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Ultrastructure of turnip crinkle- and saguaro cactus virus-infected tissues
VIROLOGY
118,
log-116
(1982)
Ultrastructure
of Turnip Crinkle- and Saguaro Cactus Virus-Infected Tissues
MARCELLO RUSSO AND GIOVANNI P. MARTELLI’ Istituto
di Pat&g&x
vegetde,
Univereitd de& Studi, Bari, It&
Received August 5, 1981; accepted Nuvemher
12, 1981
An electron microscope study of different hosts infected with turnip crinkle (TCV) and saguaro cactus (SCV) viruses, two tentative members of the tombusvirus group, was carried out. Particles of both viruses were readily detected in cells of different tissues, in the cytoplasm of which they occurred in great numbers, though not in crystalline arrays. Cytological modifications of various types were also observed. The most striking of these was an extensive peripheral vesiculation of mitochondria in TCV-infected cells, which was accompanied by plastic activity of the organelles that often engulfed portions of ground cytoplasm and virus particles. Cytoplasmic inclusion bodies were not present. Likewise, none of the cytopathological features characterizing tombusvirus infections was observed. No indications were obtained to support the idea that TCV and SCV may continue to be considered members, even though tentative, of the tombusvirus group. INTRODUCTION
The tombusvirus group comprises seven definitive and two possible members, turnip crinkle (TCV) and saguaro cactus (SCV) viruses, both of which have been tentatively assigned to the group because of their physicochemical properties. However, these viruses differ sufficiently from typical members in their biological properties to make their retention in the group questionable (Martelli, 1931). In infected cells, true tombusviruses elicit cytological alterations and formation of cytoplasmic inclusions (e.g., “multivesicular bodies”) which are regarded as group specific and of diagnostic value (Martelli et al, 1977; Martelli, 1981). The presence of such intracellular modifications in tissues of several hosts infected with Cymbidium ringspot virus (CyRSV), has propitiated the inclusion of this virus, previously regarded as a possible member (Matthews, 1979), among definitive tombusviruses (Martelli and Russo, 1981;Russo and Martelli, 1981). The ultrastructure of TCV- and SCV-in1To whom reprint requests should be addressed.
fected plant cells was investigated to establish if these viruses exhibit an intracellular behavior comparable to that of tombusviruses. The results of these observations are reported in the present paper. MATERIALS
AND METHODS
I+u.s source. The viruses used in this study were: a British isolate of TCV, obtained from Dr. R. Hull and the Chamucereus sylvestrii aureus strain of SCV, supplied by Dr. W. Welvaert. Both viruses were maintained in a glasshouse at 22-24’ in herbaceous hosts, to which they were periodically transferred by mechanical inoculation. Experimental hosts. The ultrastructure of TCV was investigated in systemically invaded Nicotiana benthamiana and BrasS&Xpekinensis and in local lesion on Chenopodium amaranticolor. Samples of N. benthamiana were taken from symptomsbearing leaves, 1,2, and 3 cm in diameter. SCV was studied in locally infected C&nopodium guinea plants. Electron microscopy. Tissue fragments were excised in a drop of 4% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.0, 109
0042-6822/82/050109-08$02.00/O Copyright All righta
0 1982 by Academic Press, Inc. of reproduction in any form resewed.
