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Tubules with viruslike particles in leaf cells infected with bean pod mottle virus
43, 329-337 (1971)
VIROLOGY
Tubules
with
Viruslike
Particles Pod
Mottle
K. S. KIM Virology
and Biocontrol
AND
Cells
Infected
with
Bean
Virus’ J. P. FULTON
Laboratory, Department of Plant Pathology, Arkansas, Fayetteville, Arkansas YBYOl Accepted
’
in Leaf
University
of
October IS, 19YO
Ultrastructural studies of plant tissues infected with bean pod mottle virus (BPMV) revealed viruslike particles within tubules as a prominent feature of infection. Tubules with particles were found between the plasmalemma and the cell wall in a local lesion host, Pinto bean. In a systemic host, Cherokee Wax bean, the tubules with particles were embedded within the cell wall. Many irregularities and protrusions of the cell wall were noted in the systemically infected host.
INTRODUCTION This paper reports the occurrence of virusSmall, polyhedral plant viruses (25-30 nm like particles within tubules in tissues inin diameter) often evoke only limited cyto- fected with bean pod mottle virus (BPMV) logical changes in infected cells, and the (Cryptogram R/l 2.4/35 S/S S/Cl). Alvirus particles are difficult to locate in though the tubules are similar to those electron micrographs unlessthey have accu- reported above there is a striking difference mulated in large amounts (Matsui and in the location of the tubules in infected Yamaguchi, 1966). In some cases,however, cells. viruslike particles occur within tubules in MATERIALS AND METHODS infected cells. Roberts and Harrison (1970) BPMV (obtained from H. J. Walters) was described tubules in young leaf cells of Chenopodium quinoa infected with straw- used throughout this study. The virus was berry latent ringspot virus (SLRV). Similar inoculated to the primary leaves of young tubules were present in meristematic tissues plants of Phaseolusvulgaris L., cvs. “Cheroof various plants infected with cherry leaf kee Wax” and “Pinto.” Cherokee Wax was roll virus (CLRV) and tobacco ringspot virus systemically infected and Pinto developed (TRSV) as well as SLRV (Walkey and local lesions. Strips of inoculated tissue and systemically Webb, 1970). Davidson (1969) reported infected tissue were fixed at various times evidence of a file of TRSV particles conafter inoculation in 4% glutaraldehyde in tained within tubules which passedthrough the plasmodesmata and into the cytoplasm 0.1 M cacodylate buffer, pH 7.2, for 2 hr at of neighboring cells. DeZoeten and Gaard room temperature. After several washings (1969) made similar observations in tomato with buffer solution, the tissues were postringspot virus (TomRS) infected cells. fixed in 1% osmium tetroxide for 2 hr, then stained in bulk overnight at O-4” in 0.5 % Tubules with virus particles were evident only in phloem sieve tubes of plants infected uranyl acetate in distilled water. The tissues with citrus infectious variegation virus were dehydrated in an ethanol series, embedded in a mixture of Epon 812, 1 part (Gerola et al., 1969). Epon A:1 part Epon B, and sectioned with 1 Supported in part by Cooperative States glassknives on an LKB Ultrotome. Sections Research Services Grant No. 816-15-16. were double stained in 2 % uranyl acetate
329 Copyright
@ 1971 by Academic
PI-S
Inc.
Printed
in
U.S.A.
