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Distribution of viruslike particles in leaf cells of Dianthus barbatus infected with carnation vein mottle virus
VIROLOGY 40, 868-881 (1970)
Distribution of Viruslike Particles in Leaf Cells of Dianthus barbatus Infected with Carnation Vein Mottle Virus M . WEINTRAUB
H . W . J . RAGETLI
ANn
Research Station, Canada Department of Agriculture 6660 . .147 ITarine Drive, Vancouver, Canada N Accepted December 1, 1969
Leaf exudates and partially purified preparations of Dianthus barbatus infected with carnation vein mottle virus showed the presence of relatively few elongated flexuous rods, one-half of which were between 675 and 850 tire long, and one-third between 750 and 825 Mo . Electron microscopic examination of ultrathin sections of infected leaf cells showed that nearly all viruslike particles were present in a single thin layer enclosed by two membranes, lying along the surface of the cytoplasm just inside the tonoplast_ Sometimes this peripheral area of the cytoplasm appeared to have been torn away or separated from the main body of cytoplasm, so that strands or bridges of virus particles extended into the central vacuole . The maximum length of particles in sections was about 750 TIM . Many cytoplasmic inclusions were observed, consisting of dense bands, loops, and circles . However, the occurrence of these inclusions does not necessarily permit the conclusion that carnation vein mottle virus is a member of the potato virus Y group . Coll organelles appeared normal, except for the nuclei, which were elongated, and displayed an intense electron density of the chromatin . INTRODUCTION
Carnation vein mottle virus (CVMV) in Dianthus barbatus L ., and in D. caryophyllus seedlings, was described by Kassanis (1955), who attempted to purify the virus, but with only partial success . He found long filamentous particles in some preparations, presumably similar to those lie observed in sap from infected leaves, but not consistently enough to identify these particles with the virus . CVMV is aphid transmissible (Hakkaart, 1964 ; Kassanis, 1955), and mechanically transmissible not only to Dianthus species but also, according to Hollings (1956, 1959), to C,henopodiur amaranticolor . In the latter host the virus is said (Hollings, 1956) to produce diagnostically characteristic lesions which separate it from other virus infections . CVl-1V can also be distinguished by serology from other carnation viruses for which autisera can be made . We also have attempted to purify CVMV, and although filamentous particles were
found in preparations from infected leaves only, their number was unsatisfactorily small . However, since one-half of the particles measured were between 675 and 850 nm long, and one-third between 750 and 825 nm (Fig . 2), there appeared to be another way of determining whether these long filamentous particles were actually the virus causing carnation vein mottle . A series of studies has shown that viruses of about this length induce characteristic inclusions in the cytoplasm, and sometimes in the nuclei of infected leaf cells. One group of cytoplasmic inclusions consists of a variety of forms that have been named dense bands (Bos and Rubio-Huertos, 1969 ; Christie et al., 1968 ; Kamei et al ., 1969 ; Lee, 1965 ; Matsui and Yamaguchi, 1964a ; Weintraub and Ragetli, 1966), cylindrical inclusions, bundles, laminated aggregates, fibrous masses, plates, oblong inclusions, pinwheels, starlike inclusions, circles, and loops (Bos and RubioHuertos, 1969 ; Christie et al ., 1968 ; Cremer 868
DISTRIBUTION OF CVMV IN D . BARBATUS LEAF CELLS and van der Veken, 1964 ; Edwardson, 1966a, b ; Edwardson et al ., 1968 ; Goethals el al ., 1969 ; Hoefert, 1969 ; Kamei et al ., 1969 ; Kim and Milton, 1969 ; Krass and Ford, 1969 ; Lee, 1965 ; Purcifull and Edwardson, 1967 ; Rubio-Huertos and Lopez-Abella, 1966 ; Shepard and Carroll, 1967 ; Stein-Margolina et al., 1969 ; Yamaguchi et al ., 1963) . Some of these terms seem to be derived from the two-dimensional appearance of a threedimensional structure sectioned in various planes . This structure yields pinwheels, circles, or loops in cross section, and bundles or tubes of various widths in longitudinal or tangential section . A three-dimensional reconstruction was first proposed by RubioHuertos and Lopez-Abella (1966) based on serial sections of virus-infected pepper cells . This was later called a "cylindrical inclusion" by Edwardson (1966b) . It is also possible that "dense bands" are the same structures as "laminated aggregates ." The cytoplasmic inclusions described as "dense bands" in bean yellow mosaic infection, arid as "pinwheel and bundle inclusions" with beet mosaic infection have been shown, by digestion with pepsin and trypsin, to be composed of protein and, as far as could be detected, devoid of nucleic acids (Hoefert, 1969 ; Weintraub and Ragetli, 1968 ; Weintraub el al ., 1969) . Another group of cytoplasmic inclusions, usually called "comblike," consist of lilamentous particles arranged arid attached perpendicularly to the tonoplast on one side and often to endoplasmic reticulum on the other side . These have been observed in cells infected with lettuce mosaic virus (Goethals el al ., 1969), a virus of Amaranthus lividus (Rubio-Huertos and Vela Corncjo, 1966), turnip mosaic virus (Kamei et al ., 1969), and papaya ringspot virus (Herold and Weibel, 1962) . A third group of inclusions, which are crystals, have been found in the cytoplasm and nuclei of cells infected with tobacco etch virus (Matsui arid Yamaguchi, 1964b ; Rubio-Huertos and Hidalgo, 1964), bean yellow mosaic virus (Weintraub and Ragetli, 1966), and pea necrosis virus (Bos and Rubio-Huertos, 1969) . The composition of the crystals of bean yellow mosaic virus has been found to be similar to that of the dense
869
bands, although some differences were detected in the composition of the crystalline protein (Weintraub and Ragetli, 1968 ; Weintraub et al ., 1969) . It seemed reasonable, therefore, to expect that, with such a variety of inclusions characteristically and consistently induced by certain elongated viruses, an electron microscopic examination of leaf cells infected with CVMV might answer the question whether the filamentous particles observed in crude sap and in purified preparations could be identified with the causal agent of the disease . MATERIALS AND METHODS
Puriication and electron microscopy of CVY1V . Exudates from CVBIV-infected and healthy D . barbatus leaves were prepared for electron microscopic examination by touching the cut leaf surface, including the central vein, to a drop of water on the specimen grid . This was allowed to dry, and was then shadowed with palladium . Attempts to purify CVMV by differential centrifugation in phosphate buffer, or in buffers containing sodium bisulfite, or sodium diethyldithiocarbamate, yielded preparations that were not infectious when tested on D . barbatus and on Chenopodium quinoa (Hollings and Stone, 1965), nor could particles, other than those found in healthy control preparations, be seen with the electron microscope . Unlike Hollings (1956, 1959), we were not able to transmit CVMV mechanically to C . anzarcudicoloi, arid so could not use it as an additional test plant . Hakkaart (1964) also found it difficult to transmit CVMV to C . amaranticolor . Preparations that appeared to be partially purified, were infectious, and contained some particles not found in healthy leaves were finally produced by the butanol-chloroform method of Steere (1956) . The final preparation was concentrated about 15-fold on a weight basis and was tested for infectivity on 1) . barbatus and C . quinoa, as well as being examined in a Philips EMI . 100, or E .M . 200, after the specimens were shadowed with palladium . A portion of this preparation was further centrifuged at high speed ; the pellet was dissolved in one-tenth the original volume,
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WEINTRAUB AND RAGETLI
