2006. Total nucleic acids were extracted from leaf samples by the cetyl trimethyl ammonium bromide method (Doyle and Doyle, 1990). A partial clone of the begomovirus was obtained by PCR amplification with “universal primers” (Mansoor et al., 1998) and partially sequenced (results not shown). No amplification products were produced in PCR reactions with nucleic acids extracted from non-symptomatic samples collected from glasshouse grown plants. Specific abutting primer pairs (MT-V 5 -ATCGATGTGAAACTGACCCCAGTCG-3 ; MT-R 5 -ATCGATGGAAGATATGGTCGAGGAGGCAAC-3 , for isolate

a The genes of the virus are denoted as encoding the coat protein (CP), the replication associated protein (Rep), the transcriptional activator protein (TrAP), the replication enhancer protein (REn). The functions of the proteins encoded by V2 and C4 genes remain unclear.

563–201 563–201 562–200 85 (9.4) 85 (9.2) – 2452–2195 2452–2195 – 134 (15.7) 134 (15.7) – 1476–1072 1476–1072 – 134 (15.2) 134 (15.2) – 1621–1217 1621–1217 – 361 (40.5) 361 (40.2) – 2609–1524 2609–1524 – 118 (13.5) 121 (13.8) – 145–501 145–510 –

Coding capacity (no. of amino acids/kDa) Coordinates Coding capacity (no. of amino acids/kDa)

Coding capacity (no. of amino acids/kDa)

Coordinates

Coding capacity (no. of amino acids/kDa)

Coordinates

Coding capacity (no. of amino acids/kDa)

C4 REn TrAP

Coding capacity (no. of amino acids/kDa)

256 (29.6) 256 (29.6) –

Coordinates Coordinates Coordinates

305–1075 305–1075 – CAGL CAL CAGS

␤C1 CP

Coordinates

Betasatellite

Rep V2 Begomovirusa Isolate

Table 1 Positions and coding capacity of predicted genes for the begomoviruses and betasatellites isolated from Capsicum.

120 (13.8) 120 (13.8) 120 (13.6)

M. Tahir et al. / Virus Research 149 (2010) 109–114

Coding capacity (no. of amino acids/kDa)

