Journal of General Virology (1996), 77, 531-540. Printed in Great Britain
531
Long-distance movement of cherry leaf roll virus in infected
tobacco plants
P a l o m a M d s and V i c e n t e P a l h i s *
Departamento de Mejora y Patologia Vegetal, C E B A S (CSIC), Avenida de la Farna 1, 30003 Murcia, Spain
The long-distance movement of cherry leaf roll virus
(CLRV) in tobacco plants was studied using a tissue
printing technique with non-isotopic RNA probes.
Time-course analysis revealed that CLRV RNA accumulated in the inoculated leaf at an early stage, such as 20 h
post-inoculation. The virus accumulation reached a
peak at 8-10 days post-inoculation (d.p.i.) and then
progressively decreased. The virus RNA signal was
detected before the appearance of symptoms. The virus
invaded stem vascular tissues at 3 d.p.i., moving towards
the roots before moving to the upper leaves. In
systemically infected leaves, the virus appeared first in
the basal regions and then moved to the distal parts
through the vascular system. The distribution pattern of
the virus coat protein in systemically infected leaves was
parallel to that observed for the virus RNA, suggesting
that CLRV requires the coat protein for long-distance
movement. The movement of the virus was influenced by
the phyllotactie position of the leaves. The viral
symptoms and the virus RNA signal in older systemically
infected leaves were asymmetrically distributed, being
localized in the side of the lamina closest to the
inoculated leaf. Virus distribution in infected plants as
well as the susceptibility of the plant to systemic infection
were also influenced by the developmental stage of the
inoculated leaves. Inoculation of leaves at 95 % of their
final size resulted in virus replication but no systemic
infection. In fully mature leaves the virus did not
replicate.
Introduction
al., 1991 ; Taliansky & Garcia-Arenal, 1995) and potexvirus (Chapman et al., 1992) genera. Assembled virions
are required for efficient systemic transport of cowpea
mosaic virus (CPMV; Wellink & van Kammen, 1989)
and tobacco mosaic virus (TMV; Siegel et al., 1962;
Dawson et al., 1988; Saito et al., 1990). In the case of
tobacco etch potyvirus (TEV), the CP possesses distinct
functions required for virion assembly, cell-to-cell movement and long-distance transport (Dolja et al., 1994).
The dispensability of the CP for other viruses has been
documented for tobra- (Harrison & Robinson, 1986),
hordei- (Petty & Jackson, 1990), tombus- (Rochon et al.,
1991; Dalmay et al., 1992; Scholthof et al., 1993) and
dianthoviruses (depending on the environmental conditions for red clover necrotic mosaic virus; Xiong et al.,
1993). In addition, it has been recently shown that longdistance movement can be regulated by sequences within
the RNA 2 in the case of brome mosaic virus (BMV;
Traynor et al., 1991) or within RNA 1 as described for
cucumber mosaic virus (Gal-On et al., 1994), previously
thought to be involved only in virus replication.
Less is known about how the plant influences the
pattern of accumulation of viruses in their susceptible
hosts. Classical experiments of Samuel (1934) showed the
rapid long-distance movement of TMV in tomato plants.
To systemically invade a plant, viruses must use two
different forms of spread, short- and long-distance
movement, in a compatible interaction. In short-distance
movement, a virus moves from cell-to-cell through an
active process in which the involvement of a specific
virus-coded protein (movement protein; MP) is well
documented (for reviews see Citovsky & Zambryski,
1991; Maule, 1991; Deom et al., 1992; Mushegian &
Koonin, 1993). In long-distance movement, the virus
uses the plant vascular system to invade the rest of the
plant (Atabekov & Dorokhov, 1984; Hull, 1989;
Matthews, 1991 ; Maule, 1991). In contrast to cell-to-cell
movement, many factors are involved in long-distance
movement; the requirements are different depending on
the virus group. In addition to the MP, a functional coat
protein (CP) is required for long-distance movement by
some viruses belonging to the tobamo- (Takamatsu et
al., 1987), bromo- (Allison et al., 1990; Flasinski et al.,
1995), carmo- (Heaton et al., 1991), cucumo- (Suzuki et
* Author for correspondence. Fax +34 68 266613.
e-mail [email protected]
0001-3503 © 1996SGM
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532
P. Mds and V. Pallds
Bennett (1940) demonstrated that virus spread is influenced by the flow of metabolites in the plant. Studies
with tobacco ringspot virus (TRSV) led Schneider (1965)
to suggest that the virus reaches the distant parts of the
plant via the phloem transport system. More recently, in
cauliflower mosaic virus (CaMV)-infected turnips, Leisner et al. (1992) showed that the pattern of long-distance
movement was similar to the translocation of photoassimilates, indicating that the virus is apparently swept
along with the flow of photoassimilates from source
leaves to sink leaves. In a later study, the same authors
found that developmental changes had a dramatic impact
on the long-distance movement of CaMV in Arabidopsis
(Leisner et al., 1993).
