J. gen. Virol. (198o), 51, 317-326
3i 7
Printed in Great Britain
Replication of RNA-1 of Tomato Black Ring
Virus Independently of R N A - 2
By D. J. R O B I N S O N , H. B A R K E R , B. D. H A R R I S O N AND
M. A. M A Y O
Scottish Horticultural Research Institute, lnvergowrie, Dundee DD2 5DA, Scotland
(Accepted 3 July I98O)
SUMMARY
In hybridization experiments, using complementary DNA (cDNA) copies of the
two genome parts of tomato black ring virus (TBRV RNA-I and RNA-2), no
sequence homology between the two RNA species was detected.
When tobacco mesophyll protoplasts were inoculated with purified middle
component particles, which contain only RNA-2, no replication of TBRV RNA
could be detected. However, when they were inoculated with purified bottom
component particles, which contain only RNA-L extracts made 24 or 48 h later
contained RNA that had the same mobility as RNA-~ in polyacrylamide-agarose
gels, and that hybridized to cDNA copies of RNA-I. Thus RNA-1 can replicate in
protoplasts that do not contain RNA-2. Moreover, this RNA-1 was capable,
when mixed with nucleoprotein particles containing RNA-2, of inducing the
formation of local lesions in Chenopodium amaranticolor leaves, and therefore was
intact and attached to the genome-linked protein. The genome-linked protein of
nepoviruses is probably virus-coded, and its production in protoplasts inoculated
with bottom component particles therefore suggests that RNA-t contains the gene
for this protein.
INTRODUCTION
Particles of nepoviruses contain two major RNA species. The larger (RNA-l) has a mol.
wt. of about 2.8 x lO6, estimated by electrophoresis of denatured RNA in agarose gels, and
the smaller (RNA-2) has a mol. wt. of 1.4 >( I06 to 2.4 x lO6, depending on the virus (Mutant
et al. 1979). In experiments with three members of this group, raspberry ringspot, tobacco
ringspot and tomato black ring (TBRV) viruses, preparations of RNA-I or nucleoprotein
particles containing RNA-I, and preparations of RNA-2 or nucleoprotein particles containing RNA-2, induced very few lesions in test plants, whereas mixtures of preparations
containing the two kinds of RNA induced many lesions (Harrison et al. 1972a; Randles
et al. I977). Moreover, further work, in which pseudo-recombinant isolates were produced
by mixing RNA-1 and RNA-2 from different virus strains of either raspberry ringspot virus
or TBRV, indicated that genetic determinants for various properties could be assigned to
either RNA-1 or RNA-2 (Harrison et al. 1972b, 1974; Harrison & Mutant, I977)- It was,
therefore, concluded that the genome of these viruses is in two parts, both of which are
needed for infection.
A few experiments have been done to test the possibility that either RNA-t or RNA-2
of raspberry ringspot virus could replicate without inducing lesions in inoculated leaves,
but the results did not support the idea that either RNA species can replicate independently
of the other (Harrison et aL I972a ).
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D . J.
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There are several general similarities in the genome structure and properties of nepoviruses
and comoviruses (Bruening, i977). For example, comoviruses, such as cowpea mosaic virus,
resemble nepoviruses in producing two major RNA species, which are packaged in different
nucleoprotein particles; both RNA species are needed to induce lesions in inoculated leaves
(Wood & Bancroft, I965; Bruening & Agrawal, 1967; van Kammen, I968; Harrison et al.
I972a), both have polyadenylate at their 3' ends (El Manna & Bruening, I973; Mayo et al.
I979a) and both are covalently linked to a small polypeptide (Daubert et al. t978; Stanley
et al. t978; Mayo et al. 1979b ).
