Download Nucleotide sequences from tomato leaf curl viruses from different

Journal of General Virology (1995), 76, 2043 2049. Printed in Great Britain
2043
Nucleotide sequences from tomato leaf curl viruses from different
countries: evidence for three geographically separate branches in evolution
of the coat protein of whitefly-transmitted geminiviruses
Y. G. Hongl~ " and B. D. Harrison z*
1 Scottish Crop Research Institute, bTvergowrie, Dundee D D 2 5 D A and 2 Department o f Biological Sciences,
University o f Dundee, Dundee DD1 4 H N , U K
The coat protein (CP) gene-containing circular DNA
molecule of an isolate of tomato leaf curl geminivirns
(ITmLCV; 2749 nt) obtained from southern India, and
the CP genes of tomato yellow leaf curl geminivirus
isolates from Nigeria and two regions of Saudi Arabia
were sequenced. ITmLCV DNA had the same arrangement o f ORFs, and the same pattern of repeats in
the large intergenic region as is found in DNA-A of
other whitefly-transmitted geminiviruses (WTGs) from
the Old World. However, the sequence of ITmLCV
DNA and the sequences of its predicted translation
products differed substantially from those of other
WTGs, including one isolate obtained from a tomato
plant in northern India. Comparison of the four CP
sequences deduced here with those of 18 WTGs
previously studied indicated that their relationships can
be represented by a tree with three branches that are
unrelated to plant host species but which contain viruses
from the Americas, Africa to the Middle East, and Asia
to Australia, respectively. It is suggested that WTG CP
evolution has proceeded along different paths in these
three main regions, and that WTGs have adapted freely
to new hosts in each region. Indeed, the virus isolates
causing similar diseases of tomato plants in the different
continents are, with few exceptions, not closely related
and warrant recognition as separate species.
Introduction
appear to have only one, which closely resembles
DNA-A. Moreover, the nucleotide sequences of the
different isolates, although related, are no more similar
to one another than to the sequences of WTGs from
other plant species.
Evidence of antigenic differences, which parallel these
genomic differences, was provided by the results of tests
in which extracts of naturally infected tomato leaves
from many countries were allowed to react with panels of
MAbs raised against the particles of two other WTGs,
African cassava mosaic and Indian cassava mosaic
viruses. The WTGs from tomato plants in different
geographical regions proved to have consistently
different epitope profiles, whereas those from the same
region had similar profiles (Harrison et al., 1991;
Muniyappa et al., 1991; Macintosh et al., 1992). To
obtain further information on genomic and antigenic
differences among tomato-infecting WTGs, we have
determined the nucleotide sequences of a DNA molecule
(equivalent to DNA-A of other WTGs) of a tomato leaf
curl virus isolate from southern India (ITmLCV;
Muniyappa et al., 1991) and of the coat protein (CP)
genes of tomato yellow leaf curl virus isolates from
Nigeria and two regions of Saudi Arabia. Comparison of
the nucleotide sequence of DNA-A of ITmLCV with the
equivalent sequence of a virus isolate from northern
Diseases described as tomato leaf curl and tomato yellow
leaf curl occur in many parts of the tropics and subtropics, ranging from Central America to the
Mediterranean region, Africa, Asia and Australia. They
are important causes of loss in yield of tomato crops
(Cohen & Harpaz, 1964; Makkouk & Laterrot, 1983;
Saikia & Muniyappa, 1989) and have become
increasingly prevalent in recent years. The diseases are
caused by whitefly-transmitted geminiviruses (WTGs)
but the genomes of different virus isolates differ in
complexity and nucleotide sequence. Virus isolates from
Thailand (Rochester et al., 1994) and northern India
(Padidam et al., 1995a) have genomes consisting of two
molecules of circular ssDNA (DNA-A and DNA-B),
whereas isolates from Israel (Navot et al., 1991), Sardinia
(Kheyr-Pour et al., 1992) and Australia (Dry et al., 1993)
* Author for correspondence. Fax +44 1382 562426. e-mail
[email protected]
~ Present address: Department of Virus Research, John Innes
Centre, ColneyLane, NorwichNR4 7UH, UK.