CLRV belongs to the nepovirus group of plant viruses
(Jones, 1985). Its genome is composed of two positivesense polyadenylated ssRNAs which carry a genomelinked protein (VPg) at their 5' ends (Hellen & Cooper,
1987). The walnut strain of CLRV (wCLRV) is consistently associated with the causal agent of the lethal
disease of graft incompatibility in walnuts known as
blackline (Mircetich et al., 1980).
This paper deals with the pattern of accumulation of
CLRV in a systemic infection of tobacco plants and the
influence of leaf ontogeny and the phyllotactic position
of the leaves on susceptibility to virus infection.
Methods
Virus andplant material. The walnut isolate of CLRV was purified
from frozen leaf tissue as described (Rowhani et al., 1985). Plants of
Nicotiana tabacum cv. 'Xanthi nc' were grown in a growth chamber
with a day length of 16 h, average day and night temperatures of 24 °C
and 20 °C, respectively, and relative humidity of 80%. Plants were
inoculated with 10 ng/ml of wCLRV by rubbing the leaves with
carborundum as an abrasive. In time-course experiments and tissue
print immunoblotting, two leaves per plant were inoculated, while for
the study of the developmental and phyllotactic stage of the leaves on
virus distribution, only one leaf per plant was inoculated.
Tissue print R N A - R N A hybridization and non-radioactive RNA
probe. Tissue prints were performed basically as described by Song et
al. (1993) and Mils & Pallils (1995). The upper surfaces of the leaves
were printed on a membrane using a roller and applying a uniform
pressure, beginning from the tip of the leaf. The stem and petiole prints
were prepared after sectioning with a razor blade and the surfaces were
pressed manually to the membranes for 15 30 s. Nylon membranes
(Hybond; Boehringer Mannheim) were used for RNA hybridization.
After printing, the membranes were exposed for 3 rain to the UV light
of a transilluminator. Prehybridization was carried out for 2-3 h at
68 °C in 50% deiouized formamide, 5 x SSC (1 x SSC is 0.15 M-NaCI,
0.015 M-sodium citrate pH 7-0), 2 % (w/v) blocking reagent (Boehringer
Mannheim), 0.1% (w/v) N-lauroylsarcosine and 0.01% (w/v) SDS.
Plasmid clone pCL2.16 was used to synthesize digoxigenin-labelled
RNA probe using T7 RNA polymerase as described by Mils et al.
(1993). Hybridization was performed at 68 °C for 12 h. Membranes
were washed after hybridization twice in 2 x SSC and 0.1% (w/v) SDS
at room temperature for 5 min and twice in 0.1 x SSC and 0.1% (w/v)
SDS at 68 °C for 15 min. The chemiluminescent detection of virus
RNA was carried out as described (M~is & Pallils, 1995).
Tissue print immunoblotting. The second systemically infected leaf
and stems of both infected and mock-inoculated tobacco plants were
processed at 5 days post-inoculation (d.p.i.; data not shown) and
10 d.p.i, for CP detection. The leaves and stems were printed, as
described above, onto nitrocellulose membranes (Schleicher and
Schuell). The membranes were washed at room temperature in 3 %
BSA in TBS (20 mM-Tris-HC1 pH 7-5 and 150 mM-NaC1) for 20 min,
3 % BSA and 0.1% Nonidet P-40 in TBS for 20 min and 3 % BSA in
TBS for 20 min. Primary antibody against wCLRV (kindly provided by
A. Rowhani, University of California, Davis, Calif., USA) was diluted
1:2000 in 3 % BSA in TBS. The primary antibody (2 gl) was preincubated with 0.5 ml of a healthy plant protein extract for 30 min on
ice. The protein extract was made by homogenizing 1 g of tissue in 1 ml
of TBS. After 2 h incubation at room temperature with the primary
antibody the membranes were washed and then incubated for 2 h with
anti-rabbit IgG alkaline phosphatase (Boehringer Mannheim) diluted
1:1000 in 3 % BSA in TBS. Membranes were further washed and
colorimetric substrate solution (NBT/BCIP; Boehringer Mannheim)
was added. The membranes were incubated in substrate solution until
a purple colour appeared (approximately 20 30 rain).
