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Ultrastructure of potato infected with potato virus M
VIROLOGY
C&238-%2
(1970)
Ultrastructure
of Potato
Infected
Paracrinkle, a potato disease caused by a strain of potato virus 1I (PVM) was first reported from England (WO), where King Edward potato (Solunum tuberosum L.) was believed to be universally infected with PVM (14). PVM, however, infects a large number of potato variet,ies and many other plants (16). Among the poOato variet,ies, pm can cause various symptoms ranging from a diffuse interveinal mottling, mosaic with or without rolling, and/or deformation of leaves to severe stunting, leaf deformation, and clumping (3, 8, 19). In the field, we observed PVM-infected King Edward potato plants growing without apparent symptoms except mild ruffling and slight leaf-rolling. This variety was imported from England to western Canada more than 40 years ago, and since then it, has been maintained in some privat,e gardens in central Alberta. Crude sap from potato plants infected with the Alberta isolate (AP-1) of PVM remained infectious even at a dilution of 1: 10,000. The high dilution end point (DEP) contradicted with previously reported DEP 1:40 to 1: 160 (3, 4). The high virus titer in infected leaf tissues may reflect a particular virus-host’ cell interact,ion, such as possible formation of virus aggregates, crystals, and/or inclusions in the infected cells. The association of PVM and its host tissues, however, has not been investigated especially at the ultrastructure level. The present investigation was init,iated in an attempt to understand the ultrastructure of potato infected with PVM. PVM (AP-1) used in this investigation was isolated from and maintained in King Edward potato under conditions previously described (12). The virus was ident,ified on the basis of differential host, symptomatology, morphology, and serology (Hiruki, unpublished data). Leaves and stems were sampled from linch tall seedlings which emerged from a PVM-infected potato tuber. The DEP of
with
Potato
Virus
M
PVM obtained from such a sample was 1: 10,000. Samples were cut into approximately 2-mm squares and fixed in 1.5% formalin-glutaraldehyde mixture in 0.1 M phosphate buffer, pH 7.0, at 4” for 10 hours. The fixative was prepared by mixing equal volumes of 3 % formalin and 3 % glutaraldehyde. The fixed materials were washed and postfixed in 2% osmium tetroxide for 7 in ethanol-propylene hours, dehydrated oxide series, and embedded in Araldite. Sections, 60-90 rnp thick, were cut with a diamond knife on a Reichert or a MT-2 Servall ultramicrotome and were picked up with 400-mesh copper grids without Formvar coating. Thin sections were stained with 2 % aqueous uranyl acetat.e for 2 hours and poststained with 0.2 % aqueous lead citrate for l-2 minutes at room temperature. Observations were made in a Philip 200 electron microscope (60 kv). Negatively stained virus preparations were made on loo-mesh copper grids with carbon-coated Formvar and were prepared by the leaf-dip technique (5). Small droplets of leaf exudate obtained from the cutting edge were touched directly to a droplet of 2 % sodium phosphotungstate, pH 7.0 (7). The preparations mere blotted dry and observed immediately in an electron microscope. In dip-preparations, numerous scattered rigid rods, averaging about 650 rnp long, believed to be PVM particles, were observed but no apparent aggregation was noticed (Fig. 1). Abundant individual virus rods and aggregates were found in the cytoplasm of infected cells (Fig. 2-6). In the sieve tubes, however, only individual virus rods but no aggregates were seen. Structures consisting of multiple membranes, frequently associated with an internal core(s), were often observed in the vacuoles (Fig. 7-10) and the cytoplasm (Fig. 7) of infected cells. The similar structures were not observed in tissues of healthy 238
~Prcs. 14. Electron micrographs of potato virus M (PI’M) particles and of portions of infected cells. (1) Negatively stained PVM particles in a dip-preparation of leaf exudates. (2) Abundant PT:M rods (R) in cytopIasm of an infected cell. (3) Small PVM aggregates (VA) in cyt.oplasm. (-1) PI’M particles aggregated in an old parenchymatous cell which has large vacuoles (1:) with thin Layer of cyt.oplasm around the cell wall. M = mitochondrion.