The nucleotide sequence data presented in this paper have been
submitted to the EMBL database under accessionnumber Z48182.
0001-3203 © 1995 SGM
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2044
Y. G. Hong and B. D. Harrison
India shows that there are many differences between the
two isolates, although the deduced amino acid sequences
of their CPs are similar. Comparison of the four new CP
sequences with other published sequences identifies a
consistent pattern of geographical variation among
WTGs from tomato plants.
Methods
Virus isolates. Four virus isolates were studied. ITmLCV was
originally obtained from a leaf curl-affected tomato plant from
Bangalore, southern India; it was transmitted initially by a single
whitefly (Bemisia tabaci) and subsequently was cultured in graftinoculated tomato plants (Muniyappa et al., 1991). The other three
isolates were obtained from yellow leaf curl-affected tomato plants
growing at Ibadan, Nigeria (TYLCV-NIG), A1 Soudieri, central Saudi
Arabia (TYLCV-NSA) and Najran, southern Saudi Arabia (TYLCVSSA) (I. A1-Shahwan, B. D. Harrison & P. F. McGrath, unpublished),
and were cultured in graft-inoculated tomato cv. Moneymaker plants.
All inoculated plants were kept in containment conditions under licence
from the Scottish Office Agriculture and Fisheries Department.
D N A extraction and P C R . Total DNA was extracted as described by
Hong et al. (1993) from young leaves of tomato plants that had recently
developed systemic symptoms. Viral CP genes were obtained by PCR
using degenerate primers P1 and P2, and these genes were then cloned
in pUC19, as previously described (Hong et al., 1993). Full-length
DNA-A of ITmLCV was amplified by PCR using total DNA from
infected leaf tissue as the template, and the following two primers based
on sequences in the CP gene. In primer Pa [5' d(cgggatcc ACAGCCTCTAGGAACATCAG) Y], the upper-case letters represent nucleotides complementary to residues 504-485 in the viral sequence, and in
primer Pb [5' d(cgggatcc GAGGGTCCATGTAAGGTCC) 3'] they
represent residues 505 523. The lower-case octanucleotides in each
primer contain a B a m H I site. Reaction mixtures (100 lal) contained
1 5 lag total DNA, 75 pmol each of Pa and Pb, 100 laM of each dNTP,
2.5 mM-MgC12, 50 mM-KC1, 0.1 rag/m1 gelatin, 10 mM-Tri~HCI
(pH 8.0) and 2.5 units of Taq DNA polymerase (Cambio). The reaction
mixture was overlaid with 50 lal of light oil. PCR was initiated with one
cycle at 94 °C for 2 min, 52 °C for 1.5 min, 72 °C for 3 min, followed by
five cycles at 94 °C for 30 s, 55 °C for 1 min, 72 °C for 3 min, then by
30 cycles at 94 °C for 30 s, 58 °C for 1 rain, 72 °C for 3 min, and finally
by one cycle at 72 °C for 5 min, in a Cambio Intelligent Heating Block.
After electrophoresis of the PCR products in a 1-0 % LMP agarose
(Gibco BRL) gel, the required fragment was recovered and cloned into
the B a m H I site of pUCI9.
