Download Variation in biological properties of cauliflower mosaic virus clones

Journal of General Virology (1994), 75, 3137-3145.
3137
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Variation in biological properties of cauliflower mosaic virus clones
Nadia AI-Kaff and Simon N. Covey*
Department of Virus Research, John Innes Centre, Colney Lane, Norwich N R 4 7UH, U.K.
Infectous clones were prepared from virion DNA of
three cauliflower mosaic virus (CaMV) isolates, 11/3,
Xinjiang (XJ), and Aust, to investigate pathogenic
variation in virus populations. Of 10 infectious clones
obtained for isolate 11/3, four pathotypes were identified, each producing symptoms in turnip that differed
from those of the 11/3 wild-type. Virus from two clonal
groups of 11/3 was transmissible by aphids whereas that
from two others was not. Of the five infectious clones
obtained from isolate X J, two groups were identified,
one of which differed symptomatically from the wildtype. Only one infectious clone was obtained from
isolate Aust and this had properties similar to the wildtype. Restriction enzyme polymorphisms were found in
some clonal groups and these correlated with symptoms.
Other groups with different pathogenic properties could
not be distinguished apart by restriction site polymorphisms. Further variation was observed in the
nucleotide sequences of gene II (coding for aphid
transmission factor) from these viruses as compared
with other CaMV isolates. In the aphid non-transmissible clones of isolate 11/3, one had a Gly to Arg
mutation in gene II similar to that of other non-deleted
non-transmissible CaMV isolates. The second had a
322 bp deletion at the site of a small direct repeat similar
to that of isolate CM4-184 although occurring in a
different position. The gene II deletion of isolate 11/3
produced a frame-shift that separated genes II and III by
60 bp. Most CaMV clones studied remained biologically
stable producing similar symptoms during subsequent
passages. However, one clone (11/3-7) produced two
new biotypes during its first passage suggesting that it
was relatively unstable. Our results show that wild-type
populations of CaMV contain a range of infectious
genome variants with contrasting biological properties
and differing stability. We suggest that a variety of
significant viral phenotypic changes can occur during
each infection cycle resulting from relatively small
genome changes.
Introduction
considerable variation has been observed in the character
of the disease caused by CaMV depending on the genetic
background of both host and virus (Daubert, 1988;
Covey et al., 1991). CaMV isolates obtained from
infected plants worldwide have been shown to cause
infections with a range of different pathogenic characters
(Melcher, 1989; A1-Kaff & Covey, 1994). Some of the
pathogenic differences between CaMV isolates, and other
viral functions such as aphid transmissibility, have been
mapped to specific parts of the CaMV genome (Woolston
et al., 1983; Armour et al., 1983; Daubert et al., 1984;
Baugbman et al., 1988; Stratford & Covey, 1989; Qiu &
Schoelz, 1992; Wintermantel et al., 1993). CaMV
genome variation has also been analysed at the
nucleotide sequence level and has been shown to have
isolate-specific restriction enzyme site polymorphisms
(Lebeurier et al., 1978 ; Volovitch et al., 1979 ; Gardner et
al., 1980; Hull, 1980) and differences in more extensive
tracts of nucleotide sequence, including those in the
complete sequences of several different CaMV isolates
determined since the Strasbourg isolate (Franck et al.,
1980) was sequenced.
Cauliflower mosaic virus (CaMV) is a pararetrovirus
that packages a DNA genome that is replicated by
reverse transcription of an RNA intermediate (Hull &
Will, 1989). The CaMV genome encodes six proteins that
have been identified in plants. Structural proteins include
the coat protein (gene IV) and a second protein (gene III)
found in virions. The functions of other CaMV genes
include those involved in virus movement within (gene I)
and between (gene II) plants, in virus replication (gene
V), and a probable multifunctional protein required in
translational transactivation (gene VI). In addition to
viral polypeptides, there are various cis-acfing nucleic
acid sequences involved in viral replication, transcription
and translation, and there may be further sequences
required for packaging and other, as yet, uncharacterized
functions (Gronenborn, 1987; Hohn & Futterer, 1991;
Covey & Hull, 1992).
