STUDIES ON TOBAOOO MOSAIC VIRUS USINE NYPERSENSITIVE TOBAOOO TISSUE OUlTURE I. IOOAI lESION FORMATION III OULTUREO OEIIS II. EEEEBT OE OYOIONEXINIOE ON TNV SYNTHESIS III. ONANBES IN SOLUBLE PROTEINS IN TNV INEEUTEII UAIIUS . Thesis for the Degree of PILD. MICHIGAN STATE umvsksm ROGER NEIL BEACHYQ I973 This is to certify that the thesis entitled STUDIES ON TOBACCO MOSAIC VIRUS USING HYPERSENSITIVE’ TABACCO TISSUE CULTURES presented by Roger Neil Beachy has been accepted towards fulfillment of the requirements for _BZLQ.____degree in WPlant Pathology ' Major professor 0-7639 ‘séi-HE—esflfl Awhwu , ABSTRACT STUDIES ON TOBACCO MOSAIC VIRUS USING HYPERSENSITIVE TOBACCO TISSUE CULTURES I. LOCAL LESION FORMATION IN CULTURED CELLS II. EFFECT OF CYCLOHEXIMIDE ON TMV SYNTHESIS III. CHANGES IN SOLUBLE PROTEINS IN TMV INFECTED CALLUS BY ROGER NEIL BEACHY Callus cultures derived from Nicotiana tabacum cvs Xanthi-nc, Samsun NN, and Burley NN, and from Nicotiana glutinosa reacted with necrotic local lesions 36-44 hours after inoculation with tobacco mosaic virus (TMV). The necrotic response occurred only in cells cul- tured in medium containing the Murashige and Skoog mineral and vitamin stocks, coconut water, 2,4-dichlorophenoxyacetic acid (2,4-D), and naphthaleneacetic acid (NAA). More than 90% of the virus synthesis in Xanthi-nc callus, as determined by the incorporation of 3H-uridine and 14C-leucine into TMV purified by sucrose gradient centrifugation, occurred after lesions became visible. Xanthi-nc callus was used to study the effect of the protein syn- thesis inhibitor, cycloheximide, on the synthesis of TMV-ribonucleic acid (RNA) and complete virus. Within one hour of treatment cyclohex- imide inhibited TMV synthesis by 90%, as shown by incorporation of 3H-uridine and 14C-leucine into purified virus. The synthesis of TMV- RNA, as measured by the incorporation of 3H-uridine into viral RNA Roger Neil Beachy separated by polyacrylamide gel electrophoresis, was not affected by 4 hours of cycloheximide treatment. The data suggest that the inhibition of complete virus is caused by the inhibition of virus protein synthe- sis and/or of virus assembly. Soluble proteins were extracted from TMV-infected and non-infected Xanthi-nc callus at 24 hour intervals after inoculation, and were sep- arated by polyacrylamide disc gel electrophoresis. Changes in extracted proteins (determined by staining with amido black) were evident from 48 to 144 hours after inoculation. Two bands of TMV coat protein, identi- fied antigenically, were evident 48-144 hours after inoculation. An additional, slower migrating protein was extracted from the infected callus but could not be positively correlated to virus infection. The results suggest that callus cultures may be useful in additional studies to identify viral proteins or changes in host proteins caused by virus infection. STUDIES ON TOBACCO MOSAIC VIRUS USING HYPERSENSITIVE TOBACCO TISSUE CULTURES I. LOCAL LESION FORMATION IN CULTURED CELLS II. EFFECT OF CYCLOHEXIMIDE ON TMV SYNTHESIS III. CHANGES IN SOLUBLE PROTEINS IN TMV INFECTED CALLUS By Roger Neil Beachy A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1973 N5\ ACKNOWLEDGEMENTS Sincere thanks go to my advisor, Dr. H. H. Murakishi, for his guidance and encouragement, especially during the early stages of this research. His ready help in evaluation of the manuscript was extremely valuable. His kindness and consideration as a teacher and friend will be remembered. I gratefully acknowledge the other members of the guidance com- mittee, Drs. R. S. Bandurski, A. H. Ellingboe, and R. S. Scheffer, for their enthusiasm of the research and for their critical evalua- tion of the thesis. Thanks to the faculty of the A. E. C. Plant Research Laboratory, especially Drs. H. Kende, and M. Jost, for the use of instruments, without which a great deal of this research could not have been pos- sible. The discussions with other faculty members and graduate stu- dents of the Department of Botany and Plant Pathology were most help— ful. A fellow graduate student, L. E. Pelcher, deserves a special word of appreciation for his instruction in techniques and for helpful dis- cussion and brain-storming throughout the course of the research. Financial support which made this research possible was given by the Michigan Agricultural Experiment Station. ii DEDICATION To my late father, and to my mother, whose influence and guidance contributed greatly to the shaping of personal goals; and above all to my dear wife, Terry, whose love, patience, and understanding gave me the confidence that I needed to reach toward those goals. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS . DEDICATION . LIST OF TABLES . LIST OF FIGURES. INTRODUCTION . CHAPTER 1. Local Lesion Formation in Tobacco Tissue Culture . References . CHAPTER 2. Effect of Cycloheximide on Tobacco Mosaic Virus Synthesis in Callus from Hypersensitive Tobacco. Summary. Introduction . Materials and Methods Callus cultures . Virus inoculation . Incorporation of 3H—uridine and 14C- leucine in Xanthi-nc callus. Extraction of labeled TMV from infected callus. RNA extraction and polyacrylamide gel electro- phoresis . . . . . Results Efficiency of virus infection . TMV synthesis in Xanthi- -nc callus . . Effect of cycloheximide on incorporation of C- leucine and 3H-uridine . . Effect of cycloheximide on complete virus synthesis. . Effect of cycloheximide on TMV- RNA synthesis. Discussion . References . iv Page ii iii vi vii \lO‘ 11 11 12 l4 l4 17 20 25 CHAPTER 3. SUMMARY AND APPENDIX . Proteins from Cultured Cells of Xanthi-nc Tobacco Inoculated with Tobacco Mosaic Virus . Summary. Introduction . Materials and Methods Callus cultures and TMV inoculation . Extraction buffer and electr0phoresis solutions . Soluble protein extraction and electr0phoresis. Immunodiffusion . . . . . . . . . . . Results and Discussion . References . RECOMMENDATIONS. Local lesion and viral crystal formation in tissue culture cells of different species of Nicotiana . Cultural procedures to increase the efficiency of virus infection . . . . . . . . . . An alternative inoculation procedure . The medium . Growth of Xanthi-nc callus on MS-W medium. SUPPLEMENTAL REFERENCE LIST. . . . . . . . . . . . Page 28 28 29 32 32 33 34 34 45 48 51 52 S3 57 58 64 6S LIST OF TABLES Table Page 1. Components of the basic media . . . . . . . . . . . . . . . 59 2. Effect of culture media on the number of local lesions formed on Burley NN callus inoculated with TMV. . . . . . . 61 vi Figure 10. 11. 12. 13. 14. 15. 16. LIST OF FIGURES Cell clumps of NN Burley tobacco with and without lesions 7 days after inoculation with tobacco mosaic virus . Comparison of the dilution curves of tobacco mosaic virus (TMV) inoculated on NN Burley tissue culture and Xanthi— nc leaves. 