330
KIM
AND
FULTON
BPMV
IN
TUBULES
for 1 hr and lead citrate (Reynolds, 1963) for 10 min and examined with a Siemens Elmiskop IA. Noninoculated control tissues were fixed, embedded, and sectioned in the same manner. RESULTS
In the local lesion host, Pinto bean, viruslike particles were observed within tubules located between the cell wall and the plasmalemma (Fig. la, b, c). In some cases the tubules were in deep invaginations of the plasmalemma, but they were separated from the cytoplasm by the invaginated plasmalemma. Tubules without particles were also present in the same region between the cell wall and the plasmalemma (Figs. la and 2). They were usually in masses and gave the appearance of a tangled network of tubules or a group of vesicles. Tubules without particles were also a feature of normal, uninfected cells but occurred in much smaller numbers than in infected tissue. Tubules with particles were usually associated with masses of tubules without particles (Figs. la and 2). The tubules with particles were relatively straight whereas those without particles were bent and twisted. In the systemic host, Cherokee Wax, tubules with and without particles were also noted between the cell wall and the plasmalemma (Figs. 4 and 7). However, the most common location of tubules with particles was within the cell wall. The majority of these tubules did not appear to be associated with plasmodesmata but were embedded in the cell wall (Fig. 6a, b). Xost of the embedded tubules were aligned parallel with the plane of the cell wall. In Fig. 6a one may question whether the tubules are embedded in the cell wall or closely associated with the surface of the cell wall. In viewing many sections cutting the tubules at different angles, it is evident that the tubules are
IN
INFECTED
CELLS
331
within the cell wall rather than closely associated with it, Figure 8 shows cross sections of tubules embedded in the cell wall and Fig. 6b (CPI) shows the cross section of a cell wall protrusion containing an embedded tubule with virus particles. Irregular thickenings and protrusions (Fig. 8) of cell walls also occurred in infected Cherokee Wax bean. These protrusions invariably contained tubules with particles (Fig. 7). Protrusions sometimes penetrated deeply into invaginated cytoplasm (Fig. 8). In some of the sections the protrusions appeared as isolated pieces of cell wall material surrounded by plasmalemma and cytoplasm. If the protruded cell wall was sectioned through a longitudinal plane, the isolated cell wall fragment appeared as an elongated bar (Fig. 6a) while it appeared as a circular body in cross section (Fig. 6b). Tubules with virus particles were commonly observed embedded in such pseudoisolated cell walls (Fig. 6a,b). There were no noticeable cell wall protrusions or thickenings in infected Pinto bean leaf cells. Some tubules with particles were noted in plasmodesmata of both hosts (Figs. 3 and 5). In all cases, however, tubules in plasmodesmata did not extend into the cytoplasm but abutted onto an area of tubules without particles or were continuous with cell wall protrusions. The tubules in both hosts consisted of an outer unit membrane, an inner layer of material, and the line of particles (Fig. la, c). Tubules with virus particles were 60-70 nm in diameter. There was no evidence of an extra envelope as with the tubules reported by Roberts and Harrison (1970). Since the membrane of the tubule is typical unit membrane it seemslikely that the tubules represent fingerlike projections of cytoplasm bounded by the plasmalemma. It is assumed that tubules with particles originate in the
Abbreviations for figures: CW, cell wall; P, plastid; TwV, tubule with virus particles; !I’, tubule without virus particle; N, nucleus; M, mitochondria; PM plasmalemma; CPI, circular pseudoisolated cell wall; EPI, elongated pseudoisolated cell wall; PR, cell wall protrusion; CH, chloroplast. FIG. 1. BPMV-infected leaf cells of Pinto beans. (a) Tubules with virus particles between plasmalemma and cell wall. A portion of plasmalemma is invaginated deeply into cytoplasm. Invagination includes tubule with virus particles and tubules without particles. (b) Cross section of tubules between cell wall and plasmalemma. (c) Higher magnification of obliquely sectioned tubule with virus particles showing outer unit membrane, inner layers (arrows), and a file of virus particles.
332
FIG. without FIG.
KIM
2. BPMV-infected particles. Tubules 3. BPMV-infected
leaf cell of Pinto are between the leaf cells of Pinto
AND
FULTON
bean. Tubules with virus particles associated with tubules cell wall and plasmalemma. bean with tubule with virus particles in the plasmadesma.
BPMV
FIG. 4. BPMV-infected
IN
TUBULES
IN
INFECTED
333
CELLS
leaf cells of Cherokee Wax bean showing tangled Some tubules contain virus particles. FIG. 5. BPMV-infected leaf cell of Cherokee Wax bean. A tubule with virus
masses
of tubules
without
virus.
particles
in a plasmadesma.
and
an elongated
FIG. 6. BPMV-infected
plasm;
leaf cell of Cherokee Wax bean. (a) A tubule with virus particles embedded pseudoisolated cell wall wifh embedded tubules with virus particles.,jb)
in cell wall; a protrusion Circular, pseudoisolated
of the cell cell wall.
wall
into
cyto-
BPMV
FIG. 7. BPMV-infected tubule
with
virus
particles
IN
TUBULES
IN
INFECTED
335
CELLS
leaf cell of Cherokee Wax bean showing cell wall protrusion with embedded and also a tubule with virus particles between the cell wall and plasmalemma.