110

CAGL, and SonAF 5 -GGGCCCCCATGAACTCTTTAAAGTG-3 and SonAR 5 -GGGCCCAAAGGGACTGGCAATC-3’, for isolate CAL), for amplification of the full-length genome, were designed to the sequences of the partial clones. Betasatellites were amplified using universal primer pair Beta01/Beta02 (Briddon et al., 2002). Potentially full-length amplification products for both begomovirus and betasatellite were cloned into the pTZ57R/T vector (Fermentas). Sequences were determined by dideoxynucleotide chain-termination sequencing using GenomeLab Dye Terminator Cycle Sequencing kits (Beckman Coulter) on a Beckman Coulter automated sequencer (CEQ 8000). Sequences were assembled and analysed using DNASTAR (Lasergene). Multiple sequence alignments and phylogenetic trees were produced using MegAlign (DNASTAR) and Clustal X (Thompson et al., 1997). Phylogenetic trees were manipulated and printed using Treeview (Page, 1996). The full-length nucleotide sequences of begomovirus genomes (or DNA A components) obtained from isolates CAGL and CAL were determined to be 2747 bp and 2686 bp, respectively, in length (database accession nos. AM404179 and AM491589). Attempts to amplify a begomovirus genome (or DNA A component) from isolate CAGS using the two primer pairs were uniformly negative. This may indicate that the virus associated with isolate CAGS is distinct from that of isolates CAGL and CAL. Analysis of the two begomovirus components showed the presence of a predicted hairpin structure with the sequence TAATATTAC forming part of the loop. This structure is typically part of the origin of virion-strand replication of geminiviruses (Fontes et al., 1994). The final adenine nucleotide of the nonanucleotide sequence is, by convention, used to start nucleotide numbering. Further examination of the sequences using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) showed the presence of six predicted genes with a coding capacity greater than 12 kDa, two in the virion-sense and four in the complementarysense, diverging from a non-coding sequence (the intergenic region) that contains the predicted hairpin structure. The positions and coding capacity of the predicted genes are given in Table 1. This arrangement of genes is typical of the genomes (or DNA A components) of begomoviruses originating from the Old World. The complete nucleotide sequences of the begomoviruses cloned from isolates CAGL and CAL show 99.4% sequence identity. Based on the presently applicable species demarcation threshold for begomoviruses (89%; Fauquet et al., 2008), this indicates that the two begomoviruses are isolates of the same species. Likely the clone from isolate CAL is a defective molecule, since the final 61 nucleotides (coordinates 2687–2747, relative to the clone from CAGL), spanning the left arm of the hairpin structure, are missing. Comparison of the begomovirus sequences obtained here to sequences available in the databases showed them to have the highest levels of identity to isolates of Pepper leaf curl Bangladesh virus (PepLCBDV) (Table 2). The highest level of identity (87.7% for CAGL and 87.4% for CAL) was with PepLCBDV-PK[PK:Kha:04]. All other sequences showed less than 87% identity. This indicates that the virus identified here is a new species in the genus Begomovirus, for which we propose the name Pepper leaf curl Lahore virus (PepLCLV), with the isolate descriptors PepLCLV[Pakistan:Lahore1:2004] and PepLCLV-[Pakistan:Lahore2:2004] for isolates CAGL and CAL, respectively. A phylogenetic tree, based upon an alignment of the full-length sequences (or DNA A components) of selected begomoviruses is shown in Fig. 1A. This shows the sequences of CAGL and CAL to be most closely related to isolates of PepLCBDV, but basal to them. Together these two species form part of a clade which also includes Croton yellow vein mosaic virus (CYVMV) and Papaya leaf curl virus (PaLCuV). The relative positions of these isolates are well supported by bootstrapping. A more detailed analysis of the sequences of PepLCLV, either by sequence alignment (results not shown), or using Plotcon

Table 2 Highest and lowest percentage nucleotide sequence identities for pairwise comparisons of the sequences of the begomovirus clones from isolates CAGL and CAL with sequences available in the database. ToLCJoV (1)a

a

TbCSV (4)

RaLCV (1)

PepLCV (2)

PepLCIV (2)

PepLCBDV (2)

PaLCuV (2)

CYVMV (1)

CLCuKV (4)

ChiLCV (6)

78.3–80.0

76.2

73.9–74.8

76.1

64.8–67.9

58.7–59.9

87.3–87.4

83.9–84.6

82.2

71.7–72.7

79.0–84.3

77.2–79.6

75.9

73.4–74.4

74.8

64.8–67.9

58.7–59.9

86.6–87.7

83.6–84.0

81.7

71.5–72.4

77.6–83.4

83.4–88.5

81.685.9

77.5–83.6

78.1–81.6

67.5–73.1

60.4–62.3

78.2–83.5

74.5–78.4

70.9–74.2

73.7–76.5

74.9–76.8

78.4–79.1

77.1–79.2

81.4–82.0

64.0–68.2

60.0–60.6

72.2–74.6

76.5–80.3

72.8–74.1

73.8–73.8

75.4

73.6–74.7

75.3

63.4–67.3

59.4–60.5

80.2–80.9

83.4–83.6

76.5–78.0

79.7–79.8

77.0–81.9

82.6–83.5

64.7–68.1

58.0–59.4

83.0–85.1

80.8–84.2

76.8–78.0

74.9–76.5

76.5–77.8

63.4–68.2

58.3–60.5

60.5–61.5

61.3–62.3

59.7–60.9

61.8–62.4

57.0–62.0

61.5–74.2

69.4–73.8

70.0–76.3

66.6–70.4

80.9–81.6

84.4

80.8–83.9

80.1–83.7

85.2–86.7

CAGL (1) 99.2 M. Tahir et al. / Virus Research 149 (2010) 109–114

CAL (1) CAGL (1) ChiLCV (6) CLCuKV (4) CYVMV (1) PaLCuV (2) PepLCBDV (2) PepLCIV (2) PepLCV (1) RaLCV (1) TbCSV (4) ToLCBDV (1)

ToLCBDV (1)

83.8–84.6

The number of sequences available (Fauquet et al., 2008) which were used in the comparisons.