110
RUSSO AND MARTELLI
and vacuum infiltrated in the same buffered fixative for 2 hr at room temperature. After thorough washing in cacodylate buffer in the cold (4”), the samples were postfixed with 1% osmium tetroxide and stained overnight in 0.5% uranyl acetate in distilled water. Dehydration was in a graded ethanol series and embedding in Spurr’s medium. Thin sections were double stained with uranyl acetate and lead citrate and viewed with a Philips 201C electron microscope. Tissues from ‘healthy plants were similarly processed to serve as controls. RESULTS
Cytopathologg of TCV The majority of the cells of hosts systemically infected with TCV had a fairly well-preserved general architecture. Necrotic cellular elements were few and, except for mitochondria, most organelles exhibited minor or negligeable signs of alteration. Cell necrosis was more frequent in locally infected C amaranticolw tissues which, apart from this, showed the same kind of ultrastructural modifications as infected cells of other hosts. The cytopathology of N. benthamiana was the same, regardless of the age of infection. Figure 1 represents a typical view of infected mesophyll cells of. N. benthamianu. Chloroplasts were normally present although their outline was severely deformed by massive accumulation of starch. Fairly large lipid globules were interspersed with the plastids and the ground cytoplasm was packed with virus particles. The large numbers of intracellular virions was one of the most consistent characteristic of TCV infections and was common to all hosts examined. Virus particles were seen in epidermal and parenchyma cells as well as in difft:rrctiating and mature sieve tubes and tr:%,c&earyelements. Virions appeared as intensely electronopaque bodies, about 25 nm in diameter, with a smooth, rounded, or angular outline. They were scattered throughout the cytoplasm gathering in massive accumulations which, notwithstanding the tight packing, were never organized in crystal-
line arrays. Intravacuolar localization of virions was also observed, but it was not associated with the vesicular extrusions of the tonoplast that, with tombusviruses, appears to mediate transport of virus particles from the cytoplasm into the vacuole (Martelli et al, 1977). Mitochondria were the only organelles significantly affected by viral infection, displaying alterations in size and internal structure. Swollen mitochondria, as large as plastids, were sometimes encountered. More commonly, development of the major axis was observed, resulting in formation of elongated structures with a sinuous, dumbbell- or crescent-shaped appearance (Figs. 4, 5). Many of these long mitochondria were bent or deeply indented in a more or less median position (Figs. 4, 5), or extruded thin appendages that tended to fold back on the main body (Fig. 8), thus engulfing portions of ground cytoplasm. Changes of the internal structure consisted of modification and/or reduction of the cristae which were either replaced by one or more membranous rings (Figs. 2, 3) or became elongated and stacked (Fig. 8), sometimes in whorls with a myelin-like configuration. In some mitochondria, despite the depletion of the internal membranous components, an apparently normal stroma was present (Figs. 2, 3), whereas in others, especially in B. pekinensis cells, the reverse was true, the stroma being scanty and the cristae evident. The most striking alteration present in mitochondria of all hosts was the conspicuous peripheral vesiculation (Figs. 2 to 9). Single-membraned round to ovoid vesicles, measuring up to 200 nm in diameter, were consistently seen between the inner and outer membranes of the bounding envelope. Individual vesicles were either electron clear and apparently empty or contained finely granular material of low electron opacity or a network of tiny fibrils (Fig. 8, inset). These vesicles lined the whole outer contour of the organelles and appeared to develop as invaginations of the outermost membrane to which they were connected by a short neck opening to the outside (Fig. 6). Similar vesicles also
ULTRASTRUCTUREOFTCVANDSCV
FIG. 1. Section through two mesophyll cells of N. benthamtinu infected with TCV. The shape of chloroplasta (Ch) is altered because of the massive accumulation of starch; the cytoplasm contains large lipid globules (L) and is packed with virus particles. Bar = 500 nm.
111
112
RUSSO AND MARTELLI
FIGS. 2-6. Aspects of altered mitochondria. FIGS. 2 and 3. Two mitochondria from TCV-infected N. benthmiana and development of peripheral vesiculations.
showing depletion
of cristae
ULTRASTRUCTURE
lined the periphery of electron-clear enclaves containing virus particles, that were present in the center of many mitochondria, especially in C. amarant&&r cells (Figs. 7,8). The occurrence of these internal vesicles may be taken as evidence that the enclaves originated from the plastic activity of altered mitochondria that led to their elongation and folding or to the production of deep invaginations; hence to trapping of portions of cytoplasm with various particulates, including virions. Virus particles were never seen within mitochondria with an apparently intact outer envelope as in cells infected with some tombusviruses (Hatta and Francki, 1981; Martelli and Russo, 1981; Russo and Martelli, 1981). Vesicular structures similar to the perimitochondrial ones were sometimes seen free in the cytoplasm singly or in membrane-bounded groups (Fig. 9), this being the result of a possible sloughing process taking place in mitochondria in late stages of degeneration.