representing a 150-fold concentration. This preparation was also tested for infectivity and examined with the electron microscope . The concentrated preparation was then layered on a 5-30 % sucrose density gradient and centrifuged for 1 hour at 35,000 rpm in a Spinco SW 39 rotor. A narrow opalescent band about two-thirds down the gradient was syringed off . This band, and material from the column above and below it, were tested for infectivity and examined kithh the electron microscope. Electron microscopy of ultrathin sections. Leaves of D . barbatus, on plants inoculated 11 days previously with CVMV, were sampled from green areas in mature leaves, and from somewhat chlorotic areas in younger leaves . Since both areas produced similar results, no further distinction will be made . Pieces 2-3 mm' were cut and fixed in 5 % glutaraldehyde in phosphate buffer, pH 7.2 at room temperature for 1-2 hours . They were rinsed with distilled water, once before,
and 3-4 times after post-fixation for 1 hour with 1 % Palace's OsO, solution, pH 7 .2 . After dehydration in graded dilution of ethanol, they were treated with propylene oxide, and embedded in Epon 812 (Luft, 1961) . Thin sections were cut with glass knives and stained with uranyl acetate and lead citrate . RESULTS Purification and Electron Microscopy of CVMV
The CVMV-infected leaf exudates contained the usual debris, but very few elongated flexous particles (Fig . la) . Those that could be measured were 500-800 rim long . The preparations from the butanolchloroform procedure, at both concentrations, contained considerably less debris, and more elongated particles (Fig. 1b) . The particles were still sparse, so that many photographs were needed to accumulate the dimensions of 174 particles . Measurements
The scale on each figure indicates 1 µ unless otherwise noted . FIG . 1 . (a) Exudate from cut leaf of Dianthus barbatus infected with CVMV . (b) Sample of butanol-
chloroform-purified CVMV . Both shadowed with palladium .
DISTRIBUTION OF CVMV IN D . BARBATUS LEAF CELLS
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20 19 18 17161514-
1 I-
a 0.
13121110 .
^r 0
9-
=
8-
Is 7-
e
6-
350 450 550 650
750 850 950 1050 1150 1250 1350 1450 1550 1650 1750 1850 1950 2050 2150 2250 2350 2450
LENGTH
mp
Fla . 2 . Size distribution of particles seen in purified CVMV . were made from 35-mm negatives with a Gaertner tool-maker's microscope and were grouped into categories of 25 nm . The distribution of lengths of these particles is shown in Fig . 2, where it is evident that about 45-50% of the particles fall into the categories of 675-850 not . There is a smaller peak at about 1500 ma, and another even smaller at 2300-2400 nm ; these probably represent dimers and trimers . It is equally evident that although the main peak occurs at about 750-825 nm, most of the particles fall into various other categories both longer and shorter . The width of the particles was 18-19 too . Both the 15-fold and 150-fold concentrated preparations were infectious in D . barbatus, and also in C. quinoa in which they produced rather vague, small blotchy lesions .
After the butanolchloroform preparations were fractionated on the sucrose density gradients, no infectivity and no particles such as those in Fig . 1 were detected either in the narrow opalescent band or in the fractions above and below this band .
Electron Microscopy of Ultrathin Sections In D . barbatus, inclusions were found readily in sections of the cytoplasm. They consisted mainly of the dense band type (Figs . 3 and 4), although they were neither so electron dense, nor so well-formed or long as those produced by bean yellow mosaic virus (BYMV) in Vicia faba (Weintraub and Ragetli, 1966) . The striated structure readily seen in BYMV dense bands was not found in the CVRMV bands . Most of the inclusions were straight, and only occasionally showed
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the oblique filaments (Fig . 3, arrow) that were often seen to come off such bands with other viruses (e.g ., Fig . 1, Weintraub and Ragetli, 1968) . No pinwheels were seen in any of the cells, but loops and circles were frequent, either singly or in double parallel arrangements (Fig. 4) . Only occasionally was an inclusion seen that might have represented a very loose pinwheel arrangement (Fig . 5) . Single viruslike particles, or particles in small aggregates randomly oriented in the cytoplasm were seen only rarely . In this position, they usually appeared in the cytoplasm not very distant from the central vacuole (Fig. 6) . Figure 6 also shows the disposition and location of most of the elongated particles that we believe to be CV\1V, as a narrow layer or sheet along the surface of the cytoplasm and next to the tonoplast (Fig . 6, arrow) . The particles lie parallel to each other (Figs . 7-9), and also parallel to the surface of the cytoplasm (Fig . 9) . They are enclosed by membranes toward the cytoplast and toward the tonoplast, the latter membrane being quite distinct from the tonoplast . The viruslike particles were sometimes seen as narrow bridges or strands over a vacuolar region . These bridges or strands containing spherical particles are interpreted as representing a thin cross section of a sheet of particles and its enclosing membranes . In Fig . 7, the particles are seen in cross section at the left end of such a bridge, and in oblique section at the right end (Fig . 7, arrow) . Figure 8 also shows a bridge over a small vacuole next to the cytoplasm, the bridge protruding into the larger central vacuole . It is clear in Fig . 8 that there is no membrane lining the cytoplasm next to the smaller vacuole, a condition frequently seen near the bridges . However, most of the viruslike particles were not found in these bridges or strands, but lay closely apposed to the main part of the cytoplasm and the tonoplast, adjacent to the central vacuole (Fig . 9) . The appearance of the particles varied with the angle of sectioning . Figs . 7-9 show the circular aspect produced by a cross section of the particles, while the short rods seen at the right end of the bridge (Fig . 7, arrow), and in parts of
Fig . 8 are the result of angles of section not far from cross sections . The apparent length of the particles increases with the angle of sectioning (Fig . 10) . Thus, when a layer of particles is sectioned parallel to the length of the rods and in between their enclosing membranes, only the rods are seen (Fig . 11) . In sections of this type, a few of the longest particles were measured and were about 750 nor . The width of the electron dense portions of these particles was 8-9 rim, so that if they are virus particles, this presumably represents the nucleic acid moiety, and the electron transparent portion is the protein . Parallel aggregates or sheets of particles were sometimes sectioned at oblique angles near one end of the aggregate, so that only one of the membranes was seen (Fig . 12, arrow) . The tonoplast appeared as a separate membrane, loosened from the cytoplasm and the sheet of particles, and protruding into the central vacuole . Cell organelles, such as chloroplasts and mitochondria, appeared relatively normal . However, the nuclei were often extremely elongated, with the chromatin portions considerably more electron dense than in healthy leaf cells (Fig. 13) . When examining nuclei of D. barbatus leaf cells it was essential not to mistake the normal crystals for those of virus origin . Longitudinal sections of normal crystals (Fig. 14), being composed of electron dense material 10-15 rim wide and 0 .5 2 µ long, somewhat resemble aggregates of viruslike particles in CVMV-infected cells . But normal crystals are present in many seedlings of D . ba •b atus and in D . chinensis which are, as far as can be determined, virus free and healthy (Weintraub et al ., 1968) . Cross sections of such crystals, in which the components consist of electron dense material 10-15 rim wide and electron transparent centers 6-8 nor in diameter, arc totally different (Fig . 15) from cross sections of the viruslike particles associated with CVMV infection.
