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Ultrastructure of Datura stramonium leaves infected with the physalis mottle strain of belladonna mottle virus
VIROLOGY
66, 123-133 (1973)
Ultrastructure Physalis
of Datum Mottle
stramoniom
Strain
of Belladonna
HAROLD Northern
Leaves
Infected Mottle
with
the
Virus”’
E. MOLINE
Gra.in Insects Research Laboratory, Agricultural Research Service, rTSDL4, Brookings, Soxth Dakota 57006 Accepted July
26, 1973
An electron microscopic study of Datura stmmonium leaves inoculated with the physalis mottle strain (PMVj of belladonna mottle virus (BRLV) revealed modifications of chloroplast structure. Numerous vesicles develop in chloroplasts 7 days after inoculation. A study of the lat,ter at different stages after inoculation suggests that some part of PMV replication may take place within chloroplast,s. Numerous vesiculations of the outer membranes of chloroplasts further suggest, release of virus from plastids by way of these vesicles. Similar vesicles have been reported in plants infected with turnip yellow mosaic virus and wild cucumber mosaic virus, members of t.he tymovirus group, to which PlVIV may be allied. Crystalline aggregates of virions closely associated with chloroplasts and crystalline inclusions in nuclei were found in systemically infected leaf tissue INTRODUCTION
Physalis mottle (PRIV) is a newly described virus isolated from Physalis heterophylla (Rloline and Fries, 1973). Hostrange studies and physical propert,ies place the virus in the turnip yellow mosaic virus (TTRIV) group as a strain of belladonna mottle virus (BRIV) (Bercks and Querfurth, 1971; Bercks et al., 1971; Gibbs et al., 1966; Jankulowa et al., 1968). Isomet8ric particles 27-30 nm in diameter are associated with infectivity. The virus occurs in high titer in inoculated Datura stmmoniunl. leaves, causing chlorotic lesions on inoculated leaves and a yellow mottle on systemically infected leaves. By comparing leaves at different stages of 1 This work was funded in part by a National Science Foundation Presidential Internship appointment. In cooperation with the Youth L)akota -4gricultural Experiment Station, Brookings. 4 Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agricuture and does not imply its approval to the exclusion of other products that may also be suitable.
infection, I have determined the distribution and sequential spread of PRIV particles in leaf tissues and its effect, on chloroplast ulkast,ructure. These rest&s are described herein. MhTERIAIS
BND
hIETHOI)H
Healthy D. stramoni~unz seedlings were inoculated with PRIV by grinding infected tissue in 0.01 Ai phosphate buffer (pH 7.0) and rubbing inoculum onto the 3rd and 4th leaves, which had been dusted with silicon carbide. Tissue from inoculated leaves was collected and fixed 6, 7, 8, 10, 12, 16, and 31 days after inoculation. Inoculations were staggered so that tissue in all stages of infection could be harvested and fixed on the sa,me date. Leaves collected 6 days after inoculation did not have visible chlorobic lesions; all other samples were t,alcen from areas displaying chlorotic lesions. Healthy D. stramotzium leaves from uninoculated plants grown under the same condit,ions as the infect,ed ones were harveskd and studied as the controls. Other plants were inoculated and held in the greenhouse for 1 month to allow systemic spread of the virus. Systemically infect,ed leaves 123
were then harvested and prepared for electron microscopy. Specimens were fixed in 4% phosphat,ebuffered glut,araldehTde (pH 7.2), postfixed in 1% osmium tetroxlde, dehydrated through a graded series of ethanol-propylene oxide, and embedded in Araldite-Epon@. Blocks mere sectioned with a diamond knife on an LKB Ultrotome. Sections were stained with aqueous uranyl acetat’e and/or lead citrate. RESULTS