239
FIGS. 5-6. Portions of cells from PVM-infected leaves and stems, (5) A longitudinal section of a large PVM aggregate (VA). (6) A transverse and ohlique sectzion of a large PVM aggregate (VA). CW = cell wall, M = mitochondrion, V = vacuole. FIGS. 7-10. Sequential developments of multimembrane body (MB) in PVM-infected cells. (7) Initial stage (arrow). (8 and 9) Intermediate stages. Note: a MB was also found in cytoplasm. (10) An advanced stage of MB and its cross section showing details of the structure. CW = cell wall, M = mitochondrion, N = nucleus, NM = nuclear membrane, NO = nucleolus, V = vacuole. 240
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241
COMMUNICATIONS
plants. This struct,ure was tentatively named multimembrane body (MB). The sequential development,s of the MB from what appeared to be t,he initial stage to the advanced ones were observed (Fig. 7-10). At early stages of infection, these MBs resembled the “sac-like body” @a) in lacking internal material (Figs. 7 and 8). However, at advanced stages of infection, they consisted of a few cores in cross sect’ion. Each core was often densely surrounded with concentric lamellae. These MBs were circular, oval, and elliptic in shape (Fig. 7-10). Some &IBs that projected into the vacuole were connected with the cyt.oplasm by a basal stalk (Fig. 10). Neit>her pinwheels nor pinwheel-related structures were observed in infected cells. This appears t,o agree with the general concept (9, 10) that pinwheel structure occurs only in Gssue infected with viruses of the potato virus Y group (6). No virus particles and/or aggregates were found in chloroand nuclei. Occaplasts, mitochondria, sionally virus particles were closely associated with and often attached to the membrane of these organelles. Nevertheless, mitochondria and nuclei appeared to be unaltered morphologically. Chloroplasts in the infected cells were usually swollen with scattered stroma lamellae and fewer grana. These findings were similar to what is known with other viruses (2, 15, 21). Chloroplasts in PVXI-infected cells usually possessed larger and more abundant sbarch grains. Individual virus particles were usually confined to the juvenile cells where more mitochondria and fewer and smaller vacuoles were evident (Fig. 2). In the more matured cells, individual virus rods were less in number, and small aggregates were observed (Fig. 3). The cells containing large aggregates showed only a small number of individual virus rods (Figs. 5 and 6) _ A longit.udinal (Fig. 4) and cross section (Fig. 6) of an aggregate clearly illustrated the orientation of virus particles and a lattice structure. PVM particles in dip-preparations, as already mentioned, were scattered and without apparent aggregation, even though various sizes of aggregates were commonly
observed in thin sections. This suggests that the aggregates could either have been dissociated when they came int.0 cont,act w&h the droplet of neutral sodium phosphotungstate on t,he grid or were never included in the exudates of the leaf. Neither of these could reflect, the true orientation of PV;\l in the cells. Alt,hough virus aggregates have been found in a number of virus and host combinations (1, 11, 13, 17), this is t’he first time that PVM aggregat,es were found in t’hin sections of infected tissue. The time of appearance of rods, aggregates, and MB appeared t.o be related to age of infect,ed cells. This would suggest that the physiological alteration of the cells during infection may be responsible at least for the format,ion of virus aggregation. However, furt’her study is needed to assess the causal factor leading to t,he aggregation. At present, the cause, function, and constituent’s of MB in plant t.issues are not yet known. Our MB closely resembles the “sac-like body” observed by Weintraub and Ragetli (Wd) in broad bean infected with bean yellow mosaic virus at bhe initial stage of it.s development. However, these aut,hors did not, describe the subsequent st,ages. The MB also resembles carrot mottle virus (18) in t,he mode of protrusion into the vacuoles. From our observations, we believe that the formation of MB is a host reaction which is stimulated or accelerated by virus infect’ion. Furt.her study is needed to correlate MB with processes of intracellular degeneration and autolysis. ACKNOWLEDGMENT This investigation Alberta Agricultural AR-28648.
was supported in part by Research Trust Grant
REFERENCES 1. ALLEN, T. C., JR., and LYONS, A. R., PhzJtopathology 59, 1318-1322 (1969) 2. ARNOTT, H. J., and SMITH, K. M., Ultrastruct. Res. 19, 173-195 (1967) 3. BAONALL, R. H., LARSON, R. H., and WALKER, J. C., Wis. Agr. Exp. &a. Res. Bull. 198, 45 (1956). 4. BAWDEN, F. C., KASSANIS, B., and NIXON, H. L., J. Gen. Microbial. 4,21Ck219 (1950). 5. BRANDES, J., Nuchrichtenbl. Deut. P&menschutzdiensles 9, 151-152 (1957).