DISCUSSION Although the purification of CVMV was not altogether satisfactory and resulted in relatively few particles for a proper study of the length distribution of the flexous rods, sufficient data were collected on particle size
Fio . 3 . Portion of mesophyll cell of Dianthus barbatus iniceted with CVMV . Note the dense bands (DI3), singly and in bundles . Arrow points to a band with filaments that come off at an angle .
lha . 4 . As in Fig . 3, showing inclusions in the form of loops (L) and circles . Fm, . .5, As in Fig . 3, showing inclusions that may represent a loose pinwheel arrangement (arrow) . 873
Fin . B . Portion of cell showing viruslike particles (V) in cytoplasm near cell vacuole (CV) . Arrow points to the more usual disposition of particles as a sheet at the periphery of the cytoplasm . Fm, 7 . Sheet of viruslike particles, in cross section at the left end (V), and in oblique section at the right end (arrow) . Note the membrane above and below the particles . 874
Fin . S . Cytoplasm devoid of tonoplast (arrow), indicating that the strand containing the viruslike particles (V) has torn away from the main body of cytoplasm, and extends into the cell vacuole (CV) . Fin . 9 . Viruslike particles (V) apposed closely to the cytoplasm near a nucleus (N), and lying parallel to each other and to the surface of the cytoplasm . 9,5
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FIG . 10. Viruslike Particles (V) sectioned at various angles of ohliqueness . Fie. . 11 . Viruslike particles near cell vacuole (CV) sectioned parallel to the length of the particles and their enclosing membranes .
DISTRIBUTION OF CVMV IN D . BARBATUS LEAF CELLS
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FIG . 13 . Viruslike particles sectioned at an oblique angle, so that only one of the two enclosing membranes is visible (arrow) . Note the loosened tonoplast (T) protruding into the central vacuole (CV) . Fic . 13 . Portion of CVAIV-infected leaf cell, showing extreme elongation of the nucleus (N), and the abnormal electron density of the chromatin portions .
Fic . 14 . Portion of nucleus (N) in healthy leaf cell of Dianthus bathatus showing a longitudinal section of a nonviral crystalline inclusion impinging on the nucleolus (Ns) . Fic . 15 . Portion of nucleus (N) in healthy leaf cell of Dianthus barbatus showing a cross section of a nonviral crystalline inclusionn near the nucleolus (Ne) . 878
DISTRIBUTION OF CVMV IN D . BARBATUS LEAF CELLS
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to predict with some confidence that dense band inclusions would be found in the CVMV-infected leaf cells . This does not mean, however, as proposed by Edwardson (1966a), that the presence of these inclusions necessarily permits the diagnostic identification of CVJ-IV as a member of the potato virus Y (PVY) group of viruses, with lengths of 730-750 nm (Brandes and Berclcs, 1965) . With CVMV, the major peak of particles measured (Fig . 2) was in the 750-825 nm range . Furthermore, cytoplasmic inclusions of similar type have been found in cells infected with wheat streak mosaic virus (Lee, 1965 ; Shepard and Carroll, 1967 ; Stein-Margolina et al ., 1969), and in cells of virus-infected Amarant/aus lividus (RubioHu(rtos and Vela-Cornejo, 1966) . Both of these viruses are 650 nm long . Similar inclusions have also been found in cells infected with narcissus yellow stripe (Cremer and van der Veken, 1964) and papaya ringspot virus (Herold and Weibel, 1962), both of which viruses are 800 nm long . Recently, Bos and Rubio-Huortos (1969) described inclusions of pea necrotic virus, some of which resembled inclusions produced by the PVY group whereas others resembled inclusions associated with the PVX group, including potato virus X itself, clover yellow mosaic virus, and papaya mosaic virus . Unless one postulates a considerable error in published measurements of these virus particles, it would appear that the induction of dense bands, cylindrical inclusions, pinwheels, etc ., is not a simple function of an arbitrary 730-750 nm length of the virus particle . At this time, the evidence suggests that such peculiar inclusions are associated with elongated viruses that are not restricted to the PVY group, but range in length from at least 650 to 800 nm . If the rod-shaped particles seen in the CVMV-infected cells are CVM virions, their arrangement in a narrow layer or sheet along the surface of the cytoplasm, enclosed by two membranes, is unusual and differs greatly from the "comb-like" arrangement described for certain viruses (Goethals et al ., 1969 ;
izing the particles in leaf exudates and in purifying the virus . Even with a considerable cytoplasmic surface over which the particles are arranged, there must be distinctly fewer than in systemic infections such as those produced by tobacco mosaic virus, potato virus X, etc ., where massive aggregates of particles are seen throughout the cytoplasm. Another difficulty in purification may be the necessity to break the membranes that enclose the viruslike particles to free them, and this would be considerably more difficult than simply freeing unenclosed particles by disrupting the infected cells . The bridgelike strands containing viruslike particles (Figs . 7 and 8) have been observed with pokeweed mosaic (Kim and Fulton, 1969), and with beet mosaic virus (Hoefert, 1969) . Both were interpreted as being the result of an enlargement of vesicles which subsequently protruded into the central vacuole. When sectioned these appear as narrow cytoplasmic strands containing viruslike particles . In CVMV-infected cells, the particles were observed most frequently at the periphery of the cytoplasm (Fig . 9), and it appeared to us that the bridges represent a tearing away of the outer layer of cytoplasm, because of the unusually heavy mass of viruslike particles concentrated along the surface . This is substantiated by the frequency with which we observed (Fig . 8, arrow) that the cytoplasm below the bridge was totally devoid of a cytoplasmic membrane, suggesting that a tearing took place, rather than an orderly development of an enlarged vesicle in the cytoplasm. The unusual arrangement of the viruslike particles makes it difficult to understand, if these are CVM virions, where or how the virus is synthesized and assembled . It would be odd, indeed, if the components were synthesized either in the nucleus or in the main
Herold and Weibel, 1962 ; Kamei et at ., 1969 ; Rubio-Huertos and Vela-Cornejo, 1966) . This physical arrangement may account for some of the difficulties encountered in visual-
ACKNOWLEDGMENTS
body of cytoplasm, and then migrated to the surface of the cytoplasm to be assembled into their final form in this very regular, parallel arrangement .