Time course analysis" by dot-blot and tissue print R N A - R N A
hybridization. Inoculated and second systemically infected leaves
(numbered 0 and 2 in Fig. 4, respectively) and stems and petioles of
infected and mock-inoculated tobacco plants were harvested at different
times post-inoculation and processed for tissue printing R N A - R N A
and dot-blot hybridization. Dot-blot experiments were performed as
previously described (Mils & Pallils, 1995). Briefly, total nucleic acids of
the leaves, petioles and roots of infected plants were extracted by
homogenizing the tissue in a buffer containing 100 mM-Tris HC1
pH 7.5, 100 mM-NaCI, 10 mM-EDTA, 5 mM-2-mercaptoethanol and
0.1% SDS. Nucleic acids were extracted with phenol chloroform and
precipitated from the extracted aqueous phase using ethanol. The
samples were denatured with formaldehyde (White & Bancroft, 1982)
and hybridized with the non-radioactive R N A probe as described
above.
The appearance of typical ringspot symptoms in CLRV-infected
tobacco plants was recorded until 15-20 d.p.i, in both inoculated and
systemically infected leaves. At the same time, the viral RNA signal was
detected by either tissue printing or dot-blot hybridization. The
progress of systemic invasion was also recorded in experiments where
the inoculated leaf was removed at successive d.p.i.
Influence o f developmental stage and phyllotactic position o f leaves on
virus distribution. These studies were carried out in the inoculated and
systemically infected leaves, analysed by dot-blot and tissue print
R N A - R N A hybridization. The locations of the leaves in the plant are
shown in Fig. 4. In the developmental stage experiments, only one leaf
was inoculated. The size of the inoculated leaf as a percentage of the
final size ranged from 30 % (plants with four fully expanded leaves) to
100% (plants with seven fuIly expanded leaves). The percentages were
calculated by measuring the length of the inoculated leaf at the time of
its inoculation with respect to its final size. The inoculated and
systemically infected leaves were examined for the appearance of
symptoms, recorded at 12-15 d.p.i. The viral RNA signals were
determined by dot-blot experiments by extracting total nucleic acids of
the apices, systemically infected leaves (other than the first one; leaves
numbered 2 and 3 in Fig. 4), first systemically infected leaf (leaf
numbered 1 in Fig. 4) and the inoculated leaf (leaf numbered 0) at
12-15 d.p.i. Hybridization and detection were performed as described
above.
To study the influence of phyllotactic position of the leaves on virus
infection, tobacco plants were inoculated only on a leaf at either 30 %
or 65 % of its final size. The leaves were analysed by the tissue printing
R N A - R N A hybridization technique at 10 d.p.i.
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Long-distance movement of C L R V
Fig. 1. Time-course analysis of the virus RNA
distribution by tissue-printing hybridization
of inoculated (a) and the second systemically
infected (b) tobacco leaves (see Fig. 4).
Analysis was carried out at 20 h (i), 3 (ii), 4
(iii), 6 (iv), 8 (v), 12 (vi) and 15 (vii) d.p.i. A
healthy tobacco leaf at 8 d.p.i, is shown in
(viii).
(a)
iii
ii
!!ii
iiiiiiiiiiiiiil
iv
:):%
vi
vii
533
viii
(b)
iv
vii
Results
Time course analysis of virus accumulation in tobacco
leaves as judged by R N A - R N A tissue print
hybridization
Inoculated and second systemically infected leaves, stems
and petioles of tobacco plants inoculated with C L R V
were excised and analysed by tissue printing R N A R N A
hybridization at several times post-inoculation (Fig. 1).
In inoculated leaves, the virus R N A signal a p p e a r e d at
an early stage, such as 20 h post-inoculation, giving
several infection foci distributed all a r o u n d the leaf (Fig.