Although none of this work provided direct evidence that nepovirus or comovirus RNA-I
or RNA-2 can replicate independently of each other, translation products of cowpea mosaic
virus RNA-/ are synthesized in protoplasts inoculatedwith bottom component particles,
which contain only RNA-~ (Rottier, I98o; Rottier et al. I98o). This suggests that the
possibility should be examined further, and in this paper we describe tests of the ability of
TBRV RNA-I and RNA-2 to replicate in mesophyll protoplasts inoculated with bottom
component particles alone or middle component particles alone. The results show unequivocally that RNA-I can replicate independently, whereas RNA-2 is synthesized only
when the cells also contain RNA-I.
METHODS
Purification of viruspartic/es. TBRV (isolates A and GI2; Hanada & Harrison, I977) was
propagated in Nicotiana clevelandii. Preparations of virus particles were obtained using the
method described by Mayo et al. (I979a). Middle (M) and bottom (B) component particles
were obtained from preparations of virus particles as described by Harrison & Barker (1978).
All experiments were done with TBRV-A unless otherwise stated.
RNA extraction from preparations of virus particles. Preparations of the two RNA species
(RNA-~ and RNA-2) were obtained by extracting RNA from separated B and M component particles respectively, using the phenol/SDS method described by Harrison &
Barker (I978). Following precipitation with ethanol at - I 8 °C, RNA was collected by
sedimentation at 3o0o g for 5 min. The RNA was resuspended in o'I5 M-sodium acetate
plus o'5 ~ SDS, pH 6 (acetate-SDS), to which was added an equal volume of 4 M-LiC1.
After i8 h at 4 °C, the precipitated RNA was collected by sedimentation at IOOOOg for
to rain, resuspended in acetate-SDS and precipitated at - I8 °C by adding 3 vol. ethanol.
Complementary D N A - R N A hybridization. 3H-cDNA to each TBRV RNA species was
prepared essentially by the method of Taylor et al. 0976). Reaction mixtures of 5o/d
contained 5o mM-tris-HCl, pH 8"3, 8 mi-dithiothreitol, 3 m~-MgC12, o'67 mu each of
dATP, dCTP and dGTP, 5o/~Ci Me-~H-TTP (sp. act. 6o Ci/mmol; ICN Pharmaceuticals,
Irvine, Calif., U.S.A.), 33/zg/ml olivomycin (Calbiochem, San Diego, Calif., U.S.A.),
2o/lg/ml TBRV RNA-I or RNA-2, 5oo/lg/ml DNA primer mixture and 16 units of avian
myeloblastosis virus reverse transcriptase (kindly donated by Dr G. E. Houts, Bethesda,
Md., U.S.A.). Incubation was for 2 h at 37 °C. aH-cDNA was purified from the reaction
mixtures as described by Gould & Symons (I977), and dissolved in hybridization buffer
(o.I8 M-NaCt, o.oi M-tris-HC1, pH 7, I mM-EDTA, o'o5~ SDS; Gonda & Symons, z978)
to give about ~ooo ct/min//A.
For hybrid formation, RNA samples were serially diluted in hybridization buffer, and
duplicate Io/~I samples of each dilution were mixed with 2/tl aH-cDNA. The mixtures
were sealed into 25/11 capillary tubes, immersed in boiling water for 5 rain, incubated at
6o °C for 2"5 h and chilled on ice.
Hybrid formation was assayed using Sx nuclease, purified from Taka-diastase powder
(Uniscience, Cambridge, U.K.) by the method of Vogt (1973), up to and including the
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DEAE-cellulose column step. The capillaries were opened and washed out with 200/A
5o raM-sodium acetate, pH 4"5, plus Ioo mM-NaC1 plus 2 m~-ZnSO4 plus IO/~g/ml degraded DNA (Sigma, Type IV). Samples 0oo/A) were transferred on to DE-8I filter discs
(Maxwell et al. 2978) before and after digestion with $1 nuclease at 45 °C for 3o min. As
Gonda & Symons (I 978) have shown that both the extent of resistance to digestion of hybrids
and estimates of sequence homology decrease with increasing $1 nuclease concentration,
the amount of $1 nuclease used was the minimum required to digest at least 95 ~o of the
cDNA in control experiments without RNA. The DE-81 discs were washed ten times in
o'4 •-Na2HPO4, once in water, twice in ethanol and once in diethyl ether. After drying,
remaining radioactivity was counted in 3 ml toluene containing o'5~ diphenyloxazole,
using an Intertechnique SL3o liquid scintillation counter.