We are grateful to Miss Esther Lo and Miss Bea Schroeder for technical assistance, and to Mr . J . H . Severson for preparing the figures .
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REFERENCES Bes t L ., and RDDIO-HTERTOS, M. (1969) . Light
and electron microscopy of cytoplasmic and unusual nuclear inclusion bodies evoked by a virus from necrotic peas . Virology 37, 377-385 . BRANDES, J ., and BERCES, R . (1965) . Gross morphology and serology as a basis for classification of elongated plant viruses . In "Advances in Virus Research" (K. M . Smith and M . A . Lalfler, eds .), Vol . 11, pp . 1-24 . Academic Press, New York . CHRISTIE, S . R ., EDWARDSON, J . It ., and ZETTLER, F . W . (1968)- Characterization and electron microscopy of a virus isolated from Bidens and Lepidium . Plant Disease Retr . 52, 763-768 . CREMER, M . C ., and VAN DER VEEEN, J . A . (1964) . Localization of viral antigen in narcissus leaves infected with yellow stripe virus, determined by means of a fluorescein conjugated antiserum . N eth . J . Plant Pathol . 70, 105-113 . EDW ARDSON, J . R . (1966a) . Electron microscopy of cytoplasmic inclusions in cells infected with rodshaped viruses . Arn . J . Botany 53, 359-364 . EDWARDSON, J. R. (1966b) . Cylindrical inclusions in the cytoplasm of leaf cells infected with tobacco etch virus. Science 153, 883-884 . EDWARDSON, J. R ., PURCIFULL, D . E ., and CHRISTIE, R . G . (1968) . Structure of cytoplasmic inclusions in plants infected with rod-shaped viruses . Virology 34,250-203 . GOETHALS, M ., HORVATH, F ., and MEYER, J . A . (1969) . Cytoplasmic inclusions fit leaf cells infected with lettuce mosaic virus . Virology 37, 685-687 . HAEEAART, F . A. (1964) . Description of symptoms and assessment of loss caused by some vi rusesin the carnation cultivar" William Sim ." Neth . J . Plant Pathol . 70, 53-60 . HEROLD, F ., and WEIBEL, J . (1962) . Electron microscopic demonstration of papaya ringspot virus . Virology 18,302-311 . HOEFERT, L . L . (1969) . Proteinaceous and viruslike inclusions in cells infected with beet mosaic virus. Virology 3 7 , 498-5(11 . HOLLINGS. M- (1956) . Chenopodium anmaranticolor as a test plant for plant viruses . Plant Pathol . 5, 57-60 . HOLLINOS, M . (1950) . Host, range studies with fifty-two plant viruses . Ann . Appl . Biel. 47, 98108 . HOIJSNCS, M ., and STONE, 0 . M . (1965) . Chenopodium guinoa Willd . as an indicator plant, for carnation latent virus . Plant Pathol . 14, 66-OS . KAMEi, T ., hIoNDA, Y ., and MATSUI, C . (1969) . Intracellular appearance of turnip mosaic and bean yellow mosaic virus particles . Phytopathology 59,1 .30-14-4 .
KASSANIS, B . (1955) . Some properties of four viruses isolated from carnation plants . Ann . Appl . Biol. 43, 103-113 . KIM, K . S ., and FLLPON, J . P . (1969) . Electron microscopy of pokeweed leaf colts infected with pokeweed mosaic virus . I irology 37 , 297-308 . KR .ASS, C . J ., and FORD, R . E . (1969) . Ultrastructure of corn systemically infected with maize dwarf mosaic virus . Phytopathology 59, 431-439 . LEE, P . F . (1965) . Electron microscopy of inclusions associated with wheat streak mosaic virus . J. Ultrastruct . Res . 13, 359-366 . LOFT, J . H . (1961) . Improvements in epoxy resin embedding methods. J . Biophys . Biochem . Cytol . 9,409-414 . MATSUI, C ., and YAMAGUCHI, A . (1964a) . Electron microscopy of host cells infected with tobacco etch virus, I . Fine structures of leaf cells at later stages of infection . Virology 22, 40-47 . MATsun, C ., and YAMAGUCHI, A . (1964b) . Electron microscopy of host cells infected with tobacco etch virus . II . Fine structures of leaf cells before and after the appearance of external symptoms . Virology 23, 346-353, PL-RCIFULL, D . F ., and EDWARDSON, J . R . (1967) . Watermelon mosaic virus : Tubular inclusions in pumpkin leaves and aggregates in leaf extracts . Virology 32, 393-401 . R.UBio-HUERTOS, M ., and G .ARCIA HIDALGO, F . (1964) . Ultrathin sections of intranuclear and intracytoplasmic inclusions induced by severe etch virus. Virology 24,84-90 . RUBIO-HUER'rOS, M ., and LOPEZ-ADELLA, D . (1966) . Ultraestructura de celulas de pimiento infectadas con un virus y su localization en las mismas . Microbial . Espan . 19, 77-86 . RURIO-HUERTOS, M ., and VELA-CORNEJO, A . (1966) . Light and electron microscopy of virus inclusions in Amaranthus livirdus cells . Protoplasma 62, 184-193 . SHEPARD, J . F ., and CARRoLl„ T . W . (1967) . Electron microscopy of wheat streak mosaic virus particles in infected plant cells- J . Ultrastruct. Res . 21,145-152 . STEERE, R . L . (1956) . Purification and properties of tobacco ringspot virus . Phytopathology 46, 60-60 . STEIN-MARGOLINA, V . A ., CHERNY, N . E ., and RAZYV%ZKINA, G. 1M. (1969) . Phytopathogenic viruses in plant cells and in mite-vector (electron microscopic investigation) . freest . Akad . Nazdc S .S .R . Set . Biol . 1, 62-68. WEINTRAUB, M., and RAGETLI, H . W . J . (1966) . Fine structure of inclusions and organelles in Vicia fate infected with bean yellow mosaic virus . Virology 28, 290-302 . WEINTRAUB, M., and RAGETLI, H . W . J . (1968) .
DISTRIBUTION OF CVMV IN D . BARBATUS LEAF CELLS Intracellular characterization of bean yellow mosaic virus-induced inclusions by differential enzyme digestion . J . Cell Biol . 38, 316-328 . WEINTRAUB, M ., RAUETLI, H . W. J ., and VETO, M . (1968) . Fine structure of nuclear crystals in leaves of two Dianthvs species . Am . J. Botany 55,214-220 . WEINTRAUB, M., RAGETLI, H . W . J ., and VETO, M .
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(1969) . The use of glycol methacrylate for the study of the ultrastructure of virus-infected leaf cells. J . Ultrastract . Res . 26, 197-215. YA]IAGUGBI, A ., KIKUMOTO, T., and MATSUI, C . (1963) . Electron microscopy of elongated particles associated with tulip mosaic . Virology 20, 143-146 .
