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Journal of Virology, November 1998, p. 8731-8737, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Isolation of an Arabidopsis thaliana
Mutant in Which the Multiplication of both Cucumber Mosaic Virus
and Turnip Crinkle Virus Is Affected
Motoyasu
Yoshii,
Norimichi
Yoshioka,
Masayuki
Ishikawa,* and
Satoshi
Naito
Department of Applied Bioscience, Faculty of
Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan
Received 13 April 1998/Accepted 18 July 1998
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ABSTRACT |
During the systemic infection of plants by viruses, host factors
play an important role in supporting virus multiplication. To identify
and characterize the host factors involved in this process, we isolated
an Arabidopsis thaliana mutant named RB663, in which
accumulation of the coat protein (CP) of cucumber mosaic virus (CMV) in
upper uninoculated leaves was delayed. Genetic analyses suggested that
the phenotype of delayed accumulation of CMV CP in RB663 plants was
controlled by a monogenic, recessive mutation designated
cum2-1, which is located on chromosome III and is distinct
from the previously characterized cum1 mutation. Multiplication of CMV was delayed in inoculated leaves of RB663 plants,
whereas the multiplication in RB663 protoplasts was similar to that in
wild-type protoplasts. This suggests that the cum2-1 mutation affects the cell-to-cell movement of CMV rather than CMV
replication within a single cell. In RB663 plants, the multiplication of turnip crinkle virus (TCV) was also delayed but that of tobacco mosaic virus was not affected. As observed with CMV, the multiplication of TCV was normal in protoplasts and delayed in inoculated leaves of
RB663 plants compared to that in wild-type plants. Furthermore, the
phenotype of delayed TCV multiplication cosegregated with the
cum2-1 mutation as far as we examined. Therefore, the
cum2-1 mutation is likely to affect the cell-to-cell
movement of both CMV and TCV, implying a common aspect to the
mechanisms of cell-to-cell movement in these two distinct viruses.
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INTRODUCTION |
Systemic infection of plants by
viruses occurs through complex interactions between virus-encoded and
host-encoded factors. Although much information on the roles of viral
factors involved in the infection process is available, little is known
about the host factors involved. To understand the molecular mechanism
of virus multiplication in plants, it is necessary to identify and characterize such host factors. During the process of systemic infection, positive-strand RNA viruses uncoat to release genomic RNA in
the cytoplasm of host cells and replicate by using replication proteins
that are translated from the genomic RNA. Then the viruses move from an
infected cell to adjacent cells via plasmodesmata, a process mediated
by virus-encoded movement proteins (MPs). In many virus-host
combinations, cell-to-cell movement is the key step which determines
susceptibility. Finally, the viruses enter the phloem and rapidly move
to noninfected tissues at some distance from the inoculated leaf
(long-distance movement). At present, several host factors necessary
for efficient plant viral RNA replication have been identified
(31, 33), but those necessary for the local or systemic
spreading of plant viruses remain unknown.
Cucumber mosaic virus (CMV) has three individual segments of capped
messenger-sense RNA as a genome, RNAs 1, 2, and 3 (reviewed in
reference 32). RNAs 1 and 2 encode proteins 1a and
2a, respectively. Both 1a and 2a are necessary for viral RNA
replication in a single cell (15, 29), and they have amino
acid sequence similarities to the replication proteins of other
alpha-like viruses, including tobacco mosaic virus (TMV) (1, 12,
14). RNA 2 also encodes a protein designated 2b (9),
which is suggested to be involved in host-specific long-distance
movement of the virus (10). RNA 3 encodes two proteins, 3a
and the coat protein (CP). The 3a protein is translated directly from
RNA 3 and has an approximate molecular weight of 30,000, whereas CP is
translated from subgenomic RNA 4, which is synthesized during
replication (35). Both the 3a protein and CP are necessary
for efficient cell-to-cell movement of CMV (4, 6, 8, 37).
Arabidopsis thaliana is widely regarded as an ideal model
plant for genetic and molecular biological studies (reviewed in references 7, 21, 28, and 36).
Furthermore, the Y strain of CMV (CMV-Y), the Cg strain of TMV
(TMV-Cg), and the B strain of turnip crinkle virus (TCV-B) systemically
infect A. thaliana ecotype Col-0 plants without causing
visible hypersensitive reactions (17, 25, 38, 43).