242
SHORT
COMMUNICATIONS
6. BRANDES, J., and BERCKS, IL, Advan. Virus Res. 11, l-24 (1965). ?. BHENNER, S., and HOKNE, 16. W., Bioehim. Biophys. Acta 34, 103-110 (1959). 8. DYI~STRA, T. P., Phytopathology 29, 597-606 (1939). 9. EDWARDSON, J. It., Amer. J. Bot. 53, 359-364 (1966) . 10. ED~AKDSON, J. R., PUHCIFULL, I>. E., and CHRISTIE, It. G., Virology 34,25@263 (1968). 11. GEROLA, F. M., BASSI, M., and BELL; G., Carylogia 18, 567-597 (1965). 12. HIRUKI, C., Phytopathology 60, 739-740 (1970). IS. HONDA, Y., and MATSUI, C., Phytopathology 58, 1230-1235 (1968). 1.4. KASSANIS, B., Xature (London) 188, 688 (1960). 15. KRASS, C. J., and FORD, 1~. IS,, Phytopathology 59, 431439 (1969). 16. MACLEOD; D. J., Can. Dept. Agr. Publ. 1150, 79 (1962).
f7. MATSUI, C., and YAMAGUCHI, A., A&an. Virus Res .12,127-174 (1966). 18. MURANT, A. F., GOULD, R. A., ROBERTS, I. M., and CATHRO, J., J. Gen. Viral. 4, 329341 (1969). f9. ROZENDAAL, A., and VAN SLOGTEREN, D. H. M., Proc. 3rd Conf. Potato Virus Dis. Lisse-Wageningen 1957, 20-36 (1958). 20. SALAMAN, R. N., and LE PELLEY, R. H., Proc. Roy. Sot. Ser. B 106, 14@175 (1930). 21. SUN, C. N., Protoplasma 60,426-434 (1965). 22. WEINTRAUD, M., and RAGETLI, H. W. J., Virology 28, 290-302 (1966). J. C. Tu C. HIRUKI Depurtment of Plant Science University of Alberta Edmonton, Alberta, Canada Accepted May 13, 1970
C&238-%2
(1970)
Ultrastructure
of Potato
Infected
Paracrinkle, a potato disease caused by a strain of potato virus 1I (PVM) was first reported from England (WO), where King Edward potato (Solunum tuberosum L.) was believed to be universally infected with PVM (14). PVM, however, infects a large number of potato variet,ies and many other plants (16). Among the poOato variet,ies, pm can cause various symptoms ranging from a diffuse interveinal mottling, mosaic with or without rolling, and/or deformation of leaves to severe stunting, leaf deformation, and clumping (3, 8, 19). In the field, we observed PVM-infected King Edward potato plants growing without apparent symptoms except mild ruffling and slight leaf-rolling. This variety was imported from England to western Canada more than 40 years ago, and since then it, has been maintained in some privat,e gardens in central Alberta. Crude sap from potato plants infected with the Alberta isolate (AP-1) of PVM remained infectious even at a dilution of 1: 10,000. The high dilution end point (DEP) contradicted with previously reported DEP 1:40 to 1: 160 (3, 4). The high virus titer in infected leaf tissues may reflect a particular virus-host’ cell interact,ion, such as possible formation of virus aggregates, crystals, and/or inclusions in the infected cells. The association of PVM and its host tissues, however, has not been investigated especially at the ultrastructure level. The present investigation was init,iated in an attempt to understand the ultrastructure of potato infected with PVM. PVM (AP-1) used in this investigation was isolated from and maintained in King Edward potato under conditions previously described (12). The virus was ident,ified on the basis of differential host, symptomatology, morphology, and serology (Hiruki, unpublished data). Leaves and stems were sampled from linch tall seedlings which emerged from a PVM-infected potato tuber. The DEP of
with
Potato
Virus
M
PVM obtained from such a sample was 1: 10,000. Samples were cut into approximately 2-mm squares and fixed in 1.5% formalin-glutaraldehyde mixture in 0.1 M phosphate buffer, pH 7.0, at 4” for 10 hours. The fixative was prepared by mixing equal volumes of 3 % formalin and 3 % glutaraldehyde. The fixed materials were washed and postfixed in 2% osmium tetroxide for 7 in ethanol-propylene hours, dehydrated oxide series, and embedded in Araldite. Sections, 60-90 rnp thick, were cut with a diamond knife on a Reichert or a MT-2 Servall ultramicrotome and were picked up with 400-mesh copper grids without Formvar