Preparation, inoculation and culture of protoplasts. Mesophyll protoplasts were prepared
from tobacco (Nicotiana tabacum cv. Xanthi) plants grown in controlled conditions
(Kubo et al. I975b). Protoplasts were prepared and inoculated by the indirect method as
described by Kubo et al. (1975a); inocula contained M component (final concentration
o.I/~g/ml), B component (o.oi ktg/ml), or both. After inoculation, protoplasts were incubated for periods up to 48 h at zz °C in continuous light (Kubo et al. 1975a). Protoplast
samples were stained with fluorescein-conjugated antibody to TBRV particles as described
by Kubo et al. (I975a).
Radioactive labelling experiments. Protoplasts (2 × Io 5 to z × Io 5) in I ml incubation
medium were mixed with 5 to IO/zl 8H-uridine (z8 Ci/mmol, I mCi/ml; ICN Pharmaceuticals) either z h or I9 h after inoculation. RNA was extracted as described below,
usually 19 h or 44 h after inoculation. After precipitation from ethanol plus acetate-SDS
(5:2, v/v), RNA was dissolved in 15/d o'Z~o SDS plus o'036 ta-tris plus 0"o3 M-NaH~PO~
plus o.oI M-EDTA, pH 7"8 (TPE), containing 5 ~ sucrose (RNase-free grade; Sigma).
Samples of ~o #1 were analysed in I mm-thick slab gels of 2.4~o polyacrylamide plus
o'5~ agarose by electrophoresis at about 7 V/cm in TPE buffer. Gels were then fixed
overnight in methanol plus water plus acetic acid (5:5:I, by vol.) at 4 °C, infiltrated for
60 to 90 min with 4 to 5 vol. of scintillator solution (En3Hance; New England Nuclear,
Boston, Mass., U.S.A.) and for 60 min with water and then dried. Fluorographs were made
by exposing pre-flashed (Laskey & Mills, I975) Kodak XRP5 film at - 7 o °C for I to 5 days.
Extraction of RNA from protoplast samples. Protoplast samples were resuspended in
o.oI M-tris-HC1 plus I ~ SDS, pH 7"5, and emulsified with an equal volume of watersaturated phenol plus m-cresol (9:I, v/v) containing o.x~ 8-hydroxyquinoline. The
aqueous phase was re-extracted with phenol mixture and the RNA was precipitated twice
from 7o~ ethanol at - I 8 °C, once from 2 ~t-LiCl at 4°C and once again from 7O~o
ethanol, as described above.
Infectivity assay. The relative infectivity of RNA-containing extracts or TBRV particles
was estimated from the number of lesions produced in inoculated leaves of Chenopodium
amaranticolor. The samples were diluted in o-oI M-tris-HC1, o-or 5 M-NaCI, pH 7"6, conmining bentonite (2o/zg/ml); leaves were dusted 'Corundum' (UK Optical Co., London,
U.K.) before inoculation; plastic gloves were worn during inoculation; the fore-finger
was rinsed and dried after inoculating each leaf; and a Latin square design was used
to allocate treatments to different leaves on a group of plants. After inoculation, plants
were kept for 3 days in a glasshouse at 15 to 2o °C and were then transferred to a cabinet in
which they were illuminated (5o0o lux) for I6 h at z4 °C with alternating periods of 8 h in
darkness at 2o °C. These conditions favour the production of readily countable lesions
(R. L. S. Forster, personal communication).
To assay samples that contained only one species of TBRV genome RNA, the infectivity
of each sample was tested with and without added M component or B component particles.
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D.
I
100
-(a)
I
J. ROBINSON
I
I
AND
OTHERS
' _(~)
I
I
I
I
I
I
I
I
I
0
4
-3
log Rot (mol. s/l)
-2
1
0
75
.~ 50
25
I
I
I
I
-4
-3
-2
-!