FIG. 8. BPMV-infected leaf cell of Cherokee Wax bean protrusions of the cell wall. Several cross or oblique sections the cell wall. manner or are derived from tubules without particles.
same
DISCUSSION
Roberts and Harrison (1970) and Walkey and Webb (1970) suggestthat the occurrence
showing irregular of tubules with
thickeninga virus particles
and elongated are evident in
of virus particles in tubules may be characteristic of certain NEPO viruses. Bean pod mottle virus, however, is beetle transmitted (Ross, 1963). The striking similarity of NEPO viruses and certain beetle transmitted viruses has been noted (Walters, 1969).
336
KIM
AND
The tubules observed with BPMV are structurally very similar to those which have been reported (Roberts and Harrison, 1970; Walkey and Webb, 1970; Davidson, 1969; DeZoeten and Gaard, 1969). In size they have a little greater diameter. An inner layer of material between the outer unit membrane and the line of virus particles is more distinct and may account for the larger diameter. The studies of Roberts and Harrison (1970) and also those of Walkey and Webb (1970) suggest that the tubules represent a transitory stage in the appearance of virus within infected cells. Roberts and Harrison (1970) observed at an early stage in the appearance of symptoms the presence of an inclusion body. Later, tubules with virus particles were noted in an outer zone of the inclusion body. They also noted a third stage where the tubules seemed to be disintegrating. Walkey and Webb (1970) found tubules with virus particles only in meristematic and embryonic tissue. They were not evident in leaf or stem tissue. In our observations the appearance of tubules with virus particles was observed shortly after the first evidence of symptoms in the local lesion host and tubules persisted without evidence of disintegration. In the systemically infected host tubules with virus particles were present in mesophyll of leaves showing symptoms. Young and fully mature leaf tissues were included in the observations. There was no evidence of disintegration of tubules at any stage in our observations. Invaginations in the plasmalemma containing tubules or vesicles have been noted as a feature of some normal cells (Thomson, 1967; Cronshaw and Bouck, 1965; Grun, 1963; Whaley et al., 1960). Tubules have also been described in the cytoplasm near the plasmalemma (Wooding and Northcote, 1964; Cronshaw and Bouck, 1965). We did not find evidence of an increase of tubules in the cytoplasm but the tubules occurring in invaginations of the plasmalemma were a prominent feature of infected cells. It has been suggested that such structures may function in the elaboration of the cell wall materials. Such an explanation is compatible with the cell wall protrusions observed in
FULTON
infected Cherokee Wax tissues. It would appear that virus infection causes an increase in the amount and activity of such masses of tubules. In comparing the sequence of events surrounding the appearance of virus particles in tubules in the two hosts, the initial evidences are similar. That is, there is an increase in the amount of tangled masses of tubules without particles and the associated appearance of virus particles in some tubules. We would suggest that since Pinto is a local lesion host and cells quickly become necrotic, there is no further development beyond this stage. With the systemic host, however, infected cells continue to function and the increased activity of the masses of tubules without particles results in an elaboration of cell wall materials and, subsequently, irregularities and protrusions of the cell wall. In the process of such cell wall elaboration, tubules with virus particles become embedded in the cell wall. Such pronounced cell wall elaboration is well demonstrated in Fig. 8, and it is apparent that the various shapes of pseudoisolated cell walls as those in Fig. 6a and b were the result of sectioning cell wall protrusions at various angles. Others have suggested (Walkey and Webb, 1970; Roberts and Harrison, 1970; Davidson, 1969; DeZoeten and Gaard, 1969) that tubules function in the movement of virus from cell to cell through the