Sequence analysis. Nucleotide sequences were determined by the
dideoxynucleotide chain termination method (Sanger et al., 1977) in
both directions using plasmid DNA templates and the Klenow
fragment of Escherichia coli DNA polymerase I (Pharmacia) or
Sequenase Version 2.0 (United States Biochemical) with 7-deazadGTP in the sequencing reaction. Sequences were assembled and
analysed using UWGCG software (Devereux et al., 1984). Other WTG
sequences used in the comparisons were those of abutilon mosaic virus
(AbMV; Frischmuth et al., 1990), African cassava mosaic virus from
Kenya (ACMV-K; Stanley & Gay, 1983), Nigeria (ACMV-N; Morris
et al., 1990) and Ghana (ACMV-G; Y. G. Hong & B. D. Harrison,
unpublished), bean dwarf mosaic virus (BDMV; Hidayat et al., 1993),
bean golden mosaic virus (BGMV; Howarth et al., 1985), East African
cassava mosaic virus (EACMV; Hong et al., 1993), Indian cassava
mosaic virus (ICMV; Hong et al., 1993), potato yellow mosaic virus
(PYMV; Coutts et al., 1991), squash leaf curl virus (SqLCV-E;
Lazarowitz & Lazdins, 1991), tomato golden mosaic virus (TGMV;
Hamilton et al., 1984), tomato mottle virus (TMoV; Abouzid et al.,
1992), tomato leaf curl virus from Australia (TLCV-A; Dry et al., 1993)
and northern India (TLCV-IN; Padidam et al., 1995a), tomato yellow
leaf curl virus from Israel (TYLCV-ISR; Navot et al., 1991), Sardinia
(TYLCV-SAR; Kheyr-Pour et al., 1992) and Thailand (TYLCV-THI;
Rochester et al., 1994), and tomato geminivirus from Mexico (TGVMX; R. L. Gilbertson, personal communication).
A ca. 2.7 kb product was amplified from samples containing
ITmLCV DNA by using the abutting primers Pa and Pb. Two subfragments produced by treatment with B a m H I were cloned in pUC19,
and the whole 2749 nt molecule (27.2% A, 19.9% C, 22.4% G and
30.5% T) was sequenced in both orientations from these two
contiguous clones. Sequences at the junctions between the two
fragments were confirmed by sequencing the CP gene (see below) and
a fragment produced by the PCR that spanned nucleotides 125 1245.
Results and Discussion
Sequences of I T m L C V
The complete ITmLCV sequence forms a circular
molecule containing, as in other Old World WTGs such
as ACMV, ICMV and TYLCV-ISR, two ORFs in the
virus sense and four in the complementary sense (Fig. 1).
In accord with evidence indicating the site of the nick
made during replication of the DNA of other WTGs
(Stanley, 1995), nucleotide number 1 was assigned to the
A underlined in the sequence TAATATTAC, which also
occurs in the intergenic region of all other geminiviruses
(Fig. 1).
Comparison of sequences of the large intergenic
regions of five WTGs from tomato showed that ITmLCV
2749/1
2000
A ~
O0
Fig. I Arrangement of O R E s (VI, nt 301-107] ; V2, nt ]41488; CI, nt
2608 1526; C2, nt 1623 1219; C3, nt 1478-1074; C4, nt 245]-2]58) in
the 2749 nt long D N A of ITmLCV. V] encodes the CP. Potential
promoter sequences (V) and polyadeny]ation signals (V) are shown
inside the circle for virus-sense genes and outside the circle for
complementary-sense genes. IR, intergenic region.
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Tomato geminivirus CP relationships
ITmLCV
TLCV-IN
1
50
............ TTTGAATC GGTGGACACT CTAATTCTCT GTATATCGGT
IIIII
II I1 I
I
I
II III
A A A A C T T G T C G T T T T G A T T C GGCGTCCCTC ~.CTTATCTA TATGATTGGT
51
GGAA.TGGTG
II I
I
I
T T T T G C T G T C G T T C T G A A T C GGGGGACACT CA~'~GTATCC
II
ill
II I
I
I
..... T G C G T T T T A G C A A T T GGTGTCTCTC ~..CTTGGT~
I
I Ill
IIIIII
I I I II
II
....... GTT G A A A T G A A T C GGTGTCCCTC ~'~.AGC"I~TAT
I
1111
TYLCV-SAR
TLCV-A
TYLCV-ISR
ITmLCV
III
ill
AAGTTCCAGT
III
TYLCV-SAR
III
ilJll
TATTTTTTAA
I I
il
GGAATTGGGG
GGCAATATAT
I
1 I
I
TTCATAAATG
TTCATTTCAA
TTCAAAATTC AAAATTCAAA
ITmLCV
201
CTATCATGGT
CC ........
CCTCCACTAA
TLCV-IN
TAGGTGGGCC
TYLCV-SAR
AGAAGTGGGT
CC ........