1 a, i). By 4 d.p.i, the hybridization concentrated a r o u n d
the main and lateral veins (Fig. 1 a, iii). The distribution
pattern became m o r e uniform over time, with the
hybridization signal preferentially accumulating a r o u n d
the vascular system (Fig. 1 a, iv-vii). Virus accumulation
reached a peak at 8-10 d.p.i, and then progressively
decreased. N o signal was obtained when samples from
healthy plants were hybridized (Fig. 1 a, viii). Fig. 1 (b)
shows a macroscopic view of the C L R V m o v e m e n t in
systemically infected leaves at different stages. The signal
clearly first appears at the base of the leaf and then
moves towards its tip along the main vein, until it reaches
p r i m a r y veins.
Time-course analysis of the virus movement through
petioles and stems to invade the distal parts of the plant
In order to determine exactly when the virus is m o v i n g
out of the inoculated leaf, tissue printing and dot-blot
RNA-RNA
hybridization of petioles of inoculated
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534
P. M~s and V. Pallc~s
(b) Stems
(a) Petioles
tp
Inoculated
6
8
Systemic
db
2
4
tp
db
4
2
3
4
(c)Apices
5?
(d) Roots
Fig. 2. Localization of virus RNAs in petioles, stems, apices and roots. Petioles (a) from inoculated and the second systemically infected
tobacco leaves were analysed by tissue-printing (tp) or dot-blot (db) hybridization at 2, 3 or 4 d.p.i, for inoculated leaves and 4, 6 or
8 d.p.i, for systemically infected leaves. Cross- and longitudinal sections of infected tobacco stems (b) were analysed by tissue-printing
hybridization at 4, 6 and 8 d.p.i. The arrows on the right indicate the petiole insertion point of the inoculated leaf in the stem. Dotblot hybridization of apices (c) and roots (d) of infected tobacco plants at 2, 3 and 4 d.p.i, were also performed. Cross- and longitudinal
sections of petioles and stems are presented in duplicate. The petiole side closest to the leaf is indicated by the upper arrow in tp 3. The
petiole insertion point to the stem is indicated by the lower arrow. Note that signal is more evident at this time in the side closest to
the leaf.
leaves at 2, 3 and 4 d.p.i, were performed (Fig. 2a). A
very weak signal was observed at 2 d.p.i. (Fig. 2a, tp 2).
This signal was stronger at 3 d.p.i., mainly on the side
closest to the leaf (Fig. 2a, tp 3). A more evident signal
appeared at 4 d.p.i., being homogeneously distributed
along the petiole (Fig. 2a, tp 4). The same results were
obtained when dot-blot hybridization, a more sensitive
technique, was used (Fig. 2a, column db), confirming
that the virus exits the inoculated leaf at around 3 d.p.i.
When petioles of systemically infected leaves were
analysed at 6 and 8 d.p.i., using either tissue printing or
dot-blot hybridization (Fig. 2a), a clear signal was
obtained while no hybridization signal was detected at
4 d.p.i.
Longitudinal stem sections, covering approximately
2 cm up and down from the petiole insertion point of
inoculated leaves, were analysed by tissue printing
RNA RNA hybridization (at 4 6 d.p.i, this size includes
the whole stem). By 4 d.p.i, the hybridization signal
appeared initially at the lower part of the section (Fig.
2b, 4). At 6 d.p.i, the virus was present at both the lower
and the upper part of the stem section (Fig. 2b, 6) and at
8 d.p.i, the hybridization signal had considerably increased (Fig. 2b, 8). These results indicate that CLRV
moved downwards to the root before going up to the
apex. When we analysed roots and apices of infected
plants at 2, 3 and 4 d.p.i.by dot-blot hybridization (Fig.
2 c, d), the signal firstly appeared in roots, suggesting that
the virus accumulated there before invading the rest of
the plant.
A summary of the time-course analysis of the
relationship between the symptomatology, systemic
infection and virus hybridization signal is shown in Table
1. The appearance of symptoms was delayed in relation
to the virus signal. The typical ringspot symptoms were
clearly seen at 2 d.p.i, and 6 d.p.i, on inoculated and
systemically infected leaves, respectively. However, by
tissue printing and dot-blot hybridization, the viral
signal can be detected at 20 h p.i. on inoculated leaves
and 5 d.p.i, on systemically infected ones.
As stated above, the virus exits the inoculated leaf at
2-3 d.p.i., since viral signal is detected in petioles at
3 d.p.i. (Fig. 2a). This result was confirmed in experiments where the inoculated leaf was detached at 1 day
intervals from 1 to 15 days (Table 1). Those plants in
which the inoculated leaf was detached at 1, 2 or 3 d.p.i.
showed no signs of systemic infection.