Expression of these basic viral functions is required to
establish a systemic infection in host plants and to ensure
the propagation of the virus in other plants. However,
0001-2613 © 1994 SGM
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3138
N. Al-Kaff and S. N. Covey
Genetic variation is an essential component of the
adaptive potential of a virus. Variation in the CaMV
genome has been attributed to the high error rate of
reverse transcriptase generating point mutations (Steinhauer & Holland, 1987), to strand-switching during
reverse transcription (Dixon et al., 1986; Grimsley et al.,
1986), and to deletions resulting from cryptic splicing
(Hirochika et al., 1985; Scholthof et al., 1991) or from
recombination at short direct repeats (Howarth et al.,
1981; Pennington & Melcher, 1993). Since such mutagenic processes appear to be relatively frequent, it
would be expected that natural populations of CaMV
might gradually accumulate variants over time. However, the composition of DNA in preparations of some
CaMV isolates appears to be fairly uniform as shown by
the coherent restriction maps and genome sequences
suggestive of single molecular species that have been
derived (e.g. Franck et al., 1980). In contrast, infections
by certain virus isolates or those resulting from coinoculations by different molecular types have been
shown to contain mixtures of progeny molecules some of
which have had major deletions generated in vivo
(Hirochika et al., 1985; Zhang & Melcher, 1989; Vaden
& Melcher, 1990; Scholthof et al., 1991 ; Pennington &
Melcher, 1993). However, systemic infections initiated
by mechanical inoculation appear to result from mobilization of one or a small number of molecules from the
inoculation site (Riederer et al., 1992) suggesting that
virus genome variants arise de novo during each infection
cycle and are maintained as a minor proportion of an
otherwise uniform population. Some of these new
variants should possess novel biological properties that
are either suppressed by the predominating genome type
or appear as infections causing altered symptoms during
passage. Indeed, from our studies of some 36 CaMV
isolates we have observed the emergence, during passage,
of three new CaMV variants with novel biological
properties (A1-Kaff & Covey, 1994). We have investigated further the relationship between genome
variation and the generation of new viral phenotypes
by studying different infectious molecular clones isolated
from wild-type CaMV isolates.
Methods
Viruses and plants. Cauliflower mosaic virus isolates 11/3, Aust,
Cost-2 and Braunschweig were originally provided by Dr R. Hull, and
isolate Xinjiang (X J) was obtained from Dr R. Fang, each being
provided as infected leaf material. Other CaMV isolates used were
Cabb B-JI and Bari-1 (Stratford et al., 1988). Virus was propagated in
turnip plants (Brassica rapa-rapifera cv 'Just Right'), following
mechanical inoculation, under glasshouse conditions as previously
described (Stratford et al., 1988). Virus and viral DNA was purified
from infected plants using the method of Gardner & Shepherd (1980).
DNA analysis and cloning. CaMV virion DNA was mapped by
digestion with a variety of restriction endonucleases (as described later
in Results) using conditions recommended by the manufacturers.
Digested DNA was analysed by agarose gel electrophoresis with DNA
fragments detected by staining with ethidium bromide or Southern blot
hybridization using a radiolabelled CaMV DNA probe.
For cloning, virion DNA was linearized at the unique SalI site and
ligated to SalI-cut vector DNA (pGEM5). Subsequent steps of
transformation, colony analysis and purification of plasmid DNA were
as described by Sambrook et al. (1989). Clones were initially
characterized as 'minipreps' and full-length clones were tested for
infectivity following 'mini-' or 'midi-' plasmid purification and release
of viral sequences from the vector by Sa/l-digestion. Following
digestion, 1 to 2 lag of insert DNA (in the presence of vector sequences)
was mechanically inoculated onto the second true leaf of turnip
seedlings in the presence of Celite abrasive in a total volume of 10 lal of
10 mM-sodium phosphate buffer, pH 7.0. Infectivity was tested on a
minimum of five plants.