111

112

M. Tahir et al. / Virus Research 149 (2010) 109–114

Fig. 1. Phylogenetic dendrograms based upon alignments of the complete nucleotide sequences of the begomoviruses (A) and betasatellites (B) identified here with selected sequences available in the databases. Horizontal distances are proportional to mutation distances whereas vertical distances are arbitrary. The numbers at each branch indicate the percentage bootstrap confidence scores (1000 replicates). Begomovirus genome sequences used are Ageratum enation virus (AEV), Ageratum leaf curl virus (ALCuV), Ageratum yellow vein virus (AYVV), Chili leaf curl virus (ChiLCV), Cotton leaf curl Alabad virus (CLCuAV), Cotton leaf curl Bangalore virus (CLCuBV), Cotton leaf curl Kokhran virus (CLCuKV), Cotton leaf curl Multan virus (CLCuMV), Croton yellow vein mosaic virus (CYVMV), Euphorbia leaf curl virus (EuLCV), Indian cassava mosaic virus (ICMV), Luffa yellow mosaic virus (LYMV), Malvastrum leaf curl virus (MaLCV), Malvastrum yellow leaf curl virus (MaYLCV), Papaya leaf curl virus (PaLCuV), Pedilanthus leaf curl virus (PedLCV), Pepper leaf curl Bangladesh virus (PepLCBDV), Pepper leaf curl virus (PepLCV); Pepper yellow leaf curl Indonesia virus (PepLCIV), Radish leaf curl virus (RaLCV), Tobacco curly shoot virus (TbCSV), Tobacco leaf curl Yunnan virus (TbLCYnV), Tomato leaf curl Bangalore virus (ToLCBV), Tomato leaf curl Bangladesh virus (ToLCBDV), Tomato leaf curl Guangxi virus (ToLCGxV), Tomato leaf curl Gujarat virus (ToLCGV), Tomato leaf curl Joydebpur virus (ToLCJoV) and Tomato leaf curl Pune virus (ToLCBV). The betasatellite sequences used are Ageratum yellow leaf curl betasatellite (AYLCB), Ageratum yellow vein betasatellite (AYVB), Bean leaf curl China betasatellite (BLCCNB), Chili leaf curl betasatellite (ChLCB), Cotton leaf curl Multan betasatellite (CLCuMB). Croton yellow vein mosaic betasatellite (CroYVMB), Papaya leaf curl betasatellite (PaLCuB), Sida leaf curl betasatellite (SiLCuB), Tobacco leaf curl betasatellite (TbLCB), Tomato leaf curl Bangladesh betasatellite (ToLCBDB), Tomato leaf curl betasatellite (ToLCB), Tomato leaf curl Java betasatellite (ToLCJB), Tomato leaf curl Joydebpur betasatellite (ToLCJoB), Tomato leaf curl Karnataka betasatellite (ToLCKB), Tomato yellow leaf curl China betasatellite (TYLCCNB), and Tomato yellow leaf curl Yunan betasatellite (TYLCYnB). In each case the database accession number of the isolate is given. Isolate codes are as given in Fauquet et al. (2008) and Briddon et al. (2008) for the begomoviruses and betasatellites, respectively. The sequences corresponding to the northern (N) and southern (S) isolates of ChiLB, identified by Hussain et al. (2009) are highlighted.

