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Chattopadhyay2008 Article InfectivityOfTheClonedComponen
Chattopadhyay2008 Article InfectivityOfTheClonedComponen
DOI 10.1007/s00705-007-0017-2
BRIEF REPORT
Received: 19 July 2007 / Accepted: 22 November 2007 / Published online: 4 January 2008
Ó Springer-Verlag 2007
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534 Arch Virol (2008) 153:533–539
chilli leaf curl etiology has been established during the For cloning of the DNA-b satellite, universal primers
1960s in the country [9, 19], pathogenicity of the cloned located in the highly conserved region found in all DNA-b
DNA components has not been demonstrated. Based on satellite sequences were used for amplification [5]. An
partial DNA-A sequences, tomato leaf curl New Delhi approximately 1.3-kb fragment was amplified from infec-
virus (ToLCNDV) and chilli leaf curl virus (ChiLCV) were ted chilli plants and was cloned in the pTZ57R/T vector
reported to be associated with ChiLCD [15, 24]. Although (INSTA cloning kit, MBI Fermentas, USA). In spite of
full-length genome sequence suggested association of chilli several attempts, we could not isolate a DNA-B component
leaf curl virus—India [India::05] (GenBank accession from the infected samples. PCR reactions using different
number DQ673859) with ChiLCD in India [26], no combinations of primers based on the DNA-A intergenic
detailed information is available. Here we demonstrate the region and DNA-B [21] did not yield a DNA-B fragment.
association of chilli leaf curl virus (ChiLCV) with a This shows that there is no DNA-B component for this
DNA-b satellite from leaf curl infected chilli samples from virus.
Varanasi, which together cause typical leaf disease To assess infectivity, the viral genome and DNA-b
symptoms in chilli. satellite were subcloned as partial tandem repeats in the
Leaves from chilli plants, cultivar Kashi Anmol, show- binary vector, pCAMBIA2301. The BamHI (147)—PstI
ing yellowing and leaf curling were collected from a (1,444) fragment of the ChiLCV genome (pBCVA8) was
severely affected field in the experimental farm of the first cloned into pCAMBIA2301 and the full-length BamHI
Indian Institute of Vegetable Research, Varanasi, Uttar fragment was then inserted to produce a 1.5mer tandem
Pradesh, India, in 2005. Total DNA from symptomatic repeat named pTChVA. For the DNA-b satellite
leaves was isolated by the cetyl-trimethylammonium bro- (pBCVb4), the KpnI (1,111)-BamHI (1,148) fragment was
mide method [27] followed by concentration of the super- cloned into pCAMBIA2301, followed by ligation at the
coiled DNA by the alkaline lysis method [1]. The viral KpnI site to produce a 1.9mer tandem repeat named
replicative form (RF) was used as a template for poly- pTChVb. Insert orientation within partial tandem repeats
merase chain reaction using degenerate primers was confirmed by restriction digestion with the appropriate
(PAL1v1978/PAR1c496) used for detection of begomovi- enzymes. The partial tandemly repeated inserts of clones of
ruses [21], and begomovirus infection was initially viral genome and DNA-b satellite were agroinoculated into
detected. RF DNA was digested with restriction endonu- chilli (Capsicum annuum cv. Kashi Anmol), N. benthami-
cleases (BamHI, EcoRI, HindIII, PstI) and cloned into ana and tomato (Lycopersiscon esculentum cv. Kashi
pUC18 previously linearized with the same enzymes. Four Amrit). Seeds were surface sterilized after soaking in
full-length clones were obtained. Cross-hybridization HgCl2 for 1 min and sown on sterilized soil. Equimolar
among the four ChiLCV isolates, partial sequencing of concentrations of viral genome and DNA-b satellite were
several clones, and sequences comparisons with other mixed and agroinoculated onto 15-day-old test plants by
begomoviruses revealed that all four clones comprised the stem agroinoculation (by crown needle puncture). Symp-
complete viral genome (at Bam HI site) and yielded similar toms were recorded periodically.