0 O O O O O O O O O O O O O O Radioactivity profiles of sucrose gradients. TMV growth curve in Xanthi-nc callus . Effect of increasing concentrations of cycloheximide on protein synthesis in Xanthi-nc callus. Effect of cycloheximide on the incorporation of 14C- leucine and 3H-uridine into TCA insoluble fractions of Xanthi-nc callus . Effect of cycloheximide on the incorporation of 3H-uridine into TMV in Xanthi-nc callus . Effect of cycloheximide (CHX, 1 pg per m1) on the incor- poration of 14C-leucine and 3H-uridine into TMV. . . Effect of cycloheximide (CHX, 1 pg per ml) on the incor- poration of 3H-uridine in RNA of virus infected Xanthi-nc callus . Soluble proteins from Xanthi-nc callus separated on 7% and 10% polyacrylamide disc gels . Soluble proteins from infected and non—infected Xanthi-nc callus after inoculation with TMV: 24 hours after inoculation . 48 hours after inoculation . 72 hours after inoculation . 96 hours after inoculation . 120 hours after inoculation. 144 hours after inoculation. vii Page 10 13 15 16 18 19 21 36 38 38 39 39 4O 4O Figure Page 17. Identification of TMV protein by antigenic reaction. . . . 42 18. Increase of TMV in callus tissue from cultivars of Nicotiana tabacum, and other Nicotiana species . . . . . . 55 19. Growth of Xanthi—nc callus sn solidified MS-W medium . . . 63 viii INTRODUCTION INTRODUCTION Studies of the hypersensitive or necrotic response to virus infec- tion have previously been made only in whole leaves or leaf discs: these studies were complicated by problems of metabolite uptake, sample variability, microbial contamination, etc. (Matthews, 1970). A cell system without these problems would be valuable for studies of the virus-cell interaction which leads to the necrotic or local lesion reaction. Use of plant tissue cultures is a possible solution to such prob- lems. Tissue cultures are known to take up radioactive precursors and specific metabolic inhibitors readily. In addition, use of relatively homogenous cell populations and aseptic conditions would eliminate some of the problems posed by Matthews (1970). Researchers in several lab- oratories have attempted to induce the necrotic reaction by inoculating cultured cells from known hypersensitive hosts, but none have been suc- cessful (Wu 93.3}; 1960; Hirth and Lebeurier, 1965; Nims et_a13 1967; Kassanis, 1967). Venketeswaran (1968) inoculated cultured cells of Nicotiana glutinosa with tobacco mosaic virus (TMV) and observed death of many cells and virus multiplication. A relatively high concentra- tion of inoculum (10 pg TMV per ml) was required, however, to trigger the response, which was a ”general" cell killing rather than the local- ized response seen on inoculated leaves (Holmes, 1929). 2 Recently Murakishi et_al_(l970) successfully inoculated callus derived from systemic tobacco with TMV, and showed that this method produced high virus titers and virus crystals. Murakishi's results indicated the potential usefulness of the technique for TMV infection of, and, perhaps, local lesion formation in, callus derived from hyper- sensitive tobacco cultivars. The first aim of my study was to culture callus tissue from sev- eral hypersensitive tobacco cultivars and to test the callus for the ability to form necrotic lesions in response to TMV infection. Next, I hoped to apply the system to studies in plant virology. After es- tablishing the cultural conditions for lesion formation (Appendix) and characterizing the response (Chapter 1), the system was used in the following studies: (1) to elucidate the TMV growth curve in the hyper- sensitive callus in relation to symptom development (Chapter 2); (2) to examine the effect of the protein synthesis inhibitor, cycloheximide, on the synthesis of TMV-RNA and complete virus during the period of rapid virus accumulation (Chapter 2); and (3) to evaluate the usefulness of the callus-TMV system for identifying changes in host and viral pro- teins which may occur as a result of virus infection (Chapter 3). CHAPTER 1 LOCAL LESION FORMATION IN TOBACCO TISSUE CULTURE Reprinted from PHYTOPATHOLOGY, Vol. 61, No. 7, 877-878, July 1971 ' ' S. A. Local Lesion Formation in Tobacco Tissue Culture R. N. Beachy and H. H. Murakishi Graduate Assistant and Professor, respectively, De partment of Botany and Plant Pathology, Michigan State University, East Lansing 48823. Michigan Agricultural Experiment Station Journal Paper 5114. The advice of L. E. Pelcher and J. X. Hartmann is gratefully acknowledged. ABSTRACT Callus cultures derived from Nicotiana glutinosa and N. tabacum, ‘NN Samsun’, ‘NN Burle ’,and ‘Xanthi- -nc’ responded with reddish— brown local le- sions when infected with tobacco mosaic virus (TMV). Cultured cells of NN Burley inoculated with 0.08.3 to 83 pg TMV/ml gave a virus dilution curve similar to that obtained with intact leaves of Xanthi- -nc tobacco plants Lesion formation on tissue cultures occurred only when cultures were grown on Murashige an Skoog medium supplemented with 2,-4 dichlorophenoxyacetic acid, naphthaleneacetic acid, and coconut water. Phytopathology 61:877- 878. Tobacco mosaic virus (TMV) infection of tobacco leaves usually results in systemic mosaic symptoms or in localized necrotic lesions. The latter “hypersensitive” reaction is governed by the “NN” gene, which is pres- ent in Nicotiana glutinosa L. (2) and N. tabacum L., ‘NN Samsun’ (3), ‘NN Burley’ (9), and ‘Xanthi-nc’ (8). Earlier work in this laboratory (5, 6) showed that TMV infection of tissue culture from a systemic host resulted in a high virus titer and a high percentage of cells with inclusions, but no macroscopic symptoms. In subsequent studies with tissue cultures from 13 species and cultivars of Nicotiana, local lesions were produced only in cell cultures from N. tabacum ‘NN Samsun’ (1). We now find that cell cultures from each of the hyper- sensitive hosts mentioned develop local lesions when the culture medium is suitably modified. Interveinal leaf segments (1-1.S cm?) of the hyper- sensitive hosts were surface-sterilized and washed in sterile, distilled water. Tissue explants were placed on a basic Murashige and Skoog (MS) medium (7) modi- fied by the addition of 240 11g of 2.4-dichlorophenoxy- acetic acid (2,4-D), 40 pg of naphthaleneacetic acid (NAA), and 50ml of coconut water/liter. and solidi- fied with 1% agar. Callus formed from the explants was transferred to a similarly modified liquid MS medium and grown in flasks on a rotary shaker (120 rpm). Cell suspensions (150-250 mg fresh wt) in the log phase of growth were aseptically inoculated by diSpers- in a TMV solution throughout the tissues with a Vortex mixer (Scientific Industries New York) as pre- viously described (6). The cells were then spread over the surface of modified MS agar and incubated at 22- 24C under 90 ft-c of fluorescent light. Reddish-brown necrotic lesions were macroscopically visible on cell clumps as early as 39 hr after inoculation (1), and were Fig. 1. without lesions 7 days after inoculation with tobacco mosaic virus. L : local lesions; S :symptomless tissues. Bar rep- resents 1 cm. Cell clumps of NN Burley tobacco with and 1-2 mm in diam after 6 days (Fig. l). Cultures inocu- lated with heat- or ultraviolet-inactivated TMV, with phosphate buffer. or with southern bean mosaic virus (which does not infect tobacco) never developed le- sions. To determine whether