Gene II sequences were determined from clones of various CaMV
isolates. Double-stranded DNA was sequenced using a Pharmacia T7
Sequencing kit. Oligonucleotide primers used for sequencing the minus
strand were at nucleotides (i) 1280 to 1299 and (ii) 1566 to 1586, and
for the plus strand at (iii) 1883 to 1863 and (iv) 1586 and 1566. All four
primers were used to determine the gene II sequences of the following
clones: Aust, 11/3-1, -2, -7, and -8; primers (i) and (iii) were used for
clones 11/3-3 and -9; primers (ii) and (iii) for completing the sequence
of isolate Campbell (Woolston et al., 1987); primers (i) and (iii) for
Bari-1. The above primers were synthesized from the sequence of
isolate Cabb B-JI but these did not prime sequencing reactions for the
XJ clones. Therefore, an 880 bp Nsil BamHl fragment of the XJ clones
containing gene II was sub-cloned into pGEM7 and sequenced using
Universal primers (Pharmacia). From the partial sequence obtained,
further oligonucleotide primers were designed to complete the gene II
sequence.
Aphid transmission. Aphid transmissibility of CaMV isolates and
clones was tested by feeding starved aphids (Myzus persicae) for 2 to
2.5 h before transfer to non-infected seedlings for a further 24 h after
which time the aphids were destroyed by fumigation. Each transmission
test consisted of 10 plants with 50 aphids per plant.
Results
Cloning virus from wild-type populations
We attempted to obtain infectious clones from passaged
virus preparations of five CaMV isolates with different
pathogenic characteristics. Table I shows the relative
success of obtaining infectious clones (in those molecules
with a single SalI site) for the different isolate preparations. In general, we found that from 5 to 25 % of fulllength clones were infectious. However, we were not able
to obtain infectious clones from preparations of CaMV
isolates Cost-2 and Braunschweig.
Table 1. Preparation of infectious C a M V clones
CaMV
isolate
11/3
XJ
Aust
Cost-2
Braunschweig
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Full-length
insert
Infectious
clones
60
19
16
11
23
10
5
1
0
0
Biological variation of CaMV clones
3139
Table 2. Biological properties of CaMV clones expressed in turnip plants
Systemic s y m p t o m s
Isolate/clone
Local Symptom
lesions
timing
Early
Middle
Late
Aphid
transmissible
Aust (wt)*
pAust*
N
N
VC + + +
Chl VB
+ +
Chl VB
Stunting
Rugosity
+
+ + +
+ + +
XJ (wt)
N
Late
VC + + +
Chl VB
+
Green VB
Stunting
Cupping
+ + +
+ +
+ +
N
Late
VC + + +
Chl VB
+
Green VB
Stunting
Rugosity
+ +
+
+
pXJ-l*
X J-2
N
Late
VC + +
Chl VB < +
Green VB
Stunting
Cupping
11/3 (wt)
N
N
VC + +
Chl VB
+ +
Chlorosis
Stunting
Rugosity
+
+ +
pll/3-1*
pl 1/3-4
pl 1/3-6
pl 1/3-8
pll/3-10
N
N
VC + + +
Chl VB
+ + +
Chlorosis
Stunting
Rugosity
+ + +
+ +
+
pl 1/3-2"
N
N
VC + + +
Chl VB
+ +
Chlorosis
Stunting
Rugosity
+ + + +
+ +
+ +
V
pl 1/3-3"
pl 1/3-5
pl 1/3-9
N
N
VC + + +
Chl VB
+ + +
Chlorosis
Stunting
Rugosity
+ +
+
+
X
pl 1/3-7"
Small
N
VC + + +
Chl VB
+
+ + +
-
passaged 11/3-7:
plant 1
Small
N
VC + + +
Chl VB
+ +
Dark green
Stunting
Rugosity
Chl VB
Stunting
Rugosity
plant 2
Small
N
VC + + +
Chl VB
+
plant 3
Small
N
VC + + +
Chl VB
+ + +
pXJ-2*
pXJ- 1*
pXJ-3*
pXJ-4
pXJ-5
+
< +
+
ND
ND
X
+ +
+
ND
Dark green
Stunting
Rugosity
+ + +
-
biD
Chlorosis
Stunting
Rugosity
+ + +
+
ND
* Clones tested for aphid transmissibility: ,/, transmissible; × , non-transmissible; ND, not determined.
wt, wild-type; N, normal; VC, vein clearing; Chl VB, chlorotic vein banding.