Therefore, a complete set of host factors which support efficient
multiplication of CMV-Y, TMV-Cg, and TCV-B are present in this ecotype.
As a first step to the identification of host factors which support
virus multiplication, we have previously isolated mutants of A. thaliana in which TMV-Cg or CMV-Y cannot multiply efficiently
(17, 30, 43). The tom1 and tom2
(tobamovirus multiplication) mutations affect TMV multiplication within
a single cell, whereas the cum1 (cucumovirus multiplication)
mutation affects the local spreading of CMV within the inoculated leaf.
Each mutation is single, recessive, nuclear, and virus specific; i.e.,
tom1 and tom2 mutations do not affect the
multiplication of CMV or TCV, and the cum1 mutation does not
affect that of TMV or TCV. These characteristics suggest that the
corresponding wild-type gene product supports virus multiplication
through specific interactions with virus-encoded factors.
In this study, we have analyzed a second mutant of A. thaliana, RB663, in which the multiplication of CMV is delayed and
show that the causal mutation, cum2, is likely to affect the
local spreading of both CMV and TCV. The genome of TCV is thought to encode two separate nonstructural proteins with molecular weights of
ca. 8,000 and 9,000, which are both necessary for virus movement (13, 24). Despite the differences in molecular weight and number of movement proteins between CMV and TCV, our results suggest that the wild-type CUM2 gene product is involved in the
cell-to-cell movement of these two distinct viruses.
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MATERIALS AND METHODS |
Viruses and antisera.
CMV-Y (37), TMV-Cg
(26, 42), and TCV-B (16) were propagated, and
their virus particles and virion RNAs were purified as described by
Ishikawa et al. (17, 18). Rabbit antiserum against TMV-Cg
was obtained from E. Shikata, rabbit antiserum against TCV-M was
provided by A. E. Simon and C. Zhang, and rabbit antiserum against
CMV-D was obtained from American Type Culture Collection (Manassas,
Va.). The rabbit antiserum against either CMV-D and TCV-M efficiently
cross-reacts with the CP of CMV-Y and TCV-B, respectively.
Plant materials and growth conditions.
A.
thaliana (L.) Heynh. Columbia (Col-0) and Landsberg
erecta (Ler) were used as the wild-type strains. A. thaliana RB568 (the cum1-1 mutant) and RB663, in which
the accumulation of CMV-Y CP is delayed, were isolated from an
M2 population derived from ethyl
methanesulfonate-mutagenized A. thaliana Col-0 seeds as described by Yoshii et al. (43).
Seeds were sown on rockwool soaked in distilled water, incubated in the
dark for 2 days at 4°C, and grown at 22 to 23°C under continuous
fluorescent illumination. The plants were watered with a nutrient
medium as described by Fujiwara et al. (11).
Plant inoculation.
Mechanical inoculation of A. thaliana plants with CMV-Y, TMV-Cg, and TCV-B was carried out as
described by Yoshii et al. (43). Growth conditions after
virus inoculation for examination of viral CP accumulation in
inoculated or uninoculated leaves were as described by Yoshii et al.
(43).
Detection and quantification of viral CP.
Extraction of
total protein from A. thaliana plants and protoplasts,
analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and immunoblotting, and quantification of viral CP were
performed as described by Yoshii et al. (43). The dot
enzyme-linked immunosorbent assay (ELISA) method was also carried out
as described by Yoshii et al. (43) and used to determine the
CMV multiplication phenotype of the F2 lines in Table 1. To
determine the CMV multiplication phenotype of F2 lines as
shown in Table 2 or of F2 mapping lines, we inoculated 10 to 12 F3 plants per line with either CMV-Y or TCV-B, bulk
harvested the aerial parts of the inoculated plants at 5 or 6 days
postinoculation (p.i.), and determined the concentration of viral CPs
in the protein extract by using SDS-PAGE and Coomassie brilliant blue
R-250 (CBB) staining. By using this bulk-harvesting assay, lines
homozygous for the cum2-1 mutation showed CMV-Y or TCV-B CP
accumulation similar to that of infected RB663 plants (i.e., almost
undetectable [see Fig. 4]) and those with either
cum2-1/CUM2 or CUM2/CUM2 genotypes showed a
higher accumulation of viral CPs (see Fig. 4). This method was
relatively quick and easy, and as mentioned in the footnote to Table 2,
gave equivalent results to those obtained by the dot ELISA method shown
in Table 1.
