IN VITRO Vol. 12, No. 7, 1976
CHANGES IN S O L U B L E P R O T E I N S IN CALLUS CELLS OF H Y P E R S E N S I T I V E TOBACCO INOCULATED WITH TOBACCO MOSAIC VIRUS i R. N. BEACHY~ AND H. H. MURAKISHP
Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824 SUMMARY
Protein changes occurred in callus cells of hypersensitive tobacco (Nicotiana tabacum var. Xanthi-nc) 72 hr after inoculation with tobacco mosaic virus and incubation on a minimal growth medium. Two protein bands, serologically related to viral coat protein, were obtained from extracts of infected cells following electrophoresis on 7% and 10% polyacrylamide gels. An additional, slower migrating protein, perhaps due to virusinduced stimulation of a host protein, also was detected. Although local lesions appeared on callus after 40 hr of incubation, four proteins previously reported in lesion-bearing hypersensitive tobacco leaves were not found. The possible significance of this and the usefulness of a callus-TMV system as a tool to study virus-induced protein changes are discussed.
Key words: plant viruses; virus cultivation; tobacco callus; plant culture; plant pathology. INTRODUCTION
Leaves of Nicotiana tabacum varieties that possess the N gene for hypersensitivity produce local lesions (1) when rubbed with a solution of tobacco mosaic virus (TMV) in the presence of an abrasive such as carborundum or Celite. Callus cultures of hypersensitive tobacco respond similarly to TMV but do not require abrasives (2); abrasion may cause severe shock to leaf cells (3). Since callus cells can be cultured aseptically under controlled nutritional and environmental conditions (4) and since they are more easily disrupted than leaf cells, it seemed to us that callus cells would be suitable for studies of a biochemical nature. Several workers (5-7) have studied TMVinduced changes in soluble proteins in leaves or in leaf discs of hypersensitive tobacco by means of disc gel electrophoresis. They interpreted their results on the basis of acquired resistance (8, 9)
or the production of TMV coat protein. A similar virus-induced acquired resistance has been found in Xanthi-nc callus (White, unpublished data). The objective of the work reported herein was to characterize the Xanthi-nc tobacco callus-TMV system with respect to changes in soluble proteins. M A T E R I A L S AND M E T H O D S
Culture and inoculation of callus with TMV. Callus was induced from interveinal leaf segments of N. tabacum var. Xanthi-nc and maintained on a combined Murashige and Skoog (10), and White's (11) agar medium (MS-W) as previously described (12). Mannitol medium was similarly prepared except that mannitol was substituted for sucrose and glucose in equimolar amounts. Callus clumps consisting of 20 to 30 cells per aggregate were grown in Erlenmyer flasks of liquid MS-W medium on a rotary shaker (120 rpm) at 22 to 24°C. Cultures of fewer cells per ag1 Michigan Agricultural Experiment Station Journal gregate or single cells produced few lesions (2). Paper No. 7191. z Present address: Department of Plant Pathology, Recently, Otsuki and co-workers (13) found that protoplasts from Xanthi-nc leaves did not reCornell University, Ithaca, New York 14853. 3 To whom reprint request should be addressed. spond necrotically. Apparently surrounding ceils 517
518
BEACHY AND MURAKISHI