Distribution of Viruslike Particles in Leaf Cells of Dianthus barbatus Infected with Carnation Vein Mottle Virus M . WEINTRAUB
H . W . J . RAGETLI
ANn
Research Station, Canada Department of Agriculture 6660 . .147 ITarine Drive, Vancouver, Canada N Accepted December 1, 1969
Leaf exudates and partially purified preparations of Dianthus barbatus infected with carnation vein mottle virus showed the presence of relatively few elongated flexuous rods, one-half of which were between 675 and 850 tire long, and one-third between 750 and 825 Mo . Electron microscopic examination of ultrathin sections of infected leaf cells showed that nearly all viruslike particles were present in a single thin layer enclosed by two membranes, lying along the surface of the cytoplasm just inside the tonoplast_ Sometimes this peripheral area of the cytoplasm appeared to have been torn away or separated from the main body of cytoplasm, so that strands or bridges of virus particles extended into the central vacuole . The maximum length of particles in sections was about 750 TIM . Many cytoplasmic inclusions were observed, consisting of dense bands, loops, and circles . However, the occurrence of these inclusions does not necessarily permit the conclusion that carnation vein mottle virus is a member of the potato virus Y group . Coll organelles appeared normal, except for the nuclei, which were elongated, and displayed an intense electron density of the chromatin . INTRODUCTION
Carnation vein mottle virus (CVMV) in Dianthus barbatus L ., and in D. caryophyllus seedlings, was described by Kassanis (1955), who attempted to purify the virus, but with only partial success . He found long filamentous particles in some preparations, presumably similar to those lie observed in sap from infected leaves, but not consistently enough to identify these particles with the virus . CVMV is aphid transmissible (Hakkaart, 1964 ; Kassanis, 1955), and mechanically transmissible not only to Dianthus species but also, according to Hollings (1956, 1959), to C,henopodiur amaranticolor . In the latter host the virus is said (Hollings, 1956) to produce diagnostically characteristic lesions which separate it from other virus infections . CVl-1V can also be distinguished by serology from other carnation viruses for which autisera can be made . We also have attempted to purify CVMV, and although filamentous particles were
found in preparations from infected leaves only, their number was unsatisfactorily small . However, since one-half of the particles measured were between 675 and 850 nm long, and one-third between 750 and 825 nm (Fig . 2), there appeared to be another way of determining whether these long filamentous particles were actually the virus causing carnation vein mottle . A series of studies has shown that viruses of about this length induce characteristic inclusions in the cytoplasm, and sometimes in the nuclei of infected leaf cells. One group of cytoplasmic inclusions consists of a variety of forms that have been named dense bands (Bos and Rubio-Huertos, 1969 ; Christie et al., 1968 ; Kamei et al ., 1969 ; Lee, 1965 ; Matsui and Yamaguchi, 1964a ; Weintraub and Ragetli, 1966), cylindrical inclusions, bundles, laminated aggregates, fibrous masses, plates, oblong inclusions, pinwheels, starlike inclusions, circles, and loops (Bos and RubioHuertos, 1969 ; Christie et al ., 1968 ; Cremer 868
DISTRIBUTION OF CVMV IN D . BARBATUS LEAF CELLS and van der Veken, 1964 ; Edwardson, 1966a, b ; Edwardson et al ., 1968 ; Goethals el al ., 1969 ; Hoefert, 1969 ; Kamei et al ., 1969 ; Kim and Milton, 1969 ; Krass and Ford, 1969 ; Lee, 1965 ; Purcifull and Edwardson, 1967 ; Rubio-Huertos and Lopez-Abella, 1966 ; Shepard and Carroll, 1967 ; Stein-Margolina et al., 1969 ; Yamaguchi et al ., 1963) . Some of these terms seem to be derived from the two-dimensional appearance of a threedimensional structure sectioned in various planes . This structure yields pinwheels, circles, or loops in cross section, and bundles or tubes of various widths in longitudinal or tangential section . A three-dimensional reconstruction was first proposed by RubioHuertos and Lopez-Abella (1966) based on serial sections of virus-infected pepper cells . This was later called a "cylindrical inclusion" by Edwardson (1966b) . It is also possible that "dense bands" are the same structures as "laminated aggregates ." The cytoplasmic inclusions described as "dense bands" in bean yellow mosaic infection, arid as "pinwheel and bundle inclusions" with beet mosaic infection have been shown, by digestion with pepsin and trypsin, to be composed of protein and, as far as could be detected, devoid of nucleic acids (Hoefert, 1969 ; Weintraub and Ragetli, 1968 ; Weintraub el al ., 1969) . Another group of cytoplasmic inclusions, usually called "comblike," consist of lilamentous particles arranged arid attached perpendicularly to the tonoplast on one side and often to endoplasmic reticulum on the other side . These have been observed in cells infected with lettuce mosaic virus (Goethals el al ., 1969), a virus of Amaranthus lividus (Rubio-Huertos and Vela Corncjo, 1966), turnip mosaic virus (Kamei et al ., 1969), and papaya ringspot virus (Herold and Weibel, 1962) . A third group of inclusions, which are crystals, have been found in the cytoplasm and nuclei of cells infected with tobacco etch virus (Matsui arid Yamaguchi, 1964b ; Rubio-Huertos and Hidalgo, 1964), bean yellow mosaic virus (Weintraub and Ragetli, 1966), and pea necrosis virus (Bos and Rubio-Huertos, 1969) . The composition of the crystals of bean yellow mosaic virus has been found to be similar to that of the dense
869
bands, although some differences were detected in the composition of the crystalline protein (Weintraub and Ragetli, 1968 ; Weintraub et al ., 1969) . It seemed reasonable, therefore, to expect that, with such a variety of inclusions characteristically and consistently induced by certain elongated viruses, an electron microscopic examination of leaf cells infected with CVMV might answer the question whether the filamentous particles observed in crude sap and in purified preparations could be identified with the causal agent of the disease . MATERIALS AND METHODS
Puriication and electron microscopy of CVY1V . Exudates from CVBIV-infected and healthy D . barbatus leaves were prepared for electron microscopic examination by touching the cut leaf surface, including the central vein, to a drop of water on the specimen grid . This was allowed to dry, and was then shadowed with palladium . Attempts to purify CVMV by differential centrifugation in phosphate buffer, or in buffers containing sodium bisulfite, or sodium diethyldithiocarbamate, yielded preparations that were not infectious when tested on D . barbatus and on Chenopodium quinoa (Hollings and Stone, 1965), nor could particles, other than those found in healthy control preparations, be seen with the electron microscope . Unlike Hollings (1956, 1959), we were not able to transmit CVMV mechanically to C . anzarcudicoloi, arid so could not use it as an additional test plant . Hakkaart (1964) also found it difficult to transmit CVMV to C . amaranticolor . Preparations that appeared to be partially purified, were infectious, and contained some particles not found in healthy leaves were finally produced by the butanol-chloroform method of Steere (1956) . The final preparation was concentrated about 15-fold on a weight basis and was tested for infectivity on 1) . barbatus and C . quinoa, as well as being examined in a Philips EMI . 100, or E .M . 200, after the specimens were shadowed with palladium . A portion of this preparation was further centrifuged at high speed ; the pellet was dissolved in one-tenth the original volume,