I I
II
II
I
i
CCCCCCACGT
[
I
TLCV-A
AAAGTGGTCC
TYLCV-ISR
TATGTGGTCC
CCACGAGGGT
TACACAGATG
ITmLCV
301
CGCCAAGTTT
TACCATTAAA
324
TAAA
TLCV-IN
CACCAAGTTT
I llilill
It
I
1 1111ILl1
CCCTAAGTTT
TLCV-A
CCCTAAGTAT
TYLCV-ISR
CCCCAAGTTT
llil
]
I
il
CCAATGAAAT
I l]lll
I
.ACCTGTCGA CGAATGAGAA
II]I
CTCAAGCGGC
II
I
TAGTTGGACA
Ill
II
IIIIII
IIIIIll
CTAAATGGCA
TAGATGTAAT
I
I
IIIIII
CCGTATGGCA
tl
Illll
tlllt
TTTTGGTAAT
lllllllll
IIIIIIIII
Illlllllll
CATTCGTATA
ATATTACCGA
ATGGCCGCGC
III
Illllllll
IIIIIIllll
CATCCGTATA
ATATTACCGG
ATGGCCGCGC
illllll
iillilltll
llIllllll
ATATTACCGG
ATGGCCGCG .... AAAAAAT
IIIIII
II
CATCCGTCTA
IIIIIII
I
TCCCCGATAA
I i
iiiiiiii11
llIlillIi
CATCCGTATA
ATATTACCGG
ATGGCCGCGC
CTTTTCCTTT
251
GTCACGCTGA
AAGCTTAATT
ATTTATTTTT
GTCCT.TATA
300
TAAACTTAGT
i
TCACGCTACA
Ii
il
il
II
I
...AAATTTT
TTAAAGCGGC
LI It
I I
TGGCCTATTT
II
II
I
AGTGCGTGGG
i I
II
TGCAGCCTCA AAGCTTAAAT AACT.GTTCA
II III
illil
t I
II
CCGCGCGTCA TCGCTTATTT A.AGTTTT..
I I
III
CCAATCAAAT
TGCATTCTCA
i I III
TTATTGTCAA
I
III
I I 111
AACGTTAGAT
II
A.AGTGTTCA
I
I
I
t II
11
IIII
I
TAGACTTGCT
I I
l II I I I l l l l
GCTGT.GTTA TAAACTTGCT
GATCAATAAA
II
I[
illll
TTGTCGTATA
TATACTTGGG
II
IIIIIIIII
TATACTTGGT
I II
TTTGTCTTTA
II
I
TYLCV-SAR
III
II
GGATCCACAA
1 I JJlJlJ
ll[llil[
GATTGATGTG
Ililtll
Ill
GTAAAGCGGC
I
I II[IIIIIII
TTTCTGTCGA
IIIII
I llillll
f lillill
AATCAAATCA
250
CCAATCATAT
II i IIIII I [I
TCAA CCAATGAAAT
II
i
IIIIII
I llllill
ATCATTTCCA
C .........................
II
CTAAAGCGGC
1
ATGATGCCCC
I
GTCTTATTTA
IIIII1111
ATTCAAATCC
[ IIIIIIII
TACGCAGTAG
II
III
GTC..T.GGG
i[
TTTTTTTTTG
II
TYLCV-ISR
i
IIIIII
200
CTTTCTGTCA
CTCA ......... AAAGTAA ATAATTCAAA
il
I
ATGGCCGCGC
TCGGAGAATT
I
1 IIIIIIit
TTATTGTAAT
[ Illlll
ATATTACCGG
TLCV-A
1
I
CATCCGTATA
i
i
IIIIII
t
I
151
AAATTGCGGC
GTAATAAATT
III
III
CCATATGGCA
I
150
CA . . . . . . . . . A T A T C C C G G
II
ill
II
AGGTAAGACA
ATTTGGTAAT
TTCAAATTCC
III
I
IIIIII
CCTAATGGCT
I I I
I
I
TAATTGTAAT
TACTTGGACA
AAAGAAA ATTACTTTAA
II
Illlll
TCCTATATAT
I
CCAAATGGCA
GTCTTACTTA
[
I
I
ACCCTGTCCA
GTA..TCGGT
TTTGA ..................