Pathogenic variants of CaMV clones
The biological properties of infectious clones of the three
CaMV isolates were compared following infection of
turnip plants (Table 2). The single infectious clone of
isolate Aust had biological properties indistinguishable
from those of the wild-type isolate in that it produced a
very severe reaction in turnip. Isolate Aust was also
found to be aphid-transmissible. Infection of turnip
plants with DNA from the five infectious clones obtained
from virion DNA of isolate XJ produced two distinct
groups of symptom variants. One clone (XJ-2) was
similar to the wild-type whereas the other four clones
(XJ-1, -3, -4 and -5) produced milder stunting and a
different type of leaf wrinkling than wild-type XJ
infections. Representatives of both clonal groups of XJ
were found to be aphid transmissible (Table 2). Since
clone XJ-1 and the other group of clones originated from
the same population, we attempted to determine the
effect of mixed inoculation of representatives of the two
groups. However, when equal quantities of the DNAs of
X J-1 and X J-2 were inoculated onto plants, the symptoms produced had characteristics of both groups but
were much milder than either (Table 2). We tested several
combinations of clones from different isolates and always
found the symptoms to be milder than when clones were
inoculated individually (data not shown).
Isolate 11/3 was more complex in that four groups of
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3140
N. Al-Kaff and S. N. Covey
1
2
3
4
Fig. 1. Clonal variants of CaMV isolate 11/3 showing symptom
phenotypic differences in turnip leaves infected with 11/3 wild-type (1),
11/3 clone-3 (2), 11/3 clone-1 (3) and 11/3 clone-2 (4).
clones could be distinguished apart on the basis of their
biological properties. Moreover, all of the clonal variants
of 11/3 produced symptoms that were different from
those of the wild-type. Three of the groups produced
symptoms that were more severe than the wild-type
(Table 2) and each with quite different leaf coloration
patterns (Fig. 1). For instance, clone 11/3-3 produced
wide vein banding and little chlorosis or stunting (Table
2; Fig. 1 leaf 2) in contrast to 11/3-1 which caused more
severe stunting and chlorosis (Table 2; Fig. 1 leaf 3).
Clone 11/3-2 caused the most severe chlorosis and
bleaching (Table 2; Fig. 1 leaf 4). Clone 11/3-7 was
different again in that it produced uncharacteristically
small local lesions with very mild systemic symptoms and
dark green leaf tissue (Table 2). The ability of clonal
groups of isolate 11/3 to be transmitted by aphids also
differed with two groups found to be aphid-transmissible
and two non-transmissible (Table 2). The stability of
different clones during subsequent passage was tested.
Most clones shown in Table 2 when serially-passaged at
least four times produced symptoms similar to the
original clones. However, clone 11/3-7 produced two
new symptomatic variants (Table 2) on the first passage
following inoculation of cloned DNA.
Restriction enzyme site polymorphisms
Since different clones of the CaMV isolates produced a
variety of biological properties, we then attempted to
characterize genome differences. Maps of nine different
restriction enzymes sites of the clonal variants were
compared with those of selected sequenced isolates, and
with isolate Bari-1 which we have previously observed to
be least typical of CaMV isolates (Stratford & Covey,
1989). By comparing restriction site polymorphisms we
found that, in general, groupings of clones with similar
maps produced similar symptoms as might be expected.
For instance, clones XJ-1 and XJ-3 produced identical
symptoms and restriction maps. Clone X J-2 had a
different map from the other clones and produced
different symptoms (Fig. 2 a and Table 2). However, both
clonal groups of isolate XJ had maps that were different
from that predicted by the published nucleotide sequence
of this isolate (Fang et al., 1985).
Three clonal groups (cg 1 to 3) were identified for
isolate 11/3 on the basis of restriction enzyme mapping.