virus multiplication had occurred in the necrotic areas. lesions were excised and assayed for infectious virus. Excised lesions and sur- rounding symptomless tissues were ground separately 1:50 (w/v) in 0.1 M phosphate buffer. pH 7.3, and the homogenates were rubbed on opposite half-leaves of Xanthi-nc tobacco plants. Homogenates of excised lesions and of symptomless tissues produced an average of 433 and 21 lesions/haIf-Ieaf. respectively. Moreover. microscopic examination revealed that 25% of the cells within a 0.5-mm radius of the lesions contained the characteristic hexagonal crystals of TMV. Symptomlcss areas more distant from the lesions were free of inclu— sions. To determine the TMV dilution curve of cultured cells from hypersensitive tobacco, NN Burley cell sus- pensions were inoculated with 0.083-8311g TMV/ml (12 replicates /dilution). The log of the number of lesions produced per g fresh wt of cells increased lin- early with the Iog of the virus concentration between 0.083 and lOug virus ’ml. At higher virus concentra- tions, the slope of the curve leveled off. suggesting near saturation of the infectible sites. To determine the TMV dilution curve on intact plants, 24 half-leaves of greenhouse-grown Xanthi-nc tobacco were inoculated in a Latin square randomization scheme with 0.083 to 8311;! TMV/ml. Dilution curves of the tissue culture and leaf systems were similar in the range of virus con- centrations tested (Fig. 2). Tissue cultures of Xanthi-nc, NN Samsun, and N. glutinosa also produced lesions when inoculated with 3 878 3 U 1” I I ’I {a / U 5 f 3 2 $0 .1 '5 U 0 d O p a 3 11—15 NN Barley cells a - D Xanthi-ne leaves 0.] 1.0 ‘0 TMV Concentration [pg/ml] Fig. 2. Comparison of the dilution curves of tobacco mosaic virus (TMV) inoculated on NN Burley tissue cul- ture (Logro number of local lesions per g cells inoculated) and Xanthi-nc leaves (Logm number of local lesions per half-leaf). TMV. In previous studies [8. Venketeswaran, personal communication; and (4)], local lesions were not formed in cultured cells of N. glutinosa nor in hybrids of N. glutinosa and N. tobacum. Our positive results with tissues from N. glutinosa and the NN hybrids suggested that either the culture medium or the inoculation tech- nique we used was important for lesion formation. In earlier experiments with nonsupplemented MS medium and with White’s medium (10) supplemented with 2,4-D, NAA, and coconut water, no lesions were pro- duced on inoculated cells. Clearly, both the supplements and the substances present in the basic MS medium are crucial for lesion formation. In addition, the method of cell culture following inoculation is important. In repeated experiments, NN Burley cells were inoculated with 511g TMV/m1 and were incubated on liquid or agar media. Incubation in the liquid medium resulted PHYTOPATHOLOGY [V0]. 61 in 6 i 6 lesions/g, whereas incubation on the agar me- dium resulted in 125 1- 14 lesions/g. In more recent tests, the number of lesions produced in cultured cells increased fourfold over that of pre- vious experiments. Maintaining this higher efficiency of inoculation has not been consistent, however. Param- eters such as the growth medium, the cell culture tech- niques, and the physiologic state of the cells may be critical for optimizing the efiiciency of inoculation. Ex- periments are in progress to more precisely determine the extent that these factors are involved in lesion for- mation. The plant tissue culture-virus system we de- scribe should be a useful tool for studies of the virus- induced hypersensitive reaction. LITERATURE CITED 1. Bmcnv, R. N., 8: H. H. MURAKLSHI. 1970. Local lesion and viral crystal formation in tissue culture cells of different species and cultivars of Nicotiana. Phytopathology 60:1283 (Abstr.). 2. HOLMES, F. 0. 1929. Local lesions in tobacco mosaic. Bot. Gaz. 87:39-55. .3. HOLMES, F. O. 19.38. Inheritance of resistance to to- bacco mosaic disease in tobacco. Phytopathology 28: 553-561. 4. KASSANIS, B. 1967. Plant tissue culture, p. 537—56611: K. Maramorosch & H. Koprowski [ed]. Methods in Virology, Vol. 1. Academic Press, N.Y. 5. MURAKISHI, H. H., J. X. HARTMANN, R. N. Bucm‘. & L. E. PELCHER. 1971. Growth curve and yield of tobacco mosaic virus in tobacco callus cells. Virology 43:62-68. 6. MURAKism, H. H., J. X. HART‘MANN, L. E. PELCHER. & R. N. BEACHY. 1970. Improved inoculation of cul- tured plant cells resulting in high virus titer and crystal formation. Virology 41:365-367. 7. MURASIIIGE, T. 81 F. SKOOG. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physio]. Plantarum 15:473-497. 8. TAKAHASHI, W. N. 1956. Increasing the sensitivity of the local~lesion method of virus assay. Phytopathol- ogy 46:654-656. 9. VALLEAU, W. D. 1952. Breeding tobacco for disease resistance. Econ. Bot. 6:69-102. 10. WHITE, P. R. 1963. The cultivation of animal and plant cells. Ronald Press, N. Y. 228 p. CHAPTER 2 EFFECT OF CYCLOHEXIMIDE ON TOBACCO MOSAIC VIRUS SYNTHESIS IN CALLUS FROM HYPERSENSITIVE TOBACCO EFFECT OF CYCLOHEXIMIDE ON TOBACCO MOSAIC VIRUS SYNTHESIS IN CALLUS FROM HYPERSENSITIVE TOBACCO SUMMARY Local lesions were formed on callus derived from Nicotiana tabacum cv Xanthi-nc 36-44 hours following inoculation with tobacco mosaic virus (TMV). The necrotic lesions, however, had little or no inhibitory effect on virus synthesis. The greatest accumulation of 3H-uridine and 14C-leucine labeled virus occurred 48-84 hours after inoculation. When infected callus tissue was treated with cyclohexi- mide (1 pg per m1) during the period of rapid virus accumulation, the synthesis of host protein and complete virus was reduced by 90% within 1 hour. The synthesis of TMV-RNA was not affected even after 4 hours of treatment. The results suggest that cycloheximide prevented the formation of complete virus by inhibiting the synthesis of viral pro- tein and/or by preventing the assembly of virus particles. INTRODUCTION Cycloheximide (CHX) is a potent inhibitor of protein synthesis OWcKeehan and Hardesty, 1969) in plant tissue cultures (Nudel and Bam- berger, 1971). Reports indicate that it may stimulate or inhibit to- bacco mosaic virus (TMV) synthesis in inoculated leaves of Nicotiana tabacum. Ouchi g£_al_(1969) reported that 1 pg CHX per m1 stimulated virus synthesis in infected leaf discs, whereas higher concentrations 5 reduced synthesis. Takebe g£_al_(197l) and Zaitlin g£_al_(l968), using infected leaf protoplasts and leaf discs, respectively, reported near complete inhibition of TMV synthesis by CHX at 10-15 pg per ml. We re- examined the effect of CHX on the synthesis of TMV in tobacco tissue cultures and also studied the effects of CHX on the synthesis of TMV- RNA. Tissue cultures have certain advantages over leaf discs or proto- plasts used in previous work. Callus tissue can be cultured in a meta— bolically active state under aseptic conditions; thus, problems caused by tissue senescence or by microbial contaminants GMatthews, 1970) are avoided. We used the TMV and Xanthi-nc callus cultures previously described by Beachy and Murakishi (1971 a). The degree of virus infec- tion in such cultures is indicated by the number of necrotic lesions produced after inoculation. The conditions for maximum efficiency of infection were determined in a preliminary study. In addition, the virus growth curve was characterized because this had not previously been done for a hypersensitive callus culture. MATERIALS AND METHODS Callus cultures The culture medium was a combination of the media described by White (W) (1963) and Murashige and Skoog (MS) (1962). The W and MS media were prepared separately, each were brought to pH 5.9 and com- bined in a ratio of 3 MS:2 W. The combined medium contained 50 ml of coconut water 30 ug of kinetin, 250mg of 2,4-dichlorophenoxyacetic acid, and 40 pg of naphthaleneacetic acid per liter. The medium (MS-W) was sterilized by autoclaving. Solid medium was prepared by adding agar (1% by weight). 