TATTCAAA ............
I
GTC..TGGAG
I
TLCV-IN
I00
GACAATATAT
IIIII
AGC~TTG~
I
III
II
ATG~TCC-GT
IIIIIII
GGCAATC~T
II
i01
TACCATTA ............
I
2045
AAAAAATATA
I
q
CAAA
II
TTAGGCCCAT
l il
AC..
I
i i
TTTGTCTTGC
AAA.
iP
AAT.
Fig. 2. Comparisonof the sequences of the intergenic regions of fiveWTGs derived from
tomato. Dots represent spaces inserted to improvethe match. The sixth nucleotide in the
ITmLCV sequence is residue 2614 in the complete sequence.
differed not only from TYLCV-ISR, T Y L C V - S A R and
TLCV-A (61%, 65% and 53 % identity, respectively),
but also from T L C V - I N (60 % ; Fig. 2). However, despite
these differences, the 34 nucleotide sequence G C G G C C A T c C G T a T A A T A T T A C C G G A T G G C C G C G , which
can form the well-known stem-loop structure, was the
same in all five viruses except that the lower case ' c '
is T in T L C V - I N and the ' a ' is C in TLCV-A. This
sequence, or only slightly different versions of it, also
occur in other WTGs, such as ICMV, E A C M V and
ACMV, which are obtained from hosts other than
tomato. Like those of other W T G s (Argfiello-Astorga et
al., 1994), the large intergenic region of I T m L C V
contains short nucleotide sequence repeats. The sequence
A T C G G T G G A at nucleotides 2614-2622 is repeated at
nucleotides 2641-2649, and the overlapping sequence
G G T G G A C A (2617-2624) also occurs at nucleotides
2652-2659 and in the complementary sense at nucleotides
2670-2677. This arrangement of iterations is similar to
that found in other W T G s from the Old World; most of
the sequence that is repeated also occurs in TYLCV-ISR,
T L C V - A and a Brazilian form of B G M V (ArgfielloAstorga et al., 1994). The comparable repeats in TYLCVSAR and T L C V - I N differ to a greater extent from
those in ITmLCV. Analysis of the intergenic region of
I T m L C V therefore suggests that the virus is distinct from
T L C V - I N and also from the other W T G s obtained
from tomato plants. Comparison of the deduced amino
Table 1. Percentage amino acid sequence identity of
putative products of open reading frames of 11 WTGs
compared with ITmLCV
Open reading frame
Virus
V1
V2
C1
C2
C3
C4
TYLCV-ISR
TYLCV-SAR
TLCV-A
TLCV-IN
ICMV
ACMV-K
AbMV
BGMV
PYMV
SqLCV-E
TGMV
74
69
74
86
87
71
76
72
74
73
75
69
64
59
61
69
62
78
74
80
77
74
73
62
63
69
56
67
67
62
63
58
62
66
50
54
53
49
50
68
61
63
64
67
64
53
50
54
48
53
72
48
52
58
51
31
55
53
23
54
13
-
-
acid sequences of the gene products of I T m L C V and 11
other W T G s provides further support for this proposition (Table 1). The products of O R F s C2, C3 and C4
are most similar to those of T Y L C V - I S R whereas the CI
product is most like that of T L C V - A ; the V1 product
(CP) is most like that of I C M V and the V2 product is
similar to the same extent to those o f I C M V and
TYLCV-ISR. With the possible exception of CP, the
gene products of I T m L C V differ from those of other
W T G s to about the same extent that other WTGs differ
from one another. However, the C1, C2 and C3 products
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2046
Y. G. Hong and B. D. Harrison
1
TYLCV-NSA
50
MSKRPGDIII
STPVSKVRRR
LNFDSP__YSSR~VAPIVPGIN
TYLCV-ISR
...............................
TYLCV-NIG
TYLCV-SAR
...........................
.P..T.,.L
..................