FIG. 1. Clumps of callus cells of N. tabacum Xanthi-nc 96 hr after inoculation with tobacco mosaic virus showing necrotic lesions. Bar represents 1 cm. are necessary for the expression of the N gene in hypersensitive tobacco. As previously described (4), about 1 g of callus cells was transferred to a large test tube containing 3 ml of MS-W medium and 300 /zg of purified TMV, common strain. To inoculate cells, the tube was subjected to a vibratory motion on a Vortex mixer (800 rpm) for 10 sec. The inoculated cells were transferred to a Miracloth filter-lined funnel, washed with mannitol liquid medium and incubated on mannitol agar in Petri plates at 22 to 24°C under diffuse fluorescent light (1000 lx). Local lesions were first visible 36 to 40 hr after inoculation and continued to enlarge to about 1 mm in diam after 96 hr (Fig. 1). Protein extraction and gel electrophoresis. Cell samples were harvested at 24 hr intervals after inoculation, weighed and cooled at 4°C. They were homogenized in a conical, glass tissue grinder in 5 volumes of buffer containing 0.1 M Tris, pH 8.0 (at room temperature), 0.5 M sucrose, 55 mM ascorbic acid, and 75 mM 2-mercaptoethanol. The homogenate was immediately passed through cheesecloth, centrifuged at 10,000 x g for 30 min, and the supernatant fluid was centrifuged at 144,000 × g for 2.5 hr in the Spinco 39L
rotor. The final supernatant was removed carefully with a pasteur pipet and the fluid made 5% with respect to sucrose. Bromphenol blue was added to serve as a marker and the solutions were subjected to electrophoresis on 7% or 10% polyacrylamide disc gels prepared according to Davis (14). After placing 0.1 ml of protein extract on the gel surface, electrophoresis was performed at 4°C with 2 mA/gel for 20 min, and 3 mA/gel until the dye marker reached the end of the gel. Gels were stained in a solution of amido black in 7% acetic acid, electrophoretically destained in 7% acetic acid, and scanned at 660 nm with a Gilford 2400 spectrophotometer equipped with a gel transporter. Immunodiffusion. To identify TMV coat protein, 7% polyacrylamide gels containing the extracted proteins were sliced lengthwise, and placed flat side down in a Petri plate. Antiserum to TMV, diluted 1:4 with saline in 0.9% agar at 37 to 40°C, was poured around the sliced gels and left at room temperature for 2 to 3 days. RESULTS AND DISCUSSION In preliminary tests inoculated cells, incubated on MS-W medium containing sucrose and glucose,
519
PROTEINS IN TMV-INFECTED CALLUS showed no consistent changes in extractable proteins following lesion development. Since changes in virus-induced proteins in infected ceils might be obscured by changes in host proteins in adjacent noninfected cells, mannitol was substituted for the usual carbon sources. Under these conditions cells became progressively greener over a 120-hr period, but little or no fresh weight increase in these cells occurred. Mannitol was not metabolized by tobacco cells (15). Extraction of TMV from these cultures (12) showed that virus increased between 24 and 72 hr after inoculation. The amount of TMV per g of callus grown on mannitol medium was the same as that from callus grown on sucrose and glucose-containing medium during 72 hr incubations. After 72 hr, however, no additional virus was extracted from cells on mannitol medium, but cells grown on the combined sucrose and glucose medium displayed a second peak of synthesis (12). This second smaller burst of synthesis may be due to spread of the infection and virus synthesis in neighboring, newly produced daughter cells (16). When protein extracts from healthy and TMVinfected callus cultures were compared by disc electrophoresis on 10% polyacrylamide gels, only 72
hrs. P.I.
.2! .$$ ~.00 .048
/
A~
~
Control ,,o0
Rf FIG. 2. Densitometrie profile of soluble proteins from Xanthi-ne callus infected with TMV or from noninoculated callas. Protein extraets were separated on 11)% polyaerylamide disc gels (3), stained with amido black, and scanned at 660 nm after destaining. Migration was from left to right. Rf values were determined by setting the migration of bromphenol blue equal to 1.00. The extracts were prepared 72 hr postinoeulation.
144
hrs.
P.I.