plasmodesmata. Lawson and Hearon (1970) reported that chrysanthemum aspermy virus could be observed within plasmodesmata without any evidence of t,ubules. Our observations indicate that although tubules with virus particles can be observed in plasmodesmata, this may not represent the mechanism of cell to cell movement of the virus. In all cases the tubules in the plasmodesmata abutted onto masses of tubules or onto cell wall protrusions rather than being in contact with cytoplasm. Roberts and Harrison (1970) suggest that the tubules function in virus replication. From our observations it could not be determined whether the tubules functioned in virus replication or simply represented a site of virus accumulation. Virus infection increases the number of masses of tubules
BPMV
IN
TUBULES
without particles and the associated thickenings and protrusions of the cell walls of the systemically infected host. The tubules, therefore, represent an active membrane system and it may be that some of this activity is associated with virus replication. It is interesting to note that although BPMV is a three-component virus (Bancroft, 1962) there is no evidence of empty shells, or top component, occurring within tubules. The full particles, either middle or bottom component, occur in a regularly spaced file in the tubules. REFERENCES BANCROFT, J. B. (1962). Purification and properties of bean pod mottle virus and associated centrifugal and electrophoretic components. Virology 16, 419427. CRONSHAW, J., and BOUCK, G. B. (1965). The fine structure of differentiating xylem elements. J. Cell Biol. 24,415-431. DAVIDSON, E. M. (1969). Cell to cell movement of tobacco ringspot virus. Virology 37, 694-696. DEZOETEN, G. A., and GAARD, G. (1969). Possibilities for interand intracellular translocation of some icosahedral plant viruses. J. Cell Biol. 40,814-823. GEROLA, F. M., LOMBARDO, G., and CATARA, A. (1969). Histological localization of citrus infectious variegation virus (CVV) in Phaseolus vulgaris. Protoplasma 67,319-326. GRUN, P. (1963). Ultrastructure of plant plasma and vacuolar membranes. J. Ultrastruct. Res. 9, 198-208.
IN
INFECTED
CELLS
337
LAXSON, R. H., and HEARON, S. (1970). Subcellular localization of chrysanthemum aspermy virus in tobacco and chrysanthemum leaf tissue. vi?-ozogy 41, 30-37. MATSUI, C., and YAMAGUCHI, A. (1966). Some aspects of plant viruses in situ. Advan. Virus Res. 12, 127-174. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-213. ROBERTS, I. M., and HARRISON, B. I>. (1970). Inclusion bodies and tubular structures in Chenopodium amaranticolor plants infected with strawberry latent ringspot virus. J. Gen. Virol. 7,47-54. Ross, J. P. (1963). Transmission of bean pod mottle virus in soybeans by beetles. Plant Dis. Reptr. 47, 1049-1050. THOMSON, W. N. (1967). Electron microscope studies on some modifications of the plasmalemma in oranges. J. Ultrastruct. Res. 17, 475486. WALKEY, D. G. A., and WEBB, M. J. W. (1970). Tubular inclusion bodies in plants infected with viruses of the NEPO type. J. Gen. Viral. 7, 159-166. WALTERS, H. J. (1969). Beetle transmission of plant viruses. Advan. Virus Res. 15, 339-363. WHALEY, W. G., MOLLENHAUER, H. H., and LEECH, J. II. (1960). The ultrastructure of the meristematic cell. Amer. J. Bot. 47, 401-419. WOODING, F. B. P., and NORTHCOTE, D. H. (1964). The development of the secondary wall of the xylem in Acer pseudoplatanus. J. Cell Biol. 23, 327-337.
VIROLOGY
Tubules
with
Viruslike
Particles Pod
Mottle
K. S. KIM Virology
and Biocontrol
AND
Cells
Infected
with
Bean
Virus’ J. P. FULTON
Laboratory, Department of Plant Pathology, Arkansas, Fayetteville, Arkansas YBYOl Accepted
’
in Leaf
University
of
October IS, 19YO
Ultrastructural studies of plant tissues infected with bean pod mottle virus (BPMV) revealed viruslike particles within tubules as a prominent feature of infection. Tubules with particles were found between the plasmalemma and the cell wall in a local lesion host, Pinto bean. In a systemic host, Cherokee Wax bean, the tubules with particles were embedded within the cell wall. Many irregularities and protrusions of the cell wall were noted in the systemically infected host.
