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Cauliflower mosaic virus in the nucleus of Nicotiana
VIROLOGY
146,141-145
(1985)
Cauliflower
Mosaic Virus in the Nucleus of Nicotima
OLGA
GRACIA
AND R. J. SHEPHERD’
Estacim Exp. Agrop. Mendoza, INTA, 5507 Lujan de Cuyo, Mendma, Argentina, and Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546 Received March 12, 1985;accepted June 11, 1985 An isolate of cauliflower mosaic virus that occurs in nuclei of Niwtiam is reported. Affected nuclei become greatly enlarged and filled with virions without the electron-dense matrix material characteristic of cytoplasmic sites of virus assembly. Conventional cytoplasmic inclusions with embedded virus occurred in other cells of the same tissue. 0 1985 Academic
Press. Inc.
The ultrastructure of cells infected with cauliflower mosaic virus (CaMV) has been studied extensively (1-6). Cytoplasmic inclusions with spherical virions embedded in an electron-dense matrix are characteristic for CaMV and other members of the caulimovirus group (4, 7). They are believed to be sites of virus assembly in the cytoplasm. In addition to the typical inclusion bodies present in the cytoplasm, a uniformly electron-dense inclusion body was found in chloroplasts induced by the CM4-184 strain of CaMV (8). Other cytopathological effects are enlarged plasmodesmata in cell wall extrusions, masses of tubules or vesicles associated with the cell wall, and morphological alterations in nuclei and mitochondria (9). The virions of CaMV in infected cells of Brassica and other cruciferae are mainly associated with inclusion bodies; only occasionally are a few virions found free in the cytoplasm (.2,&s). In addition there is considerable evidence that viral DNA replicates in inclusion bodies in the cytoplasm (10). Consequently, it was unexpected when we recently found CaMV virions in the nu’ Author addressed.
to whom requests for reprints
should be
141
cleus of infected Nicotianu clevelandii. This seemed to be an exceptional observation for a virus that may replicate and assemble in the cytoplasm. During a survey of viruses affecting cruciferous hosts in Mendoza province of Argentina, CaMV was frequently encountered. These virus isolates were readily sap transmissible to various members of the Cruciferae. Surprisingly, some solanaceous plants also were found to be susceptible, viz., Datura stramonium and N. cleuelandii gave local reactions, and in some cases systemic infections. Other solanaceous species such as N. glutinosa, N. rustica, and N. tabacum reacted with chlorotic lesions on inoculated leaves. Since strains of CaMV that systemically infect noncruciferous plants are rare, the identity of these virus isolates was confirmed by serological reactions in agar-gel diffusion tests with antiserum to the CM4-184 strain of CaMV (11). Three isolates of CaMV that induced systemic infections in D. stramonium and N. clevehndii were selected for further study. These isolates, designated W260, W262, and W283, were examined for the type of inclusion bodies. For this purpose systemically infected tissue, 4-6 weeks after infection, was fixed in cacodylate-buffered 5% glutaraldehyde, postfixed in 1%
0042-6822185$3.00 Copyright All rights
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
142
SHORT COMMUNICATIONS
FIG. 1. Ultrastructure of CaMV-infected h? cfevelundii cells. (a) Nucleus containing a large number of virions uniformly distributed in the nucleoplasm. (b) A nucleus with a cluster of virions. (c) A cluster of virions (left center) and dispersed virions (upper right) in nuclei. A cell wall swelling with enlarged plasmodesmata is visible at the upper left. The scale bars in each case represent 500 nm.
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FIG. 2. Enlarged views of CaMV-infected scattered virions. (b) Cytoplasmic inclusion ‘esents 150 nm.