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representing a 150-fold concentration. This preparation was also tested for infectivity and examined with the electron microscope . The concentrated preparation was then layered on a 5-30 % sucrose density gradient and centrifuged for 1 hour at 35,000 rpm in a Spinco SW 39 rotor. A narrow opalescent band about two-thirds down the gradient was syringed off . This band, and material from the column above and below it, were tested for infectivity and examined kithh the electron microscope. Electron microscopy of ultrathin sections. Leaves of D . barbatus, on plants inoculated 11 days previously with CVMV, were sampled from green areas in mature leaves, and from somewhat chlorotic areas in younger leaves . Since both areas produced similar results, no further distinction will be made . Pieces 2-3 mm' were cut and fixed in 5 % glutaraldehyde in phosphate buffer, pH 7.2 at room temperature for 1-2 hours . They were rinsed with distilled water, once before,
and 3-4 times after post-fixation for 1 hour with 1 % Palace's OsO, solution, pH 7 .2 . After dehydration in graded dilution of ethanol, they were treated with propylene oxide, and embedded in Epon 812 (Luft, 1961) . Thin sections were cut with glass knives and stained with uranyl acetate and lead citrate . RESULTS Purification and Electron Microscopy of CVMV
The CVMV-infected leaf exudates contained the usual debris, but very few elongated flexous particles (Fig . la) . Those that could be measured were 500-800 rim long . The preparations from the butanolchloroform procedure, at both concentrations, contained considerably less debris, and more elongated particles (Fig. 1b) . The particles were still sparse, so that many photographs were needed to accumulate the dimensions of 174 particles . Measurements
The scale on each figure indicates 1 µ unless otherwise noted . FIG . 1 . (a) Exudate from cut leaf of Dianthus barbatus infected with CVMV . (b) Sample of butanol-
chloroform-purified CVMV . Both shadowed with palladium .
DISTRIBUTION OF CVMV IN D . BARBATUS LEAF CELLS
871
20 19 18 17161514-
1 I-
a 0.
13121110 .
^r 0
9-
=
8-
Is 7-
e
6-
350 450 550 650
750 850 950 1050 1150 1250 1350 1450 1550 1650 1750 1850 1950 2050 2150 2250 2350 2450
LENGTH
mp
Fla . 2 . Size distribution of particles seen in purified CVMV . were made from 35-mm negatives with a Gaertner tool-maker's microscope and were grouped into categories of 25 nm . The distribution of lengths of these particles is shown in Fig . 2, where it is evident that about 45-50% of the particles fall into the categories of 675-850 not . There is a smaller peak at about 1500 ma, and another even smaller at 2300-2400 nm ; these probably represent dimers and trimers . It is equally evident that although the main peak occurs at about 750-825 nm, most of the particles fall into various other categories both longer and shorter . The width of the particles was 18-19 too . Both the 15-fold and 150-fold concentrated preparations were infectious in D . barbatus, and also in C. quinoa in which they produced rather vague, small blotchy lesions .
After the butanolchloroform preparations were fractionated on the sucrose density gradients, no infectivity and no particles such as those in Fig . 1 were detected either in the narrow opalescent band or in the fractions above and below this band .
Electron Microscopy of Ultrathin Sections In D . barbatus, inclusions were found readily in sections of the cytoplasm. They consisted mainly of the dense band type (Figs . 3 and 4), although they were neither so electron dense, nor so well-formed or long as those produced by bean yellow mosaic virus (BYMV) in Vicia faba (Weintraub and Ragetli, 1966) . The striated structure readily seen in BYMV dense bands was not found in the CVRMV bands . Most of the inclusions were straight, and only occasionally showed
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the oblique filaments (Fig . 3, arrow) that were often seen to come off such bands with other viruses (e.g ., Fig . 1, Weintraub and Ragetli, 1968) . No pinwheels were seen in any of the cells, but loops and circles were frequent, either singly or in double parallel arrangements (Fig. 4) . Only occasionally was an inclusion seen that might have represented a very loose pinwheel arrangement (Fig . 5) . Single viruslike particles, or particles in small aggregates randomly oriented in the cytoplasm were seen only rarely . In this position, they usually appeared in the cytoplasm not very distant from the central vacuole (Fig. 6) . Figure 6 also shows the disposition and location of most of the elongated particles that we believe to be CV\1V, as a narrow layer or sheet along the surface of the cytoplasm and next to the tonoplast (Fig . 6, arrow) . The particles lie parallel to each other (Figs . 7-9), and also parallel to the surface of the cytoplasm (Fig . 9) . They are enclosed by membranes toward the cytoplast and toward the tonoplast, the latter membrane being quite distinct from the tonoplast . The viruslike particles were sometimes seen as narrow bridges or strands over a vacuolar region . These bridges or strands containing spherical particles are interpreted as representing a thin cross section of a sheet of particles and its enclosing membranes . In Fig . 7, the particles are seen in cross section at the left end of such a bridge, and in oblique section at the right end (Fig . 7, arrow) . Figure 8 also shows a bridge over a small vacuole next to the cytoplasm, the bridge protruding into the larger central vacuole . It is clear in Fig . 8 that there is no membrane lining the cytoplasm next to the smaller vacuole, a condition frequently seen near the bridges . However, most of the viruslike particles were not found in these bridges or strands, but lay closely apposed to the main part of the cytoplasm and the tonoplast, adjacent to the central vacuole (Fig . 9) . The appearance of the particles varied with the angle of sectioning . Figs . 7-9 show the circular aspect produced by a cross section of the particles, while the short rods seen at the right end of the bridge (Fig . 7, arrow), and in parts of
Fig . 8 are the result of angles of section not far from cross sections . The apparent length of the particles increases with the angle of sectioning (Fig . 10) . Thus, when a layer of particles is sectioned parallel to the length of the rods and in between their enclosing membranes, only the rods are seen (Fig . 11) . In sections of this type, a few of the longest particles were measured and were about 750 nor . The width of the electron dense portions of these particles was 8-9 rim, so that if they are virus particles, this presumably represents the nucleic acid moiety, and the electron transparent portion is the protein . Parallel aggregates or sheets of particles were sometimes sectioned at oblique angles near one end of the aggregate, so that only one of the membranes was seen (Fig . 12, arrow) . The tonoplast appeared as a separate membrane, loosened from the cytoplasm and the sheet of particles, and protruding into the central vacuole . Cell organelles, such as chloroplasts and mitochondria, appeared relatively normal . However, the nuclei were often extremely elongated, with the chromatin portions considerably more electron dense than in healthy leaf cells (Fig. 13) . When examining nuclei of D. barbatus leaf cells it was essential not to mistake the normal crystals for those of virus origin . Longitudinal sections of normal crystals (Fig. 14), being composed of electron dense material 10-15 rim wide and 0 .5 2 µ long, somewhat resemble aggregates of viruslike particles in CVMV-infected cells . But normal crystals are present in many seedlings of D . ba •b atus and in D . chinensis which are, as far as can be determined, virus free and healthy (Weintraub et al ., 1968) . Cross sections of such crystals, in which the components consist of electron dense material 10-15 rim wide and electron transparent centers 6-8 nor in diameter, arc totally different (Fig . 15) from cross sections of the viruslike particles associated with CVMV infection.