I
Fig. I. Hybridization o f (a) c D N A - I a n d (b) c D N A - 2 with R N A - J ( O - - O )
of T B R V .
and RNA-2 (A--&)
RESULTS
Hybridization of cDNA to purified TBRV RNA species
Fig. I shows the kinetics of hybridization of each of the cDNA preparations to the
two TBRV genome R N A species in excess. At the highest concentrations of RNA, about
6 o ~ of the homologous cDNA became resistant to S~ nuclease, and this value is taken to
represent I o o ~ hybridization. Conversely, in the absence of RNA, tess than 5 ~ of the
cDNA was resistant, and this is taken to represent o ~ hybridization.
For the hybridization of each cDNA to its homologous RNA species, a single sharp
transition was observed. Values of Rot~ (initial RNA concentration×time, for 5 o ~
hybridization) were estimated from experiments similar to those shown in Fig. I but
including more determinations near the midpoint of the curve, and were I-42 x Io .2 mol.
s/1 for cDNA-I versus RNA-I and I-29 x IO -2 mol. s/1 for cDNA-2 versus RNA-2.
Reaction of cDNA-I with RNA-2 and of cDNA-2 with RNA-I resulted in very little
hybridization, and this occurred only at high values of Rot. The curves are consistent with
mutual contamination of each RNA preparation with the other RNA species to the extent
of between o.I and o'5 9/0. No evidence for sequences common to the two RNA species was
obtained, and it is estimated that such sequences, if present at all, account for less than
5 ~ of either species.
Hybridization of cDNA to RNA extracted from protoplasts
Hybridization of cDNA to RNA preparations extracted from protoplasts followed
kinetics similar to those shown in Fig. I. Concentrations of TBRV genome RNA species
in the extracts were estimated by multiplying the concentration of purified RNA (R0)
required to hybridize a chosen percentage of the cDNA by the dilution of the protoplast
extract required to reach the same percentage hybridization. Results of three typical
experiments are given in Table I. As far as possible, calculations were based on 5 o ~
hybridization of the cDNA, but with extracts that contained only small amounts of the
TBRV RNA, this extent of hybridization was not reached, and estimates were based on the
lower part of the curves. Such estimates are less reliable and are indicated by parentheses in
Table I.
It is clear that in protoplasts inoculated with both M and B components, substantial
replication of both RNA species takes place. For RNA-I, at least, more synthesis occurs in
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Table I. Virus RNA content, estimated from hybridization kinetics, of extracts from
protoplasts inoculated with M and B particles, separately or together
Copies ( x io -4)
Protoplasts
inoculated
with*
Time
extracted
(h after
inoculation)
No. o f
viable
protoplasts
( x [o -~)
Wt. (ng) of
,
--J~
--~
RNA-~
RNA-2
Expt. I
M+ B
B
M
Expt. 2
M+B
M+ B
B
B
24
24
24
I4
1"3
t "3
325
353
(2)
24
48
24
48
0'8
0-7
o.8
o.6
134
455
16o
93
Expt. 3
M
M
1-5
24
J5
0.8
346
([2):~
52
per infected
protoplastt o f
c- - . - - - ~ - - - RNA-I
RNA-2
5'8
7"1
Io'5
(0"4)
(<oq)
J.8
5'7
22"2
7'5
5"5
6I
07)
-
-
2'5
0'4)
* Inocula contained M c o m p o n e n t at o' t p g / m l a n d / o r B c o m p o n e n t at o.o~/zg/ml.
"i" The proportion o f viable protoplasts infected in each experiment was assumed to be equal to the proportion that were stained with fluorescein-conjugated antibody to TBRV 48 h after inoculation with both
components. This proportion was 84% (expt. I), 6o% (expt. 2) and 59% (expt. 3)- Less than I ~oo o f protoplasts inoculated with either component separately could be stained.