COMMUNICATIONS
N. clevelundii cells. (a) Portion of a nucleus enelor sing showing the same type of particles. The scale bar I“ep-
144
SHORT
COMMUNICATIONS
osmium tetroxide, treated with 2% uranyl acetate, dehydrated in acetone, and embedded in an Epon-Araldite mixture. Thin sections were stained with uranyl acetate and lead citrate prior to examination in a Siemens Elmiscop electron microscope. Typical inclusion bodies were induced by two of the isolates in both cruciferous and solanaceous hosts. Infections induced by W262 and W233 in Brct.~sicacampestris and N. clevelandii showed typical inclusion bodies with large amounts of virus. Mitochondria were swollen with disrupted cristae. In addition nuclei were observed to have enlarged nucleoli; however, no virions were seen inside these nuclei. In contrast many nuclei of N. clevelandii infected with the W260 isolate showed heavily stained virus particles about 50 nm in diameter. These were loosely spread throughout the nucleoplasm where they occurred in great numbers, either uniformly filling the nucleoplasm where it was not occupied by heterochromatin (Fig. 1A) or dispersed over a less dense background (Fig. lC, 2A). In some areas virions were clustered but did not form crystalline arrays (Fig. lB, 1C). No association of virus particles with the nucleolus was observed. In W260-infected N. cleuelundii not every cell contained virus in the nucleus. Instead many cells contained typical cytoplasmic inclusion bodies with an electron-dense matrix and embedded virions. Adjoining cells might contain virus in the nucleus, however, although such cells were never found to have cytoplasmic inclusions as well. The appearance of virus in nuclei and cytoplasmic inclusion bodies was identical (Fig. 2A, B). W260 in B. campesttis and D. stramonium was present in typical inclusion bodies. The proportion of nuclei-containing virus particles varied from relatively few in one batch of plants (examined 48 days after infection) to approximately 50% in another batch of plants (34 days after infection). Enzyme-linked immunosorbent tests were carried out with several N. clevelandii plants infected with W260 to ensure that the infection was CaMV in origin. Small
discs of tissue (lo-12 in number) were removed from each plant with a cork borer and homogenized, after weighing, in phosphate-buffered saline at a tissue:buffer ratio of 1:9 (1 g/9 ml). Aw values of triplicate wells of each sample were compared with purified CaMV at a known concentration and the values were converted to amount of virus per gram of fresh tissue. Extracts of six infected N. clevelundii plants gave values of 1.04,0.30,0.73,0.62,0.70, and 0.59 pg virus per gram tissue. Infected B. campestris (turnip var. “Just Right”) gave a value of 0.88 pg per gram when infected with W260 and 3.34 pg per gram when infected with CaMV strain CM1341. The spherical particles observed in the nuclei of N. clevelundii are believed to be vlrions of CaMV because of their characteristic size. Their appearance is identical to virions in cytoplasmic inclusion bodies contained in the same tissue. Although no definitive labeling procedure (e.g., ferritinlabeled antibody) was carried out to establish that the spherical particles in the nuclei were bona fide CaMV particles, their unique size suggests they were. For this reason it seems unlikely the particles were those of a contaminating virus. It also seems unlikely that the spherical particles are a newly induced cytological structure arising as a consequence of perturbed plant metabolism. Other investigators have reported the virions of caulimoviruses to occur in the nuclei of infected cells. Hearon and Lawson (I.@, for example, found particles of carnation etched ring virus in the nuclei of Saponaria vwcaria The presence of large amounts of virus in nuclei without accompaniment by cytoplasmic inclusion bodies in the cells suggests CaMV DNA might replicate in nuclei of cells of some hosts. An alternative explanation is that virus replicates in the cytoplasm and is then transported into the nucleus. If the latter applies, the cytoplasmic inclusion bodies in which virus DNA is believed to replicate (10) are missing from those cells in which virus is transported into the nucleus.
SHORT
COMMUNICATIONS
REFERENCES 1. FUJISAWA, I., RUBIO-HUERTOS, M., MATSUI, C., and 57,1130-1132 YAMAGUCHI, A., Phgtopatho~ (1967). 2. RUBIO-HIJERTOS, M., MATSUI, C., YAMAGUCHI, A., and KAMEI, T., Phytopathology 58, 548-549 (1968). 3. MARTELLI, G. P., and CASTEUANO, M. A., J. Ga viral. 13,133-140 (1971). 4. CHRISTIE, R. G., and EDWARDSON, J. R., Fla Agr. Exp. Stat. Maogr. Ser. 9,121-122 (1977). 5. SHWA, T. A., SHEPHERD, R. J., and PETERSEN, L. J., V&-w 102.381-388 (1980). 6. XIONG, C., BALAZS, E., LEBEURIER, G., HINDELANG,
7.
8. 9. 10.
11. 12.
145
C., STOECKEL,M. E., and PORTE, A., J. Gen V&l 61,75-81 (1982). SHEPHERD, R. J., In “The Atlas of Insect and Plant Viruses” (K. Maramorosch, ed.), pp. 159-166. Academic Press, New York, 1977. SHEPHERD, R. J., RICHINS, R., and SHALLA, T. A., Virw 102,389-400 (1980). CONTI, G. G., VEGEITI, G., BASSI, M., and FAVALI, M. A., virdogy 47,694-700 (1972). MODJTAHEDI, N., VOLOVITCH, M., Sossomzov, L., HARBRICOT, Y., BONNEVILLE, J. M., and YOT, P., virdogy 133,289-300 (1984). HOWARTH, A. J., GARDNER, R. C., MELDING, J., and SHEPHERD, R. J., I&-o&v 112,678-685 (1981). HEARON, S. S., and LAWSON, R. H., PhytopatholosV 71,645-652 (1981).