Rf FIG. 3. Densitometer profiles of soluble proteins from Xanthi-nc callus infected with TMV or from noninoculated eaUus. The extracts were prepared 144 hr postinoeulation and analyzed as described under Fig. 2. minor changes in stained proteins were observed 24 and 48 hr after inoculation. There were slight increases in the staining of two bands having migrations of 0.21 and 0.28 relative to the migration of the bromphenol blue dye. The only consistent changes in stainable proteins were in the top third of the gels; i.e. proteins which migrated relatively slowly (Fig. 1). At 72 hr after infection definitive changes in soluble proteins having Rf values of 0.048, 0.21, and 0.28 were observed. Immunodiffusion tests demonstrated that the latter two bands were serologically related to TMV coat protein. The 0.21 value is similar to the value of 0.22 reported for TMV coat protein produced in systemically infected leaves of Samsun tobacco (7). The 0.28 band may represent smaller subunits of X protein (17). The 0.048 band did not react with TMV antiserum and may reflect the stimulated synthesis of a host protein since a similar band was observed in the noninoculated controls, but at a lower intensity (Fig. 2). When extracts prepared from cells 120 and 144 hr after inoculation were similarly analyzed, a stained band with an Rf of 0.05 was greatly increased in extracts from infected tissues as compared to those from healthy tissues (Fig. 3). This protein, therefore, may represent a virus coded, or a virus induced protein. Kajita and Matsui (6), studying the formation of X protein in hypersensitive hosts, induced an initial systemic spread of infection in Xanthi-nc tobacco leaf discs by incubating at 38°C
520
BEACHY AND MURAKISHI
lesion formation in tobacco tissue culture. for 24 hr followed by incubation at 25°C for Phytopathology 61: 877-878. 24 hr to enhance lesion formation. They found 3. Matthews, R. E. F. 1970. Plant Virology. Academic a large excess of X protein which is generally Press, New York, pp. 165-171. considered the same as TMV coat protein. 4. Murakishi, H. H., J. X. Hartmann, L. E. Pelcher, Gianinazzi and Vallee (5) also found coat protein and R. N. Beachy. 1970. Improved inoculation of cultured plant cells resulting in high virus in Xanthi-nc tobacco leaves after TMV inoculatiter and crystal formation. Virology41: 365- 367. tion and incubation for 72 hr at 30° to 32°C. At 5. Gianinazzi, S., and J. C. Vailed. 1%9. Temptrature 20°C, no new protein bands were visible on their et synth~se de mattriel prottique viral ehez le densitometer tracings of electrophoretic patterns. Nicotiana Xanthi n. c. infect$ par le virus de la Mosaique du Tabac. C. R. Acad. Sei. [D] (Paris) Similarly, Van Loon and Van Kammen (7) de269: 593-595. tected very little coat protein in Samsun NN 6. Kajita, S., and C. Matsui. 1972. Effect of temperaor N. glutinosa leaves inoculated with TMV. On ture on the synthesis of tobacco mosaic virus the other hand, we found that large amounts of and X-protein in Nicotiana tabacum "Xanthi soluble viral coat protein were produced in callus nc." Phytopathology 62: 615-620. 7. Van Loon, L. C., and A. Van Kammen. 