Genetic mapping.
To map the cum2-1 mutation,
reciprocal crosses were performed between RB663 (derived from Col-0)
and wild-type Ler plants. Individual F2 mapping lines were
established by harvesting F3 seeds from each of the
self-pollinated F2 plants. From these lines, we selected 14 lines in which the accumulation of TCV-B was reduced at 5 or 6 days
p.i., by using the bulk-harvesting method mentioned above. In these 14 lines, CMV-Y accumulation was also reduced at 6 days p.i. We then
extracted genomic DNA (2) from approximately 18 combined
F3 plants per selected line. The simple sequence length polymorphism (SSLP) or cleaved amplified polymorphic sequence (CAPS)
markers nga63 and nga280 on chromosome I, nga168 on chromosome II,
nga162, GAPA, GL1, nga112, and nga6 on chromosome III, nga8 on
chromosome IV, and nag106 and nga76 on chromosome V were subsequently used to examine the genotype of each line. Information on the nucleotide sequences of PCR primers and map positions of DNA markers were obtained from the A. thaliana database
(http://genome-www.stanford.edu/Arabidopsis) in March 1998 or from the
literature (3, 20). Primers for the DNA markers were
purchased from Research Genetics (Huntsville, Ala.). The PCR conditions
were as described by Bell and Ecker (3) when SSLP markers
were used and as described by Konieczny and Ausubel (20)
when CAPS markers were used.
Preparation and inoculation of A. thaliana
protoplasts.
A. thaliana protoplasts were prepared from
suspension-cultured calli and inoculated with virion RNAs by
electroporation as described by Ishikawa et al. (18).
Northern blot analysis.
Total nucleic acids were extracted
from protoplasts, purified, denatured with glyoxal, separated in 1%
agarose gels, transferred onto GeneScreen membranes (DuPont-NEN,
Boston, Mass.), and hybridized with 32P-labelled probes, as
described by Ishikawa et al. (18). Probes used for detection
of virus-related RNAs were prepared as described by Yoshii et al.
(43). A ubiquitin gene-specific probe was prepared as
follows. UBQ5-specific DNA was amplified by PCR from genomic DNA of A. thaliana Col-0 with the primers
dGTGGTGCTAAGAAGAGGAAGA and dTCAAGCTTCAACTCCTTCTTT
(34) and cloned into pCR2.1 with an Original TA
cloning kit (Invitrogen, San Diego, Calif.) to obtain pCR-UBQ5.
pGEM-UBQ5 was then constructed by inserting the 266-bp EcoRI
fragment from pCR-UBQ5 into the EcoRI site of pGEM-7Zf(+). The nucleotide sequence of the insert DNA was confirmed by dye terminator cycle sequencing with an automated DNA sequencer (PRISM 373;
Applied Biosystems, Foster City, Calif.). 32P-labelled
ubiquitin gene-specific RNA probe was synthesized from XbaI-digested pGEM-UBQ5 with SP6 RNA polymerase
(27). After hybridization and washing, Northern blots were
analyzed with a Bio Imaging Analyzer (BAS1000; Fuji Photo Film, Tokyo,
Japan).
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RESULTS |
Isolation of an A. thaliana mutant, RB663, in which the
accumulation of CMV-Y CP is delayed.
To identify host factors
involved in CMV multiplication, we previously screened for A. thaliana mutants in which the accumulation of CMV-Y CP in upper
uninoculated leaves was reduced to low levels and isolated two such
mutants from 4,800 M2 plants derived from ethyl
methanesulfonate-mutagenized Col-0 seeds (43). In this paper, we report the characterization of one of these mutants, RB663.
RB663 plants grew normally under our growth conditions. To examine the
accumulation pattern of CMV-Y in RB663 plants, aerial
tissues of
CMV-infected plants were harvested at 1, 3, 5, 8, 10,
and 14 days p.i.
and divided into four regions as illustrated
in Fig.
1A (the first to third leaves [R1], the
fourth to sixth
leaves [R2], the seventh to ninth leaves [R3], and
a mixture of
cauline leaves, stems, and unexpanded rosette leaves that
lack
petioles [Cau]), and the accumulation of the CP in each region
was determined (Fig.
1). Inoculated leaves withered within a few
days
after inoculation and were excluded from this analysis. In
wild-type
Col-0 plants, CMV-Y CP was first detected in the combined
region of Cau
and R3 at 3 days p.i. The concentration of CMV-Y
CP in the upper
regions of infected Col-0 plants was higher at
5 days p.i. but lower at
14 days p.i. than in the lower regions
(Fig.