DISCUSSION Although the purification of CVMV was not altogether satisfactory and resulted in relatively few particles for a proper study of the length distribution of the flexous rods, sufficient data were collected on particle size
Fio . 3 . Portion of mesophyll cell of Dianthus barbatus iniceted with CVMV . Note the dense bands (DI3), singly and in bundles . Arrow points to a band with filaments that come off at an angle .
lha . 4 . As in Fig . 3, showing inclusions in the form of loops (L) and circles . Fm, . .5, As in Fig . 3, showing inclusions that may represent a loose pinwheel arrangement (arrow) . 873
Fin . B . Portion of cell showing viruslike particles (V) in cytoplasm near cell vacuole (CV) . Arrow points to the more usual disposition of particles as a sheet at the periphery of the cytoplasm . Fm, 7 . Sheet of viruslike particles, in cross section at the left end (V), and in oblique section at the right end (arrow) . Note the membrane above and below the particles . 874
Fin . S . Cytoplasm devoid of tonoplast (arrow), indicating that the strand containing the viruslike particles (V) has torn away from the main body of cytoplasm, and extends into the cell vacuole (CV) . Fin . 9 . Viruslike particles (V) apposed closely to the cytoplasm near a nucleus (N), and lying parallel to each other and to the surface of the cytoplasm . 9,5
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WEINTRAUB AND RAGETLI
FIG . 10. Viruslike Particles (V) sectioned at various angles of ohliqueness . Fie. . 11 . Viruslike particles near cell vacuole (CV) sectioned parallel to the length of the particles and their enclosing membranes .
DISTRIBUTION OF CVMV IN D . BARBATUS LEAF CELLS
877
FIG . 13 . Viruslike particles sectioned at an oblique angle, so that only one of the two enclosing membranes is visible (arrow) . Note the loosened tonoplast (T) protruding into the central vacuole (CV) . Fic . 13 . Portion of CVAIV-infected leaf cell, showing extreme elongation of the nucleus (N), and the abnormal electron density of the chromatin portions .
Fic . 14 . Portion of nucleus (N) in healthy leaf cell of Dianthus bathatus showing a longitudinal section of a nonviral crystalline inclusion impinging on the nucleolus (Ns) . Fic . 15 . Portion of nucleus (N) in healthy leaf cell of Dianthus barbatus showing a cross section of a nonviral crystalline inclusionn near the nucleolus (Ne) . 878
DISTRIBUTION OF CVMV IN D . BARBATUS LEAF CELLS
879
to predict with some confidence that dense band inclusions would be found in the CVMV-infected leaf cells . This does not mean, however, as proposed by Edwardson (1966a), that the presence of these inclusions necessarily permits the diagnostic identification of CVJ-IV as a member of the potato virus Y (PVY) group of viruses, with lengths of 730-750 nm (Brandes and Berclcs, 1965) . With CVMV, the major peak of particles measured (Fig . 2) was in the 750-825 nm range . Furthermore, cytoplasmic inclusions of similar type have been found in cells infected with wheat streak mosaic virus (Lee, 1965 ; Shepard and Carroll, 1967 ; Stein-Margolina et al ., 1969), and in cells of virus-infected Amarant/aus lividus (RubioHu(rtos and Vela-Cornejo, 1966) . Both of these viruses are 650 nm long . Similar inclusions have also been found in cells infected with narcissus yellow stripe (Cremer and van der Veken, 1964) and papaya ringspot virus (Herold and Weibel, 1962), both of which viruses are 800 nm long . Recently, Bos and Rubio-Huortos (1969) described inclusions of pea necrotic virus, some of which resembled inclusions produced by the PVY group whereas others resembled inclusions associated with the PVX group, including potato virus X itself, clover yellow mosaic virus, and papaya mosaic virus . Unless one postulates a considerable error in published measurements of these virus particles, it would appear that the induction of dense bands, cylindrical inclusions, pinwheels, etc ., is not a simple function of an arbitrary 730-750 nm length of the virus particle . At this time, the evidence suggests that such peculiar inclusions are associated with elongated viruses that are not restricted to the PVY group, but range in length from at least 650 to 800 nm . If the rod-shaped particles seen in the CVMV-infected cells are CVM virions, their arrangement in a narrow layer or sheet along the surface of the cytoplasm, enclosed by two membranes, is unusual and differs greatly from the "comb-like" arrangement described for certain viruses (Goethals et al ., 1969 ;
izing the particles in leaf exudates and in purifying the virus . Even with a considerable cytoplasmic surface over which the particles are arranged, there must be distinctly fewer than in systemic infections such as those produced by tobacco mosaic virus, potato virus X, etc ., where massive aggregates of particles are seen throughout the cytoplasm. Another difficulty in purification may be the necessity to break the membranes that enclose the viruslike particles to free them, and this would be considerably more difficult than simply freeing unenclosed particles by disrupting the infected cells . The bridgelike strands containing viruslike particles (Figs . 7 and 8) have been observed with pokeweed mosaic (Kim and Fulton, 1969), and with beet mosaic virus (Hoefert, 1969) . Both were interpreted as being the result of an enlargement of vesicles which subsequently protruded into the central vacuole. When sectioned these appear as narrow cytoplasmic strands containing viruslike particles . In CVMV-infected cells, the particles were observed most frequently at the periphery of the cytoplasm (Fig . 9), and it appeared to us that the bridges represent a tearing away of the outer layer of cytoplasm, because of the unusually heavy mass of viruslike particles concentrated along the surface . This is substantiated by the frequency with which we observed (Fig . 8, arrow) that the cytoplasm below the bridge was totally devoid of a cytoplasmic membrane, suggesting that a tearing took place, rather than an orderly development of an enlarged vesicle in the cytoplasm. The unusual arrangement of the viruslike particles makes it difficult to understand, if these are CVM virions, where or how the virus is synthesized and assembled . It would be odd, indeed, if the components were synthesized either in the nucleus or in the main
Herold and Weibel, 1962 ; Kamei et at ., 1969 ; Rubio-Huertos and Vela-Cornejo, 1966) . This physical arrangement may account for some of the difficulties encountered in visual-
ACKNOWLEDGMENTS
body of cytoplasm, and then migrated to the surface of the cytoplasm to be assembled into their final form in this very regular, parallel arrangement .