1970. tissues bearing local necrotic lesions. These Polyacrylamide disc electrophoresis of soluble results suggest that in the Xanthi-nc callus tissue leaf proteins from Nicotiana tabacum var. "Samthe production of necrotic lesions and the consun" and "Samsun NN." II. Changes in protein tinued production of viral coat protein and of constitution after infection with tobacco mosaic virus. Virology 40: 199-211. complete virus particles as shown earlier (12) may be dissimilar to the situation in intact leaf 8. Ross, A. F. 1961a. Localized acquired resistance to plant virus infection in hypersensitive hosts. tissues. Van Loon and Van Kammen (7) found Virology 14: 329-339. four new proteins with Rf values of 0.55 to 0.84 9. Ross, A. F. 1961b. Systemic acquired resistance in 10% gels of extracts from Samsun NN leaves induced by localized virus infection in plants. Virology 14, 340-358. bearing local lesions. They suggested that the new proteins might be involved in the phenomenon of 10. Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with acquired resistance (8). We did not observe tobacco tissue culture. Physiol. Plant 15: similar protein bands from Xanthi-nc callus. 473-497. Perhaps the absence of the four proteins in 11. White, P. R. 1963. The Cultivation of Animal and Plant Cells. 2nd ed. Ronald Press Co., callus tissue and the continued synthesis of large New York, pp. 57-63. amounts of viral RNA and coat protein in callus 12. Beachy, R. N., and H. H. Murakishi. 1973. Efeven after necrotic lesions are formed are somefects of cycloheximide on tobacco mosaic virus how related. synthesis in callus from hypersensitive tobacco. Virology 55: 320-328. Hypersensitive tobacco callus inoculated with TMV and incubated on mannitol medium should 13. Otsuki, Y., T. Shimomura, and T. Takebe. 1972. Tobacco mosaic virus multiplication and expresbe useful to study viral proteins or virussion of the N gene in necrotic responding induced host proteins under controlled cultural tobacco varieties. Virology 50: 45-50. and nutritional conditions. Since cell growth is 14. Davis, B. J. 1964. Disc electrophoresis--II. Method and application to human serum prominimal on mannitol medium, it should be teins. Ann. N.Y. Acad. Sci. 121: 404-427. possible to study changes that occur primarily in 15. Trip, P., G. Krotkov, and C. D. Nelson. 1%4. virus-affected cells and to better relate such Metabolism of mannitol in higher plants. Amer. changes to the appearance of local lesions and J. Bot. 51, 828-835. 16. Pelcher, L. E., H. H. Murakishi. and J. X. viral crystals (2). Hartmann. 1971. Kinetics of TMV-RNA synthesis and its correlation with virus accumulaREFERENCES tion and crystalline viral inclusion formation in 1. Holmes, F. O. 1938. Inheritance of resistance to tobacco tissue culture. Virology 47: 787-796. tobacco mosaic disease in tobacco. Phyto- 17. Takahashi, W. N., and M. Ishii. 1953. An abnormal pathology 28: 553-561. protein associated with tobacco mosaic virus in2. Beachy, R. N., and H. H. Murakishi. 1971. Local fection. Nature 169: 419-420.
CHANGES IN S O L U B L E P R O T E I N S IN CALLUS CELLS OF H Y P E R S E N S I T I V E TOBACCO INOCULATED WITH TOBACCO MOSAIC VIRUS i R. N. BEACHY~ AND H. H. MURAKISHP
Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824 SUMMARY
Protein changes occurred in callus cells of hypersensitive tobacco (Nicotiana tabacum var. Xanthi-nc) 72 hr after inoculation with tobacco mosaic virus and incubation on a minimal growth medium. Two protein bands, serologically related to viral coat protein, were obtained from extracts of infected cells following electrophoresis on 7% and 10% polyacrylamide gels. An additional, slower migrating protein, perhaps due to virusinduced stimulation of a host protein, also was detected. Although local lesions appeared on callus after 40 hr of incubation, four proteins previously reported in lesion-bearing hypersensitive tobacco leaves were not found. The possible significance of this and the usefulness of a callus-TMV system as a tool to study virus-induced protein changes are discussed.