Comparison between the spatial distribution of virus CP
and virus RNA in infected tobacco plants
In order to study the accumulation pattern of virus CP in
infected tissue and its relationship to virus RNA
distribution, tissue prints in the second systemically
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Long-distance movement of CLRV
535
T a b l e 1. Time-course of symptoms, systemic infection and virus replication
signal in infected tobacco leaves
Inoculated leaves
Time (d.p.i.)
Viral
signal~
Symptoms
0
Viral
signal
Symptoms
Systemic infection*
.
.
.
.
+
+
+
+
+
.
.
1
+
2
3
5
6
+
+
+
+
+
8
+
+
12
15
+
+
+
+
4
Systemic leaves
-
.
.
-
--
--
+
+
+
-/+
+
+
+
+
+
+
+
+
+
+
+
+
* Systemic infection visualized at 10 15 d.p.i, after the inoculated leaf was removed.
~ Viral signal was recorded by either tissue printing or dot-blot hybridization.
(a)
(b)
(¢)
RNA
CP
(e)
RNA
(a)
(f)
I
(g)
cP
. . . . . .
(h)
Fig. 3. Comparison of the accumulation patterns of virus RNAs (RNA) or coat protein
(CP) by tissue-printing RNA-RNA hybridization or tissue-printing immunolocalization.
The second systemicallyinfected leaves (a, c; see Fig. 4) or longitudinal stem sections of
healthy (e, g) or infected (J; h) tobacco plants were transferred to nylon or nitrocellulose
membranes as described in Methods. The filmsin (b), (e) and 64)were exposed for 20 rain.
The membranes in (d), (g) and (h) were developed for the colour reaction for
approximately 30 min. Note that because the colorimetric detection method was used,
the viral foci in (d) are the mirror image of those observed in the leaf, so that the right
part of the leaf must be compared with the left part of the membrane whereas in (b) the
film can be superimposed on the leaf.
infected leaves a n d stems were performed. Fig. 3 shows
the d i s t r i b u t i o n p a t t e r n of virus R N A (b,J) a n d CP
(d, h). The d i s t r i b u t i o n p a t t e r n of C P in inoculated leaves
was parallel to the signal o b t a i n e d for the virus R N A . I n
i n o c u l a t e d leaves, the virus infection foci were u n i f o r m l y
distributed (data n o t shown). I n systemically infected
leaves, b o t h the virus R N A (Fig. 3b) and the CP (Fig.
3 d) accumulated along the veins a n d s y m p t o m areas. N o
signal was o b t a i n e d when m o c k - i n o c u l a t e d plants were
analysed (data n o t shown).
L o n g i t u d i n a l stem sections of infected a n d m o c k inoculated plants were also hybridized against R N A
p r o b e (Fig. 3e, J) or i n c u b a t e d with C L R V a n t i s e r u m
(Fig. 3g, h). A very clear a c c u m u l a t i o n signal was
observed whether R N A or C P was assayed,
The pattern of virus infection depends on the
phyllotactic position of the leaves
The d i s t r i b u t i o n a n d a p p e a r a n c e of s y m p t o m s a n d virus
a c c u m u l a t i o n was studied in relation to the p o s i t i o n of
the i n o c u l a t e d leaf. Fig. 5 shows the i n o c u l a t e d a n d the
first, second a n d third systemically infected leaves of a
p l a n t inoculated in a leaf at 65 % of its final size. The
spatial location o f these leaves in the infected p l a n t are
s h o w n in Fig. 4. The leaves were analysed by the tissue
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536
P. M~s and V. Pallas
Fig. 4. Diagram of a CLRV-infected tobacco plant showing the
location of the leaves at 10 d.p.i. The inoculated leaf is indicated by the
number 0. The leaves situated below the inoculated one are numbered
negatively (leaves - 1 and - 2) and the leaves above the inoculated one
are numbered 1-5. The leaf numbered 1 is situated immediately above
the inoculated leaf and corresponds to the first systemic leaf in Figs 5
and 6. The second and third systemically infected leaves are numbered
2 and 3, respectively and correspond to the systemically infected leaves
(not including the first systemic leaf) in Fig. 6. Note that in time-course
experiments where two leaves per plant were inoculated, the inoculated
leaves were the ones numbered 0 and - 1.
printing R N A - R N A hybridization technique at 10 d.p.i.