Virus particles were sparse in symptomless leaves collected 6 days after inoculation. Occasionally part,icles were found in the cytoplasm adjacent to plastids and nuclei. There were no abnormalities in plastid membranes at this stage of infect,ion. Seven days after inoculation, chlorotic lesions began to appear in inoculated leaves. Cells in these chlorotic areas contained chloroplasts that had begun to develop membranous vesiculations. These vesiculations were double membrane bound and appeared to originate from the outer limitsing membranes of the chloroplast (Fig. 1). Chloroplasts were spherical and smaller than t#hose of healthy controls (Fig. 2). BJ 10 days, virus particles had become more numerous in the cytoplasm, and chloroplasts had developed numerous vesiculations (Fig. 3). Although more palisade parenchyma cells were infected than other tissue, based on cytological observations, the virus was not restricted to any tissue. Nuclei of infected cells were granular and appeared to contain less chromatin than did healthy nuclei. Nucleoli stained densely, but a major portion of the nucleoplasm failed to take up stain (Fig. 3). Twelve days after inoculation the proportion of vesiculated chloroplasbs increased, as did bhe amount of virus present in the cytoplasm of infected cells (Fig. 4). As infect,ion proceeded, vesiculation increased and extended to include the thylakoid membranes. These membranes were distended and filled with viral particles. Some starch granules were still present, in chloroplasts, however, in observations of many sections they were generally reduced in size. The presence of viral particles increased total cytoplasmic contents. RIit,ochondria appeared less numerous and smaller than those found in
healthy cells, and fen- microbodies \\-rre visible. Chloroplast membrane degeneration was minimal; plastids appeared more rounded with fewer &arch granules and exhibited a reduction in the number of grana stacks as compared to healt,hy tissue of t,he same age. Sixteen days after inoculation viral particles had aimost complet.ely replaced the cellular cyt,oplasmic cont.ents. llembrane inbegrity was preserved, and although chloroplast membrane area had been increased by the formation of many vesicles, plastids had not, broken down (Fig. 5). Vacuolation appeared to be limited to 1 or more large central vacuoles in each cell, which were generally free of viral particles. No crystalline viral inclusions were found in inoculated leaves. Examination of systemically infected tissue revealed manv crystalline aggregates of virus particles (Fig. 6). An increase in t#he size and number of osmiophilic bodies within chloroplasts was also observed and vesiculat#ion of chloroplasts had increased. Numerous small vacuoles, formed as a result of chloroplast, membrane hypert#rophy, were found filled wit,h viral particles (Fig. 7). Tubules occupied a large area in chloroplasts (Fig. 8). Many nuclei contained crystalline aggregates. Even t.hough the particles did not stain well in nulcei, the large inclusions of viruslike particles were easily visualized as they occupied a large portion of the nuclei (Fig. 9). Membrane proliferation was quite apparent in systemically invaded tissue. Chlorop1ast.sbecame irregular in shape, with many lobes and numerous vesicles (Figs. S and 9). The few mitochondria that were observed in sysrstemically infected tissue appeared swollen, with few cristae (Fig. 10). DISCUSSION
The thesis that chloroplasts may serve as a site of replication for a number of viruses has Received an increased amount of support recently (Favali and Ccmti, 1970; Gerola et al., 1966, 1969; Milne, 1967; Ushiyama and Matthews, 1970). Chalcroft and Matthews (1966, 1967) suggested that some part of t’urnip yellow mosaic (TYRIV) viral synthesis t,akes place wit,hin chloroplasts of infected Chinese cabbage. Although they mere
FIG. 1. Chloroplasts (Ch) and nucleus (IV) in a palisade parenchyma cell 7 days after inoculation with PMV. Vesiculations (arrows) in outer membranes of swollen chloroplasts are the first indications of PMV infection. iIf, mitochondria; Vat, vacuole. Bar = 1 pm. FIG. 2. Chloroplasts (C/L) and nucleus (:V) in healthy leaf. M, mitochondria; S, starch granules; Vuc, vacuole; W, cell wall. Bar = 1 Grn. 125
126
MOLINE
FIG. 3. Part of a palisade parenchyma cell 10 days after inoculation with PMV. Nucleus (AT) contains little chromatin; with the exception of t.he nucleolus (&ju), virus particles (5’) are numerous in cytoplasm. Chloroplasts (Ch) contain many vesicles (Ve). Bar = 1 pm. FIG. 4. Typical palisade cell 12 days after inoculat,ion, with rows of tubrdes (T) developing in chloroplasts (Ch) and virus (V) throughout t,he cyt,oplasm. Vat, vacuole. Bar = 1 pm.