Key words: plant viruses; virus cultivation; tobacco callus; plant culture; plant pathology. INTRODUCTION
Leaves of Nicotiana tabacum varieties that possess the N gene for hypersensitivity produce local lesions (1) when rubbed with a solution of tobacco mosaic virus (TMV) in the presence of an abrasive such as carborundum or Celite. Callus cultures of hypersensitive tobacco respond similarly to TMV but do not require abrasives (2); abrasion may cause severe shock to leaf cells (3). Since callus cells can be cultured aseptically under controlled nutritional and environmental conditions (4) and since they are more easily disrupted than leaf cells, it seemed to us that callus cells would be suitable for studies of a biochemical nature. Several workers (5-7) have studied TMVinduced changes in soluble proteins in leaves or in leaf discs of hypersensitive tobacco by means of disc gel electrophoresis. They interpreted their results on the basis of acquired resistance (8, 9)
or the production of TMV coat protein. A similar virus-induced acquired resistance has been found in Xanthi-nc callus (White, unpublished data). The objective of the work reported herein was to characterize the Xanthi-nc tobacco callus-TMV system with respect to changes in soluble proteins. M A T E R I A L S AND M E T H O D S
Culture and inoculation of callus with TMV. Callus was induced from interveinal leaf segments of N. tabacum var. Xanthi-nc and maintained on a combined Murashige and Skoog (10), and White's (11) agar medium (MS-W) as previously described (12). Mannitol medium was similarly prepared except that mannitol was substituted for sucrose and glucose in equimolar amounts. Callus clumps consisting of 20 to 30 cells per aggregate were grown in Erlenmyer flasks of liquid MS-W medium on a rotary shaker (120 rpm) at 22 to 24°C. Cultures of fewer cells per ag1 Michigan Agricultural Experiment Station Journal gregate or single cells produced few lesions (2). Paper No. 7191. z Present address: Department of Plant Pathology, Recently, Otsuki and co-workers (13) found that protoplasts from Xanthi-nc leaves did not reCornell University, Ithaca, New York 14853. 3 To whom reprint request should be addressed. spond necrotically. Apparently surrounding ceils 517
518
BEACHY AND MURAKISHI
FIG. 1. Clumps of callus cells of N. tabacum Xanthi-nc 96 hr after inoculation with tobacco mosaic virus showing necrotic lesions. Bar represents 1 cm. are necessary for the expression of the N gene in hypersensitive tobacco. As previously described (4), about 1 g of callus cells was transferred to a large test tube containing 3 ml of MS-W medium and 300 /zg of purified TMV, common strain. To inoculate cells, the tube was subjected to a vibratory motion on a Vortex mixer (800 rpm) for 10 sec. The inoculated cells were transferred to a Miracloth filter-lined funnel, washed with mannitol liquid medium and incubated on mannitol agar in Petri plates at 22 to 24°C under diffuse fluorescent light (1000 lx). Local lesions were first visible 36 to 40 hr after inoculation and continued to enlarge to about 1 mm in diam after 96 hr (Fig. 1). Protein extraction and gel electrophoresis. Cell samples were harvested at 24 hr intervals after inoculation, weighed and cooled at 4°C. They were homogenized in a conical, glass tissue grinder in 5 volumes of buffer containing 0.1 M Tris, pH 8.0 (at room temperature), 0.5 M sucrose, 55 mM ascorbic acid, and 75 mM 2-mercaptoethanol. The homogenate was immediately passed through cheesecloth, centrifuged at 10,000 x g for 30 min, and the supernatant fluid was centrifuged at 144,000 × g for 2.5 hr in the Spinco 39L
rotor. The final supernatant was removed carefully with a pasteur pipet and the fluid made 5% with respect to sucrose. Bromphenol blue was added to serve as a marker and the solutions were subjected to electrophoresis on 7% or 10% polyacrylamide disc gels prepared according to Davis (14). After placing 0.1 ml of protein extract on the gel surface, electrophoresis was performed at 4°C with 2 mA/gel for 20 min, and 3 mA/gel until the dye marker reached the end of the gel. Gels were stained in a solution of amido black in 7% acetic acid, electrophoretically destained in 7% acetic acid, and scanned at 660 nm with a Gilford 2400 spectrophotometer equipped with a gel transporter. Immunodiffusion. To identify TMV coat protein, 7% polyacrylamide gels containing the extracted proteins were sliced lengthwise, and placed flat side down in a Petri plate. Antiserum to TMV, diluted 1:4 with saline in 0.9% agar at 37 to 40°C, was poured around the sliced gels and left at room temperature for 2 to 3 days. RESULTS AND DISCUSSION In preliminary tests inoculated cells, incubated on MS-W medium containing sucrose and glucose,
519
PROTEINS IN TMV-INFECTED CALLUS showed no consistent changes in extractable proteins following lesion development. Since changes in virus-induced proteins in infected ceils might be obscured by changes in host proteins in adjacent noninfected cells, mannitol was substituted for the usual carbon sources. Under these conditions cells became progressively greener over a 120-hr period, but little or no fresh weight increase in these cells occurred. Mannitol was not metabolized by tobacco cells (15). Extraction of TMV from these cultures (12) showed that virus increased between 24 and 72 hr after inoculation. The amount of TMV per g of callus grown on mannitol medium was the same as that from callus grown on sucrose and glucose-containing medium during 72 hr incubations. After 72 hr, however, no additional virus was extracted from cells on mannitol medium, but cells grown on the combined sucrose and glucose medium displayed a second peak of synthesis (12). This second smaller burst of synthesis may be due to spread of the infection and virus synthesis in neighboring, newly produced daughter cells (16). When protein extracts from healthy and TMVinfected callus cultures were compared by disc electrophoresis on 10% polyacrylamide gels, only 72
hrs. P.I.