coating. Thin sections were stained with 2 % aqueous uranyl acetat.e for 2 hours and poststained with 0.2 % aqueous lead citrate for l-2 minutes at room temperature. Observations were made in a Philip 200 electron microscope (60 kv). Negatively stained virus preparations were made on loo-mesh copper grids with carbon-coated Formvar and were prepared by the leaf-dip technique (5). Small droplets of leaf exudate obtained from the cutting edge were touched directly to a droplet of 2 % sodium phosphotungstate, pH 7.0 (7). The preparations mere blotted dry and observed immediately in an electron microscope. In dip-preparations, numerous scattered rigid rods, averaging about 650 rnp long, believed to be PVM particles, were observed but no apparent aggregation was noticed (Fig. 1). Abundant individual virus rods and aggregates were found in the cytoplasm of infected cells (Fig. 2-6). In the sieve tubes, however, only individual virus rods but no aggregates were seen. Structures consisting of multiple membranes, frequently associated with an internal core(s), were often observed in the vacuoles (Fig. 7-10) and the cytoplasm (Fig. 7) of infected cells. The similar structures were not observed in tissues of healthy 238
~Prcs. 14. Electron micrographs of potato virus M (PI’M) particles and of portions of infected cells. (1) Negatively stained PVM particles in a dip-preparation of leaf exudates. (2) Abundant PT:M rods (R) in cytopIasm of an infected cell. (3) Small PVM aggregates (VA) in cyt.oplasm. (-1) PI’M particles aggregated in an old parenchymatous cell which has large vacuoles (1:) with thin Layer of cyt.oplasm around the cell wall. M = mitochondrion.
239
FIGS. 5-6. Portions of cells from PVM-infected leaves and stems, (5) A longitudinal section of a large PVM aggregate (VA). (6) A transverse and ohlique sectzion of a large PVM aggregate (VA). CW = cell wall, M = mitochondrion, V = vacuole. FIGS. 7-10. Sequential developments of multimembrane body (MB) in PVM-infected cells. (7) Initial stage (arrow). (8 and 9) Intermediate stages. Note: a MB was also found in cytoplasm. (10) An advanced stage of MB and its cross section showing details of the structure. CW = cell wall, M = mitochondrion, N = nucleus, NM = nuclear membrane, NO = nucleolus, V = vacuole. 240
SHORT
241
COMMUNICATIONS
plants. This struct,ure was tentatively named multimembrane body (MB). The sequential development,s of the MB from what appeared to be t,he initial stage to the advanced ones were observed (Fig. 7-10). At early stages of infection, these MBs resembled the “sac-like body” @a) in lacking internal material (Figs. 7 and 8). However, at advanced stages of infection, they consisted of a few cores in cross sect’ion. Each core was often densely surrounded with concentric lamellae. These MBs were circular, oval, and elliptic in shape (Fig. 7-10). Some &IBs that projected into the vacuole were connected with the cyt.oplasm by a basal stalk (Fig. 10). Neit>her pinwheels nor pinwheel-related structures were observed in infected cells. This appears t,o agree with the general concept (9, 10) that pinwheel structure occurs only in Gssue infected with viruses of the potato virus Y group (6). No virus particles and/or aggregates were found in chloroand nuclei. Occaplasts, mitochondria, sionally virus particles were closely associated with and often attached to the membrane of these organelles. Nevertheless, mitochondria and nuclei appeared to be unaltered morphologically. Chloroplasts in the infected cells were usually swollen with scattered stroma lamellae and fewer grana. These findings were similar to what is known with other viruses (2, 15, 21). Chloroplasts in PVXI-infected cells usually possessed larger and more abundant sbarch grains. Individual virus particles were usually confined to the juvenile cells where more mitochondria and fewer and smaller vacuoles were evident (Fig. 2). In the more matured cells, individual virus rods were less in number, and small aggregates were observed (Fig. 3). The cells containing large aggregates showed only a small number of individual virus rods (Figs. 5 and 6) _ A longit.udinal (Fig. 4) and cross section (Fig. 6) of an aggregate clearly illustrated the orientation of virus particles and a lattice structure. PVM particles in dip-preparations, as already mentioned, were scattered and without apparent aggregation, even though various sizes of aggregates were commonly