INTRODUCTION This paper reports the occurrence of virusSmall, polyhedral plant viruses (25-30 nm like particles within tubules in tissues inin diameter) often evoke only limited cyto- fected with bean pod mottle virus (BPMV) logical changes in infected cells, and the (Cryptogram R/l 2.4/35 S/S S/Cl). Alvirus particles are difficult to locate in though the tubules are similar to those electron micrographs unlessthey have accu- reported above there is a striking difference mulated in large amounts (Matsui and in the location of the tubules in infected Yamaguchi, 1966). In some cases,however, cells. viruslike particles occur within tubules in MATERIALS AND METHODS infected cells. Roberts and Harrison (1970) BPMV (obtained from H. J. Walters) was described tubules in young leaf cells of Chenopodium quinoa infected with straw- used throughout this study. The virus was berry latent ringspot virus (SLRV). Similar inoculated to the primary leaves of young tubules were present in meristematic tissues plants of Phaseolusvulgaris L., cvs. “Cheroof various plants infected with cherry leaf kee Wax” and “Pinto.” Cherokee Wax was roll virus (CLRV) and tobacco ringspot virus systemically infected and Pinto developed (TRSV) as well as SLRV (Walkey and local lesions. Strips of inoculated tissue and systemically Webb, 1970). Davidson (1969) reported infected tissue were fixed at various times evidence of a file of TRSV particles conafter inoculation in 4% glutaraldehyde in tained within tubules which passedthrough the plasmodesmata and into the cytoplasm 0.1 M cacodylate buffer, pH 7.2, for 2 hr at of neighboring cells. DeZoeten and Gaard room temperature. After several washings (1969) made similar observations in tomato with buffer solution, the tissues were postringspot virus (TomRS) infected cells. fixed in 1% osmium tetroxide for 2 hr, then stained in bulk overnight at O-4” in 0.5 % Tubules with virus particles were evident only in phloem sieve tubes of plants infected uranyl acetate in distilled water. The tissues with citrus infectious variegation virus were dehydrated in an ethanol series, embedded in a mixture of Epon 812, 1 part (Gerola et al., 1969). Epon A:1 part Epon B, and sectioned with 1 Supported in part by Cooperative States glassknives on an LKB Ultrotome. Sections Research Services Grant No. 816-15-16. were double stained in 2 % uranyl acetate
329 Copyright
@ 1971 by Academic
PI-S
Inc.
Printed
in
U.S.A.
330
KIM
AND
FULTON
BPMV
IN
TUBULES
for 1 hr and lead citrate (Reynolds, 1963) for 10 min and examined with a Siemens Elmiskop IA. Noninoculated control tissues were fixed, embedded, and sectioned in the same manner. RESULTS
In the local lesion host, Pinto bean, viruslike particles were observed within tubules located between the cell wall and the plasmalemma (Fig. la, b, c). In some cases the tubules were in deep invaginations of the plasmalemma, but they were separated from the cytoplasm by the invaginated plasmalemma. Tubules without particles were also present in the same region between the cell wall and the plasmalemma (Figs. la and 2). They were usually in masses and gave the appearance of a tangled network of tubules or a group of vesicles. Tubules without particles were also a feature of normal, uninfected cells but occurred in much smaller numbers than in infected tissue. Tubules with particles were usually associated with masses of tubules without particles (Figs. la and 2). The tubules with particles were relatively straight whereas those without particles were bent and twisted. In the systemic host, Cherokee Wax, tubules with and without particles were also noted between the cell wall and the plasmalemma (Figs. 4 and 7). However, the most common location of tubules with particles was within the cell wall. The majority of these tubules did not appear to be associated with plasmodesmata but were embedded in the cell wall (Fig. 6a, b). Xost of the embedded tubules were aligned parallel with the plane of the cell wall. In Fig. 6a one may question whether the tubules are embedded in the cell wall or closely associated with the surface of the cell wall. In viewing many sections cutting the tubules at different angles, it is evident that the tubules are
IN
INFECTED
CELLS
331