Clone 11/3-2 was the only member of clone group (cg) 1
(Fig. 1) and produced a map and symptoms different
from the other 11/3 clones and from the wild-type 11/3
(Table 2). Clone group 2 of 11/3 comprised six clones,
five of which (11/3-1, -4, -6, -8 and -10) produced
symptoms that were similar to one another. The other
clone in cg 2 (11/3-7) produced symptoms that were
unlike those in any other map group. The third clone
group had three members with identical restriction maps
and these produced symptoms that were similar to one
another. Each of the clones in cg 3 lacked a XhoI site and
appeared to contain a deletion in this region. Subsequently, the presence of a deletion of two of the 11/3
clones (11/3-3 and -9) was confirmed by sequencing. The
deletion removed the XhoI site as in isolate CM4-184 (see
Fig. 4). It is interesting to note that the members of cg 2
and cg 3 apparently differed only in the region of the
XhoI site (in gene II) and yet they produced different
symptoms. The very severe symptom-producing isolate
Aust, had a unique restriction map but was somewhat
similar to that of the Strasbourg isolate which caused
relatively mild symptoms. In contrast, the map of the
mild symptom-producing isolate Bari-1 had the greatest
number of restriction enzyme site changes (Fig. 2a).
Since the clonal variants identified above were derived
from single virus preparations of the respective isolates,
we then determined whether a range of variants could be
detected by restriction enzyme analysis of virus DNA
preparations. We chose passaged material of isolate 11/3
from which the three clonal groups described above (Fig.
2a) originated. The ancestry of the 11/3 clones is shown
in Fig. 2(b). Viral DNAs were digested with four
restriction enzymes to detect variations. Sap inoculum
was made from the original infected material [wild-type
(wt) 0] and used to infect two sets of plants. Virus was
prepared from single leaves from each set (wt 1a and 1b)
and assayed for restriction sites. In these, and in all other
virus preparations analysed, we observed a single
predominant restriction pattern suggesting the presence
of one major variant type. Minor bands were observed
that could have represented other types (data not shown).
After a single passage, we found that the SpeI site was
absent from one of the virus preparations (Fig. 2c)
suggesting that a change had occurred during a single
passage although we were not able to check the
composition of the original material. The material from
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Biological variation o f CaM V clones
(a)
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Ill
PR
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R
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R
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R
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SpMRC
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wt o
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wt 2
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wt la
wt lb
wl 2
wt 3
Wt 4
~,
wt 3
i
cgl
wt 4
cgl
cg2
cg 2
X J-l, -2, -3
11/3-l, -8
11/3-2
11/3-7
11/3-3, -9
Aust
Campbell
Cabb B-JI
Bari-1
9
12
13
13
A332
15
14
12
71
2
3
2
6
A130
5
5
3
24
the wt lb passage was further passaged and assayed from
single leaves three more times and we observed no
further changes in the restriction pattern. The 11/3
clones were obtained from virus isolated from leaves
pooled from several plants. Although the material (wt 4)
from which the clones were isolated contained virion
DNA with one predominating restriction pattern (Fig.
2 c), it gave rise to clones with three different restriction
patterns (Fig. 2a) and other variants with different
biological properties (Table 2).
Variations in nucleotide sequence of gene H
(b)
wtlb
Amino
acid
changes*
* Changes relative to the Strasbourg isolate properties.
A, Deletions.
V
D
Clone
Base
changes*
i
R
11~3:,~ 1
Bari-1
6
Sp
xJ.2
CM4-184 °
5
V
xa-~,-3
1113: cg 3
>
R
xa,
11/3: Cg 2
4
XCRRC
[ |
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0
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Table 3. Gene H sequence variation
VI
--~
3141
Sal
Xho
Mlu
1
1
1
2
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
1
1
1
0
1
cg3
Fig. 2. (a) Restriction maps of CaMV isolates and clones in linear
configuration aligned at the virion D N A negative strand gap. The
position of viral genes (horizontal arrows labelled with Roman
numerals) is shown. The map for the Strasbourg (Stras) isolate for
restriction enzymes EcoRl (R), ClaI (C), NsiI (N), XhoI (X), SalI (S),
Pstl (P), MluI (M), DraII (D), Spel (Sp) is shown. The maps below this
show additional (~') or lost ( V ) sites relative to those in the Strasbourg
isolate. Open boxes represent deletions. Clonal groups (cg) of isolate
11/3 with similar restriction maps are cg 1 (11/3-2), cg 2 (11/3-1, -4,
-6, -7, -8, -10) and cg 3 (11/3-3, -5, -9). Maps of some isolates, indicated
by an asterisk, were derived from the published nucleotide sequences.