.2! .$$ ~.00 .048
/
A~
~
Control ,,o0
Rf FIG. 2. Densitometrie profile of soluble proteins from Xanthi-ne callus infected with TMV or from noninoculated callas. Protein extraets were separated on 11)% polyaerylamide disc gels (3), stained with amido black, and scanned at 660 nm after destaining. Migration was from left to right. Rf values were determined by setting the migration of bromphenol blue equal to 1.00. The extracts were prepared 72 hr postinoeulation.
144
hrs.
P.I.
Rf FIG. 3. Densitometer profiles of soluble proteins from Xanthi-nc callus infected with TMV or from noninoculated eaUus. The extracts were prepared 144 hr postinoeulation and analyzed as described under Fig. 2. minor changes in stained proteins were observed 24 and 48 hr after inoculation. There were slight increases in the staining of two bands having migrations of 0.21 and 0.28 relative to the migration of the bromphenol blue dye. The only consistent changes in stainable proteins were in the top third of the gels; i.e. proteins which migrated relatively slowly (Fig. 1). At 72 hr after infection definitive changes in soluble proteins having Rf values of 0.048, 0.21, and 0.28 were observed. Immunodiffusion tests demonstrated that the latter two bands were serologically related to TMV coat protein. The 0.21 value is similar to the value of 0.22 reported for TMV coat protein produced in systemically infected leaves of Samsun tobacco (7). The 0.28 band may represent smaller subunits of X protein (17). The 0.048 band did not react with TMV antiserum and may reflect the stimulated synthesis of a host protein since a similar band was observed in the noninoculated controls, but at a lower intensity (Fig. 2). When extracts prepared from cells 120 and 144 hr after inoculation were similarly analyzed, a stained band with an Rf of 0.05 was greatly increased in extracts from infected tissues as compared to those from healthy tissues (Fig. 3). This protein, therefore, may represent a virus coded, or a virus induced protein. Kajita and Matsui (6), studying the formation of X protein in hypersensitive hosts, induced an initial systemic spread of infection in Xanthi-nc tobacco leaf discs by incubating at 38°C
520
BEACHY AND MURAKISHI
lesion formation in tobacco tissue culture. for 24 hr followed by incubation at 25°C for Phytopathology 61: 877-878. 24 hr to enhance lesion formation. They found 3. Matthews, R. E. F. 1970. Plant Virology. Academic a large excess of X protein which is generally Press, New York, pp. 165-171. considered the same as TMV coat protein. 4. Murakishi, H. H., J. X. Hartmann, L. E. Pelcher, Gianinazzi and Vallee (5) also found coat protein and R. N. Beachy. 1970. Improved inoculation of cultured plant cells resulting in high virus in Xanthi-nc tobacco leaves after TMV inoculatiter and crystal formation. Virology41: 365- 367. tion and incubation for 72 hr at 30° to 32°C. At 5. Gianinazzi, S., and J. C. Vailed. 1%9. Temptrature 20°C, no new protein bands were visible on their et synth~se de mattriel prottique viral ehez le densitometer tracings of electrophoretic patterns. Nicotiana Xanthi n. c. infect$ par le virus de la Mosaique du Tabac. C. R. Acad. Sei. [D] (Paris) Similarly, Van Loon and Van Kammen (7) de269: 593-595. tected very little coat protein in Samsun NN 6. Kajita, S., and C. Matsui. 