We are grateful to Miss Esther Lo and Miss Bea Schroeder for technical assistance, and to Mr . J . H . Severson for preparing the figures .
WEINTRAUB AND RAGETLI
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REFERENCES Bes t L ., and RDDIO-HTERTOS, M. (1969) . Light
and electron microscopy of cytoplasmic and unusual nuclear inclusion bodies evoked by a virus from necrotic peas . Virology 37, 377-385 . BRANDES, J ., and BERCES, R . (1965) . Gross morphology and serology as a basis for classification of elongated plant viruses . In "Advances in Virus Research" (K. M . Smith and M . A . Lalfler, eds .), Vol . 11, pp . 1-24 . Academic Press, New York . CHRISTIE, S . R ., EDWARDSON, J . It ., and ZETTLER, F . W . (1968)- Characterization and electron microscopy of a virus isolated from Bidens and Lepidium . Plant Disease Retr . 52, 763-768 . CREMER, M . C ., and VAN DER VEEEN, J . A . (1964) . Localization of viral antigen in narcissus leaves infected with yellow stripe virus, determined by means of a fluorescein conjugated antiserum . N eth . J . Plant Pathol . 70, 105-113 . EDW ARDSON, J . R . (1966a) . Electron microscopy of cytoplasmic inclusions in cells infected with rodshaped viruses . Arn . J . Botany 53, 359-364 . EDWARDSON, J. R. (1966b) . Cylindrical inclusions in the cytoplasm of leaf cells infected with tobacco etch virus. Science 153, 883-884 . EDWARDSON, J. R ., PURCIFULL, D . E ., and CHRISTIE, R . G . (1968) . Structure of cytoplasmic inclusions in plants infected with rod-shaped viruses . Virology 34,250-203 . GOETHALS, M ., HORVATH, F ., and MEYER, J . A . (1969) . Cytoplasmic inclusions fit leaf cells infected with lettuce mosaic virus . Virology 37, 685-687 . HAEEAART, F . A. (1964) . Description of symptoms and assessment of loss caused by some vi rusesin the carnation cultivar" William Sim ." Neth . J . Plant Pathol . 70, 53-60 . HEROLD, F ., and WEIBEL, J . (1962) . Electron microscopic demonstration of papaya ringspot virus . Virology 18,302-311 . HOEFERT, L . L . (1969) . Proteinaceous and viruslike inclusions in cells infected with beet mosaic virus. Virology 3 7 , 498-5(11 . HOLLINGS. M- (1956) . Chenopodium anmaranticolor as a test plant for plant viruses . Plant Pathol . 5, 57-60 . HOLLINOS, M . (1950) . Host, range studies with fifty-two plant viruses . Ann . Appl . Biel. 47, 98108 . HOIJSNCS, M ., and STONE, 0 . M . (1965) . Chenopodium guinoa Willd . as an indicator plant, for carnation latent virus . Plant Pathol . 14, 66-OS . KAMEi, T ., hIoNDA, Y ., and MATSUI, C . (1969) . Intracellular appearance of turnip mosaic and bean yellow mosaic virus particles . Phytopathology 59,1 .30-14-4 .
KASSANIS, B . (1955) . Some properties of four viruses isolated from carnation plants . Ann . Appl . Biol. 43, 103-113 . KIM, K . S ., and FLLPON, J . P . (1969) . Electron microscopy of pokeweed leaf colts infected with pokeweed mosaic virus . I irology 37 , 297-308 . KR .ASS, C . J ., and FORD, R . E . (1969) . Ultrastructure of corn systemically infected with maize dwarf mosaic virus . Phytopathology 59, 431-439 . LEE, P . F . (1965) . Electron microscopy of inclusions associated with wheat streak mosaic virus . J. Ultrastruct . Res . 13, 359-366 . LOFT, J . H . (1961) . Improvements in epoxy resin embedding methods. J . Biophys . Biochem . Cytol . 9,409-414 . MATSUI, C ., and YAMAGUCHI, A . (1964a) . Electron microscopy of host cells infected with tobacco etch virus, I . Fine structures of leaf cells at later stages of infection . Virology 22, 40-47 . MATsun, C ., and YAMAGUCHI, A . (1964b) . Electron microscopy of host cells infected with tobacco etch virus . II . Fine structures of leaf cells before and after the appearance of external symptoms . Virology 23, 346-353, PL-RCIFULL, D . F ., and EDWARDSON, J . R . (1967) . Watermelon mosaic virus : Tubular inclusions in pumpkin leaves and aggregates in leaf extracts . Virology 32, 393-401 . R.UBio-HUERTOS, M ., and G .ARCIA HIDALGO, F . (1964) . Ultrathin sections of intranuclear and intracytoplasmic inclusions induced by severe etch virus. Virology 24,84-90 . RUBIO-HUER'rOS, M ., and LOPEZ-ADELLA, D . (1966) . Ultraestructura de celulas de pimiento infectadas con un virus y su localization en las mismas . Microbial . Espan . 19, 77-86 . RURIO-HUERTOS, M ., and VELA-CORNEJO, A . (1966) . Light and electron microscopy of virus inclusions in Amaranthus livirdus cells . Protoplasma 62, 184-193 . SHEPARD, J . F ., and CARRoLl„ T . W . (1967) . Electron microscopy of wheat streak mosaic virus particles in infected plant cells- J . Ultrastruct. Res . 21,145-152 . STEERE, R . L . (1956) . Purification and properties of tobacco ringspot virus . Phytopathology 46, 60-60 . STEIN-MARGOLINA, V . A ., CHERNY, N . E ., and RAZYV%ZKINA, G. 1M. (1969) . Phytopathogenic viruses in plant cells and in mite-vector (electron microscopic investigation) . freest . Akad . Nazdc S .S .R . Set . Biol . 1, 62-68. WEINTRAUB, M., and RAGETLI, H . W . J . (1966) . Fine structure of inclusions and organelles in Vicia fate infected with bean yellow mosaic virus . Virology 28, 290-302 . WEINTRAUB, M., and RAGETLI, H . W . J . (1968) .
DISTRIBUTION OF CVMV IN D . BARBATUS LEAF CELLS Intracellular characterization of bean yellow mosaic virus-induced inclusions by differential enzyme digestion . J . Cell Biol . 38, 316-328 . WEINTRAUB, M ., RAUETLI, H . W. J ., and VETO, M . (1968) . Fine structure of nuclear crystals in leaves of two Dianthvs species . Am . J. Botany 55,214-220 . WEINTRAUB, M., RAGETLI, H . W . J ., and VETO, M .
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(1969) . The use of glycol methacrylate for the study of the ultrastructure of virus-infected leaf cells. J . Ultrastract . Res . 26, 197-215. YA]IAGUGBI, A ., KIKUMOTO, T., and MATSUI, C . (1963) . Electron microscopy of elongated particles associated with tulip mosaic . Virology 20, 143-146 .