q&pathology of scv As illustrated in Fig. 10, SCV induced various types of alterations in infected cells. Except for nuclei, which were apparently unaffected, other organelles and the ground cytoplasm were modified to different extents. Chloroplasts were severely damaged, degenerative changes consisting of swelling and modification of the shape, which became globose, and of the thylakoids. Stroma lamellae were much reduced in number, had circular profiles, or ran in diverging directions. Grana were fewer than in cells of healthy plants and showed frequent dilations of the lamellae. The stroma was uniformly granular with a texture quite different from that of normal chloroplasts and contained numerous large plastoglobuli. Mitochondria seemed to retain their basic structure but displayed some modi-
OF TCV AND SCV
113
fications (i.e., decreased electron opacity, reduction and dilations of cristae) unlike the changes observed in TCV-infected cells. Many membranous vesicles were interspersed with the ground cytoplasm scattered at random or, on occasion, forming small aggregates. Lipid globules were unusually frequent, clustering together, especially in necrosing cells (Fig. 10, inset). Virus particles were also extremely abundant. Their outward aspect and size was similar to that of TCV virions and, like them, were disorderly scattered throughout the cytoplasm without forming crystals. DISCUSSION
The results of the present investigation demonstrate that neither TCV nor SCV evoke intracellular modifications comparable to those consistently present in tombusvirus infections. The only similarity lay in the abundance of intracellular virions which, however, in TCV- and SCVinfected cells were not found inside nuclei, as with most tombusviruses (Martelli et al, 19’7’7),nor were they associated with the vesicular extrusions of the tonoplast bulging into the central vacuole. Moreover the “multivesicular bodies,” so typical of all tombusvirus infections (Martelli, 1981; Martelli and Russo, 1981; Makkouk et al., 1981; Russo and Martelli, 1981) were totally absent and other cytoplasmic inclusions were not seen. No ultrastructural features of special interest were apparently associated with SCV infections. The intracellular modifications elicited by this virus were, in fact, of a kind common to a number of other isometric plant viruses. Conversely, in TCV-infected cells, remarkable alterations of mitochondria were observed which recalled previously recorded abnormalities of these organelles. Vesiculation of mitochondria has been observed in cells
FIGS. 4 and 5. Two elongated mitochondria from TCV-infected C. amaronk&r. The organelles are indented and bent and bear extensive peripheral vesiculations (arrows). FIG. 6. Detail of perimitochondrial vesicles showing their connection with the outer membrane of the mitochondrial envelope. The cytoplasm surrounding mitochondrion contains numerous viruus particles. All bars = 350 nm.
FIGS. 7-9. Aspects of altered mitochondria. FIG. ‘7. A group of coalescing mitochondria in a TCV-infected C. amaranticolor cell. Vesiculations are visible on outside (arrows) and internally, lining the electron-clear enclaves which contain virus particles. FIG. 8. A severely altered and extensively vesiculated mitochondrion (M) in a TCV-infected C’. ama~antacolor cell. The organelle has developed a long appendix which is folding back on the main body thus engulfing ground cytoplasm and virus particles (V). Arrows indicate the narrow gap where junction is taking place. Inset shows a fibril-containing perimitochondrial vesicle. FIG. 9. Detail of a mitochondrion (M) in a B. pekinensis cell showing peripheral vesicles. Similar vesicles are present in the cytoplasm (arrow heads), some being bounded by a membrane (arrow). All bars = 250 nm; bar of inset = 50 nm.
ULTRASTRUCTURE
OF TCV AND SCV
FIG. 10. Section through a C. quinou cell infected with SCV showing a severely damaged chloroplast (Ch), mitochondria (m), lipid globules (L), and many virus particles scattered at random. Inset shows a cluster of lipid globules in a necrosing cell. Bars = 250 nm.