observed in thin sections. This suggests that the aggregates could either have been dissociated when they came int.0 cont,act w&h the droplet of neutral sodium phosphotungstate on t,he grid or were never included in the exudates of the leaf. Neither of these could reflect, the true orientation of PV;\l in the cells. Alt,hough virus aggregates have been found in a number of virus and host combinations (1, 11, 13, 17), this is t’he first time that PVM aggregat,es were found in t’hin sections of infected tissue. The time of appearance of rods, aggregates, and MB appeared t.o be related to age of infect,ed cells. This would suggest that the physiological alteration of the cells during infection may be responsible at least for the format,ion of virus aggregation. However, furt’her study is needed to assess the causal factor leading to t,he aggregation. At present, the cause, function, and constituent’s of MB in plant t.issues are not yet known. Our MB closely resembles the “sac-like body” observed by Weintraub and Ragetli (Wd) in broad bean infected with bean yellow mosaic virus at bhe initial stage of it.s development. However, these aut,hors did not, describe the subsequent st,ages. The MB also resembles carrot mottle virus (18) in t,he mode of protrusion into the vacuoles. From our observations, we believe that the formation of MB is a host reaction which is stimulated or accelerated by virus infect’ion. Furt.her study is needed to correlate MB with processes of intracellular degeneration and autolysis. ACKNOWLEDGMENT This investigation Alberta Agricultural AR-28648.
was supported in part by Research Trust Grant
REFERENCES 1. ALLEN, T. C., JR., and LYONS, A. R., PhzJtopathology 59, 1318-1322 (1969) 2. ARNOTT, H. J., and SMITH, K. M., Ultrastruct. Res. 19, 173-195 (1967) 3. BAONALL, R. H., LARSON, R. H., and WALKER, J. C., Wis. Agr. Exp. &a. Res. Bull. 198, 45 (1956). 4. BAWDEN, F. C., KASSANIS, B., and NIXON, H. L., J. Gen. Microbial. 4,21Ck219 (1950). 5. BRANDES, J., Nuchrichtenbl. Deut. P&menschutzdiensles 9, 151-152 (1957).
242
SHORT
COMMUNICATIONS
6. BRANDES, J., and BERCKS, IL, Advan. Virus Res. 11, l-24 (1965). ?. BHENNER, S., and HOKNE, 16. W., Bioehim. Biophys. Acta 34, 103-110 (1959). 8. DYI~STRA, T. P., Phytopathology 29, 597-606 (1939). 9. EDWARDSON, J. It., Amer. J. Bot. 53, 359-364 (1966) . 10. ED~AKDSON, J. R., PUHCIFULL, I>. E., and CHRISTIE, It. G., Virology 34,25@263 (1968). 11. GEROLA, F. M., BASSI, M., and BELL; G., Carylogia 18, 567-597 (1965). 12. HIRUKI, C., Phytopathology 60, 739-740 (1970). IS. HONDA, Y., and MATSUI, C., Phytopathology 58, 1230-1235 (1968). 1.4. KASSANIS, B., Xature (London) 188, 688 (1960). 15. KRASS, C. J., and FORD, 1~. IS,, Phytopathology 59, 431439 (1969). 16. MACLEOD; D. J., Can. Dept. Agr. Publ. 1150, 79 (1962).
f7. MATSUI, C., and YAMAGUCHI, A., A&an. Virus Res .12,127-174 (1966). 18. MURANT, A. F., GOULD, R. A., ROBERTS, I. M., and CATHRO, J., J. Gen. Viral. 4, 329341 (1969). f9. ROZENDAAL, A., and VAN SLOGTEREN, D. H. M., Proc. 3rd Conf. Potato Virus Dis. Lisse-Wageningen 1957, 20-36 (1958). 20. SALAMAN, R. N., and LE PELLEY, R. H., Proc. Roy. Sot. Ser. B 106, 14@175 (1930). 21. SUN, C. N., Protoplasma 60,426-434 (1965). 22. WEINTRAUD, M., and RAGETLI, H. W. J., Virology 28, 290-302 (1966). J. C. Tu C. HIRUKI Depurtment of Plant Science University of Alberta Edmonton, Alberta, Canada Accepted May 13, 1970