7 Stem pith cores (eXplants) of Nicotiana tabacum cv Xanthi-nc were removed aseptically with a cork borer, placed on solidified MS-W medium and grown under Gro-lux fluorescent lamps (80 foot candles). Callus growth usually became green pigmented within 2 weeks and was maintained by monthly transfers on solid medium. To prepare cells for inoculation, 1 gram of agar-grown callus was transferred to 20 m1 of liquid medium in 125 ml flasks and placed on a rotary shaker (120 rpm). After 6-9 days, 1-2 ml of resulting cell suspension was transferred with a large mouth pipette (3 mm opening) to fresh liquid medium. After 6-9 days of addi- tional growth, the cells had developed into small aggregates, 20-75 cells per aggregate, and were inoculated. Virus inoculation A common strain of TMV was purified according to Knight (1962) from leaves of N: tabacum cv Havana-38 inoculated 2-4 weeks earlier. The virus was freed of microorganisms by passage through an ultra- fine sintered glass filter. Suspension cell cultures were inoculated with 150 pg TMV per ml by the vibratory technique of Murakishi gt_al, (1970; 1971). Cells from several flasks were combined on funnels lined with Miracloth filters, washed with fresh medium and dispensed in tubes containing 3 m1 of MS-W medium. After adding the TMV suspension the tubes were vibrated for 10 seconds on a Vortex mixer (Scientific Products Corp., Evanston, I11.), the cells were collected on Miracloth-lined funnels, and washed with fresh liquid medium. The cell clumps were gently stirred to ensure randomization, transferred to filter pads resting on solid medium, and incubated at 24°C under the Gro-lux lamps (Pelcher 2.2.9.1., 1972). 8 Incorporation of 3H-uridine and l4C-leucine in Xanthi-nc callus The incorporation of radioactive metabolites into Xanthi-nc callus was determined by placing cell samples into 2 ml of liquid medium con- taining 3H-uridine (specific activity 9-27 curies per millimole) or 14C-L-leucine (specific activity 230 millicuries per millimole) from New England Nuclear. Cell samples were harvested, washed with ice cold 0.25 M sucrose containing 10'4 M uridine and/or 10‘3 M L-leucine, and ground in a conical glass homogenizer in 0.1 M Tris-HCl (pH 8.0) containing 0.25 M sucrose, 10'4 M uridine, 10‘3 M L—leucine, and 75 mM 2-mercaptoethanol. The homogenate was centrifuged at 1,000 g_for 10 minutes and aliquots of the supernatant were precipitated at 4°C in 10% trichloroacetic acid (TCA), collected on glass fiber filters, and washed with cold 5% TCA, 95% ethanol and ethanol:ether (1:1). The fil- ter-trapped materials were digested at 40°C for 4 hours with 0.5 m1 of a solution containing 20% NCS Solubilizer (Amersham/Searle, Des Plaines, 111.), 3.75% water, and 76.25% toluene. Ten milliliters of toluene- based POPOP-PPO solution was added and samples were counted in a Packard Tri-Carb, or Beckman LS-133 liquid scintillation counter with a counting efficiency of 17% for 3H, and 51% for 14C. Doubly labeled samples were corrected for bleedover using 14C-leucine standards prepared in an iden- tical manner. To determine the efficiency of the labeling procedure, randomly mixed callus samples were placed in liquid medium containing 2.5 pCi 3H-uridine per ml and 0.1 pCi 14C-leucine per m1. Callus samples were harvested after 1, 3, 6, 9, and 12 hours, and the acid-insoluble radio- activity was determined as described above. Both isotopes were incor- porated at a constant rate under these labeling conditions (Figure 6). 9 Cycloheximide (Sigma Chem. Co., St. Louis, Mo.) was prepared in liquid MS~W medium and sterilized by Millipore filtration. Extraction of labeled TMV from infected callus Infected cells were incubated in 3H-uridine (12.5-25 pCi per m1) and/or 14C-leucine (0.5-1.0 pCi per ml) in liquid medium, harvested by washing with 0.25 M sucrose containing 10'4 M uridine and 10'3 M leu- cine, weighed, and frozen. Thawed cells were ground in a glass homo- genizer in 0.1 M Tris-HCl, pH 8.0, containing 10'4 M uridine, 10‘3 leucine, and 75 mM 2-mercaptoethanol and centrifuged at 1,000 g_for 10 minutes. The supernatant fluid was centrifuged at 12,000 g_for 15 minutes. The final supernatant was layered on 1 m1 of 30% sucrose in the Tris buffer and centrifuged in the SW 39L rotor in the Spinco Model L ultracentrifuge at 35,000 rpm for 90 minutes. The final pellet was resuSpended in 1 m1 of water and given a low-speed centrifugation, and the resultant supernatant was layered on 10-40% linear sucrose gra- dients prepared in 0.01 M potassium phosphate buffer (pH 7.3). After centrifugation at 23,000 rpm for 90 minutes in the SW 25.1 rotor, gra- dients were fractionated with an ISCO Model D fractionator coupled to a Model UA-2 UV (254 nm) analyzer. One milliliter fractions were col— lected and co-precipitated with 0.5 mg bovine serum albumin by 10% TCA. Precipitates were trapped on glass fiber filters and radioactivity was determined as described above. Radioactivity profiles of sucrose gradients from extracts of TMV- infected and non-infected cells labeled with 3H-uridine were used to determine incorporation into TMV (Figure 3). The peak of radioactivity representing 3H-labeled TMV was found in fractions 9-11. TMV purified by the differential centrifugation procedure (Knight, 1962) sedimented to the same level under these conditions. 10 . 