within the cell wall rather than closely associated with it, Figure 8 shows cross sections of tubules embedded in the cell wall and Fig. 6b (CPI) shows the cross section of a cell wall protrusion containing an embedded tubule with virus particles. Irregular thickenings and protrusions (Fig. 8) of cell walls also occurred in infected Cherokee Wax bean. These protrusions invariably contained tubules with particles (Fig. 7). Protrusions sometimes penetrated deeply into invaginated cytoplasm (Fig. 8). In some of the sections the protrusions appeared as isolated pieces of cell wall material surrounded by plasmalemma and cytoplasm. If the protruded cell wall was sectioned through a longitudinal plane, the isolated cell wall fragment appeared as an elongated bar (Fig. 6a) while it appeared as a circular body in cross section (Fig. 6b). Tubules with virus particles were commonly observed embedded in such pseudoisolated cell walls (Fig. 6a,b). There were no noticeable cell wall protrusions or thickenings in infected Pinto bean leaf cells. Some tubules with particles were noted in plasmodesmata of both hosts (Figs. 3 and 5). In all cases, however, tubules in plasmodesmata did not extend into the cytoplasm but abutted onto an area of tubules without particles or were continuous with cell wall protrusions. The tubules in both hosts consisted of an outer unit membrane, an inner layer of material, and the line of particles (Fig. la, c). Tubules with virus particles were 60-70 nm in diameter. There was no evidence of an extra envelope as with the tubules reported by Roberts and Harrison (1970). Since the membrane of the tubule is typical unit membrane it seemslikely that the tubules represent fingerlike projections of cytoplasm bounded by the plasmalemma. It is assumed that tubules with particles originate in the
Abbreviations for figures: CW, cell wall; P, plastid; TwV, tubule with virus particles; !I’, tubule without virus particle; N, nucleus; M, mitochondria; PM plasmalemma; CPI, circular pseudoisolated cell wall; EPI, elongated pseudoisolated cell wall; PR, cell wall protrusion; CH, chloroplast. FIG. 1. BPMV-infected leaf cells of Pinto beans. (a) Tubules with virus particles between plasmalemma and cell wall. A portion of plasmalemma is invaginated deeply into cytoplasm. Invagination includes tubule with virus particles and tubules without particles. (b) Cross section of tubules between cell wall and plasmalemma. (c) Higher magnification of obliquely sectioned tubule with virus particles showing outer unit membrane, inner layers (arrows), and a file of virus particles.
332
FIG. without FIG.
KIM
2. BPMV-infected particles. Tubules 3. BPMV-infected
leaf cell of Pinto are between the leaf cells of Pinto
AND
FULTON
bean. Tubules with virus particles associated with tubules cell wall and plasmalemma. bean with tubule with virus particles in the plasmadesma.
BPMV
FIG. 4. BPMV-infected
IN
TUBULES
IN
INFECTED
333
CELLS
leaf cells of Cherokee Wax bean showing tangled Some tubules contain virus particles. FIG. 5. BPMV-infected leaf cell of Cherokee Wax bean. A tubule with virus
masses
of tubules
without
virus.
particles
in a plasmadesma.
and
an elongated
FIG. 6. BPMV-infected
plasm;
leaf cell of Cherokee Wax bean. (a) A tubule with virus particles embedded pseudoisolated cell wall wifh embedded tubules with virus particles.,jb)
in cell wall; a protrusion Circular, pseudoisolated
of the cell cell wall.
wall
into
cyto-
BPMV
FIG. 7. BPMV-infected tubule
with
virus
particles
IN
TUBULES
IN
INFECTED
335
CELLS
leaf cell of Cherokee Wax bean showing cell wall protrusion with embedded and also a tubule with virus particles between the cell wall and plasmalemma.
FIG. 8. BPMV-infected leaf cell of Cherokee Wax bean protrusions of the cell wall. Several cross or oblique sections the cell wall. manner or are derived from tubules without particles.
same
DISCUSSION
Roberts and Harrison (1970) and Walkey and Webb (1970) suggestthat the occurrence
showing irregular of tubules with
thickeninga virus particles
and elongated are evident in
of virus particles in tubules may be characteristic of certain NEPO viruses. Bean pod mottle virus, however, is beetle transmitted (Ross, 1963). The striking similarity of NEPO viruses and certain beetle transmitted viruses has been noted (Walters, 1969).