115
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RUSSO AND MARTELLI
infected with two anisometric plant viruses: tobacco rattle (TRV) (Harrison et aL, 1970) and cucumber green mottle mosaic (CGMMV) (Hatta and Ushiyama, 1973). In both instances, rounded to ovoid vesicles similar to those found in TCV-infected cells were seen in perimitochondrial positions. The origin of these vesicles was not ascertained but they appeared to be produced internally to the mitochondrial envelope. As with TCV some of the perimitochondrial vesicles induced by CGMMV were continuous with the outermost mitochondrial membrane (Hatta et aL, 1971). Also, in all these cases, at least some of the vesicles contained a network of fine fibrils the nature of which was not experimentally established, although they were very reminiscent of nucleic acid strands. In this connection it is worth pointing out that vesiculated mitochondria of TRV take part in the formation of cytoplasmic inclusion bodies that were indicated as possible sites of viral RNA synthesis (Harrison et aL, 1970) and that the development of perimitochondrial vesicles in CGMMVinfected cells was concomitant or preceded the appearance of virus particles (Hatta and Ushiyama, 1973). According to Esau (1979), the occurrence of vesicles with fibrils in mitochondria of CGMMV-infected cells is “the best indication thus far that plant mitochondria may be involved in viral synthesis.” On this account, it is tempting to speculate that a similar situation may also occur with vesiculated mitochondria of TCV. Mitochondrial vesiculation has never been found associated with tombusvirus infections. This is therefore an additional point of differentiation that strengthens the conclusion that there are fewer reasons for assigning TCV and SCV to the tombusvirus group.
ACKNOWLEDGMENTS Grateful thanks are expressed to Dr. R. Hull, John Innes Institute, Norwich, United Kingdom, and Dr. W. Welvaert, Laboratory of Plant Virology, University of Gent, Belgium, for kindly supplying the viruses used in this study and to Mr. R. Lafortezza for the very valuable technical assistance. REFERENCES ESAU, K. (1979). Beet yellow stunt virus in cells of Sonchus deraceus L. and its relation to host mi-
tochondria. virdogl
98, 1-8.
HARRISON, B. D., STEFANAC,Z., and ROBERTS, I. M.
(1970). Role of mitochondria in the formation of X-bodies in cells of Nicottinu &?&on&~ infected by tobacco rattle virus. J. G~TL vird 6,127-140. HATTA, T., and USHIYAMA, R. (1973). Mitochondrial vesiculation associated with cucumber green mottle virus-infected plants. J. Gen. V&L 21, S-17. HATTA, T., and FRANCKI,R. I. B. (1981). Identification of small polyhedral virus particles in thin sections of plant cells by an enzyme cytochemical technique. J. Vltrastrud Bea 74.116-129. HATTA, T., NAKAMOTO, T., TAGAKI, Y., and USHIYAMA, R. (1971). Cytological abnormalities of mitochondria induced by infection with cucumber green mottle mosaic virus. virdogll45, 292-297. MAKKOUK, K. M., KOENIG, R., and LESEYANN, D. E. (1981). Characterization of a tombusvirus isolated from eggplant. Phgtupathdogy 71,572-577. MARTELLI, G. P. (1981). Tombusviruses. In “Handbook of Plant Virus Infections and Comparative Diagnosis” (E. Kurstak, ed.), pp. 61-90. Elsevier/ North-Holland, Amsterdam. MARTELLI,G. P., and RUSSO,M. (1981). The fine structure of Cymbidium ringspot virus in host tissues. I. Electron microscopy of systemic infections. J. lJZtra&uct
Res 77,93-104.
MARTELLI, G. P., Russo, M., and QUACQUARELLI,A. (1977). Tombusvirus (tomato bushy stunt virus) group. In “The Atlas of Inset and Plant Viruses” (K. Maramorosch, ed.), pp. 257-279. Academic Press, New York. MA~EWS, R. E. F. (1979). Classiilcation and nomenclature of viruses. IM* 12,133~2%. Ru880, M., and MARTELLI, G. P. (1981). The fine structure of Cymbidium ringspot virus in host tissues. II. Light and electron microscopy of localized infections. J. Ultroetra& Rea 77,105-118.