5 o\‘ TMV 0 ”won x10" . Q . ) 5 i8 B ib is rucnow (ml) Figure 3. Radioactivity profiles of sucrose gradients. TMV-infected and non-infected Xanthi-nc callus were incubated in 12.5 pCi 3H-uridine per m1 of medium and extracted for TMV as described in the text. The extracts were centrifuged in 10-40% sucrose gradients, and radioactiv- ity in 1 ml fractions was determined. Non-infected, 0--O, TMV-infected, o-—o. 11 RNA extraction and polyacrylamide gel electrophoresis Inoculated callus was incubated in 20 pCi 3H-uridine per ml, har- vested by washing with the cold sucrose solution, weighed, and RNA ex- tracted at 4°C by the sodium-dodecyl-sulfate (SDS)-phenol procedure described by Pelcher g£_§l_(l972). The RNA was taken up in the electro- phoresis buffer (0.04 M Tris, 0.02 M sodium acetate, 0.001 M EDTA and brought to pH 7.8 with glacial acetic acid) made 5% with respect to sucrose. The RNA concentration of the solution was determined assuming an Eg6éfim = 30' The 2.4% polyacrylamide gels were prepared as described by Bishop g£_al_(1967). Polymerized gels were allowed to stand in the electro- phoresis buffer containing 0.2% SDS (w/v) at 4°C for 72 hours prior to use. The gels were pre-run for 30 minutes at 5 mA per gel, and then loaded with 15 pg of the RNA in volumes of 15-25 pl. Following electro- phoresis for 90 minutes at 5 mA per gel (10 V per cm), the gels were scanned at 260 nm in a Gilford 2400 spectrophotometer with attached gel scanner. The gels were frozen in dry ice, sliced into 1 mm sections and the radioactivity was determined as previously described (Pelcher et_al, (1972). Specific activities (CPM per pg RNA) were determined by calculating the pg of RNA from optical density profiles of gels, and by establishing the CPM in RNA from the corresponding radioactivity profiles of the gels. RESULTS Efficiency of virus infection The efficiency of TMV infection of Xanthi-nc callus is expressed by the numbers of lesions produced (Beachy and Murakishi, 1971 a). The greatest number of lesions was produced on callus subcultured 2 times 12 in liquid medium prior to inoculation while fewer lesions were obtained from callus continuously subcultured. Rapidly growing cultures con- sisting mainly of single cells produced fewer local lesions than did slowly growing cultures composed of small (0.5-2 mm in diameter) cell aggregates with a "grainy" texture. Cultures composed of grainy cell aggregates were produced by carefully regulating the amount of cells transferred in the second liquid subculture. New explants were taken when the efficiency of infection began to drop. TMV synthesis in Xanthi-nc callus The TMV growth curve in Xanthi-nc callus was determined by measur- ing the incorporation of 3H-uridine (12.5 pCi per m1) and 14C-leucine (0.5 pCi per ml) into virus over 12 hour intervals until 144 hours after inoculation. TMV was purified from 1.5 grams of freshly harvested cells. The results are expressed in counts per minute (cpm) in TMV for each 12 hour interval. The rates of virus synthesis and total yields of TMV from inoculated Xanthi-nc callus varied slightly with each experiment but in general the growth curves were similar to the curve given in Figure 4. Virus synthesis was first detected 12-24 hours after inocu- 1ation, and the rate of synthesis increased until 60-72, or 72-84 hours. The high rate of synthesis was followed by a sharp decline (12-24 hours in duration) and then by a second peak of virus synthesis. The second burst of synthesis lasted for 12 or 24 hours and was followed by a drop in the rate of synthesis and the maintenance of the low rate through the remainder of the experiment. Local lesions were observed 36-44 hours after inoculation in all experiments and continued to enlarge until 84-96 hours (Figure 4). More than 90% of the extracted virus was produced after lesions appeared. Figure 4. extracted for TMV as described in the text. ification, radioactivity in TMV was determined as described. 5 :5 ’H-CPM x 10”2 in TMV/12 h (u—o) ? 13 ‘Lfi 0.57;» a). ll. diam 5., O a. "c-CPM x ld’iu TMV/l2 h (--) v N L 24 4a ' 72 96 120 HOURS AFTER INOCULATION I44 TMV growth curve in Xanthi-nc callus. for 12 hours in 12.5 pCi H-uridine and 0.5 pCi 14C-leucine per m1, and Cells were incubated After sucrose gradient pur- The upper portion of the figure indicates the diameter (mm) of the local lesions (with standard deviation) over the experimental period. 14 To determine whether or not lesions affected virus extraction, callus containing lesions and callus without lesions were homogenized in the presence of purified 3H-uridine-TMV. Analysis of sucrose gradients of the two extracts showed that extractability of virus was not affected by lesions in the callus. Effect of cycloheximide on incorporation of 14C-leucine and 3H-uridine The sensitivity of Xanthi-nc callus to cycloheximide (CHX) was de- termined by placing randomly mixed cell samples in liquid medium con- taining 14C-leucine (0.1 pCi per m1) and various concentrations of CHX for 3 hours. Radioactivity of acid insoluble fractions was determined. CHX at 1 pg per ml gave near maximum inhibition of 14C-leucine incor- poration (Figure 5) without cell damage. This concentration was used in all later experiments unless otherwise indicated. The effect of CHX on the incorporation of 14C-leucine and 3H-uri- dine into the acid-insoluble fraction as a function of time was deter- mined (Figure 6). CHX gave 50% inhibition of 3H-uridine incorporation in 12 hours and about 90% inhibition of 14C-leucine within 1 hour. There was no inhibition of the incorporation of either isotope into the acid soluble fraction. Under these conditions, CHX remained an effective inhibitor of protein synthesis for 16-20 hours. Thereafter, the rate of 14C-leucine incorporation approached that of the non-treated controls. The recov- ery of Tetrahymena and rat cells from CHX inhibition was observed pre— viously (Frankel, 1970; Yeh and Shils, 1969). Effect of cycloheximide on complete virus synthesis Forty-eight hours after inoculation, Xanthi-nc callus was treated with 20 pCi 3H-uridine per ml in the presence or absence of CHX. 15 0’ % INHIBITION .o ' J ' i ' ib CYCLOHEXIMIDE couc- Pg [M I. Figure 5. Effect of increasing concentrations of cycloheximide on protein synthesis in Xanthi-nc callus. Cells were incubated with 4C-leucine (0.1 pCi per ml) for 3 hours, washed with cold 0.25 M sucrose containing 10'3 M leucine, and homogenized in 0.1 M Tris- HCl, pH 8.0, with 0.25 M sucrose, 10-3 M leucine, and 75 mM 2-mer- captoethanol. The homogenate was centrifuged and aliquots were pre- cipitated with 10% trichloroacetic acid (TCA); the precipitate was collected on glass filters and washed with 5% TCA, ethanol, and ether. Radioactivity was determined by liquid scintillation. Per- cent inhibition was determined by comparing CPM in CHX treated and non-treated samples. Non-treated cells incorporated 1500-2000 CPM per 0.06 gram of cells in 4 experiments. ‘ CONTROL 3 I I ? 1 9 u '° 0 q 2 9 f‘ ‘\ O I‘ r0 '6 ; cux “ I. ‘ I E '1’ 1’ U I U n 3 IT Figure 6. Effect of cycloheximide (CHX, 1 ug Per m1) on the incorpora- tion of 14C—leucine (0.1 pCi per ml) (A) and 3H-uridine (1.0 pCi per ml) (0) into TCA insoluble fractions of Xanthi-nc callus. Radioactive ma— terials and CHX were added simultaneously. See Figure 5 for details. 