(Fig. 2), shows the first ∼2160 nucleotides to have high levels of sequence identity (92.8% for CAGL) to Chili leaf curl virusPakistan[Pakistan:Multan:1998] (ChLCV-PK[PK:Mul:98]) but only low identity (44.6%) for the remaining sequence. An initial BLAST comparison to the databases with the remaining 586 nucleotides of PepLCLV identified Papaya leaf curl virus-India[India:Lucknow] (PaLCuV-IN[IN:Luc]) to have the highest levels of similarity. MegAlign alignments showed the sequence of isolate CAGL to have 83.6% overall nucleotide sequence identity to PaLCuVIndia[India:Lucknow], 88.7% over the last 586 nucleotides but only 82.2% identity to nucleotides 1–2160. In contrast, ChLCVPK[PK:Mul:98] showed 75.8% overall nucleotide sequence identity to PaLCuV-IN[IN:Luc] but 82.7% to the first ∼2160 nucleotides and 45.8% to the remaining sequence. This thus suggests that PepLCLV is a recombinant species, consisting of sequences derived from ChLCV and PaLCuV. However, since all these virus species are, at least to some degree, recombinant, the ultimate origin of the sequences that make up PepLCLV remains obscure.

The sequence of PepLCLV originating from PaLCuV includes the entire C4 gene and intergenic sequences upstream of the origin of replication. Consistent with this, PapLCuV and PepLCLV share the same predicted iteron sequences (GGGGA; the sequences to which the replication associated protein [Rep] binds that form part of the origin of replication; Argüello-Astorga et al., 1994) and iteron related domains of Rep (FCVN; the amino acid sequences in the N-terminus of Rep that are predicted to interact with iterons; Argüello-Astorga and Ruiz-Medrano, 2001) whereas those of ChLCV differ (GGTGG and GRFNIN, respectively). The complete nucleotide sequences of betasatellites from isolates CAGL, CAL (acc. nos. AM260466 and AM258978, respectively) were determined to be 1385 bp while that of CAGS (AM849549) was 1372 bp in length. These sequences contain all the features typical of this group of DNA satellites (Briddon et al., 2003); a region of sequence rich in adenine, a single predicted gene in the complementary-sense with the capacity to encode a 120 amino acid protein with a predicted molecular weight of 13.8 kDa and a region of sequence conserved between all betasatellites (known as

M. Tahir et al. / Virus Research 149 (2010) 109–114

Fig. 2. Plotcon similarity plot between the complete nucleotide sequence of the begomovirus cloned from isolate CAGL with ChiLCV-Mul[PK:Mul:98] (upper panel) and PaLCuV-IN[IN:Luc] (lower panel). The approximate positions (with respect to the nucleotide coordinates of the alignment shown at the base of each plot) of the predicted genes (indicated as encoding the V2 protein, the coat protein [CP], the replication associated protein [Rep], the transcriptional activator protein [TrAP], the replication enhancer protein [REn] and the C4 protein) are shown between the two plots. The scanning window size for the plots was 10 nucleotides and the comparison matrix was the default (EDNAFULL).

the satellite conserved region) of approximately 118 bp which contains at it 3 end a predicted hairpin structure containing in the loop the sequence TAATATTAC with similarity to the origin of replication of geminiviruses. The betasatellites cloned from isolates CAGL and CAL share 100% nucleotide identity, whereas that from CAGS shows 92.3% identity to the other two clones. Comparison of these betasatellite sequences to sequences available in the databases showed them to have the highest levels of identity to isolates Chili leaf curl betasatellite (ChLCB) with sequence identities between 86.5% and 94.5% for the 13 sequences available in the databases. Based on the recently proposed species demarcation threshold of 78% for betasatellites (Briddon et al., 2008), this indicates that all three betasatellites identified here are isolates of ChLCB. To all other betasatellite sequences available in the databases the sequences obtained here showed less than 75.5% identity, the level of identity