unable to visualize viral particles in chlorc,plasts, t.hey obtained evidence for chloroplast, involvement in viral synthesis at the lightand electron-microscopic levels. Ushiyama and Matthews (1970) suggest that viral nucleic acid may be synthesized in T‘I’RIVinfected chloroplast vesicles.
Allen (1972) found wild cucumber mosaic virus in mesophyll chloroplasts of dlarah oregaws, which contained vesicles similar to t,hose produced by TYAIV in Chinese cabbage (Chalcroft and Pllatthews, 1966; Gerola ef al., 1966). This type of vesicle was also found in PAN-infected D. st~arno?~iwn leaves. In addition, another more complex tubule network was found in chloroplasts in lat,er stages of infection. Both types of chloroplast vesiculations are associated with P1IV infection. The small vesicles seen attached to the outer chloroplast membrane in Fig. 5b appear to have a different origin than the tubule network that. ramifies throughout the chloroplast, in Fig. 5a. When sectioned in various planes, the vesicles bound to t,he outer membranr appear to be cup-shaped depressions of the membranes resembling pinocytot,ic vesicles (Figs. 3, 5b). While the t,ubular networks may arise from t,he outer membrane, they are not restricted in shape or size (Fig. .~a, 8). The possibility also exists that’ the tubular networks are formed from thylakoid membranes t,hat have been modified as a result of PMV infection. Indeed, some of the larger tubules contai’ned vesiclrs attached to the membrane system. The t,ubes t,hat, Hitchborn and Hills (Matthews, 1970) obtained from negative13 stained extracts of Chinese cabbage leaves infected with a necrotic strain of TYRIV resemble the tubules found in PJIV-infected D. stra~nor~iu~~z chloroplasts (Fig. 5a). The extent of chloroplast vesiculation ma) be expressed quite differently in t,wo hosts infwted with the same virus (Chalcroft and IIIatthews, 1966; Matthews, 1970). PMVinfected Sicotiana glutinosa chloroplasts show only the small outer membrane-bound vesicles (RIoline, 1973), as opposed to the two types of vesicles found in PRIV-infect,ed D. strmno~~izm.
5b FIG. 5. Two types of vesiculations in PhW infected chloroplasts (Ch) 16 days after inoculation. (aj Tubular (7’) networks t.hat appear to have arisen from swelling of the thylakoid membranes. (b) vesicles (I’ej attached to the outer chloroplast limiting membranes. I’, \‘irtls. Bar = 200 nm.
The association of PRIV particles with chloroplasts indicat,cs that some part of viral replication or assembly occurs in these organelles. The sequential observations of infected chloroplasts have shown that vesiculation proceeds as lesions develop on inoculated leaves. Examination of systemically infected leaves that arc yellow and severely deformed as a result of viral infection shows cells filled with \iruslikc particle.
128
MOLINK
FIG. 6. Viral inclusion (V) adjacent to chloroplasts (CH) in a systemically infected cell. Chloroplast degeneration is evident from destruction of thylakoid (Th) membrane system and grana stacks (G). Numerous vesicles (Ve) and large osmiophillic granules (Og) are also present in chloroplasts. W, cell wall; Vat, vacuole. Bar = 500 nm.
FIG. 7. Stages of degeneration of PM-infected chloroplasts (Ch). (a) V’ lrus-filled vesicles (Ve) originating from degenerating chloroplast. (b) Vesicles and tubules (T) filled with virus. (c) Virus-filled vesicles that have budded off from a degenerating chloroplast. Vat, vacuole. Bar = 500 nm. 129
130
MOLINE:
FIG. 8. Chloroplast (Ch) from systemically infected cell, showing tubular (T) network veloped within the infected plastid. Og, osmiophilic granules; l'h, thylakoid membrane mitochondria; V, virus; Vat, vacuole; G, grana stacks. Bar = 200 nm.
t,hat has de network; .lf
CYTOLOGY
OF BMV
IN
DATURA STRAMONIUM
FIG. 9. Nucleus (N) from systemically infected cell, showing crystalline inclusion take up stain, and degenerating organelles surround the nucleus. Ch, chloroplast; Nu, nucleolus; W, cell wall. Bar = 500 nm.