17 After 2, 4, and 6 hours, 3.0 gram cell samples were harvested and analyzed for 3H incorporation into completed virus. Cycloheximide reduced the rate of 3H-uridine incorporation into TMV throughout the 6 hour period, and complete virus formation was inhibited by approximately 90% at the end of this time (Figure 7). Ouchi et_al_(l969) reported that the inhibition of TMV synthe- sis in leaf discs by CHX at 1 pg per ml was bipartite in nature; treatment at 0-12 hours after inoculation resulted in decreased virus synthesis, and treatment beyond 12 hours caused a 2-fold in- crease in virus titer. To determine whether or not CHX inhibition of TMV synthesis in our system was dependent upon the time of treat- ment, inoculated tissue cultures were incubated in 0.4 pCi 14C- leucine per ml and 12.5 pCi 3H—uridine per ml in the presence or absence of CHX for 12 hour intervals at 4 time periods after inocu- lation. When CHX was applied at 24-36, 36-48, 48-60, and 60-72 hours after inoculation, the incorporation of both isotopes into TMV was inhibited approximately 90% (Figure 8). To determine whether or not TMV extractibility was affected by the CHX treatment, callus cultures were placed in a solution of CHX or in growth medium alone, harvested, and homogenized with purified 3H-uridine-TMV. After pur- ification by sucrose gradient centrifugation, equal amounts of 3H-TMV were recovered from tissues given both treatments. When inoculated callus was treated with CHX at 5 pg per m1 (0-30 minutes after inocu- lation), virus synthesis and local lesion formation were inhibited for at least 84 hours. Effect of cycloheximide on TMV-RNA synthesis The results suggested that synthesis of complete virus was in- hibited by CHX (1 pg per ml) to a degree similar to the inhibition 18 CONTROL 0 3H-CPM x 10" IN now/3.0 g Figure 7. Effect of cycloheximide (CHX, 1 pg per m1) on the incorpor— ation of 3H-uridine (20 pCi per m1) into TMV in Xanthi-nc callus. At 48 hours after inoculation, cells were treated simultaneously with 3H and CHX. TMV was extracted from 3.0 grams of cells homogenized in Tris buffer, followed by differential centrifugation, and 10-40% sucrose gradient centrifugation. Gradients were monitored at 254 nm and radio- activity in 1 ml fractions was determined. Incorporation into TMV was determined by comparing optical density and radioactivity profiles. 19 14c 3" - Dcomnon. 5w =:::::cux '5 U! D 2 L. n, \ l— n 'o 2 v; F n F x x E E ”I 'U .I F 24'36 36'48 48‘60 60-72 TREATMENT PERIOD (HPI) Figure 8. Effect of cycloheximide (CHX, 1 pg per m1) on incorporation of 14C-leucine (1 pCi per ml) and H-uridine (20 pCi per m1) into TMV. Each sample was treated for 12 hours during 24-72 hours post inocula- tion (HPI) and radioactivity in TMV was determined as described for Figure 5. 20 of cellular protein synthesis. To determine the effect of CHX on the synthesis of TMV-RNA, inoculated cells (at 48 hours after inoculation) were incubated in 20 pCi 3H-uridine per ml with and without CHX for 2, 4, and 6 hours. The specific activity (CPM per pg RNA) of the 25 S and 18 S ribo- somal RNA (Loening and Ingle, 1967) increased throughout the 6 hour period in the untreated cells (Figure 9). Cycloheximide, however, re- duced the rate of incorporation of 3H-uridine into 25 S and 18 S riboso- mal RNA and caused a 90% reduction in their specific activities by 2 hours of treatment. There was no inhibition of 3H-uridine into 4~5 8 RNA. Similar effects of CHX on the synthesis of cellular RNA were re- ported in Neurospora (Viau and Davis, 1970) and in rat liver (Muramatsu 2E.Els 1970). The synthesis of TMV-RNA, expressed as specific activity, was not affected by CHX treatment. The specific activity of TMV-RNA from the CHX-treated and non-treated cells was approximately 2.5 times that of ribosomal RNA from non-treated cells by the fourth hour of labeling. DISCUSSION Leaves of tobacco cultivars such as Samsun NN and Xanthi-nc con- tain the N_gene (Holmes, 1939), and react by producing necrotic local lesions after inoculation with TMV. Callus cultures from these culti- vars also produce lesions following TMV infection (Beachy and Murakishi, 1971 a). The necrotic response to virus infection has not been demon- strated, however, in isolated cells (Jackson and Zaitlin, personal com- munication) nor in protoplasts (Otsuki 93.2}: 1972) from Samsun NN and Xanthi-nc. Otsuki et_al_(l972) suggested that the necrotic reaction 21 o counm 0. I A CHX l 3H-CPM x IO'°IP9 RNA Figure 9. Effect of cycloheximide (CHX, 1 ug Per ml) on the incorpor- ation of 3H-uridine in RNA of virus infected Xanthi-nc callus. RNA was extracted from 3.0 grams of cells in SDS-phenol and separated by elec- trophoresis on 2.4% polyacrylamide gels. Gels were scanned at 260 nm, and radioactivity in 1 mm slices was determined. CPM in each RNA spe- cies was determined by comparing optical density and radioactive pro- files. 22 depends upon the interactions between infected and neighboring cells. Our results support their suggestion since the reaction occurred only in callus cultures composed of cell aggregates. The interactions could occur by plasmodesmatal connections between cellinn tobacco callus aggregates (Hartmann, 1971). Tobacco mosaic virus synthesis in Xanthi-nc callus occurred in 2 major bursts during 144 hours and was similar to TMV synthesis in callus from a systemic tobacco (Pelcher 35.312 1972). The necrotic reaction appeared to have little or no inhibitory effect on virus synthesis since more than 90% of the virus was synthesized after lesions were evident. In leaf systems it is well established that virus synthesis occurs in advance of the spreading necrotic lesion (Osawa and Yamaguchi, 1970; Hayashi and Matsui, 1965). Recently Otsuki gt_al_(1972) compared the synthesis of TMV in leaf discs of Samsun tobacco (a systemic host) and Samsun NN, and reported that the rate of virus synthesis slowed after lesions became apparent. Their data showed about a lO-fold dif- ference in extractible virus (as measured by infectivity assays) by 96 hours after inoculating the two cultivars. The possibility that the lower infectivity could have resulted from decreased virus extractibil- ity from the local lesion host was apparently not explored. From all of these data it appears that the effects of the necrotic response on TMV synthesis in the initially infected and immediately adjacent cells cannot be specified at this time. Cycloheximide inhibits protein synthesis by preventing peptide elongation (McKeehan and Hardesty, 1969) and is effective in tobacco callus cultures (Figure 5; Nudel and Bamberger, 1971). We found that tobacco callus recovered from the inhibition caused by low concentra- tions of CHX (0.01-1.0 pg per ml) but not from higher concentrations. 