Restriction enzyme polymorphisms provide a useful tool
for analysing variation in relatively large sequences of
DNA. However, we required more detail of sequence
variation in a defined region of the viral genome. Gene II
(480 bp) was chosen since it has a precise function in
aphid transmission but one which is not required to
establish a normal infection by mechanical inoculation.
We also wanted to characterize aphid non-transmissible
variants isolated during our cloning of isolate 11/3. We
determined the gene II sequence in both strands of three
clones of isolate XJ, six clones of isolate 11/3, one clone
each of isolates Bari-1 and Aust, and clones of two
isolates for which partial sequence data was available:
Cabb B-JI (J. Stanley, unpublished sequence data) and
the aphid non-transmissible isolate Campbell (Woolston
et al., 1987). The sequences were compared with that of
the published Strasbourg isolate in a pair-wise fashion
(Table 3). The level of variation between most clones was
similar to that between isolates with 2"3 % nucleotide and
1.4% amino acid variations compared with the Strasbourg isolate (Table 3). In contrast, isolate Bari- 1 differed
greatly with 15 % nucleotide and amino acid variation
(b) Ancestry of the l l / 3 clonal groups from the original infected
material (wt 0) through subsequent sap passages and together with
restriction enzyme polymorphisms (c).
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N. Al-Kaff and S. N. Covey
3142
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x
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Fig. 3. Amino acid sequences of CaMV gene II (aphid transmission factor) from various isolates and clones. Published sequences are:
S, Strasbourg (Franck et al., 1980); PV147 (Modjtahedi et al., 1985); XJ, Xinjiang (Fang et al., 1985); CMV-I (Melcher & Chenault,
1992); NY8153 (Chenault et al., 1992); BBC (Chenault & Melcher, 1993); CM1841 (Gardner et al., 1981); CM4-184 (Howarth et al.,
1981); D / H (Bal~.zs et al., 1982). Sequences marked with an asterisk were partially or completely determined in this work. The identity
of an amino acid is shown as a dash; deletions are shown as dots; !, termination codons. Aphid non-transmissibility, X, and
transmissibility, ,/, are indicated; those in parenthesis are suspected to be transmissible on the basis of their amino acid sequence.
even though it retained aphid transmissibility. Of the
aphid non-transmissible clones, 11/3-3 and -9 had an
identical 322 bp deletion whereas gene II of 11/3-7 was
intact (Fig. 3).
Discussion
Only a relatively small proportion of the apparently fulllength clones of CaMV virion DNA proved to be
infectious. Infectivity could have been lost during
purification of virion DNA and cloning by introduction
of minor deletions or re-arrangements. Delseny and Hull
(1983) analysed the structure of three non-infectious
clones of CaMV isolate Cabb B-JI and found deletions in
viral and vector sequences in one clone. Two other clones
were found to contain deletions only in the viral DNA
sequences (Delseny & Hull, 1983). For two isolates
(Cost-2, Braunsweig) we were unable to obtain any
infectious clones although for Cost-2 the proportion of
full-length clones was very low (Table 1). Non-viable
genomes could also have arisen from point mutations or
minor deletions during viral replication in plants.
From the 10 clones of isolate 11/3, we obtained three
different restriction maps and identified four biological
variants that differed from the wild-type. In general,
symptom phenotypes correlated with restriction site
polymorphisms. The variety of pathotypes cloned from
isolate 11/3 suggests that it might be less stable than
other isolates. In fact, one clone (11/3-7) produced two
new symptom variants during its first passage. However,
the original virion preparation from which clone 11/3-7
was derived remained relatively stable during passage
(see Fig. 2) suggesting that this population contained a
mixture with a predominant stable form and minor
unstable genome types. This level of variation in a
population could be a property of some CaMV isolates
and not others since we have re-cloned our type isolate
Cabb B-JI and passaged it many times and observed only
one spontaneous biological variant.