1B). In contrast, the
accumulation of CMV-Y CP in RB663
plants was almost negligible until 5 days p.i. The concentration
of CMV-Y CP in the upper regions of
infected RB663 plants was
higher than in the lower regions at 8, 10, and 14 days p.i. The
accumulation pattern of CMV-Y CP in RB663 plants
at 10 and 14
days p.i. was similar to that in wild-type Col-0 plants at
5 days
p.i. Thus, CMV multiplication in RB663 plants was delayed
compared
to that in wild-type plants. Under our growth conditions, no
apparent
local necrotic lesions that would indicate one of the
hypersensitive
responses were observed on inoculated leaves of RB663
plants (data
not shown).
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FIG. 1.
Time course of accumulation of CMV-Y CP in wild-type
Col-0 and RB663 plants. (A) The aerial tissues of four inoculated
individuals were harvested separately at various times p.i. and
dissected into four regions: R1 (the first to third leaves), R2 (the
fourth and sixth leaves), R3 (the seventh to ninth leaves), and Cau (a
mixture of cauline leaves, stems, and unexpanded rosette leaves that
lack petioles). Prior to harvesting, CMV-Y was inoculated on the fifth
leaf, indicated by I. (B and C) The accumulation of CMV-Y CP in Col-0
(B) and in direct descendants (M6 generation) of RB663 (C)
was quantitated for each region (Cau, R1, R2, and R3) as described by
Yoshii et al. (43). Briefly, total-protein samples were
prepared and separated by SDS-PAGE, CMV CP was detected by CBB staining
or the immunoblotting method with anti-CMV CP antibodies, and the
concentration of the CP was estimated by comparing the band intensity
with that of a known amount of purified CMV CP standards. Means and
standard deviations of CMV-Y CP concentrations calculated from the data
obtained with four individuals are shown. Similar results were obtained
in triplicate experiments. The asterisks in panels B and C indicate
that the regions Cau and R3 could not be harvested separately and hence
were combined for analysis. Inoculated leaves withered at 2 to 3 days
p.i. under the conditions used for this experiment, and thus the
accumulation of CMV-Y CP in inoculated leaves was not examined.
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Delayed accumulation of CMV CP in RB663 plants is controlled by a
monogenic, recessive trait which is distinct from the cum1
mutation.
To determine the genetic basis of the phenotype of
delayed accumulation of CMV-Y CP in RB663 plants, crosses were carried out between RB663 and wild-type Col-0 plants. The level of CMV-Y CP
accumulation in F1 plants was similar to that in Col-0
plants at 6 days p.i. (Fig. 2),
indicating that the mutant allele is not dominant. We then examined the
segregation of the phenotype in the F2 generation
(F2 plants were obtained by self-pollination of the
F1 plants). Since plants infected with CMV-Y have poor fertility, the phenotype of F2 plants cannot be reconfirmed
in the F3 generation if F2 plants are directly
inoculated. Therefore, we harvested F3 seeds from each
F2 individual separately (hereafter, we refer to each pool
of F3 seeds or plants derived from a single F2
individual as an F2 line) and determined the phenotype of
these F2 lines as follows: 10 to 12 F3 plants
per F2 line were inoculated with CMV-Y, the R3 leaves (Fig.
1A) were harvested separately at 6 days p.i., and the accumulation of
CMV-Y CP was detected separately by the dot ELISA method. When RB663
direct descendants were assayed for CMV accumulation by this method,
all the plants showed signals similar to that derived from noninfected
plants, or only 1 or 2 of the 10 plants showed positive signals weaker than that of CMV-infected Col-0 plants at 6 days p.i. In approximately one-fourth of the F2 lines, all F3 plants
accumulated similar levels of CMV-Y CP to that in RB663 plants (these
F2 lines are classified as low level), whereas in the other
F2 lines, more than half of the F3 plants
accumulated similar levels of CMV-Y CP to that in infected Col-0 plants
(these F2 lines are classified as high level) (Table
1). These results are consistent with the hypothesis that the phenotype of delayed accumulation of CMV-Y CP in
RB663 plants is controlled by a monogenic, recessive trait.
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FIG. 2.
Accumulation of CMV-Y CP in F1 plants.