113

between Tomato leaf curl Bangladesh betasatellite (AJ542489) and the betasatellite from isolate CAGL. A recent study by Hussain et al. (2009) has shown that a single species of betasatellite (ChLCB) is prevalent in chilies across north central Pakistan and that this shows phylogeographic segregation. ChLCB isolates from the northern areas sampled were more diverse and genetically distinct from southern isolates. Only one isolate, not originating from the study of Hussain et al. (2009), ChLCB[PK:MC:97] (AJ316032), did not fit into this grouping, despite being a geographically southern isolate. A phylogenetic tree based on fulllength betasatellites that includes those obtained here is shown in Fig. 1B. The north/south segregation of the isolates of Hussain et al. (2009) is evident, as is the greater diversity of the northern isolates (longer branch lengths). The ChLB identified by Mubin et al. (2009) in potato groups with the southern isolates, correlating with its southern origin. However, the betasatellite obtained from isolate CAGS, despite being a geographically southern isolate, segregates with the northern isolates. Those from CAGL and CAL, in common with ChLCB-[PK:MC:97], fall outside the north/south grouping, being basal to all ChLCBs except ChLCB-[PK:MC:97]. The reasons for this are unclear. Possibly the movement of the viruses within this area is greater than previous studies indicated and/or the genetic diversity of the betasatellites is greater than previously estimated. Whether the sampling here of both sweet/bell pepper as well as chili pepper, whereas the study of Hussain et al. (2009) considered only chili pepper, is a factor in this is unclear. Nevertheless, these studies indicate that the range of betasatellite species in Capsicum is low; only a single species (ChLCB) identified so far. The previous studies that identified ChLCB in Capsicum did not identify the begomovirus with which this betasatellite is associated (Briddon et al., 2003; Hussain et al., 2009), although Mubin et al. (2009) did identify it in association with ChiLCV in potato originating from Pakistan. Ours is thus the first study to identify the begomovirus which is associated with ChLCB in leaf curl disease affected Capsicum in Pakistan. However, it would be premature to conclude that PepLCLV is the only begomovirus occurring with ChLCB in Capsicum, since betasatellites can be transreplicated by numerous begomoviruses (Briddon et al., 2003; Saunders et al., 2008). Thus other begomoviruses and/or other betasatellites may be associated with chili leaf curl disease in Pakistan. Certainly other begomoviruses and other betasatellites have been identified in association with leaf curl disease of Capsicum across other parts of Asia (Hussain et al., 2004; Chattopadhyay et al., 2008). Both PepLCLV and ChLCB have so far only been identified in Pakistan. These components may thus represent a geographically distinct, Capsicum adapted, begomovirus–betasatellite complex. References Ali, M. (Ed.), 2006. Chili (Capsicum spp.) Food Chain Analysis: Setting Research Priorities in Asia. Shanhua, Taiwan: AVRDC—The World Vegetable Center, Technical Bulletin No. 38, AVRDC Publication 06-678, 253 pp. Argüello-Astorga, G.R., Guevara-González, L.R., Herrera-Estrella, L.R., RiveraBustamante, R.F., 1994. Geminivirus replication origins have a group-specific organization of iterative elements: a model for replication. Virology 203, 90–100. Argüello-Astorga, G.R., Ruiz-Medrano, R., 2001. An iteron-related domain is associated to motif 1 in the replication proteins of geminiviruses: identification of potential interacting amino acid–base pairs by a comparative approach. Arch. Virol. 146, 1465–1485. Briddon, R.W., Bull, S.E., Mansoor, S., Amin, I., Markham, P.G., 2002. Universal primers for the PCR-mediated amplification of DNA ␤; a molecule associated with some monopartite begomoviruses. Mol. Biotechnol. 20, 315–318. Briddon, R.W., Bull, S.E., Amin, I., Idris, A.M., Mansoor, S., Bedford, I.D., Dhawan, P., Rishi, N., Siwatch, S.S., Abdel-Salam, A.M., Brown, J.K., Zafar, Y., Markham, P.G., 2003. Diversity of DNA ␤: a satellite molecule associated with some monopartite begomoviruses. Virology 312, 106–121. Briddon, R.W., Brown, J.K., Moriones, E., Stanley, J., Zerbini, M., Zhou, X., Fauquet, C.M., 2008. Recommendations for the classification and nomenclature of the DNA-␤ satellites of begomoviruses. Arch. Virol. 153, 763–781. Briddon, R.W., Stanley, J., 2006. Sub-viral agents associated with plant-infecting single-stranded DNA viruses. Virology 344, 198–210.

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