131
(V) which fails to AJ, mitochondria;
132
MOLINE
may be of different composition than t,he virus that crystallizes in the cytoplasm (Fig. 6). Since the virus has been shown to be multicomponent in nature (RIoline and Fries, 1973), one could postulate that one of the components could be assembled within nuclei. REFERENCES
FIG. 10. hIitochondria (M) and adjacent chloroplasts (Ch) in a systemically infected cell. v, virus; T-e, vesicles. Bar = 500 nm.
The close association of virus particles with nuclei could not be demonstrated during early stages of infectsion, as part,icles failed t’o take up stain if present in nuclei; however, the cryst,allization of viruslike particles in D. stranzonium nuclei suggests that PNV may have been assembled there also. The failure of viruslike particles to take up stain in the nuclei and their poor staining characteristics in chloroplasts may be the result of differential permeability of membranes to the osmium fixative that is responsible for the major part of the initial staining of cellular contents. Att,empts to increase contrast of particles within nuclei by staining tissue with uranyl acet.ate during the dehydration process (Hills and Plaskitt, 1968) were unsuccessful. The Inarticles within the nuclei (Fig. \ u 9) -I
ALLEN, T. C. (1972). Subcellular responses of mesophyll cells to wild cucumber mosaic virus. F’irology 47, 467474. BERCKS, R., and QUERFURTH, Cr. (1971). The use of the latex t.est for the detection of distant serological relationships among plant viruses. J. Get,. Viral. 12, 25-32. BERCBS, R., HUTH, W., KOENIG, IL, LESEM~NN, 1). PAUL, H. L., and QUERFURTH, G. (1971). Scrophularia mottle virus: Charakterisierung und Vergleich mit anderen Viren der turnip yellow mosaic virus-Gruppe. Phytopathol. Z. 71, 341-356. CHALCROFT, J., and R~.ITTHEws, R. E. F. (1966). Cytological changes induced by turnip yellow mosaic virus in Chinese cabbage leaves. Virology 28, 555-562. CX~LCROFT, J., and M.ITTHEWS, R. E. F. (1967). Role of virus strains and leaf ontogeny in the production of mosaic patterns by turnip yellow mosaic virus. TTiroZogy 33, 659673. F~v.4~1, M. A., and CONTI, G. G. (1970). Ultrastruct,ural observations on the chloroplasts of basil plants either infected with different viruses or treated with 3-amino-1,2,4-triazole. Protoplasma
TO, 153-166.