Thirteen individuals each of RB663 (M6), RB568
(M6), wild-type Col-0, and F1 plants derived
from the reciprocal crosses between Col-0 and RB663 (M5)
plants and 20 individuals of F1 plants derived from the
reciprocal crosses between RB568 (M5) and RB663
(M5) plants were inoculated with CMV-Y. At 6 days p.i., one
of the R3 leaves (Fig. 1A) was harvested separately from each plant,
and the level of CMV-Y CP accumulation was determined, as in Fig. 1.
Means and standard deviations of CP concentrations are shown. The
M6 plants of RB568 and RB663 were direct descendants of the
M5 plants of RB568 and RB663 used for the crosses.
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Next, crosses were carried out between RB663 and RB568, another mutant
plant with delayed multiplication of CMV-Y which carries
a monogenic,
recessive mutation designated
cum1-1 (
43). The
accumulation of CMV-Y CP in the F1 plants was similar to that
in
wild-type Col-0 plants (Fig.
2), suggesting that the causal
mutation in
RB663 plants is distinct from
cum1. We named the causal
mutation in RB663 plants
cum2-1.
The map position of the
cum2 locus was determined by using
another set of F
2 lines (these F
2 lines are
referred to below as
the mapping lines) that were derived from
F
1 plants obtained by
the crosses between the RB663 plants
(derived from Col-0) and
Ler, a wild-type ecotype distinct from Col-0.
The multiplication
of CMV-Y and TCV-B in Ler plants was similar to that
in Col-0
plants. We selected mapping lines in which the accumulation of
CMV-Y CP was reduced to low levels at 6 days p.i. (for details,
see
Materials and Methods) and prepared genomic DNA from each
of these
lines. The genotype at PCR-based polymorphic DNA markers
covering the
A. thaliana genome (see Materials and Methods) was
then
determined for the DNA preparations. The numbers of chromatids
recombined between a DNA marker and
cum2-1 mutation with
respect
to the total numbers of chromatids examined were 13 of 28, 13
of 28, 12 of 28, 8 of 28, 8 of 28, 2 of 28, 1 of 28, 1 of 28,
13 of 28, 10 of 28, and 9 of 28 for DNA markers nga63, nga280,
nga168, nga162,
GAPA, GL1, nga112, nga6, nga8, nag106, and nga76,
respectively. As
shown in Fig.
3, some of the markers on
chromosome
III showed linkage with the
cum2-1 mutation. The
other markers
showed no significant linkage with the mutation. From
these data,
the
cum2-1 mutation was suggested to be located
within a region
between markers GL1 (map position 44.7 centimorgans
[cM]) and
nga112 (map position 77.0 cM) on chromosome III (Fig.
3).
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FIG. 3.
Map location of cum2-1 mutation on chromosome
III. The map positions (in centimorgans) of the markers are based on
the data of Camilleri et al. (5) and are shown on the left.
The markers on chromosome III that we tested for linkage analysis are
indicated on the right. SSLP and CAPS markers are shown by open boxes
and open circles, respectively. The number of the chromatids recombined
between a marker and the cum2-1 locus with respect to the
total number of chromatids examined is shown in parentheses.
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Susceptibility of RB663 plants to viruses other than CMV-Y.
The accumulation of TMV-Cg and TCV-B CPs in RB663 mutant plants was
examined to determine whether the mutation also affects the
multiplication of viruses other than CMV. TCV and TMV belong to
different taxonomic groups from CMV and have been shown to multiply
systemically in wild-type Col-0 plants (17, 25). The time
course of TMV-Cg CP accumulation in RB663 plants was similar to that in
wild-type Col-0 plants (Fig. 4; TMV-Cg CP
accumulation was also similar at 13 days p.i. [data not shown]),
whereas the accumulation of TCV-B CP was lower than that in Col-0
plants at 6 or 8 days p.i. (Fig. 4). At 10 or 13 days p.i., TCV-B CP
accumulation was still lower in RB663 plants than in Col-0 plants, but
the difference was smaller than that in the earlier stages (data not shown), suggesting that TCV-B CP accumulation was delayed in RB663 plants.
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FIG. 4.
Accumulation of CP of CMV-Y, TMV-Cg, and TCV-B in
wild-type Col-0 and RB663 plants. Ten individuals each of Col-0 (A) and
RB663 (B) plants were inoculated with CMV-Y, TMV-Cg, or TCV-B virions.