146,141-145
(1985)
Cauliflower
Mosaic Virus in the Nucleus of Nicotima
OLGA
GRACIA
AND R. J. SHEPHERD’
Estacim Exp. Agrop. Mendoza, INTA, 5507 Lujan de Cuyo, Mendma, Argentina, and Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546 Received March 12, 1985;accepted June 11, 1985 An isolate of cauliflower mosaic virus that occurs in nuclei of Niwtiam is reported. Affected nuclei become greatly enlarged and filled with virions without the electron-dense matrix material characteristic of cytoplasmic sites of virus assembly. Conventional cytoplasmic inclusions with embedded virus occurred in other cells of the same tissue. 0 1985 Academic
Press. Inc.
The ultrastructure of cells infected with cauliflower mosaic virus (CaMV) has been studied extensively (1-6). Cytoplasmic inclusions with spherical virions embedded in an electron-dense matrix are characteristic for CaMV and other members of the caulimovirus group (4, 7). They are believed to be sites of virus assembly in the cytoplasm. In addition to the typical inclusion bodies present in the cytoplasm, a uniformly electron-dense inclusion body was found in chloroplasts induced by the CM4-184 strain of CaMV (8). Other cytopathological effects are enlarged plasmodesmata in cell wall extrusions, masses of tubules or vesicles associated with the cell wall, and morphological alterations in nuclei and mitochondria (9). The virions of CaMV in infected cells of Brassica and other cruciferae are mainly associated with inclusion bodies; only occasionally are a few virions found free in the cytoplasm (.2,&s). In addition there is considerable evidence that viral DNA replicates in inclusion bodies in the cytoplasm (10). Consequently, it was unexpected when we recently found CaMV virions in the nu’ Author addressed.
to whom requests for reprints
should be
141
cleus of infected Nicotianu clevelandii. This seemed to be an exceptional observation for a virus that may replicate and assemble in the cytoplasm. During a survey of viruses affecting cruciferous hosts in Mendoza province of Argentina, CaMV was frequently encountered. These virus isolates were readily sap transmissible to various members of the Cruciferae. Surprisingly, some solanaceous plants also were found to be susceptible, viz., Datura stramonium and N. cleuelandii gave local reactions, and in some cases systemic infections. Other solanaceous species such as N. glutinosa, N. rustica, and N. tabacum reacted with chlorotic lesions on inoculated leaves. Since strains of CaMV that systemically infect noncruciferous plants are rare, the identity of these virus isolates was confirmed by serological reactions in agar-gel diffusion tests with antiserum to the CM4-184 strain of CaMV (11). Three isolates of CaMV that induced systemic infections in D. stramonium and N. clevehndii were selected for further study. These isolates, designated W260, W262, and W283, were examined for the type of inclusion bodies. For this purpose systemically infected tissue, 4-6 weeks after infection, was fixed in cacodylate-buffered 5% glutaraldehyde, postfixed in 1%
0042-6822185$3.00 Copyright All rights
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
142
SHORT COMMUNICATIONS
FIG. 1. Ultrastructure of CaMV-infected h? cfevelundii cells. (a) Nucleus containing a large number of virions uniformly distributed in the nucleoplasm. (b) A nucleus with a cluster of virions. (c) A cluster of virions (left center) and dispersed virions (upper right) in nuclei. A cell wall swelling with enlarged plasmodesmata is visible at the upper left. The scale bars in each case represent 500 nm.
SHORT
FIG. 2. Enlarged views of CaMV-infected scattered virions. (b) Cytoplasmic inclusion ‘esents 150 nm.