The symptoms and the virus R N A hybridization signal
on the second and third systemically infected leaves were
localized on the half-leaf closest to the inoculated leaf. As
can be observed in leaf 2, the foci of hybridization were
present in the first and second lateral veins and in a
symptom area on the left side of the leaf. In leaf 3 some
hybridization foci on the opposite side to the inoculated
leaf can be observed, although the foci of hybridization
of both secondary veins and symptom area are mainly
concentrated on the side closest to the inoculated leaf.
The asymmetrical distribution was more evident in older
systemic leaves than in the upper and younger leaves
(fourth and fifth systemic leaves) where the infection
became more uniformly distributed (data not shown).
No signal was observed in the first systemic leaf (leaf
numbered 1 in Fig. 4) on the opposite side of the plant to
the inoculated leaf.
Influence of developmental stage of leaves on the virus
infection
In order to study the potential influence of the leaf
developmental stage on its susceptibility to the systemic
infection, dot-blot analysis of the different parts of the
Fig. 5. Analysis by tissue-printing R N A - R N A hybridization of the
phyllotactic arrangement in an infected tobacco plant. Tobacco plants
were inoculated in a leaf which was at 65 % of its final size. The leaf
numbered 0 was inoculated and the leaves numbered 1,2 and 3 were the
first, second and third systemically infected leaves, respectively. The
virus R N A distribution pattern of the fourth and fifth systemically
infected leaves is not influenced by the position of the inoculated leaf
in the plant (data not shown). Analysis was carried out at 10 d.p.i, as
described in Methods.
%
30
45
65
78
90
95 100
Apices
Systemic leaves
First systemic leaf
Inoculated leaf
Fig. 6. Dot-blot hybridization of the total nucleic acid extracts from the
apices, systemically infected leaves (other than the first one; leaves
numbered 2 and 3 in Fig. 4), first systemically infected leaf (numbered
1 in Fig. 4) and inoculated leaves (leaf numbered 0 in Fig. 4) at different
developmental stages. The percentage leaf size (shown along the top)
was calculated by measuring the length of the inoculated leaf at the
m o m e n t of its inoculation with respect to its final size. All plants were
processed at 1 ~ 1 2 d.p.i. Samples were denatured with formaldehyde
and hybridized against a digoxigenin-labelled R N A probe as described
in Methods. The film was exposed for 20 min.
plant, within very narrow limits of plant ontogeny, were
carried out.
Fig. 6 and Table 2 show the data obtained from plants
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Long-distance movement of C L R V
537
Table 2. Effect of the developmental stage of the inoculated leaf on the symptomatology and virus
accumulation in infected tobacco plants
Percentage size of inoculated leaf*
30%
45%
65%
78%
90%
Sample
S'~
V~
S
V
S
V
S
V
S
Apex
Systemic leaves§
First systemic leaf
Inoculated leaf
+
+
+
+
+
+
+
+
+
+
wsll
+
+
+
+
ws
+
+
+
+
+
ws
+
.
+
.
+
+
+
95%
V
.
S
.
+
.
.
+
V
.
.
.
+
100%
.
S
V
-
-
.
.
.
.
.
.
+
* The percentage size was calculated by measuring the length of the inoculated leaf at the time of its inoculation with respect
to its final size.
t S, symptoms, recorded at 10-15 d.p.i.
V, viral signal, determined by dot-blot hybridization (see Fig. 6) at 10 d.p.i.
§ Systemic leaves correspond to the second and third systemically infected leaves.
II ws, Weak signals, as can be observed in Fig. 6 (row 'First systemic leaf').
assayed at different inoculated leaf stages. D o t - b l o t
hybridization (Fig. 6) was performed in apices, systemically infected leaves (other than the first one), first
systemic leaf and inoculated leaves, at different developmental stages. W h e n very y o u n g leaves were
inoculated at 30 % o f their mature length (plants having
four fully expanded leaves) a general infection occurred
in the whole plant (all the dots with a strong signal in
Fig. 6, column 30 %). This invasion decreased as the leaf
became more mature. F r o m 45 78 % o f the final length,
a very weak signal was observed in the first systemic leaf
(Fig. 6). The limit o f systemic infection seems to be a
stage where the inoculated leaf had a size o f 90 % o f its
final length. A t 95 % o f the final size there was a clear
hybridization signal in the inoculated leaf, while in the
apex and systemically infected leaves no signal was
observed, indicating that at this leaf developmental stage
the virus loses its ability for systemic spread (Fig. 6).