The RB663 plant line used was obtained through two cycles of
backcrossing, i.e., selecting a cum2-1 line from
F2 lines resulting from the self-pollination of
F1 plants generated by the crossing of cum2-1
pollen to Col-0 flowers. Total protein extract was separately prepared
from aerial tissues of inoculated plants that were harvested at 3, 6, and 8 days p.i., as indicated above the panels, equal volumes of the
extracts for each inoculation period were mixed, and the samples
derived from 0.6 mg (fresh weight) of tissue were analyzed by SDS-PAGE
with 11% (for TMV-Cg and CMV-Y) or 9% (for TCV-B) polyacrylamide gels
and CBB staining. Total proteins from mock-inoculated plants were
concurrently electrophoresed (lanes M). The positions of viral CPs are
indicated by arrows. Similar results were obtained in triplicate
experiments.
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To determine the genetic basis of the phenotype of delayed accumulation
of TCV-B CP, we examined the phenotype of plants derived
from
reciprocal crosses between RB663 and wild-type Col-0 plants.
In all of
the F
1 plants, the accumulation of TCV-B CP at 6 days
p.i.,
as determined by SDS-PAGE and CBB staining of total protein,
was
similar to that in Col-0 plants (data not shown). Of the lines
randomly
selected from the F
2 lines established by performing
crosses between RB663 and Col-0 plants and used to produce the
data
shown in Table
1, approximately one-fourth showed reduced
accumulation
of TCV-B CP (Table
2; for [29:11]
segregation,
2 [3:1] = 0.133,
P > 0.05; for [42:8] segregation,
2 [3:1] = 2.16,
P > 0.05), suggesting that the causal mutation
was a
monogenic, recessive trait. Furthermore, all of the 19 F
2 lines which showed low-level accumulation of TCV-B CP showed low-level
accumulation of CMV-Y CP, and the other 71 F
2 lines which
showed
high-level accumulation of TCV-B CP also showed high-level
accumulation
of CMV-Y CP (Table
2). None of the F
2 lines
with high-level accumulation
of TCV-B CP showed low-level accumulation
of CMV-Y CP, and none
of the F
2 lines with low-level
accumulation of TCV-B CP showed
high-level accumulation of CMV-Y CP.
These results suggest that
the
cum2-1 mutation also affects
TCV-B multiplication or that
the casual mutation for the phenotype of
reduced accumulation
of TCV-B CP in RB663 plants is distinct from, but
closely linked
to the
cum2-1 mutation (within 1.2 cM).
Multiplication of CMV-Y, TCV-B, and TMV-Cg in inoculated leaves of
RB663 plants and in RB663 protoplasts.
To investigate the
mechanisms of inhibition of CMV and TCV multiplication in RB663 plants,
we first examined the CP accumulation within inoculated leaves. The
accumulation of CMV-Y and TCV-B CPs in inoculated leaves of RB663
plants was decreased compared to that in Col-0 plants, whereas the time
course and the level of CP accumulation of TMV-Cg was similar to that
in wild-type Col-0 plants (Fig. 5),
irrespective of whether the plants were inoculated with virion or
virion RNA. The accumulation of CMV-Y and TCV-B CPs at 48 h p.i.
in inoculated leaves of RB663 plants was approximately 1/5 and 1/10,
respectively, of that in wild-type Col-0 plants (average of eight
independent experiments [data not shown]). Thus, in RB663 plants, CMV
and TCV multiplication is at least affected prior to the long-distance
movement and after the uncoating of virus particles in an initially
infected cell.
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FIG. 5.
Time course of the accumulation of CMV-Y, TCV-B, and
TMV-Cg CPs in inoculated leaves of wild-type Col-0 and RB663 plants.
Col-0 or RB663 plants were inoculated with virion (A) or virion RNA (B)
of CMV-Y, TCV-B, or TMV-Cg. The RB663 plants used were from the same
lines as those in the experiment in Fig. 4. Five inoculated leaves were
harvested at time zero (immediately after the inoculum solution was
washed off with distilled water) and at 24, 48, and 72 h p.i.