118,
log-116
(1982)
Ultrastructure
of Turnip Crinkle- and Saguaro Cactus Virus-Infected Tissues
MARCELLO RUSSO AND GIOVANNI P. MARTELLI’ Istituto
di Pat&g&x
vegetde,
Univereitd de& Studi, Bari, It&
Received August 5, 1981; accepted Nuvemher
12, 1981
An electron microscope study of different hosts infected with turnip crinkle (TCV) and saguaro cactus (SCV) viruses, two tentative members of the tombusvirus group, was carried out. Particles of both viruses were readily detected in cells of different tissues, in the cytoplasm of which they occurred in great numbers, though not in crystalline arrays. Cytological modifications of various types were also observed. The most striking of these was an extensive peripheral vesiculation of mitochondria in TCV-infected cells, which was accompanied by plastic activity of the organelles that often engulfed portions of ground cytoplasm and virus particles. Cytoplasmic inclusion bodies were not present. Likewise, none of the cytopathological features characterizing tombusvirus infections was observed. No indications were obtained to support the idea that TCV and SCV may continue to be considered members, even though tentative, of the tombusvirus group. INTRODUCTION
The tombusvirus group comprises seven definitive and two possible members, turnip crinkle (TCV) and saguaro cactus (SCV) viruses, both of which have been tentatively assigned to the group because of their physicochemical properties. However, these viruses differ sufficiently from typical members in their biological properties to make their retention in the group questionable (Martelli, 1931). In infected cells, true tombusviruses elicit cytological alterations and formation of cytoplasmic inclusions (e.g., “multivesicular bodies”) which are regarded as group specific and of diagnostic value (Martelli et al, 1977; Martelli, 1981). The presence of such intracellular modifications in tissues of several hosts infected with Cymbidium ringspot virus (CyRSV), has propitiated the inclusion of this virus, previously regarded as a possible member (Matthews, 1979), among definitive tombusviruses (Martelli and Russo, 1981;Russo and Martelli, 1981). The ultrastructure of TCV- and SCV-in1To whom reprint requests should be addressed.
fected plant cells was investigated to establish if these viruses exhibit an intracellular behavior comparable to that of tombusviruses. The results of these observations are reported in the present paper. MATERIALS
AND METHODS
I+u.s source. The viruses used in this study were: a British isolate of TCV, obtained from Dr. R. Hull and the Chamucereus sylvestrii aureus strain of SCV, supplied by Dr. W. Welvaert. Both viruses were maintained in a glasshouse at 22-24’ in herbaceous hosts, to which they were periodically transferred by mechanical inoculation. Experimental hosts. The ultrastructure of TCV was investigated in systemically invaded Nicotiana benthamiana and BrasS&Xpekinensis and in local lesion on Chenopodium amaranticolor. Samples of N. benthamiana were taken from symptomsbearing leaves, 1,2, and 3 cm in diameter. SCV was studied in locally infected C&nopodium guinea plants. Electron microscopy. Tissue fragments were excised in a drop of 4% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.0, 109
0042-6822/82/050109-08$02.00/O Copyright All righta
0 1982 by Academic Press, Inc. of reproduction in any form resewed.