GEROL~, F. M., Basso, M., and GUISS~NI, G. (1966). Some observations on the shape and localization of different viruses in experimentally infected plant,s and on the fine structure of host cells. III. Turnip yellow mosaic virus in Brassica chinensis L. Caryologia 19,457479. GEROLA, F. M., Bassr, M., and BETTO, E. (1969). rl submicroscopical study of leaves of alfalfa, basil, and tobacco infected with Lucerne mosaic virus. Protoplasma 67. 307-318. GIBBS, A. J., HECHT-POINIR, E., and WOODS, R. D. (1966). Some properties of three related viruses: Bndean potato latent, Dulcamara mottle, and Ononis yellow mosaic. J. Gen. Microbial. 44, 177-193. HILLS, G. J., and PL.%SKITT, A. (1968). 9 protein stain for the electron microscopy of small isometric plant virus particles. J. Ultrastruct. Res. 25, 325-329. J.~NIXJLO\V.~, ILL, HUTH, W., WITTM.~NN, H. G., and P.~uL, H. L. (1968). Untersuchungen iiber ein neues isometrisches Virus aus Atropn bella-
CYTOLOGY
OF B&IV
IN DdTC’Rd
donna I,.. II. Serologische Reaktionen. Basenverhaltnisse der RNS und Amino-slurenzusammensetzung des Proteins. PhutopathoZ. 2. 63, 177-185. MATTHEWS, R. E. F. (1970). “Plant Virology,” pp. 289-295. Academic Press, New York. MILNE, R. CT. (1967). Electron microscopy of mosaic virus and leaves infected with sowbane other small polyhedral viruses. T’irology 32. 589-600. MOLINE, H. E. (1973). Chloroplast anomalies asso-
LS’TR~A~O~~ZI.~JZ
133
ciated wit,h physalis mottle virus infection in Datura stramo&m and Nicotiana gl,utinosa leaves. Second International Congress of Plant Pathology Abstracts. 0308. MOLINE, H. E., and FRIES, R. E. (1973). A virus isolated from Phylsalis h.eterophyZla in Iowa. PhytopathoZogy 63, in press. USHISXX~, R., and MATTHEWS, R. E. F. (1970). The significance of chloroplast abnormalities associated with infection by turnip yellow mosaic virus. I’irology 42, 293-303.
66, 123-133 (1973)
Ultrastructure Physalis
of Datum Mottle
stramoniom
Strain
of Belladonna
HAROLD Northern
Leaves
Infected Mottle
with
the
Virus”’
E. MOLINE
Gra.in Insects Research Laboratory, Agricultural Research Service, rTSDL4, Brookings, Soxth Dakota 57006 Accepted July
26, 1973
An electron microscopic study of Datura stmmonium leaves inoculated with the physalis mottle strain (PMVj of belladonna mottle virus (BRLV) revealed modifications of chloroplast structure. Numerous vesicles develop in chloroplasts 7 days after inoculation. A study of the lat,ter at different stages after inoculation suggests that some part of PMV replication may take place within chloroplast,s. Numerous vesiculations of the outer membranes of chloroplasts further suggest, release of virus from plastids by way of these vesicles. Similar vesicles have been reported in plants infected with turnip yellow mosaic virus and wild cucumber mosaic virus, members of t.he tymovirus group, to which PlVIV may be allied. Crystalline aggregates of virions closely associated with chloroplasts and crystalline inclusions in nuclei were found in systemically infected leaf tissue INTRODUCTION
Physalis mottle (PRIV) is a newly described virus isolated from Physalis heterophylla (Rloline and Fries, 1973). Hostrange studies and physical propert,ies place the virus in the turnip yellow mosaic virus (TTRIV) group as a strain of belladonna mottle virus (BRIV) (Bercks and Querfurth, 1971; Bercks et al., 1971; Gibbs et al., 1966; Jankulowa et al., 1968). Isomet8ric particles 27-30 nm in diameter are associated with infectivity. The virus occurs in high titer in inoculated Datura stmmoniunl. leaves, causing chlorotic lesions on inoculated leaves and a yellow mottle on systemically infected leaves. By comparing leaves at different stages of 1 This work was funded in part by a National Science Foundation Presidential Internship appointment. In cooperation with the Youth L)akota -4gricultural Experiment Station, Brookings. 4 Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agricuture and does not imply its approval to the exclusion of other products that may also be suitable.
