343
J. gen. Virol. (1981), 53, 343-346
Printed in Great Britain
Cytopathic Structures Associated with Tonoplasts of Plant Cells Infected
with Cucumber Mosaic and Tomato Aspermy Viruses
(Accepted 14 November 1980)
SUMMARY
Electron microscopic examination of cucumber mosaic and tomato aspermy
virus-infected leaf cells has revealed the presence of membrane-bound vesicles
associated with the tonoplasts. The vesicles measured between 50 and 90 nm in diam.
and the membranes of some were seen to be continuous with the tonoplast. Although
the significance and function of the virus-specific vesicles is not clear, evidence is
presented that they may be the sites of viral RNA synthesis.
Cucumber mosaic virus (CMV) and tomato aspermy virus (TAV), both of which belong to
the cucumovirus group (Matthews, 1979), have isometric particles about 28 to 30 nm in diam.
when negatively stained and viewed in the electron microscope (Francki et al., 1979).
However, in sections of infected cells, they appear as densely stained particles only about 20
to 23 nm in diam. and hence are difficult to distinguish from cytoplasm!c ribosomes (Hatta &
Francki, 1979). Recently, we reported that CMV and TAV can be distinguished from
ribosomes by their greater resistance to digestion with pancreatic RNase in aldehyde-fixed
tissues under appropriate conditions (Hatta & Francki, 1979). Further observations on the
cytopathology of CMV- and TAV-infected plant cells have revealed the presence of
virus-specific vesicles associated with the tonoplast. This is the subject of this communication.
Tissue pieces from healthy or systemically infected leaves of various plant species were
excised and prepared for electron microscopy as described by Hatta & Francki (1979).
Detailed studies were conducted on Nicotiana elevelandii A. Gray and Nicotiana tabaeum L.
cv. White Burley infected with six isolates of CMV and three isolates of TAV. The Q, M and
T strains of CMV and the V and N strains of TAV have been previously described (Mossop
& Francki, 1979), whereas the X, U and B isolates of CMV and the C isolate of TAV were
obtained from lettuce, banana, gladiolus and chrysanthemum respectively. Double-stranded
(ds)RNA in infected cells was identified cytochemically by its differential susceptibility to
RNase in aldehyde-fixed tissues incubated in high and low salt media (Hatta & Francki,
1978).
Examination, using the enzyme cytochemical technique of cells from leaves of N.
elevelandii and N. tabaeum individually infected with all nine strains and isolates of CMV and
TAV revealed the presence of virus particles in the cytoplasm as previously reported in plants
infected with the T strain of CMV (Hatta & Francki, 1979). The numbers of particles detected
varied with the virus strain and the host plant species. Highest concentrations of particles were
observed in cells infected with the T strain of CMV and the three TAV strains. The particles of
all the CMV and TAV strains and isolates were more numerous in ceils ofN. elevelandii than
those of N. tabaeum. The other consistent change in cells infected with all these viruses was
the presence of small vesicles associated with the tonoplast (Fig. 1 a). Similar structures were
not observed in the cells of any species of healthy control plants investigated. From
observations on sections of N. elevelandii leaf cells infected individually with the nine isolates
of CMV and TAV, we concluded that large numbers of vesicles were correlated with the
ability of the virus strains to reach high concentrations.
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Short communications
(b)
(e)
(t)
(e)
!
~-"
,7
Fig. 1. Electron micrographs of tonoplast-associated vesicles in leaf cells infected with the T strain of
cucumber nfosaic virus (TCMV). (a) General view of the tonoplast of an infected cell. (b to f ) are
individual vesicles selected to show the structure of the membrane enclosing the vesicles (a, b), the
continuation of the membrane with the tonoplast (b), fibrils inside the vesicles (d, e), and proximity of a
vesicle with a virus crystal in an infected cell vacuole ( f ) : (g) shows a vesicle-like structure associated
with the plasmalemma of a virus-infected cell which has been torn away from the cell wall. All
micrographs are of cells from tissues prepared for electron microscopy without RNase treatment.
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345
Table 1. Effect of RNase in high and low salt media on the presence of fibrils in tonoplastassociated vesicles ofN. clevelandii infected with the T strain of CMV
Incubation conditions*
f
Number of vesiclesexaminedt
Number of vesicles containing fibrils
Percentage of vesicles containing fibrils
2 x SSC buffer
only (control)
699
494
71
20/~g/mlRNase
in 2 × SSC
609
405
67
20/lg/ml RNase
in 0.01 × SSC
791
146
18
* Aldehyde-fixed leaf tissue pieces were washed in the appropriate buffers and incubated at 25 °C for 16 h in
the test solutions before osmiumfixation and preparation for electron microscopy (Hatta & Francki, 1978).
t All vesicles in sections of 10 cells of each treatment from four different tissue pieces were examined. Cells
which had been cut open after aldehyde fixation were selected for examination to ensure that their contents had
been penetrated by RNase (Hatta & Francki, 1978).