23 A similar recovery phenomenon was observed with Tetrahymena (Frankel, 1970) and rat tissues (Yeh and Shils, 1969) when low concentrations of CHX were used. The data clearly emphasize the importance of monitoring the length of time that CHX remains effective. The inhibition of complete virus synthesis by cycloheximide ap- peared to be similar to the inhibition of host cytoplasmic protein syn- thesis (compare Figures 6 and 7) in Xanthi-nc callus. The data compare favorably with those for tobacco leaf discs (Zaitlin 95.212 1968). Syn- thesis of TMV-RNA, however, was not affected by 4 hours of CHX treatment (Figure 9). The results agree with those obtained for several animal viruses (Nobel and Levintow, 1970; Friedman and Grimley, 1969). Harrison and Crockatt (1971) and McCarthy gt_al_(l972) reported that the synthesis of tobacco rattle and tobacco necrosis viral RNAs, deter- mined by measuring the infectivity associated with phenol extracts of leaf tissues, continued in the presence of CHX. To the best of my knowledge, my results represent the first such report for TMV. Fail- ure of the specific activity of TMV-RNA to increase during the fourth to sixth hour of labeling (in CHX treated and non-treated cells) is not clear. During the same time period, complete virus continued to accum- ulate in untreated cells (Figure 7). Since TMV coat protein is present in extracts from infected Xanthi-nc callus (Beachy and Murakishi, 1971 b) and in TMV-infected Samsun NN leaves (van Loon and van Kammen, 1970), it is possible that TMV-RNA synthesized in the absence of viral protein synthesis (CHX sen- sitive) would have been encapsulated in viral protein already present in the cells. This was not the case, since the incorporation of 14C- leucine and 3H-uridine into complete virus was equally inhibited by CHX treatment (Figure 8). The possibility remains, however, that the 24 techniques used were not sufficiently sensitive to detect whether or not existing proteins were used. Inhibition of complete virus formation could be caused by the inhibition of viral coat or other protein synthesis, or to the inhibi- tion of virion assembly, as suggested by the failure of viral protein present in the cells to encapsulate viral RNA. Further studies uti- lizing appropriate plant tissue—TMV-inhibitor systems may be useful in elucidating the nature of TMV assembly in_vivgj and the nature of virus protein pools. REFERENCES Beachy, R. N. and Murakishi, H. H. (1971 a). Local lesion formation in tobacco tissue culture. Phytopathology_§4, 877-878. Beachy, R. N. and Murakishi, H. H. (1971 b). Proteins from cultured cells of Xanthi-nc tobacco inoculated with tobacco mosaic virus. Phytopathology_64, 884. (Abst.). Bishop, D. H. L., Claybrook, J. R., and Spiegelman, S. (1967). Elec- trophoretic separation of viral nucleic acid on polyacrylamide gels. 4, Mol. Biol. 26, 373-387. Frankel, J. (1969). The relationship of protein synthesis to cell division and oral development in Tetrahymena pyriformis GL-C: An analysis employing cycloheximide. Cell Physiol. 14, 135-148. Friedman, R. M., and Grimley, P. M. (1969). Inhibition of arbovirus assembly by cycloheximide. g, Virol. 4, 292-299. Harrison, B. D., and Crockatt, A. A. (1971). Effects of cycloheximide on the accumulation of tobacco rattle virus in leaf discs of Nicotiana clevelandii. 4, Gen. Virol. 42, 183-185. Hartmann, J. X. (1971). Electron microscopy of plant tissue culture cells inoculated with TMV. ln_Vitro 6, 373-374. Hayashi, T., and Matsui, C. (1965). Fine structure of lesion preiph- ery produced by tobacco mosaic virus. Phytopathology_§§, 387-392. Holmes, F. 0. (1938). Inheritance of resistance to tobacco mosaic disease in tobacco. Phytgpathology_2§, 553-561. Knight, C. A. (1962). Tobacco mosaic virus. ln_"Biochemical Prepara- tions" (M. J. Coon, ed.) 9, 132-136. Wiley, New York. Loening, U. E., and Ingle, J. (1967). Diversity of RNA components in green plant tissue. Nature (London) 215 363-367. Matthews, R. E. F. (1970). "Plant Virology," p. 168. Academic Press, New York. McCarthy, D., Lander, D. E., Hawkes, S. P., and Ketteridge, S. W. (1972). Effects of cycloheximide and chloramphenicol on the multiplication of tobacco necrosis virus. 4, Gen. Virol. 11, 91-97. McKeehan, W., and Hardesty, B. (1969). The mechanism of cycloheximide inhibition of protein synthesis in rabbit reticulcytes. Biochem. Biophys. Res. Comm. 36, 625-630. Murakishi, H. H., Hartmann, J. X., Pelcher, L. E., and Beachy, R. N. (1970). Improved inoculation of cultured plant cells resulting in high virus titer and crystal formation. Virology 44, 365-367. 25 26 Murakishi, H. H., Hartmann, J. X., Beachy, R. N., and Pelcher, L. E. (1971). Growth curve and yield of tobacco mosaic virus in tobacco callus cells. Virology 43, 62-68. Muramatsu, M., Shimada, N., and Higashinakagawa, T. (1970). Effect of cycloheximide on the nucleolar RNA synthesis in rat liver. 4, Mol. Biol. 5g, 91-106. Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 45, 473-497. Nobel, J., and Levintow, L. (1970). Dynamics of poliovirus-specific RNA synthesis and the effects of inhibitors of virus replication. Virology 49, 634-642. Nudel, U., and Bamberger, B. S. (1971). Kinetin inhibition of 3H- uracil and 14C-leucine incorporation by tobacco cells in suspen- sion culture. Plant Physiol. 47, 400-403. Osawa, K., and Yamaguchi, A. (1970). Comparative studies of virus multiplication and quality of necrotic lesions in the inoculated leaves of some local lesion hosts to tobacco mosaic virus. Ann. Phytopath. Soc. Japan _3_g, 254-259. ‘— Otsuki, Y., Shimomura, T., and Takebe, T. (1972). Tobacco mosaic virus multiplication and expression of the N gene in necrotic responding tobacco varieties. Virology 59, 45-50. Ouchi, S., Furusawa, I., and Akai, S. (1969). Bipartite nature of the effects of cycloheximide and chloramphenicol on the multipli- cation of tobacco mosaic virus. Phytopath. g, 65, 287-297. Pelcher, L. E., Murakishi, H. H., and Hartmann, J. X. (1972). Kinetics of TMV-RNA synthesis and its correlation with virus accumulation and crystalline viral inclusion formation in tobacco tissue culture. Virology 42, 787-796. Takebe, I., Otsuki, Y., and Aoki, S. (1971). In_"Les Cultures de Tissus de Plantes," 503-511. Editions du Centre National de la Recherche Scientifique, Paris. van Loon, L. C., and van Kammen, A. (1970). Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var. "Samsun” and "Samsun NN" 11. Changes in protein constitution after infection with tobacco mosaic virus. Virology 40, 199-211. Viau, J., and Davis, F. F. (1970). Effect of cycloheximide on the synthesis and modification of ribosomal RNA in Neurospora crassa. Biochim. Biophys. Acta 209, 190-195. White, P. R. (1963). lg_"The Cultivation of Animal and Plant Cells," 57-63. Second edition, Ronald Press Co., New York. 27 Yeh, S. D. J., and Shils, M. E. (1969). Quantitative aspects of cyclo- heximide inhibition of amino acid incorporation. Biochem. Pharmacol. 18, 1919-1926. Zaitlin, M., Spencer, D., and Whitfield, P. R. (1968). Studies on the intracellular site of tobacco mosaic virus assembly. In "Proceedings of the International Symposium on Plant Biochemical Regfilation on Viral and Other Disease or Injury," 91-103. Kyoritsu Printing Co., Tokyo. CHAPTER 3 PROTEINS FROM CULTURED CELLS OF XANTHI-NC TOBACCO INOCULATED WITH TOBACCO MOSAIC VIRUS *PROTEINS FROM CULTURED CELLS OF XANTHI-NC TOBACCO INOCULATED WITH TOBACCO MOSAIC VIRUS SUMMARY Cell suspensions of Nicotiana tabacum Var. Xanthi-nc tissue cul- ture were inoculated with 100 pg tobacco mosaic virus (TMV) per m1, washed with fresh medium to remove excess inoculum, and incubated on agar medium under diffuse light. Local lesions were observed on ino- culated cell cultures after 40 hours. At 24-hour intervals inoculated and noninoculated control cells were harvested, and cell extracts were prepared by homogenization and centrifugation at low (15,000 g) and high (144,000 g) speeds. The supernatant from the high speed centri- fugation was subjected to electrOphoresis on 7% and 10% polyacrylamide disc gels, stained for protein in amido black, and scanned in a Gilford densitometer. On a fresh weight basis, more proteins were extracted from lesion-bearing inoculated tissue than from control tissues. Two new protein bands, characterized as TMV protein, were detected from ex- tracts of infected cells. Intensities of several normal host protein bands diminished, whereas others increased following infection. Data indicate that protein changes can be detected in virus-infected cells from tissue cultures more easily than in virus-infected leaves. *A portion of this material was presented at the Annual Meeting of the American Phytopathological Society, Aug. 15-19, 1971, held at Philadel— phia, Penn. Abstracted in Phytopathologygl, 884 (1971). 