TYLCV-SSA
....................................
TLCV-IN
ITmLCV
TLCV-A
TYLCV-THI
.A...A
.......
AV...Q.T
A.M..
.G .... Y..L
A..V ....
A ..... K
.....
A..L
...........
A ....
V.T.R..S
T..
,A..T.Q.,*
A .............
.....
.....
KRRSWTYRPM
Q.T
..........
..........
...........
GA.
..V..ARVT*
.AKA..N...
.SSI .... K.
...N..FK.A
.AVR..R.T*
.AV,T.RVT*
.GKE.AN.,.
R..T.VN...
.AV.T,RVT*
.GQV.KN..A
..................
N..
51
i00
TYLCV-NSA
TYLCV-ISR
TYLCV-NIG
YRKPRIYRMY
...........
..... M...F
RSPDVPRGCE
GPCKVQSYEQ
RDDIKHTGIV
R .............................
.T..I ..................
V..V...
TYLCV-SAR
TYLCV-SSA
TLCV-IN
..... M ..........
P ................
...........
T ...............
FDK
N .... M .....................
F.S
V .... V.
.H.L .... E.
.H.VS.I.K.
LC .......
MC .......
N
T
ITmLCV
TLCV-A
TYLCV-THI
N...MF...F
.G ...............
.... MM, .LF
.....................
....... M ........
K ..........
.H,.I.I.K.
H. VA. V. K.
KN..G.M.K.
MCI ......
LC .......
ICLY .....
T
T
I
TYLCV-NSA
TYLCV-ISR
GITHRVGKRF
CVKSIYFLGK
VWMDENIKKQ
~HTNQVMFFL
..................................................
TYLCV-NIG
TYLCV-SAR
TYLCV-SSA
SL .........
...........
.L .........
I .... I .................
I .... I...
I ..........
I .... IV ..........
VK
N .............
ISI ..............
.... TC..W
.......
.L ............
.L ............
..... T .....
V.V...
V.V,..
I .... V...
I .......
TK
I .......
TK
I...D...TR
.... S ...........
T.*.
.... S ........
...VD*K
.... T ........
..... *T
I .......
.... T.L.WI
F.S
FDA
RRVSDVTRGS
C ........
°C.°°,,,°,
.C°°°...o°
i01
TLCV-IN
ITmLCV
TLCV-A
TYLCV-THI
150
.L ............
L..V..
VK
VRDRRPYGNS
*.
T.
VT*T
.... G.T.*T
200
151
TYLCV-NSA
TYLCV-ISR
TYLCV-NIG
TYLCV-SAR
TYLCV-SSA
TLCV-IN
ITmLCV
TLCV-A
TYLCV-THI
TYLCV-NSA
TYLCV-ISR
TYLCV-NIG
TYLCV-SAR
TYLCV-SSA
TLCV-IN
~MD[GQ~__N.NM
FDNEPSTATV
KNDLRDRFQVMRKFHATVIG
..................................................
.S .... I ............
I N ...... Y..
L...S
..........
.YG..EL...
.Q...E
.Q...D
................
................
.K ........
.Y..Q .... V
Y ............
Y ............
201
VKRFFRVNSH
VTY**NHQEA
..E.VK..NY
IN..YKIYN.
IRK.Y...NY
TYLCV-NSA
TYLCV-ISR
251
260
RIYFYDSIS~
..........
TYLCV-NIG
TYLCV-SAR
TYLCV-SSA
...... AVT.
...... AVT.
.V ........
TLCV-A
TYLCV-THI
.........
.......
.......
AT.
VT.
........
Q.
.S ..... VT.
C .....
Y ..........
V .........
C.
C..
LKR.T..PEC
.QYAS...CV
MH...Y..
MH...Y..
M..G...
Q ......
AKYENHTENA
L..W
L..W
.... T.
.... T.
.TYAS
.QYAS
.....
.....
IK.WS...T.
I.R.Q...T.
.QYAS
.QYAA
.... I
.... I
LLLYMACTHA
250
SNPVYATMKI
..... KI .....