110
RUSSO AND MARTELLI
and vacuum infiltrated in the same buffered fixative for 2 hr at room temperature. After thorough washing in cacodylate buffer in the cold (4”), the samples were postfixed with 1% osmium tetroxide and stained overnight in 0.5% uranyl acetate in distilled water. Dehydration was in a graded ethanol series and embedding in Spurr’s medium. Thin sections were double stained with uranyl acetate and lead citrate and viewed with a Philips 201C electron microscope. Tissues from ‘healthy plants were similarly processed to serve as controls. RESULTS
Cytopathologg of TCV The majority of the cells of hosts systemically infected with TCV had a fairly well-preserved general architecture. Necrotic cellular elements were few and, except for mitochondria, most organelles exhibited minor or negligeable signs of alteration. Cell necrosis was more frequent in locally infected C amaranticolw tissues which, apart from this, showed the same kind of ultrastructural modifications as infected cells of other hosts. The cytopathology of N. benthamiana was the same, regardless of the age of infection. Figure 1 represents a typical view of infected mesophyll cells of. N. benthamianu. Chloroplasts were normally present although their outline was severely deformed by massive accumulation of starch. Fairly large lipid globules were interspersed with the plastids and the ground cytoplasm was packed with virus particles. The large numbers of intracellular virions was one of the most consistent characteristic of TCV infections and was common to all hosts examined. Virus particles were seen in epidermal and parenchyma cells as well as in difft:rrctiating and mature sieve tubes and tr:%,c&earyelements. Virions appeared as intensely electronopaque bodies, about 25 nm in diameter, with a smooth, rounded, or angular outline. They were scattered throughout the cytoplasm gathering in massive accumulations which, notwithstanding the tight packing, were never organized in crystal-
line arrays. Intravacuolar localization of virions was also observed, but it was not associated with the vesicular extrusions of the tonoplast that, with tombusviruses, appears to mediate transport of virus particles from the cytoplasm into the vacuole (Martelli et al, 1977). Mitochondria were the only organelles significantly affected by viral infection, displaying alterations in size and internal structure. Swollen mitochondria, as large as plastids, were sometimes encountered. More commonly, development of the major axis was observed, resulting in formation of elongated structures with a sinuous, dumbbell- or crescent-shaped appearance (Figs. 4, 5). Many of these long mitochondria were bent or deeply indented in a more or less median position (Figs. 4, 5), or extruded thin appendages that tended to fold back on the main body (Fig. 8), thus engulfing portions of ground cytoplasm. Changes of the internal structure consisted of modification and/or reduction of the cristae which were either replaced by one or more membranous rings (Figs. 2, 3) or became elongated and stacked (Fig. 8), sometimes in whorls with a myelin-like configuration. In some mitochondria, despite the depletion of the internal membranous components, an apparently normal stroma was present (Figs. 2, 3), whereas in others, especially in B. pekinensis cells, the reverse was true, the stroma being scanty and the cristae evident. The most striking alteration present in mitochondria of all hosts was the conspicuous peripheral vesiculation (Figs. 2 to 9). Single-membraned round to ovoid vesicles, measuring up to 200 nm in diameter, were consistently seen between the inner and outer membranes of the bounding envelope. Individual vesicles were either electron clear and apparently empty or contained finely granular material of low electron opacity or a network of tiny fibrils (Fig. 8, inset). These vesicles lined the whole outer contour of the organelles and appeared to develop as invaginations of the outermost membrane to which they were connected by a short neck opening to the outside (Fig. 6). Similar vesicles also
ULTRASTRUCTUREOFTCVANDSCV
FIG. 1. Section through two mesophyll cells of N. benthamtinu infected with TCV. The shape of chloroplasta (Ch) is altered because of the massive accumulation of starch; the cytoplasm contains large lipid globules (L) and is packed with virus particles. Bar = 500 nm.
111
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RUSSO AND MARTELLI
FIGS. 2-6. Aspects of altered mitochondria. FIGS. 2 and 3. Two mitochondria from TCV-infected N. benthmiana and development of peripheral vesiculations.
showing depletion
of cristae
ULTRASTRUCTURE
lined the periphery of electron-clear enclaves containing virus particles, that were present in the center of many mitochondria, especially in C. amarant&&r cells (Figs. 7,8). The occurrence of these internal vesicles may be taken as evidence that the enclaves originated from the plastic activity of altered mitochondria that led to their elongation and folding or to the production of deep invaginations; hence to trapping of portions of cytoplasm with various particulates, including virions. Virus particles were never seen within mitochondria with an apparently intact outer envelope as in cells infected with some tombusviruses (Hatta and Francki, 1981; Martelli and Russo, 1981; Russo and Martelli, 1981). Vesicular structures similar to the perimitochondrial ones were sometimes seen free in the cytoplasm singly or in membrane-bounded groups (Fig. 9), this being the result of a possible sloughing process taking place in mitochondria in late stages of degeneration.