(hpi) as indicated above the lanes. Total proteins were prepared from
each sample and separated by SDS-PAGE, and the CPs were detected by the
immunoblotting method (CBB staining was not used here because the
amount of CP was relatively small). In the panels for CMV-Y or TCV-B
virion-inoculated plants, total protein from 0.06 mg of leaf tissue was
applied to each lane. In the panels for TCV-B RNA-inoculated plants,
total protein from 0.12 mg of leaf tissue was applied to each lane. In
the other panels, total protein from 0.6 mg of leaf tissue was applied
to each lane. In lanes M, total protein from 0.6 mg of mock-inoculated
plants was used. Similar results were obtained in eight independent
experiments.
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Next, to determine whether the multiplication of CMV-Y and TCV-B within
a single cell was affected in RB663, we examined the
accumulation of
virus-related RNAs in RB663 and wild-type Col-0
protoplasts inoculated
with virion RNAs of CMV-Y, TCV-B, or TMV-Cg
by electroporation. The
time course and the level of accumulation
of these virus-related RNAs
and CPs were similar between RB663
and wild-type protoplasts. The
accumulation of viral RNAs at early
stages after inoculation is shown
in Fig.
6 (consistent results
were
obtained in three independent experiments). The accumulation
of each
viral RNA at 24 h p.i. and the accumulation of each viral
CP at 24 and 48 h p.i. were also similar between RB663 and Col-0
protoplasts (data not shown). These results suggest that the
cum2-1 mutation does not affect the multiplication of either
CMV or TCV
within a single cell but, rather, affects the local
spreading
of these viruses within an inoculated leaf.
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FIG. 6.
Time course of the accumulation of CMV-Y, TCV-B, and
TMV-Cg-related RNAs in wild-type Col-0 and RB663 protoplasts. (A)
Protoplasts were prepared from liquid-cultured calli derived from
seedlings of Col-0 or RB663 plants. The RB663 plants used were from the
same line as that in the experiment in Fig. 4 (the yield and quality of
protoplasts were low if they were prepared from direct descendants of
RB663). Approximately five million Col-0 or RB663 protoplasts were mock
inoculated (lanes M) or inoculated with either 7 µg of CMV-Y RNA, 20 µg of TMV-Cg RNA, or 5 µg of TCV-B RNA by electroporation.
Inoculated protoplasts were cultured for 2, 4, 6, and 8 h as
indicated above the panels and harvested for analysis of RNA. Duplicate
Northern blots of total RNA extracted from the protoplasts were
prepared: one set was hybridized with probes that detect either
CMV-Y-related RNAs, TMV-Cg-related RNAs, or TCV-B-related RNAs, and the
other set was hybridized with a probe that detect UBQ5 mRNA.
The positions of bands for CMV-Y RNAs 1, 2, 3, and 4, TMV-Cg genomic
RNA, 30,000-molecular-weight protein and CP subgenomic mRNAs, TCV-B
genomic RNA, or UBQ5 mRNA are indicated to the right of each
panel. (B) Graphic representation of the time course of viral RNA
accumulation in protoplasts. The intensity of bands for CMV-Y RNA 4, TMV-Cg genomic RNA, TCV-B genomic RNA, and UBQ5 mRNA on
Northern blots was quantified. Boxes and error bars show means and
standard deviations in three independent experiments of relative viral
RNA accumulation normalized by the intensity of UBQ5 mRNA
bands in corresponding lanes (intensity of UBQ5 mRNA = 1).
|
|
| |
DISCUSSION |
We have characterized an A. thaliana mutant, RB663, in
which the accumulation of CMV-Y CP in uninoculated leaves of infected plants is delayed compared to that in wild-type Col-0 plants. The
causal mutation, cum2-1, was monogenic, recessive, and
distinct from a previously identified cum1 mutation
(43). The simplest interpretation of the recessiveness of
the cum2-1 mutation is that the wild-type CUM2
gene product is necessary for the efficient multiplication of CMV and
that the cum2-1 allele has lost the function to support CMV
multiplication. However, at present, other possibilities, e.g., that
the CUM2 gene product represses the activity (such as host
defense functions induced by virus infection) that represses CMV
multiplication, cannot be excluded. In RB663 plants, TMV multiplication
was similar to that in wild-type plants. Thus, delayed multiplication
of CMV-Y was not derived from a nonspecific effect on viral
multiplication, such as the inhibition of initial invasion of virion or
virion RNAs through changes of leaf surface structure or low metabolic
activity of RB663 plants.