LFIFI
.................................
..... K..N.
.V.**..HDE
...A .......................
L .... KI.T.
.V.** .... Q
...........................
IR..YKIYN.
IV.**
.... Q G ..........................
.RK.V...NY
.V.**.Q..,
G ..........
M ...............
ITmLCV
TLCV-A
TYLCV-THI
TLCV-IN
ITmLCV
LI*MT
............
Y ........
I .......
~PSGMKEQAL
.V.**,Q...
G ..........
M ...............
C..**
................................
.V.** .....
G ..........................
L..
L..
L..
L..
L..
L..
L.V
Fig, 3. Comparison of the amino acid sequences of the CPs of nine
WTGs derived from tomato. Differences from TYLCV-NSA are
indicated and conserved residues are underlined; an asterisk indicates
that the residue is absent. Virus acronyms are given in the text.
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Tomato geminivirus CP relationships
~
--
PYMV
--
TGV-MX
BDMV
TMoV
AbMV
I
SqLCV-E
- -
TGMV
BGMV
~
ACMV-G
ACMV-N
ACMV-K
EACMV
TYLCV-SAR
TYLCV-ISR
I
TYLCV-NSA
TYLCV-NIG
2047
substitutions found, many occur between residues with
similar properties, such as V/I and R/K, and most are
clustered in specific parts of the sequence, notably
residues 2847, 78-93, 129-130, 147-157 and 174-221. In
addition, TYLCV-SAR lacks residue 163, and TYLCVISR has two residues (FI) inserted at positions 214-215.
Other regions, notably residues 222-256 in Fig. 3, are
strongly conserved.
Comparisons of pairs of sequences show that those of
TYLCV-NSA and TYLCV-ISR are 96% identical,
suggesting that these two isolates are strains of the same
virus. In contrast, the CP of TYLCV-SSA is distinct
from that of TYLCV-NSA and of the other isolates from
Mediterranean countries or West Africa (75-78 % identity), and slightly more like those of the two Indian
isolates, which are even more similar (86%) to one
another. TYLCV-NIG CP is most similar to those of
TYLCV-ISR and TYLCV-NSA (84-86 %). TLCV-A CP
does not closely resemble CP of any other isolate but is
most similar to the proteins of the Indian and Thailand
isolates (74-77%), whereas TYLCV-SAR CP most
closely resembles the CPs of TYLCV-ISR and TYLCVNSA (84-85 %).
ICMV
[-~
TLCV-IN
Geographical variation in geminivirus coat proteins
ffmLCV
The similarities and differences among these tomato
WTGs are put in perspective by including in the analysis
the CPs of 13 other WTGs from a variety of hosts. It can
then be seen that sequence divergence is independent of
the natural host of each virus (Fig. 4). Thus on the basis
of their CPs, WTGs from tomato in the New World
resemble WTGs from other hosts in New World
countries more closely than they resemble Old World
WTGs from tomato. Comparable similarities in epitope
profiles of the particles of WTGs from the Americas, and
differences between these and Old World WTGs, were
found by Swanson et al. (1992). Indeed, inspection of
Fig. 4 shows that the relationships among the CPs reflect
the geographical sources of the viruses. The dendrogram
has three main branches, representing viruses from the
Americas, Africa/Mediterranean region/Middle East
and Asia/Australia, respectively. The CPs of WTGs
from different geographical regions typically have
67-79 % sequence identity, whereas the values for WTGs
from the same region range from 80-97 % mostly.
This pattern of geographically associated variation in
WTG CP is suggestive of (i) an effect of spatial isolation,
coupled with non-adaptive genetic drift and/or adaptive
change in response to regionally specific selection
pressures, and (ii) an ability of the virus lineage in a
specific region to adapt to different hosts. Evidence for a
difference associated with spatial isolation is provided by
comparison of TYLCV-NSA and TYLCV-SSA, the
TYLCV-THI
TLCV-A
TYLCV-SSA
Fig. 4. A dendrogram produced by using the UWGCG program
PILEUP, showingrelationshipsamongthe CPs of 22 WTGs.The three
main branches contain the proteins of viruses from the Americas
(upper branch), Africa/Mediterraneanregion/Middle East (middle
branch) and Asia/Australia(lowerbranch). Virus acronymsare given
in the text.
of ITmLCV are more like those of Old World than New
World WTGs. We have no information on the possible
existence of DNA-B in ITmLCV or on the infectivity of
the molecule we have sequenced.