infection, I have determined the distribution and sequential spread of PRIV particles in leaf tissues and its effect, on chloroplast ulkast,ructure. These rest&s are described herein. MhTERIAIS
BND
hIETHOI)H
Healthy D. stramoni~unz seedlings were inoculated with PRIV by grinding infected tissue in 0.01 Ai phosphate buffer (pH 7.0) and rubbing inoculum onto the 3rd and 4th leaves, which had been dusted with silicon carbide. Tissue from inoculated leaves was collected and fixed 6, 7, 8, 10, 12, 16, and 31 days after inoculation. Inoculations were staggered so that tissue in all stages of infection could be harvested and fixed on the sa,me date. Leaves collected 6 days after inoculation did not have visible chlorobic lesions; all other samples were t,alcen from areas displaying chlorotic lesions. Healthy D. stramotzium leaves from uninoculated plants grown under the same condit,ions as the infect,ed ones were harveskd and studied as the controls. Other plants were inoculated and held in the greenhouse for 1 month to allow systemic spread of the virus. Systemically infect,ed leaves 123
were then harvested and prepared for electron microscopy. Specimens were fixed in 4% phosphat,ebuffered glut,araldehTde (pH 7.2), postfixed in 1% osmium tetroxlde, dehydrated through a graded series of ethanol-propylene oxide, and embedded in Araldite-Epon@. Blocks mere sectioned with a diamond knife on an LKB Ultrotome. Sections were stained with aqueous uranyl acetat’e and/or lead citrate. RESULTS
Virus particles were sparse in symptomless leaves collected 6 days after inoculation. Occasionally part,icles were found in the cytoplasm adjacent to plastids and nuclei. There were no abnormalities in plastid membranes at this stage of infect,ion. Seven days after inoculation, chlorotic lesions began to appear in inoculated leaves. Cells in these chlorotic areas contained chloroplasts that had begun to develop membranous vesiculations. These vesiculations were double membrane bound and appeared to originate from the outer limitsing membranes of the chloroplast (Fig. 1). Chloroplasts were spherical and smaller than t#hose of healthy controls (Fig. 2). BJ 10 days, virus particles had become more numerous in the cytoplasm, and chloroplasts had developed numerous vesiculations (Fig. 3). Although more palisade parenchyma cells were infected than other tissue, based on cytological observations, the virus was not restricted to any tissue. Nuclei of infected cells were granular and appeared to contain less chromatin than did healthy nuclei. Nucleoli stained densely, but a major portion of the nucleoplasm failed to take up stain (Fig. 3). Twelve days after inoculation the proportion of vesiculated chloroplasbs increased, as did bhe amount of virus present in the cytoplasm of infected cells (Fig. 4). As infect,ion proceeded, vesiculation increased and extended to include the thylakoid membranes. These membranes were distended and filled with viral particles. Some starch granules were still present, in chloroplasts, however, in observations of many sections they were generally reduced in size. The presence of viral particles increased total cytoplasmic contents. RIit,ochondria appeared less numerous and smaller than those found in
healthy cells, and fen- microbodies \\-rre visible. Chloroplast membrane degeneration was minimal; plastids appeared more rounded with fewer &arch granules and exhibited a reduction in the number of grana stacks as compared to healt,hy tissue of t,he same age. Sixteen days after inoculation viral particles had aimost complet.ely replaced the cellular cyt,oplasmic cont.ents. llembrane inbegrity was preserved, and although chloroplast membrane area had been increased by the formation of many vesicles, plastids had not, broken down (Fig. 5). Vacuolation appeared to be limited to 1 or more large central vacuoles in each cell, which were generally free of viral particles. No crystalline viral inclusions were found in inoculated leaves. Examination of systemically infected tissue revealed manv crystalline aggregates of virus particles (Fig. 6). An increase in t#he size and number of osmiophilic bodies within chloroplasts was also observed and vesiculat#ion of chloroplasts had increased. Numerous small vacuoles, formed as a result of chloroplast, membrane hypert#rophy, were found filled wit,h viral particles (Fig. 7). Tubules occupied a large area in chloroplasts (Fig. 8). Many nuclei contained crystalline aggregates. Even t.hough the particles did not stain well in nulcei, the large inclusions of viruslike particles were easily visualized as they occupied a large portion of the nuclei (Fig. 9). Membrane proliferation was quite apparent in systemically invaded tissue. Chlorop1ast.sbecame irregular in shape, with many lobes and numerous vesicles (Figs. S and 9). The few mitochondria that were observed in sysrstemically infected tissue appeared swollen, with few cristae (Fig. 10). DISCUSSION