q&pathology of scv As illustrated in Fig. 10, SCV induced various types of alterations in infected cells. Except for nuclei, which were apparently unaffected, other organelles and the ground cytoplasm were modified to different extents. Chloroplasts were severely damaged, degenerative changes consisting of swelling and modification of the shape, which became globose, and of the thylakoids. Stroma lamellae were much reduced in number, had circular profiles, or ran in diverging directions. Grana were fewer than in cells of healthy plants and showed frequent dilations of the lamellae. The stroma was uniformly granular with a texture quite different from that of normal chloroplasts and contained numerous large plastoglobuli. Mitochondria seemed to retain their basic structure but displayed some modi-
OF TCV AND SCV
113
fications (i.e., decreased electron opacity, reduction and dilations of cristae) unlike the changes observed in TCV-infected cells. Many membranous vesicles were interspersed with the ground cytoplasm scattered at random or, on occasion, forming small aggregates. Lipid globules were unusually frequent, clustering together, especially in necrosing cells (Fig. 10, inset). Virus particles were also extremely abundant. Their outward aspect and size was similar to that of TCV virions and, like them, were disorderly scattered throughout the cytoplasm without forming crystals. DISCUSSION
The results of the present investigation demonstrate that neither TCV nor SCV evoke intracellular modifications comparable to those consistently present in tombusvirus infections. The only similarity lay in the abundance of intracellular virions which, however, in TCV- and SCVinfected cells were not found inside nuclei, as with most tombusviruses (Martelli et al, 19’7’7),nor were they associated with the vesicular extrusions of the tonoplast bulging into the central vacuole. Moreover the “multivesicular bodies,” so typical of all tombusvirus infections (Martelli, 1981; Martelli and Russo, 1981; Makkouk et al., 1981; Russo and Martelli, 1981) were totally absent and other cytoplasmic inclusions were not seen. No ultrastructural features of special interest were apparently associated with SCV infections. The intracellular modifications elicited by this virus were, in fact, of a kind common to a number of other isometric plant viruses. Conversely, in TCV-infected cells, remarkable alterations of mitochondria were observed which recalled previously recorded abnormalities of these organelles. Vesiculation of mitochondria has been observed in cells
FIGS. 4 and 5. Two elongated mitochondria from TCV-infected C. amaronk&r. The organelles are indented and bent and bear extensive peripheral vesiculations (arrows). FIG. 6. Detail of perimitochondrial vesicles showing their connection with the outer membrane of the mitochondrial envelope. The cytoplasm surrounding mitochondrion contains numerous viruus particles. All bars = 350 nm.
FIGS. 7-9. Aspects of altered mitochondria. FIG. ‘7. A group of coalescing mitochondria in a TCV-infected C. amaranticolor cell. Vesiculations are visible on outside (arrows) and internally, lining the electron-clear enclaves which contain virus particles. FIG. 8. A severely altered and extensively vesiculated mitochondrion (M) in a TCV-infected C’. ama~antacolor cell. The organelle has developed a long appendix which is folding back on the main body thus engulfing ground cytoplasm and virus particles (V). Arrows indicate the narrow gap where junction is taking place. Inset shows a fibril-containing perimitochondrial vesicle. FIG. 9. Detail of a mitochondrion (M) in a B. pekinensis cell showing peripheral vesicles. Similar vesicles are present in the cytoplasm (arrow heads), some being bounded by a membrane (arrow). All bars = 250 nm; bar of inset = 50 nm.
ULTRASTRUCTURE
OF TCV AND SCV
FIG. 10. Section through a C. quinou cell infected with SCV showing a severely damaged chloroplast (Ch), mitochondria (m), lipid globules (L), and many virus particles scattered at random. Inset shows a cluster of lipid globules in a necrosing cell. Bars = 250 nm.