restriction patterns after digestion with BamHI, BglI, ClaI, Upon agroinoculation, the viral genome could induce
EcoRI, EcoRV, HindIII, KpnI, PstI and XhoI. All four mild curling and yellowing of leaves on chilli (Table 1). On
clones were obtained from the same sample. One clone was N. benthamiana, the viral genome alone could induce leaf
selected arbitrarily for further analysis. curling and yellowing of lamina. The tandemly repeated
Table 1 Agroinfection of selected plant species with cloned tandemly repeated copies of ChiLCD components
Plant/constructs Infected/inoculateda Symptomb Incubation periodc
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Arch Virol (2008) 153:533–539 535
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536 Arch Virol (2008) 153:533–539
100 ChiLCV-Mul[PK:Mul:98];AF336806
100 ChiLCV-PK[IN:Var:06] ;EF190217
99 PepLCPKV-[PK:Kha1:04] ;DQ116878
ToLCJoV-BD[BD] ;AJ875159
35 100 ChiLCV-IN[IN:PRM:Tom:05];DQ629103
100 ChiLCV-IN[IN::05] ;DQ673859
ToLCBDV-[BD:2] ;AF188481
42 71 AEV-[NP:01] ;AJ437618
58 TbCSV-[CN:Yn35:01] ;AJ420318
CYVMV-[IN] ;AJ507777
100 PepLCBDV-PK[PK:Kha:04] ;DQ116881
100
24 PepLCBDV-BD[BD:Bog:99];AF314531
70 PepLCPKV-[PK:Kha2:04] ;DQ116879
46 PaLCuV-IN[IN:Luc] ;Y15934
100 PaLCuV-PK[PK:Cot:02] ;AJ436992
96
ToLCKV-Ban[IN:Ban:93] ;U38239
100 ToLCGV-[IN:Var:01] ;AY190290
EuLCV-[CN:Gx35:02] ;AJ558121
97
99 CLCuKV-Man[IN:Dab] ;AY456683
100 CLCuKV-Man[PK:M806b:96] :AJ002449
100
CLCuKV-Fai[PK:Fai1] ;AJ496286
ToLCSLV-[LK:Ban:97] ;AF274349
ToLCBV-[IN:Pun:05] ;AY754814
100
81 98 ToLCBV-A[IN:Ban1] ;Z48182
99
ToLCBV-B[IN:Ban5] ;AF295401
100 ToLCBV-C[IN:Ban4:97] AF165098
99 ToLCKeV-[IN:KerII:05] ;DQ852623
MaYVV-[CN:Yn47:01] ;AJ457824
100 CLCuMV-Fai[PK:Y62:95] ;AJ002447
100
CLCuMV-IN[IN:Sri:94] ;AF363011
100 CLCuAV-[PK:K802a:96] ;AJ002455
59
100 BYVMV-IN[IN:Mad] ;AF241479
100 OYVMV-[PK:Fai201:95] ;AJ002451
SLCCNV-IN[IN:Luc:Pum] ;DQ026296
100 ToLCNDV-IN[IN:ND:Svr:92] ;U15015
64 MaLCV-[CN:Gx87:04] ;AJ971263
100
TbLCYnV-[CN:Yn143:02] ;AJ512762
SiLCuV-[CN:Hn57:04] ;AM050730
SLCMV-LK[LK:Col:98] ;AJ314737
100 ICMV-Ker[IN:Ker2:02] ;AJ575819
100 ICMV-IN[IN:Mah:88] ;AJ314739
VeYVV-[IN:Mad:05] :AM182232
MYMIV-[IN:ND:Bg3:91] ;AF126406
Fig. 2 Phylogenetic tree based on complete nucleotide sequences of to calculated mutation distances. Numbers at nodes indicate percent-
43 geminiviruses isolates. Sequences were aligned using ClustalW. age bootstrap scores. GenBank accession numbers are indicated to the
Vertical distances are arbitrary, horizontal distances are proportional right of each virus name. Abbreviations are according to [12]
PepLCBDV-PK[PK:Kha:04] for AC1 (97%), PepLCBDV- for AC4 (96%), suggesting that different regions of the viral
BD[BD:Bog:99] for AC2 (94.8%), ChiLCV-IN[IN:PRM: genome have different origins, probably from chilli-
Tom:05] for AC3 (94.8%), and ChiLCV-PK[PK:Mul:98] infecting begomoviruses (data not shown). On the basis of
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Arch Virol (2008) 153:533–539 537
Fig. 3 Dendrogram of complete nucleotide sequences of DNA-b Numbers at nodes indicate percentage bootstrap scores. Accession
satellites. The tree was generated using the neighbor-joining method numbers indicated on the right of the virus name. Abbreviations are
in Phylip. Vertical distances are proportional to sequence distances. according to [6]
sequence identity of the viral genome, and in accordance Phylogenetic analyses were performed based on a mul-
with the ICTV Geminiviridae study group guidelines tiple alignment of the present isolate and 48 other well-
[11, 12], the present virus isolate is considered to be a strain characterized begomoviruses. The present virus isolate
of ChiLCV-PK and is named ChiLCV-PK[IN:Var:06]. clusters together with begomoviruses infecting chillies in
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538 Arch Virol (2008) 153:533–539
Pakistan and India and is most closely related to Association of a DNA-B was not observed, but the viral
ChiLCV-PK[PK:Mul:98] and ChiLCV-Kha[PK:Kha1:05] genome was found to be associated with a DNA-b satellite.
(Fig. 2). However, ChiLCV-PK[IN:Var:06] is distantly Therefore, ChiLCV-PK[IN:Var:06] is a monopartite virus
related to chilli-infecting begomoviruses from Bangladesh, which causes severe leaf curl disease of chillies in Varanasi
such as PepLCBDV-BD[BD:Bog:99] and PepLCBDV- when associated with a DNA-b satellite. DNA-b satellites
PK[PK:Kha:04]. are known to be required for induction of disease symptoms
Sequence analysis demonstrated that the DNA-b satel- in several host-virus combinations [2, 4, 14, 22, 23, 30].
lite isolated in the present investigation has 1,361 Although the viral genome alone is infectious and produces
nucleotides (EF190215) and has structural features shared mild and delayed symptoms, association with a DNA-b
by other DNA-b molecules, including an adenine-rich increases symptom severity and considerably shortens the
region located at approximately nucleotides 716–878, a incubation period. Typical symptoms of ChiLCD (leaf
satellite-conserved element (SCR) with 219 nucleotides curling, yellowing of the leaf lamina, leaf distortion and leaf
(1,157–13) and one conserved bC1 ORF encoding a size reduction) were observed only when both components
putative 13.7-kDa protein on the complementary-sense were present. This is the first demonstration of Koch’s
DNA. postulates using cloned DNA of ChiLCV and the associated
Nucleotide sequence comparisons show that the DNA-b DNA-b satellite.
shares high nucleotide sequence identity with ToLCBDB-
[IN:Raj:03] and ToLCBDB-[IN:Luc:CH:05] isolated from Acknowledgments This research was supported by the Department
of Science and Technology, Government of India.
tomato and chillies, respectively. The highest nucleotide
sequence identity was found with ToLCB-[IN:Raj:03]
(87.9%). Since pBCVb4 shares nucleotide identity over References
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