The thesis that chloroplasts may serve as a site of replication for a number of viruses has Received an increased amount of support recently (Favali and Ccmti, 1970; Gerola et al., 1966, 1969; Milne, 1967; Ushiyama and Matthews, 1970). Chalcroft and Matthews (1966, 1967) suggested that some part of t’urnip yellow mosaic (TYRIV) viral synthesis t,akes place wit,hin chloroplasts of infected Chinese cabbage. Although they mere
FIG. 1. Chloroplasts (Ch) and nucleus (IV) in a palisade parenchyma cell 7 days after inoculation with PMV. Vesiculations (arrows) in outer membranes of swollen chloroplasts are the first indications of PMV infection. iIf, mitochondria; Vat, vacuole. Bar = 1 pm. FIG. 2. Chloroplasts (C/L) and nucleus (:V) in healthy leaf. M, mitochondria; S, starch granules; Vuc, vacuole; W, cell wall. Bar = 1 Grn. 125
126
MOLINE
FIG. 3. Part of a palisade parenchyma cell 10 days after inoculation with PMV. Nucleus (AT) contains little chromatin; with the exception of t.he nucleolus (&ju), virus particles (5’) are numerous in cytoplasm. Chloroplasts (Ch) contain many vesicles (Ve). Bar = 1 pm. FIG. 4. Typical palisade cell 12 days after inoculat,ion, with rows of tubrdes (T) developing in chloroplasts (Ch) and virus (V) throughout t,he cyt,oplasm. Vat, vacuole. Bar = 1 pm.
unable to visualize viral particles in chlorc,plasts, t.hey obtained evidence for chloroplast, involvement in viral synthesis at the lightand electron-microscopic levels. Ushiyama and Matthews (1970) suggest that viral nucleic acid may be synthesized in T‘I’RIVinfected chloroplast vesicles.
Allen (1972) found wild cucumber mosaic virus in mesophyll chloroplasts of dlarah oregaws, which contained vesicles similar to t,hose produced by TYAIV in Chinese cabbage (Chalcroft and Pllatthews, 1966; Gerola ef al., 1966). This type of vesicle was also found in PAN-infected D. st~arno?~iwn leaves. In addition, another more complex tubule network was found in chloroplasts in lat,er stages of infection. Both types of chloroplast vesiculations are associated with P1IV infection. The small vesicles seen attached to the outer chloroplast membrane in Fig. 5b appear to have a different origin than the tubule network that. ramifies throughout the chloroplast, in Fig. 5a. When sectioned in various planes, the vesicles bound to t,he outer membranr appear to be cup-shaped depressions of the membranes resembling pinocytot,ic vesicles (Figs. 3, 5b). While the t,ubular networks may arise from t,he outer membrane, they are not restricted in shape or size (Fig. .~a, 8). The possibility also exists that’ the tubular networks are formed from thylakoid membranes t,hat have been modified as a result of PMV infection. Indeed, some of the larger tubules contai’ned vesiclrs attached to the membrane system. The t,ubes t,hat, Hitchborn and Hills (Matthews, 1970) obtained from negative13 stained extracts of Chinese cabbage leaves infected with a necrotic strain of TYRIV resemble the tubules found in PJIV-infected D. stra~nor~iu~~z chloroplasts (Fig. 5a). The extent of chloroplast vesiculation ma) be expressed quite differently in t,wo hosts infwted with the same virus (Chalcroft and IIIatthews, 1966; Matthews, 1970). PMVinfected Sicotiana glutinosa chloroplasts show only the small outer membrane-bound vesicles (RIoline, 1973), as opposed to the two types of vesicles found in PRIV-infect,ed D. strmno~~izm.
5b FIG. 5. Two types of vesiculations in PhW infected chloroplasts (Ch) 16 days after inoculation. (aj Tubular (7’) networks t.hat appear to have arisen from swelling of the thylakoid membranes. (b) vesicles (I’ej attached to the outer chloroplast limiting membranes. I’, \‘irtls. Bar = 200 nm.