COMMUNICATIONS
N. clevelundii cells. (a) Portion of a nucleus enelor sing showing the same type of particles. The scale bar I“ep-
144
SHORT
COMMUNICATIONS
osmium tetroxide, treated with 2% uranyl acetate, dehydrated in acetone, and embedded in an Epon-Araldite mixture. Thin sections were stained with uranyl acetate and lead citrate prior to examination in a Siemens Elmiscop electron microscope. Typical inclusion bodies were induced by two of the isolates in both cruciferous and solanaceous hosts. Infections induced by W262 and W233 in Brct.~sicacampestris and N. clevelandii showed typical inclusion bodies with large amounts of virus. Mitochondria were swollen with disrupted cristae. In addition nuclei were observed to have enlarged nucleoli; however, no virions were seen inside these nuclei. In contrast many nuclei of N. clevelandii infected with the W260 isolate showed heavily stained virus particles about 50 nm in diameter. These were loosely spread throughout the nucleoplasm where they occurred in great numbers, either uniformly filling the nucleoplasm where it was not occupied by heterochromatin (Fig. 1A) or dispersed over a less dense background (Fig. lC, 2A). In some areas virions were clustered but did not form crystalline arrays (Fig. lB, 1C). No association of virus particles with the nucleolus was observed. In W260-infected N. cleuelundii not every cell contained virus in the nucleus. Instead many cells contained typical cytoplasmic inclusion bodies with an electron-dense matrix and embedded virions. Adjoining cells might contain virus in the nucleus, however, although such cells were never found to have cytoplasmic inclusions as well. The appearance of virus in nuclei and cytoplasmic inclusion bodies was identical (Fig. 2A, B). W260 in B. campesttis and D. stramonium was present in typical inclusion bodies. The proportion of nuclei-containing virus particles varied from relatively few in one batch of plants (examined 48 days after infection) to approximately 50% in another batch of plants (34 days after infection). Enzyme-linked immunosorbent tests were carried out with several N. clevelandii plants infected with W260 to ensure that the infection was CaMV in origin. Small
discs of tissue (lo-12 in number) were removed from each plant with a cork borer and homogenized, after weighing, in phosphate-buffered saline at a tissue:buffer ratio of 1:9 (1 g/9 ml). Aw values of triplicate wells of each sample were compared with purified CaMV at a known concentration and the values were converted to amount of virus per gram of fresh tissue. Extracts of six infected N. clevelundii plants gave values of 1.04,0.30,0.73,0.62,0.70, and 0.59 pg virus per gram tissue. Infected B. campestris (turnip var. “Just Right”) gave a value of 0.88 pg per gram when infected with W260 and 3.34 pg per gram when infected with CaMV strain CM1341. The spherical particles observed in the nuclei of N. clevelundii are believed to be vlrions of CaMV because of their characteristic size. Their appearance is identical to virions in cytoplasmic inclusion bodies contained in the same tissue. Although no definitive labeling procedure (e.g., ferritinlabeled antibody) was carried out to establish that the spherical particles in the nuclei were bona fide CaMV particles, their unique size suggests they were. For this reason it seems unlikely the particles were those of a contaminating virus. It also seems unlikely that the spherical particles are a newly induced cytological structure arising as a consequence of perturbed plant metabolism. Other investigators have reported the virions of caulimoviruses to occur in the nuclei of infected cells. Hearon and Lawson (I.@, for example, found particles of carnation etched ring virus in the nuclei of Saponaria vwcaria The presence of large amounts of virus in nuclei without accompaniment by cytoplasmic inclusion bodies in the cells suggests CaMV DNA might replicate in nuclei of cells of some hosts. An alternative explanation is that virus replicates in the cytoplasm and is then transported into the nucleus. If the latter applies, the cytoplasmic inclusion bodies in which virus DNA is believed to replicate (10) are missing from those cells in which virus is transported into the nucleus.
SHORT
COMMUNICATIONS
REFERENCES 1. FUJISAWA, I., RUBIO-HUERTOS, M., MATSUI, C., and 57,1130-1132 YAMAGUCHI, A., Phgtopatho~ (1967). 2. RUBIO-HIJERTOS, M., MATSUI, C., YAMAGUCHI, A., and KAMEI, T., Phytopathology 58, 548-549 (1968). 3. MARTELLI, G. P., and CASTEUANO, M. A., J. Ga viral. 13,133-140 (1971). 4. CHRISTIE, R. G., and EDWARDSON, J. R., Fla Agr. Exp. Stat. Maogr. Ser. 9,121-122 (1977). 5. SHWA, T. A., SHEPHERD, R. J., and PETERSEN, L. J., V&-w 102.381-388 (1980). 6. XIONG, C., BALAZS, E., LEBEURIER, G., HINDELANG,
7.
8. 9. 10.
11. 12.
145
C., STOECKEL,M. E., and PORTE, A., J. Gen V&l 61,75-81 (1982). SHEPHERD, R. J., In “The Atlas of Insect and Plant Viruses” (K. Maramorosch, ed.), pp. 159-166. Academic Press, New York, 1977. SHEPHERD, R. J., RICHINS, R., and SHALLA, T. A., Virw 102,389-400 (1980). CONTI, G. G., VEGEITI, G., BASSI, M., and FAVALI, M. A., virdogy 47,694-700 (1972). MODJTAHEDI, N., VOLOVITCH, M., Sossomzov, L., HARBRICOT, Y., BONNEVILLE, J. M., and YOT, P., virdogy 133,289-300 (1984). HOWARTH, A. J., GARDNER, R. C., MELDING, J., and SHEPHERD, R. J., I&-o&v 112,678-685 (1981). HEARON, S. S., and LAWSON, R. H., PhytopatholosV 71,645-652 (1981).