:~ Numbers in parentheses were calculated from less than 5o% hybridization of c D N A .
the second than in the first day after inoculation, with a fourfold increase between 24 and
48 h in the amount present. In protoplasts inoculated with B component only, and
cultured for 24 h, RNA-I was found in amounts comparable to or slightly greater than those
in protoplasts inoculated with both components. However, in these protoplasts, the amount
of RNA-I decreased slightly between 24 and 48 h; the small amount of RNA-2 found can
be attributed to the few doubly infected protoplasts, detected by fluorescent antibody
staining, that resulted from the remaining slight contamination of the inoculum with M
component.
Measurable amounts of RNA-2 were found in protoplasts 24 h after inoculation with
purified M component, but these amounts were less than those in extracts made I'5 h after
inoculation. Very little RNA-2 was found in such protoplasts 48 h after inoculation (results
not given). These measurements indicate a relatively slow degradation of RNA derived from
the inoculum, and provide no evidence for replication of RNA-2 in protoplasts inoculated
with purified M component.
Incorporation of 3H-uridine into virus RNA
RNA was extracted from protoplasts after incubation in a medium containing aHuridine, and analysed by electrophoresis in acrylamide-agarose gels. When 3H-uridine was
added 19 h after inoculation and RNA was extracted 44 h after inoculation, three major
and three minor radioactive bands were detected in gels of RNA from mock-inoculated
protoplasts (Fig. 2h). Only in RNA from protoplasts inoculated with both M and B
components (Fig. 2g) were bands of virus-specific radioactive RNA detected consistently.
These corresponded in mobility to RNA-I and RNA-2 (both arrowed) extracted from
purified virus. When 3H-uridine was added to cultures 2 h after inoculation and RNA was
extracted I9 h after inoculation a faint RNA-I band was produced by extracts from protoplasts inoculated with both M and B components (Fig. 2c) and also by extracts from
protoplasts inoculated with B component only (Fig. zb). RNA-2 was found only in extracts
from protoplasts inoculated with both M and B components but was partly obscured by a
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D.
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RNA-1
RNA-2
(a) (b) (c) (d)
(e) (f)
(g) (h)
Fig. 2. Fluorogram of polyacrylamide-agarose gel electrophoresis of RNA extracted from 3Huridine-labelled protoplasts. Protoplasts were inoculated with (a, e) M component; (b, f) B
component; (e,g) M and B components; (d, h) buffer only. For samples (a) to (d), 3H-uridine
was added 2 h, and RNA extracted 19 h after inoculation. For samples (e) to (h), 3H-uridine was
added I9 h, and RNA extracted 44 h after inoculation. Arrows mark the positions of TBRV
RNA-I and RNA-2.
band of protoplast RNA. No virus-specific bands were produced by extracts from protoplasts
inoculated with M component only (Fig. 2a, e). In some experiments a band of R N A - I was
produced by extracts of protoplasts inoculated with B component when 3H-uridine was
supplied between I9 h and 44 h after inoculation, but this band was always much less
radioactive than the RNA-I band produced by extracts from protoplasts inoculated with
both M and B components.
Similar results were obtained when 3H-labelled RNA from protoplasts inoculated with
B and M nucleoprotein components of TBRV-GT2 was analysed.
When protoplasts were transferred to non-radioactive medium after ~H-uridine had been
supplied between 2 h and I9 h after inoculation, and cultured for a further 24 h before
R N A was extracted, the fate of all-labelled R N A - t differed between protoplasts inoculated
with both M and B components and those inoculated with B component only. Whereas
RNA-~ and RNA-z remained labelled in doubly inoculated protoplasts, no labelled virusspecific R N A was detected in extracts from protoplasts inoculated with B component.
Local lesion formation by TBR V RNA from inoculated protoplasts
R N A extracted from protoplasts inoculated with either M component or B component,
or a mixture of the two, was tested for its ability to induce local lesion formation. Table z
gives the results of assays on extracts of protoplasts sampled 24 h after inoculation. In
this experiment fewer than o. I ~ of the protoplasts inoculated with either M or B component
were stained by treatment with fluorescent antibody to TBRV particles, whereas 6 o ~ of
those inoculated with the mixture of M and B components were stained in parallel tests.