However, the virus was still able to replicate in the
inoculated leaf (Fig. 6). W h e n fully mature leaves were
used, no signal was observed even in the inoculated leaf,
indicating that the virus was unable to replicate (Fig. 6).
These differences between the results at 95 % and 100 %
o f the final length o f the inoculated leaves are based on
replicated data, either in the n u m b e r o f experiments or in
the n u m b e r of plants analysed per experiment. W i t h
regard to the first systemic leaf, in all cases (except at
30 % of the length o f the mature inoculated leaf) no
apparent s y m p t o m s were observed and a very weak
signal was obtained when a dot-blot hybridization was
performed (Fig. 6). This is p r o b a b l y due to its developmental stage at the time the virus reaches it, which
results in faulty i m p o r t o f the virus into this leaf.
The relationship between the s y m p t o m a t o l o g y and the
viral signal can be observed in Table 2. W h e n plants with
leaves at 30 % o f their final length were inoculated, the
symptoms closely corresponded with the positive viral
signal (except in the apices, which did not show
s y m p t o m s although the viral signal was positive). In the
case o f the plants inoculated in leaves at 45-100 % o f
their final size, the relationship between the appearance
o f s y m p t o m s and the positive viral signal is maintained
except in apices (where no s y m p t o m s were observed) and
in the first systemic leaves (where a weak signal was
obtained by dot-blot hybridization and no s y m p t o m s
were observed).
Discussion
A macroscopic view o f virus accumulation and pattern
o f translocation in whole plants was achieved by the
application o f the tissue printing technique. In conjunction with dot-blot hybridization and biological
assays, this technique was used to follow the routes that
a plant R N A virus, C L R V , used to m o v e in a systemic
infection, and to study the influence o f the phyllotactic
position o f the leaves and their developmental stage on
susceptibility to infection.
Time-course analysis revealed that the virus R N A
signals were observed in the inoculated leaf as early as
20 h p.i. indicating rapid virus replication and cell-to-cell
movement. Both the n u m b e r and the diameter o f the
virus foci increased over time until 8 d.p.i, and then
progressively decreased. F r o m the inoculated leaves the
virus m o v e d out t h r o u g h the vascular system at 3 d.p.i.
These conclusions are d r a w n n o t only from experiments
where the inoculated leaf is detached at 1 day intervals
but also by tissue printing and dot-blot analysis. Once
C L R V invades stem vascular tissues, it moves first to the
roots, accumulating there (Fig. 2), before invading the
rest o f the plant. This invasion route seems to be different
to the one observed for viroids. Palukaitis (1987) has
shown that p o t a t o spindle tuber viroid was always
detectable first in the shoot tip and in leaves that were
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538
P. Mcis and V. Pallc~s
very close to the tip in inoculated tomato plants. No
previous replication stage in the inoculated leaf seems to
be necessary in this case for a systemic infection to occur,
indicating that cell-to-cell and long-distance movement
are very much easier events for viroids to achieve than
for viruses. Thus, in the case of viruses, a minimum input
of virus R N A would be necessary to afford an active
process such as cell-to-cell movement and a critical level
in the phloem may be necessary in establishing a systemic
infection. The high virus concentration within roots
suggests that virus multiplication occurs in this organ.
Atchison & Francki (1972) have shown that virus
replication takes place in the root tip cells of bean plants
for only about 1 day following invasion of TRSV. Using
the tissue printing technique in cross sections of stem and
petiole, CLRV RNA has previously been detected in
parenchymal and outer phloem tissues (Mils & Pall~is,
1995). This suggests that from the roots the virus moves,
probably via phloem, to the systemic leaves. CLRV
enters the non-inoculated tobacco leaves at 4-6 d.p.i.
The pattern of virus distribution in systemically infected
leaves is quite different to that in the inoculated ones.
Very clear hybridization signals were observed within the
major vein of the leaves, indicating that the virus
probably replicates in vascular tissue. This is contrary to
observations by Leisner et al. (1992) in turnip plants
infected with CaMV and by Dolja et al. (1992) in tobacco
plants infected with TEV where virus accumulation was
significantly less in major than in minor veins and
mesophyll cells. The foci of hybridization resulting from
a non-vascular symptomatic area were only present
when the lateral vein situated immediately downward
had a viral signal, indicating that minor veins are the
most frequent pathways for invasion of parenchyma
(note that in the right part of leaf 3 in Fig. 5, where no
symptom foci are present, there is no hybridization
signal in the minor vein located downwards). This would
be in agreement with the idea that the viral route of
infection is similar to the photoassimilate flow since
phloem unloading of imported photoassimilate occurs
primarily from large minor veins in sink leaves (Ding et
al., 1988).