The accumulation of CMV-Y CP within inoculated leaves of RB663 plants
infected with either CMV virion or CMV virion RNA took place slowly
compared to that in wild-type Col-0 plants (Fig. 5). In contrast, the
accumulation of CMV-Y-related RNAs in RB663 protoplasts was similar to
that in Col-0 protoplasts (Fig. 6). Such characteristics of the
cum2-1 mutation are similar to those of the
cum1-1 mutation. These results suggest that the
cum2-1 mutation does not affect the uncoating of CMV virion
in an initially infected cell or the amplification of CMV-related RNAs
within infected cells but, rather, affects the spreading from cell to cell within the inoculated leaf. At present, however, another possibility, i.e., that the host response to repress CMV multiplication is more strongly induced in RB663 plants, cannot be excluded. Furthermore, in addition to the less efficient spreading of CMV within
an inoculated leaves, it is possible that long-distance movement of CMV
is also affected in RB663 plants. Comparison of the size exclusion
limit of plasmodesmata in leaf tissue cells in the presence of CMV 3a
movement protein (MP) (either microinjected or transgenically
expressed) between wild-type and RB663 plants or determination of the
localization of CMV MP within infected tissue may provide us with
further information on the mechanisms of inhibition of CMV
multiplication by the cum2-1 mutation.
In RB663 plants, TCV multiplication was also delayed in either
inoculated or systemically infected leaves (Fig. 4 and 5). Furthermore,
in RB663 protoplasts, the multiplication of TCV was similar to that in
wild-type protoplasts (Fig. 5). These results suggest that the
cell-to-cell movement of TCV was affected in RB663 plants, as observed
with CMV. As far as we examined, the phenotypes of delayed accumulation
of CMV and TCV cosegregated without exception. Therefore, it is likely
that the cum2-1 mutation affects the multiplication of TCV
as well as that of CMV, although, at present, the remote possibility
that an independent mutation affecting TCV multiplication is present
near the cum2 locus cannot be excluded. Molecular cloning of
the wild-type CUM2 locus and introduction of a minimal DNA
segment complementing the cum2-1 mutation into RB663 plants
will test these possibilities. Provided that the cum2-1
mutation affects the multiplication of TCV, this is a unique
characteristic, since most resistance genes of plants against viruses
are specific to each virus. As far as we are aware, this is the first
report of an A. thaliana mutation affecting two distinct
viruses.
For the cell-to-cell movement of CMV, two virus-encoded proteins, 3a MP
and CP, are necessary (4, 19, 37). 3a MP is known to
localize at plasmodesmata (40), where it is able to enlarge
the size exclusion limit (8, 39) and cooperatively bind
single-stranded nucleic acids in a non-sequence-specific manner in
vitro (23). The genome of TCV encodes two nonstructural proteins, p8 and p9, which are both necessary for the cell-to-cell movement of TCV (13, 22, 24). TCV CP is necessary for the cell-to-cell movement in Nicotiana benthaniana but is not
necessary in A. thaliana, Chenopodium
amaranticolor, or Brassica campestris (13, 22,
24). TCV p8 is reported to have an RNA binding activity like that
of CMV MP (41), but any other functional or structural
similarity between the MPs of CMV and TCV has not been pointed out so
far. Despite the apparent difference in the organization of the MPs
between CMV and TCV, our results may suggest a common aspect to the
mechanisms underlying the cell-to-cell movement of these two viruses,
mediated by the CUM2 factor. At the same time, however, it is also
possible that the CUM2 factor has nonhomologous functions during the
cell-to-cell movement of CMV and TCV.
| |
ACKNOWLEDGMENTS |
We are grateful to D. Bartlem, E. Nambara, and T. Yamanaka for
insightful discussion and to K. Fujiwara for general assistance. We are
grateful for the use of the facilities of the Biopolymer Analysis
Laboratory, Faculty of Agriculture, and the Research Center for
Molecular Genetics, Hokkaido University.
This work was supported in part by Grants-in-Aid for Scientific
Research from the Ministry of Education, Science and Culture of Japan
to M.I. (grant 08680733) and S.N. (grant 06278102) and by a grant from
the Japan Society for the Promotion of Science to M.I. (grant
RFTF96L00603).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Applied Bioscience, Faculty of Agriculture, Hokkaido University,
Kita-ku, Sapporo 060-8589, Japan. Phone: 81-11-706-3887. Fax:
81-11-706-4932. E-mail:
ishikawa{at}abs.agr.hokudai.ac.jp.
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0022-538X/98/$04.00+0
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Abstract
Introduction