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Table 2. Local lesion induction by RNA extracted from protoplasts inoculated with M
and B components, separately or together
R N A extracted f r o m protoplasts inoculated with
r
•
M
component
Component
added to
inoculum*
Buffer
control
~
l/8t
,
l/40
None
B
M
B+M
13'
o
Io5o
4
19
8
.
o
2o
o
.
.
B
component
r'
~
!/8
x/40
M+B
components
~
~------~
1/8
I/40
9
27
535
565
690
7o0
l
18
I24
.
6o
2o9
t93
.
* M a n d B c o m p o n e n t particles were used at final concentrations of 0'5 a n d 0.25 a g / m l , respectively.
"}" Dilution of protoplast extract. Undiluted extract contained R N A from a b o u t 8 x io 5 protoplasts/ml,
harvested 24 h after inoculation.
N u m b e r o f lesions in three Chenopodium amaranticolor leaves.
Similarly, RNA extracted from protoplasts inoculated with either M or B component
produced few lesions, whereas that from protoplasts inoculated with both kinds of particle
produced many. However, when M particles were added to RNA from protoplasts inoculated
with B component, the number of lesions increased more than 5o-fold. No such increase
occurred when B particles were added to this RNA, or when either kind of particle was
added to RNA from protoplasts inoculated with M component. The lesion-forming ability
of RNA from protoplasts inoculated with both M and B components was also increased by
the addition of M particles, but only about threefold or less, and the number of lesions
produced by this mixture was only slightly greater than that produced by the mixture
containing M particles together with RNA from protoplasts inoculated with B component.
Indeed the lesion-forming ability of RNA from protoplasts inoculated with M and B
components was increased as much by the addition of B particles as by that of M particles.
These results indicate that the RNA-I synthesized in protoplasts inoculated with B
component is biologically active. They cannot be explained by the survival of B particles or
RNA-I from the inoculum, because RNA extracted from protoplasts immediately after
inoculation with B component induced few or no local lesions either on its own or after
adding M particles. Moreover, because the ability of RNA-I of TBRV to take part in local
lesion formation depends on the presence of a virus-specific genome-linked protein (Harrison
& Barker, I978; Mayo & Barker, i98o), our results indicate that the gene for this protein is
in RNA-I.
In similar tests on RNA extracted from protoplasts 2 days after inoculation, the results
followed the same general trends as those in Table 2 but the enhancement of lesion number
produced by adding M particles to RNA from protoplasts inoculated with B component was
somewhat smaller. Between I and 2 days after inoculation the amount of active RNA-I in
these protoplasts decreased by about half, whereas the amount of active R N A extracted
from protoplasts inoculated with both M and B components increased about fourfold.
DISCUSSION
In kinetic hybridization experiments between cDNA-I and RNA-2 and between cDNA-2
and RNA-I (Fig. I), no reaction was observed in the range of Rot values where hybridization
due to sequences common to the two R N A species would be expected to occur. We estimate
that hybridization involving no more than 5 9/0 of the cDNA sequences would be reliably
observable under our conditions, and therefore conclude that common sequences, if they
exist at all, comprise about 25o nucleotide residues or less of RNA. Both species of RNA
contain polyadenylate tracts at their 3'-ends (Mayo et al. I979a) but this 'common
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D. J. ROBINSON AND OTHERS
sequence' is probably too small to be detectable. Rezaian & Francki (I974), using RNARNA hybridization, similarly concluded that there were few sequences common to RNA-I
and RNA-2 of another nepovirus, tobacco ringspot virus, although their limit for the maximum amount of homology was about 90o nucleotide residues.