336
KIM
AND
The tubules observed with BPMV are structurally very similar to those which have been reported (Roberts and Harrison, 1970; Walkey and Webb, 1970; Davidson, 1969; DeZoeten and Gaard, 1969). In size they have a little greater diameter. An inner layer of material between the outer unit membrane and the line of virus particles is more distinct and may account for the larger diameter. The studies of Roberts and Harrison (1970) and also those of Walkey and Webb (1970) suggest that the tubules represent a transitory stage in the appearance of virus within infected cells. Roberts and Harrison (1970) observed at an early stage in the appearance of symptoms the presence of an inclusion body. Later, tubules with virus particles were noted in an outer zone of the inclusion body. They also noted a third stage where the tubules seemed to be disintegrating. Walkey and Webb (1970) found tubules with virus particles only in meristematic and embryonic tissue. They were not evident in leaf or stem tissue. In our observations the appearance of tubules with virus particles was observed shortly after the first evidence of symptoms in the local lesion host and tubules persisted without evidence of disintegration. In the systemically infected host tubules with virus particles were present in mesophyll of leaves showing symptoms. Young and fully mature leaf tissues were included in the observations. There was no evidence of disintegration of tubules at any stage in our observations. Invaginations in the plasmalemma containing tubules or vesicles have been noted as a feature of some normal cells (Thomson, 1967; Cronshaw and Bouck, 1965; Grun, 1963; Whaley et al., 1960). Tubules have also been described in the cytoplasm near the plasmalemma (Wooding and Northcote, 1964; Cronshaw and Bouck, 1965). We did not find evidence of an increase of tubules in the cytoplasm but the tubules occurring in invaginations of the plasmalemma were a prominent feature of infected cells. It has been suggested that such structures may function in the elaboration of the cell wall materials. Such an explanation is compatible with the cell wall protrusions observed in
FULTON
infected Cherokee Wax tissues. It would appear that virus infection causes an increase in the amount and activity of such masses of tubules. In comparing the sequence of events surrounding the appearance of virus particles in tubules in the two hosts, the initial evidences are similar. That is, there is an increase in the amount of tangled masses of tubules without particles and the associated appearance of virus particles in some tubules. We would suggest that since Pinto is a local lesion host and cells quickly become necrotic, there is no further development beyond this stage. With the systemic host, however, infected cells continue to function and the increased activity of the masses of tubules without particles results in an elaboration of cell wall materials and, subsequently, irregularities and protrusions of the cell wall. In the process of such cell wall elaboration, tubules with virus particles become embedded in the cell wall. Such pronounced cell wall elaboration is well demonstrated in Fig. 8, and it is apparent that the various shapes of pseudoisolated cell walls as those in Fig. 6a and b were the result of sectioning cell wall protrusions at various angles. Others have suggested (Walkey and Webb, 1970; Roberts and Harrison, 1970; Davidson, 1969; DeZoeten and Gaard, 1969) that tubules function in the movement of virus from cell to cell through the plasmodesmata. Lawson and Hearon (1970) reported that chrysanthemum aspermy virus could be observed within plasmodesmata without any evidence of t,ubules. Our observations indicate that although tubules with virus particles can be observed in plasmodesmata, this may not represent the mechanism of cell to cell movement of the virus. In all cases the tubules in the plasmodesmata abutted onto masses of tubules or onto cell wall protrusions rather than being in contact with cytoplasm. Roberts and Harrison (1970) suggest that the tubules function in virus replication. From our observations it could not be determined whether the tubules functioned in virus replication or simply represented a site of virus accumulation. Virus infection increases the number of masses of tubules
BPMV
IN
TUBULES
without particles and the associated thickenings and protrusions of the cell walls of the systemically infected host. The tubules, therefore, represent an active membrane system and it may be that some of this activity is associated with virus replication. It is interesting to note that although BPMV is a three-component virus (Bancroft, 1962) there is no evidence of empty shells, or top component, occurring within tubules. The full particles, either middle or bottom component, occur in a regularly spaced file in the tubules. REFERENCES BANCROFT, J. B. (1962). Purification and properties of bean pod mottle virus and associated centrifugal and electrophoretic components. Virology 16, 419427. CRONSHAW, J., and BOUCK, G. B. (1965). The fine structure of differentiating xylem elements. J. Cell Biol. 24,415-431. DAVIDSON, E. M. (1969). Cell to cell movement of tobacco ringspot virus. Virology 37, 694-696. DEZOETEN, G. A., and GAARD, G. (1969). Possibilities for interand intracellular translocation of some icosahedral plant viruses. J. Cell Biol. 40,814-823. GEROLA, F. M., LOMBARDO, G., and CATARA, A. (1969). Histological localization of citrus infectious variegation virus (CVV) in Phaseolus vulgaris. Protoplasma 67,319-326. GRUN, P. (1963). Ultrastructure of plant plasma and vacuolar membranes. J. Ultrastruct. Res. 9, 198-208.
IN
INFECTED
CELLS
337
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