115
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RUSSO AND MARTELLI
infected with two anisometric plant viruses: tobacco rattle (TRV) (Harrison et aL, 1970) and cucumber green mottle mosaic (CGMMV) (Hatta and Ushiyama, 1973). In both instances, rounded to ovoid vesicles similar to those found in TCV-infected cells were seen in perimitochondrial positions. The origin of these vesicles was not ascertained but they appeared to be produced internally to the mitochondrial envelope. As with TCV some of the perimitochondrial vesicles induced by CGMMV were continuous with the outermost mitochondrial membrane (Hatta et aL, 1971). Also, in all these cases, at least some of the vesicles contained a network of fine fibrils the nature of which was not experimentally established, although they were very reminiscent of nucleic acid strands. In this connection it is worth pointing out that vesiculated mitochondria of TRV take part in the formation of cytoplasmic inclusion bodies that were indicated as possible sites of viral RNA synthesis (Harrison et aL, 1970) and that the development of perimitochondrial vesicles in CGMMVinfected cells was concomitant or preceded the appearance of virus particles (Hatta and Ushiyama, 1973). According to Esau (1979), the occurrence of vesicles with fibrils in mitochondria of CGMMV-infected cells is “the best indication thus far that plant mitochondria may be involved in viral synthesis.” On this account, it is tempting to speculate that a similar situation may also occur with vesiculated mitochondria of TCV. Mitochondrial vesiculation has never been found associated with tombusvirus infections. This is therefore an additional point of differentiation that strengthens the conclusion that there are fewer reasons for assigning TCV and SCV to the tombusvirus group.
ACKNOWLEDGMENTS Grateful thanks are expressed to Dr. R. Hull, John Innes Institute, Norwich, United Kingdom, and Dr. W. Welvaert, Laboratory of Plant Virology, University of Gent, Belgium, for kindly supplying the viruses used in this study and to Mr. R. Lafortezza for the very valuable technical assistance. REFERENCES ESAU, K. (1979). Beet yellow stunt virus in cells of Sonchus deraceus L. and its relation to host mi-
tochondria. virdogl
98, 1-8.
HARRISON, B. D., STEFANAC,Z., and ROBERTS, I. M.
(1970). Role of mitochondria in the formation of X-bodies in cells of Nicottinu &?&on&~ infected by tobacco rattle virus. J. G~TL vird 6,127-140. HATTA, T., and USHIYAMA, R. (1973). Mitochondrial vesiculation associated with cucumber green mottle virus-infected plants. J. Gen. V&L 21, S-17. HATTA, T., and FRANCKI,R. I. B. (1981). Identification of small polyhedral virus particles in thin sections of plant cells by an enzyme cytochemical technique. J. Vltrastrud Bea 74.116-129. HATTA, T., NAKAMOTO, T., TAGAKI, Y., and USHIYAMA, R. (1971). Cytological abnormalities of mitochondria induced by infection with cucumber green mottle mosaic virus. virdogll45, 292-297. MAKKOUK, K. M., KOENIG, R., and LESEYANN, D. E. (1981). Characterization of a tombusvirus isolated from eggplant. Phgtupathdogy 71,572-577. MARTELLI, G. P. (1981). Tombusviruses. In “Handbook of Plant Virus Infections and Comparative Diagnosis” (E. Kurstak, ed.), pp. 61-90. Elsevier/ North-Holland, Amsterdam. MARTELLI,G. P., and RUSSO,M. (1981). The fine structure of Cymbidium ringspot virus in host tissues. I. Electron microscopy of systemic infections. J. lJZtra&uct
Res 77,93-104.
MARTELLI, G. P., Russo, M., and QUACQUARELLI,A. (1977). Tombusvirus (tomato bushy stunt virus) group. In “The Atlas of Inset and Plant Viruses” (K. Maramorosch, ed.), pp. 257-279. Academic Press, New York. MA~EWS, R. E. F. (1979). Classiilcation and nomenclature of viruses. IM* 12,133~2%. Ru880, M., and MARTELLI, G. P. (1981). The fine structure of Cymbidium ringspot virus in host tissues. II. Light and electron microscopy of localized infections. J. Ultroetra& Rea 77,105-118.