The conclusion that there is little or no sequence common to RNA-I and RNA-2 of
TBRV depends on the assumption that the cDNA copies represent the entire RNA sequence
in each instance. Although this has not been demonstrated for these cDNA preparations,
cDNA copies of other virus RNA molecules prepared under essentially similar conditions
have been shown to be fully representative (Gould & Symons, 1977)Experiments described in this paper show that, in protoplasts inoculated with purified B
component, RNA is synthesized that migrates in polyacrylamide-agarose gels at the
mobility expected for TBRV RNA-I, forms hybrids with cDNA copies of TBRV RNA-I,
and when mixed with M particles induces local lesions in C. amaranticolor. We conclude,
therefore, that RNA-I is able to replicate in protoplasts that do not contain RNA-2. This
implies that RNA-I contains all the information required for its own replication. Although
nothing is known about the enzymes involved in TBRV RNA replication, any virus-coded
replicase or replicase subunit must be coded by RNA-I. Alternatively, if TBRV RNA is
replicated by a host-coded enzyme, such activity must be induced by RNA-I but not by
RNA-2.
It has been shown previously (Harrison & Barker, I978) that the genome-linked protein
must be attached to each TBRV RNA species for them to be able to induce formation of
local lesions. Because the RNA-I synthesized in protoplasts inoculated with purified B
component is active in this respect, it too must have this protein attached to it. The genomelinked proteins of different nepoviruses can be distinguished by their mobilities in polyacrylamide gels and by the patterns of peptides they produce after treatment with proteases.
In contrast, no differences were found between the genome-linked proteins in preparations
of the same virus grown in different host plants or between the proteins attached to RNA-I
and to RNA-2 of TBRV (Mayo & Barker, I98O). These results suggest that the genomelinked protein is virus-coded and, taken together with the results reported in this paper, that
a nucleotide sequence in RNA-I codes for it. It is apparently a small protein, probably of
less than 5o amino acid residues (Mayo et al. 1979 b), so that we cannot rule out the possibility
that the coding sequence also occurs in RNA-2; such a small common sequence might be
below the limit of detection by the present methods.
The amounts of RNA-I in protoplasts 24 h after inoculation either with B component
only or with M and B components are similar. However, the further four- or fivefold increase
in RNA-I during the next 24 h in protoplasts containing both RNA species is in strong
contrast to the lack of any net synthesis of RNA-I during the second day in protoplasts
inoculated with B component only. Although the labelling experiments with 3H-uridine
show that both synthesis and degradation of RNA-I are occurring during the second day of
infection with RNA-I only, such experiments cannot be interpreted quantitatively, and it is
not possible to tell whether the true rate of synthesis of RNA-I is affected by the presence
of RNA-2. One obvious effect of the absence of RNA-2, which codes for TBRV coat protein
(Randles et al. I977), is that none of the RNA-I can be incorporated into nucleoprotein
particles and thus be protected from degradation.
It now seems likely that the ability of RNA-I to replicate on its own is yet another
similarity between nepoviruses and comoviruses, since Goldbach et al. (198o) have recently
obtained results showing that RNA-I (B RNA) of cowpea mosaic virus can replicate in
protoplasts independently of RNA-2 (M RNA). In this respect, both groups of viruses
behave like tobacco rattle virus (TRV), the RNA-I of which has long been known to be
capable of replication in cells not containing RNA-2 (Harrison & Nixon, i959). However,
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RNA-1 of TRV, unlike that of TBRV, can induce local lesions and systemic symptoms in
appropriate host plants, and is readily detected by infectivity tests on phenol extracts of
infected plants (S~inger & Brandenburg, I960. These differences could be explained if, in
infections with RNA-I only, the infective RNA of TRV can move from cell to cell whereas
that of TBRV cannot, or if the infective RNA of TRV is more protected from degradation
by cellular enzymes than is that of TBRV. In this connection it is worth noting that the
infective RNA of TRV is associated with rapidly sedimenting structures in tissue extracts
and is much more stable in these extracts than is free RNA (Cadman, ~962). The outcome
of inoculation of purified TBRV B component to whole plants will require further study.
We thank Professor A. van Kammen for sending us pre-publication copies of papers
from his laboratory, and gratefully acknowledge the expert technical assistance of Miss
Wendy Adams and Miss Susan Mahoney.
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