The association of PRIV particles with chloroplasts indicat,cs that some part of viral replication or assembly occurs in these organelles. The sequential observations of infected chloroplasts have shown that vesiculation proceeds as lesions develop on inoculated leaves. Examination of systemically infected leaves that arc yellow and severely deformed as a result of viral infection shows cells filled with \iruslikc particle.
128
MOLINK
FIG. 6. Viral inclusion (V) adjacent to chloroplasts (CH) in a systemically infected cell. Chloroplast degeneration is evident from destruction of thylakoid (Th) membrane system and grana stacks (G). Numerous vesicles (Ve) and large osmiophillic granules (Og) are also present in chloroplasts. W, cell wall; Vat, vacuole. Bar = 500 nm.
FIG. 7. Stages of degeneration of PM-infected chloroplasts (Ch). (a) V’ lrus-filled vesicles (Ve) originating from degenerating chloroplast. (b) Vesicles and tubules (T) filled with virus. (c) Virus-filled vesicles that have budded off from a degenerating chloroplast. Vat, vacuole. Bar = 500 nm. 129
130
MOLINE:
FIG. 8. Chloroplast (Ch) from systemically infected cell, showing tubular (T) network veloped within the infected plastid. Og, osmiophilic granules; l'h, thylakoid membrane mitochondria; V, virus; Vat, vacuole; G, grana stacks. Bar = 200 nm.
t,hat has de network; .lf
CYTOLOGY
OF BMV
IN
DATURA STRAMONIUM
FIG. 9. Nucleus (N) from systemically infected cell, showing crystalline inclusion take up stain, and degenerating organelles surround the nucleus. Ch, chloroplast; Nu, nucleolus; W, cell wall. Bar = 500 nm.
131
(V) which fails to AJ, mitochondria;
132
MOLINE
may be of different composition than t,he virus that crystallizes in the cytoplasm (Fig. 6). Since the virus has been shown to be multicomponent in nature (RIoline and Fries, 1973), one could postulate that one of the components could be assembled within nuclei. REFERENCES
FIG. 10. hIitochondria (M) and adjacent chloroplasts (Ch) in a systemically infected cell. v, virus; T-e, vesicles. Bar = 500 nm.
The close association of virus particles with nuclei could not be demonstrated during early stages of infectsion, as part,icles failed t’o take up stain if present in nuclei; however, the cryst,allization of viruslike particles in D. stranzonium nuclei suggests that PNV may have been assembled there also. The failure of viruslike particles to take up stain in the nuclei and their poor staining characteristics in chloroplasts may be the result of differential permeability of membranes to the osmium fixative that is responsible for the major part of the initial staining of cellular contents. Att,empts to increase contrast of particles within nuclei by staining tissue with uranyl acet.ate during the dehydration process (Hills and Plaskitt, 1968) were unsuccessful. The Inarticles within the nuclei (Fig. \ u 9) -I
ALLEN, T. C. (1972). Subcellular responses of mesophyll cells to wild cucumber mosaic virus. F’irology 47, 467474. BERCKS, R., and QUERFURTH, Cr. (1971). The use of the latex t.est for the detection of distant serological relationships among plant viruses. J. Get,. Viral. 12, 25-32. BERCBS, R., HUTH, W., KOENIG, IL, LESEM~NN, 1). PAUL, H. L., and QUERFURTH, G. (1971). Scrophularia mottle virus: Charakterisierung und Vergleich mit anderen Viren der turnip yellow mosaic virus-Gruppe. Phytopathol. Z. 71, 341-356. CHALCROFT, J., and R~.ITTHEws, R. E. F. (1966). Cytological changes induced by turnip yellow mosaic virus in Chinese cabbage leaves. Virology 28, 555-562. CX~LCROFT, J., and M.ITTHEWS, R. E. F. (1967). Role of virus strains and leaf ontogeny in the production of mosaic patterns by turnip yellow mosaic virus. TTiroZogy 33, 659673. F~v.4~1, M. A., and CONTI, G. G. (1970). Ultrastruct,ural observations on the chloroplasts of basil plants either infected with different viruses or treated with 3-amino-1,2,4-triazole. Protoplasma
TO, 153-166.
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