The virus-specific vesicles measured between 50 and 90 nm (Fig. 1 a) and appeared to be
bounded by a unit membrane about 10 nm thick (Fig. 1 b, c) which, in some instances, was
observed to be continuous with the tonoplast (Fig. 1 c). This indicates that the contents of
such vesicles are in contact with the cytoplasm through a narrow neck. Many of the vesicles
contained electron-dense fibrils (Fig. 1 d, e). The vesicles were occasionally observed adjacent
to crystals of CMV particles in the vacuole (Fig. l f ) and sometimes associated with the
plasmalemma (Fig. l g). However, the vesicles observed near plasmalemmas could be
individual vesicles of paramural bodies (plasmalemmasomes or lomasomes) which are known
to occur in cells of healthy plants (Marchant & Robards, 1968).
The electron-dense fibrils observed in tonoplast-associated vesicles (Fig. 1 d, e) were
susceptible to digestion by RNase under low but not high salt incubation conditions (Hatta &
Francki, 1978). In one experiment with the T strain of CMV-infected N. clevelandii leaf cells
sampled from young systemically infected plants inoculated 12 days previously, 70% of the
vesicles contained fibrils in tissues which had not been treated with RNase (Table 1). This
proportion is very similar to that observed in sections from tissues incubated in 2 x SSC
buffer (SSC = 0.15 M-NaC1 plus 0.015 M-sodium citrate pH 7) containing 20 ~tg/ml RNase,
but much higher than in sections from tissues incubated in 0.01 x SSC containing the same
concentration of the enzyme (Table 1). These data indicate that the tonoplast-associated
vesicles contain dsRNA which could be the viral replicative form and/or replicative
intermediate. Hence, the vesicles may be the sites of viral R N A replication analogous to the
chloroplast-associated vesicles observed in tymovirus-infected cells (Matthews, 1977) or the
cytoplasmic vesicles characteristic of cells infected with cowpea mosaic virus (de Zoeten et
al., 1974). However, the tonoplast-associated vesicles also show some resemblance to the
cytoplasmic vesicles observed near the vacuoles of barley yellow dwarf virus-infected cells
(Gill & Chong, 1975) and those observed in leaf cells of Prunus spp. with little-cherry leaf
disease (Raine et al., 1975).
We have also observed numerous tonoplast-associated vesicles similar to those described
here in CMV- and TAV-infected cells, in cells of local lesions on inoculated leaves of N x
edwardsonii infected with tobacco necrosis virus. A preliminary survey of plants infected with
a number of other viruses with small polyhedral particles and ssRNA genomes failed to reveal
the presence of tonoplast-associated vesicles in cells containing virus particles. Viruses used in
the survey included members of the comovirus, luteovirus, nepovirus, tombusvirus, tymovirus
and the southern bean mosaic virus groups as well as several unclassified viruses (T. Hatta &
R. I. B. Francki, unpublished results). Nevertheless, occasional tonoplast-associated vesicles
have been observed in some published micrographs of virus-infected cells such as the one from
Esau (1968) infected with tobacco mosaic virus (Fig. 14, p. 102). On the other hand,
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numerous vesicles have been reported to be associated with the tonoplasts of N. clevelandii
infected with carrot mottle virus (Murant et al., 1969), lettuce speckles mottle virus (Falk et
al., 1979) and animal cells infected with the group A arboviruses, Semliki Forest virus, Sindbis
virus and western equine encephalomyelitis virus (Grimley et al., 1972), all of which appear
remarkably similar to those we have observed in CMV- and TAV-infected cells. The
significance of these similarities remains to be established. It has been suggested that the
vesicles in carrot mottle virus-infected cells may be the virus particles (Murant et al., 1969,
1973; Murant, 1974) and that those associated with lettuce speckles mottle virus infection are
not virus particles but sites of synthesis of infective ssRNA which does not become
encapsidated (Falk et al., 1979). In the case of the arbovirus-infected cells, there is evidence
that the vesicles are concerned with virus replication (Friedman et al., 1972).
We thank Dr J. L. Dale for supplying the X and U isolates of cucumber mosaic virus and Mr D.
Talfourd for supply and maintenance of plants. One of us (T.H.) is supported by a Commonwealth
Special Grant from the Australian Department of Primary Industry. The work was also supported by
grants from the Australian Research Grants Committee.
Department of Plant Pathology
Waite Agricultural Research Institute
The University of Adelaide, South Australia, 5064
T. H A T T A
R. 1. B. FRANCKI*
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(Received 23 September 1980)
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