The observation that the CP distribution pattern
parallels the one observed for virus RNA in stem and leaf
tissue suggests, although it does not demonstrate
conclusively, that this virus needs CP for long-distance
movement. Wellink & van Kamlnen (1989) have shown
that virions are required for the efficient systemic
transport of CPMV, the type member of a virus genus
closely related to the genus Nepovirus, which forms part
of the family Comoviridae. In addition, CLRV has been
observed to form tubular structures in the cell walls of
the differentiated leaf cells of Nicotiana clevelandii Gray
(Jones et al., 1973) which would be in agreement with the
idea that CLRV moves as virions to distant parts of the
plant.
The virus infection is influenced by leaf developmental
stage, in accordance with the sink to source leaf
transition. If CLRV moves via phloem with photoassimilates, sink leaves (that import photoassimilates)
will be easily infected, while source leaves (exporting
photoassimilates) will not become infected. Using a
different system, Leisner et al. (1992) showed that only
those parts of the plant into which photoassimilates flow
are accessible to CaMV during systemic infection. In that
sense, the analysis of the developmental stage of infected
plants confirms this hypothesis (Fig. 6). At 4-6 d.p.i., the
virus probably reaches the first systemic leaf when the net
photosynthetic flow is directed out of this leaf, so the
virus is unable to enter and replicate (Fig. 6). Only when
the virus reaches the first systemic leaf at a very young
stage (i.e. when the inoculated leaf is only 30 % of its final
size) will this leaf become infected (Fig. 6). This could be
the reason why in Nicotiana benthamiana plants infected
with BMV, the one or two lowest uninoculated leaves
consistently escaped virus infection (Mise et al., 1993).
When leaves were inoculated at 30 90 % of their final
size, the other systemic leaves became infected, although
the plants became less susceptible to infection (Fig. 6).
However, with inoculated leaves at 95 % of final size, the
virus is unable to spread systemically. In mature leaves
(100% of final size), CLRV is apparently unable to
replicate. However, the possibility that the virus causes a
subliminal infection in the fully mature leaves cannot be
excluded even though tissue print analysis of these leaves
showed no signal at all (data not shown). Our results are
in agreement with previous observations that showed
that as soybean plants age, the chance of their becoming
systemically infected with tobacco necrosis virus decreases greatly (reviewed by Schneider, 1965). The
observed pattern of movement through the different
plant developmental stages is typical of the movement of
metabolites in the phloem. The residual virus signal in
the first systemic leaf (Fig. 6) may be due to the cell-tocell movement through non-phloem cells associated with
the vasculature. In fact, the virus accumulated not only
in phloem tissue in stem cross-sections but also in the
parenchymatous tissue (Mils & Pall~is, 1995). This
relatively abundant accumulation in non-phloem cells of
the stem tissue was also observed by Wisniewski et al.
(1990) in a stem cross-section of tobacco plants infected
with TMV. Moreover, the final distribution of the virus
in the infected plants is determined by the phyllotactic
position of the leaves; foci were mainly concentrated at
the side closest to the inoculated leaf. This phyllotactic
influence is preferentially observed in the leaves situated
immediately upward of the inoculated leaf but only when
the virus reaches those leaves in a sink stage. This viral
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Long-distance movement of CLRV
distribution pattern may be explained if the virus is
transported with the net photosynthetic flow, directed
from the source to the closest side of the sink leaves. The
upper systemically infected leaves are not influenced by
their phyllotactic position. The various observations
given above indicate that the accumulation and translocation of a RNA virus are influenced by the phyllotactic position of the leaves and plant developmental
stage.
We thank Dr R. Flores, Dr J. Carbonell and Dr J. F. Marcus for
critical comments about this manuscript and J. Martinez-Fresneda for
her technical assistance. We are also grateful to M.J. Ocio for her
helpful support. This research was supported by Grant PB91~0058
from the Spanish granting agency DGYCIT. P.M. is the recipient of a
fellowship from the Consejeria de Educaci6n, Cultura y Turismo from
Regi6n de Murcia.
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