We conclude from these observations that most
genome variants of individual CaMV clones were
probably not caused by mutations during cloning in
Escherichia coli but during replication in planta. Our
results are consistent with the view that most CaMV
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Biological variation of C a M V clones
3143
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Fig. 4. Nucleotide sequence in gene 1I (aphid transmission factor) of (a) non-deleted and deleted forms of isolate 11/3 cloned from the
same virus preparation compared with (b) the deletion in isolate CM4-184 relative to CM 1841. Repeat sequences in the non-deleted
forms (above) at which deletion occurs are shown as lower case letters linked by lines in each case with the complete nucleotide
sequences of the deleted genes shown below. Positions of the deletions are indicated in the middle diagrams relative to adjacent genes
I and III. Nucleotide numberings are shown above the sequences.
populations contain a majority of one genome type with
a minor population of variants that most likely arise de
novo upon each cycle of infection. This minor population
of variants, may vary in size, complexity and viability
depending upon the parental isolate. However, it is this
sub-population which is the source of virus adaptability.
Emergence of new CaMV pathotypes will be dependent
upon (i) the statistical likelihood of them being the first
genome to be systemically mobilized from the site of
inoculation on the next round of infection and (ii) upon
any competitive advantage they have over the existing
predominating genome type. Our data indicate that some
CaMV variants are more stable than others suggesting
that certain genotypes have a greater potential for
adaptation than others.
One of the variations in biological properties observed
in different clones was differential aphid transmissibility
specified by gene II. This is an interesting region of the
CaMV genome since it is not essential for virus infectivity
but the polypeptide that it encodes has a very precise
function in aphid transmission. Thus, the selection for
this function would be removed during mechanical
passage. However, we have no evidence that mechanical
passage of CaMV compared with passage through aphids
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3144
N. Al-Kaff and S. N. Covey
results in any greater accumulation of changes in the
aphid transmission gene than in any other part of the
genome. Of the four clonal groups of isolate 11/3, two
were found to be aphid non-transmissible. One of the
non-transmissible clones (11/3-7) had an intact gene II
sequence. Inspection of the amino acid sequence (Fig. 3)
showed a glycine to arginine substitution at position 94
in common with non-transmissible isolates Campbell
and CM 1841 which also do not have deletions (Woolston
et al., 1987). We found a second change (isoleucine to
valine) at position 105 found only in the non-deleted
non-transmissible CaMV isolates and we have preliminary evidence (N. A1-Kaff & S. Covey, unpublished)
that this second change might contribute to loss in aphid
transmissibility.
In two other non-transmissible clones (11/3-3 and -9)
we found a 322 bp deletion which resulted in a frameshift producing a truncated gene I! polypeptide of 29
amino acids (Fig. 4). This deletion is located in an 8 bp
sequence that is duplicated in a non-deleted form of
isolate 11/3 cloned from the same population of virion
DNA molecules. It is most likely that the deletion
resulted from homologous recombination at the site of
the direct repeat sequence. An alternative possibility is
that the deletion occurred by splicing at cryptic sites in
the 35S RNA pre-genome template. Such deletions have
been reported in CaMV (Hirochika et al., 1985; Vaden &
Melcher, 1990) and in a related caulimovirus figwort
mosaic virus (Scholthof et al., 1991). A deletion in gene
II has been previously reported in the non-transmissible
CaMV isolate CM4-184 (Howarth et al., 1981) although
in a different position to that in our 11/3 clones (Fig. 4).
Delseny and Hull (1983) observed a deletion of similar
size (within the limits of restriction analysis) in gene II in
one of their non-infectious clones of CaMV isolate Cabb
B-JI although its fine-structure is unknown. The deletion
in clone 11/3-3 effectively separates the end of the gene
II coding sequence from the initiation codon of the next
downstream gene (gene III) by approximately 60 bp. The
close proximity of CaMV genes in wild-type virus is
important in the relay-race mechanism of translation
linking adjacent genes (Dixon & Hohn, 1984). The viable
deletion in 11/3-3 suggests that adjacent genes can be
separated by at least 60 bp without apparently affecting
the relay-race mechanism or reducing virus pathogenicity.
The emergence of a mixture of distinct biotypes of
CaMV from a single infection cycle suggests that much
of the biological variation observed between the different
CaMV isolates could, in many cases, be caused by a
relatively small number of point mutations.
We thank Drs R. Hull and R. Fang for kindly providing us with
some of the CaMV isolates used in this study. Thanks also go to Dr
P. Markham and Mr I. Bedford for advice on aphid transmission
experimentation. We gratefully acknowledge the John Innes Foundation for giving a Research Studentship to N.A.-K.
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