1972. Effect of temperaor N. glutinosa leaves inoculated with TMV. On ture on the synthesis of tobacco mosaic virus the other hand, we found that large amounts of and X-protein in Nicotiana tabacum "Xanthi soluble viral coat protein were produced in callus nc." Phytopathology 62: 615-620. 7. Van Loon, L. C., and A. Van Kammen. 1970. tissues bearing local necrotic lesions. These Polyacrylamide disc electrophoresis of soluble results suggest that in the Xanthi-nc callus tissue leaf proteins from Nicotiana tabacum var. "Samthe production of necrotic lesions and the consun" and "Samsun NN." II. Changes in protein tinued production of viral coat protein and of constitution after infection with tobacco mosaic virus. Virology 40: 199-211. complete virus particles as shown earlier (12) may be dissimilar to the situation in intact leaf 8. Ross, A. F. 1961a. Localized acquired resistance to plant virus infection in hypersensitive hosts. tissues. Van Loon and Van Kammen (7) found Virology 14: 329-339. four new proteins with Rf values of 0.55 to 0.84 9. Ross, A. F. 1961b. Systemic acquired resistance in 10% gels of extracts from Samsun NN leaves induced by localized virus infection in plants. Virology 14, 340-358. bearing local lesions. They suggested that the new proteins might be involved in the phenomenon of 10. Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with acquired resistance (8). We did not observe tobacco tissue culture. Physiol. Plant 15: similar protein bands from Xanthi-nc callus. 473-497. Perhaps the absence of the four proteins in 11. White, P. R. 1963. The Cultivation of Animal and Plant Cells. 2nd ed. Ronald Press Co., callus tissue and the continued synthesis of large New York, pp. 57-63. amounts of viral RNA and coat protein in callus 12. Beachy, R. N., and H. H. Murakishi. 1973. Efeven after necrotic lesions are formed are somefects of cycloheximide on tobacco mosaic virus how related. synthesis in callus from hypersensitive tobacco. Virology 55: 320-328. Hypersensitive tobacco callus inoculated with TMV and incubated on mannitol medium should 13. Otsuki, Y., T. Shimomura, and T. Takebe. 1972. Tobacco mosaic virus multiplication and expresbe useful to study viral proteins or virussion of the N gene in necrotic responding induced host proteins under controlled cultural tobacco varieties. Virology 50: 45-50. and nutritional conditions. Since cell growth is 14. Davis, B. J. 1964. Disc electrophoresis--II. Method and application to human serum prominimal on mannitol medium, it should be teins. Ann. N.Y. Acad. Sci. 121: 404-427. possible to study changes that occur primarily in 15. Trip, P., G. Krotkov, and C. D. Nelson. 1%4. virus-affected cells and to better relate such Metabolism of mannitol in higher plants. Amer. changes to the appearance of local lesions and J. Bot. 51, 828-835. 16. Pelcher, L. E., H. H. Murakishi. and J. X. viral crystals (2). Hartmann. 1971. Kinetics of TMV-RNA synthesis and its correlation with virus accumulaREFERENCES tion and crystalline viral inclusion formation in 1. Holmes, F. O. 1938. Inheritance of resistance to tobacco tissue culture. Virology 47: 787-796. tobacco mosaic disease in tobacco. Phyto- 17. Takahashi, W. N., and M. Ishii. 1953. An abnormal pathology 28: 553-561. protein associated with tobacco mosaic virus in2. Beachy, R. N., and H. H. Murakishi. 1971. Local fection. Nature 169: 419-420.