Coat proteins of tomato geminiviruses
To obtain a more complete picture of the relationships
among tomato WTGs from different parts of the world,
nucleotide sequences were determined for the CP genes
of TYLCV-NIG, TYLCV-NSA and TYLCV-SSA, and
the deduced amino acid sequences were compared with
those of ITmLCV, TLCV-A, TLCV-IN, TYLCV-THI,
TYLCV-ISR and TYLCV-SAR (Fig. 3). The proteins all
consist of 256-260 residues, about 45 % of which are
identical in all nine sequences. Of the amino acid
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2048
Y. G. Hong and B. D. Harrison
Saudi Arabian sources of which are separated by a large
expanse of desert. Where no such barrier exists, as in
India, the CPs of ITmLCV and TLCV-IN are considerably more alike. Evidence that a geographically
related selection pressure operating on CP structure may
be exerted by vector whiteflies comes from three findings :
the key role of geminivirus CP specificity in effecting
transmission by insect vectors (Briddon et al., 1990), the
occurrence of different biotypes of B. tabaci in different
countries (Costa & Brown, 1991), and the greater
frequency of transmission of a WTG by a sympatric than
by an allopatric biotype (McGrath & Harrison, 1995).
The association of different WTGs with similar
diseases of the same plant species in different countries,
and the possibility that WTGs can adapt more readily
than other plant viruses to different host species, raises
the questions of how WTGs should be distinguished and
what criteria should be used to justify naming individual
virus species. For WTGs with bipartite genomes, the
ability of two viruses to form pseudo-recombinants is a
strong indication that they are best considered as strains
of the same virus and such findings appear to correlate
with an identity of the iterated sequences in the large
intergenic region of their DNA (Argfiello-Astorga et al.,
1994). However, tests for pseudo-recombinant production require considerable effort and are not applicable to
WTGs with monopartite genomes. For WTGs in general,
a convenient rule of thumb for distinguishing and naming
different viruses is that their large intergenic regions
should differ in nucleotide sequence and their putative
gene products, other than CP, should in general have
sequence identities of less than 80 %. By these criteria
TYLCV-ISR, TYLCV-SAR, TYLCV-THI, TLCV-A,
TLCV-IN and ITmLCV are different virus species.
Further data are needed to enable the status of TYLCVNIG and TYLCV-SSA to be assessed but the difference
between the CP of TYLCV-SSA and CPs of other WTGs
suggests that TYLCV-SSA is likely to represent a seventh
species.
The comparisons made in this paper and elsewhere
(Padidam et al., 1995b) imply that WTGs are evolving
rapidly and may be able to adapt more readily to new
host species than plant viruses with RNA genomes. This
is despite the general conclusion that the mutation rate
caused by copying errors during replication is several
orders of magnitude less for viral DNA than viral RNA,
probably because of the host proof-reading mechanism
that exists for DNA replication (Reanney, 1984).
However, variants were found to occur more frequently
than expected in isolates of some vertebrate-infecting
viruses with ssDNA genomes (Cotmore & Tattersall,
1987; Gottschalck et al., 1991) and their occurrence may
perhaps be characteristic of other eukaryote-infecting
viruses with genomes of this type. If so, their existence
could be a factor underlying the occurrence of swarms of
variant WTG isolates that differ to greater or lesser
extents in nucleotide sequence and so provide raw
material on which selection pressures can act.
We thank R. N. Beachy, C. M. Fauquet, R. L. Gilbertson and M.
Padidam for providing sequences before publication and our colleague
David Robinson for helpful comments. We are indebted to the EEC
[Contract TS2A-0137-C(CD)] for financial support.
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(Received 14 February 1995; Accepted 20 April 1995)
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