28 29 INTRODUCTION Tobacco mosaic virus (TMV) is a rod-shaped virus composed of a single strand of ribonucleic acid (RNA) encapsulated in coat protein (Hart, 1955; Fraenkel-Conrat, 1955). TMV-RNA contains approximately 6340 nucleotides with a total molecular weight of 2.05 x 106 daltons (Takahashi, 1967). Assuming the triplet code for translation of mess- enger RNA (Crick, 1962), there are enough nucleotides to code for 2133 amino acids. The coat protein contains 158 amino acids (Fraenkel- Conrat, 1965) coded for by 474 nucleotides. This represents only 7-8% of the total genetic material, and, clearly, additional viral proteins could be made from the TMV-RNA template. Suggestions of the possible nature of the TMV gene products come from studies of other single-stranded RNA viruses of similar size. The small RNA coliphages (1.1-1.3 x 106 daltons) are known to have several gene products including a replicase enzyme, maturation protein, and coat protein (Kozak and Nathans, 1972). Poliovirus RNA (1.9 x 106 daltons) is known to code for at least 14 proteins including 4 which assemble into the coat protein (Baltimore, 1969). These viruses are amenable to study because of their ability to shut off host RNA and protein synthe- sis, facilitating the detection of virus products. This does not occur in TMV infection, however, and studies of virus gene products are more difficult. Several methods of study have been used. One method has been the use of cell free systems as described by Nirenberg and Matthaei (1961). Repeated attempts to use E, Egli_cell- free systems primed with TMV-RNA as messenger initially produced inter- est, but it now appears that only fragments of the genetic message are being randomly translated (Reichmann and Clark, 1968; Matthews, 1970). 3O 42_yi££g_systems from plant cells have also not been successful (Reichmann and Clark, 1968; Takahashi, 1967), probably because of the large amounts of nucleases released upon homogenization. Kiho (1968) reported the isolation of TMV-specific polyribosomes from infected tobacco leaves. It may be possible to identify proteins other than coat protein by using this procedure for isolating the elon- gating peptides. Another method used has been the attempted isolation and charac- terization of specific proteins. From studies which elucidated the nature of the replication of RNA-containing bacterial and animal viruses (Kozak and Nathans, 1972; Baltimore, 1969), an RNA-dependent-RNA-poly- merase was implicated in the replication of TMV-RNA. Though efforts to demonstrate the presence of the enzyme in infected tissues have been relatively successful (Ralph and Wojick, 1969; Bradley and Zaitlin, 1971), only a single report on characterization of the enzyme has been presented (Brishammar, 1970). Recent work by Zaitlin et_§4_(personal communication) indicated that they had partially purified a host medi- ated RNA-dependent-RNA-polymerase which is stimulated by TMV infection. The role of virus infection on enzyme activity and specificity remains to be determined. The polyacrylamide gel electrophoresis technique has been used in recent studies which attempted to characterize virus proteins, or virus- induced host proteins, in infected cells. Using disc gel electrophore- sis, which separates native proteins on the basis of size and charge (Davis, 1964), van Loon and van Kammen (1970) identified the viral coat protein in cell extracts from systemically infected leaves, but other viral proteins were not detected by this procedure. They also reported 31 the appearance of 4 new proteins in extracts from leaves bearing local lesions, and it was suggested that they were produced by the host in response to virus infection. Other workers used sodium-dodecyl-sulfate (SDS)-polyacrylamide gel electrOphoresis, which denatures proteins and provides for their separ- ation on the basis of size (Maizel, 1966; Weber and Osborn, 1969), and radioactive labeling to detect virus-directed or stimulated proteins in tobacco leaves systemically infected with TMV. Zaitlin and Hariharasu- bramanian (1971, 1972) and Singer (1971, 1972) fed TMV-infected leaves with 3H-amino acid and non-infected leaves with 14C-amino acid. The 2 samples were combined, homogenized, and subjected to co-electrophoresis. The ratio of the isotopes (CPM 3H%CPM l4C) throughout the gel remained constant in the proteins not affected by virus infection, but stimulated or repressed protein synthesis resulted in a change of the isotope ratio. Zaitlin and Hariharasubramanian (1971, 1972) reported finding the coat protein and several larger molecular weight proteins; they suggested that one of these might represent the replicase. Singer (1971), however, reported finding only the coat protein. Plant tissue cultures capable of being infected with virus have been developed in this laboratory and it was hoped that the tobacco callus system would provide a useful tool for the identification of viral proteins. Since local lesions were produced on callus taken from hypersensitive tobacco cultivars following TMV infection (Beachy and Murakishi, 1971), I wished to re-examine the occurrence of the 4 new proteins reported by van Loon and van Kammen (1970). Therefore, ex- tracts of TMV-infected and non-infected Xanthi-nc callus were made at 24 hour intervals until 144 hours after inoculation and compared for soluble proteins by disc gel electrophoresis. 32 MATERIALS AND METHODS Callus cultures and TMV inoculation Nicotiana tabacum Xanthi-nc callus was prepared from aseptically removed cores of stem pith grown on the solid Murashige and Skoog - White (MS-W) medium (Appendix). Pigmented callus was maintained on solidified medium, and prepared for inoculation as previously des- cribed (Chapter 2, Materials and Methods). Callus tissue grown in liquid medium was inoculated with 100-120 pg TMV per ml by the vibra- tory technique of Murakishi et_al_(1970), and incubated on solidified medium under Gro-lux lamps. A mannitol-containing medium (MS-W-mannitol) was prepared in the MS-W formula (Appendix) by substituting equimolar amounts of mannitol for dextrose and glucose in the separately prepared media. When this medium was to be used, callus was produced in the normal MS-W medium, then inoculated and washed with MS-W-mannitol, and incubated on solidi- fied mannitol medium. Extraction buffer and electrophoresis solutions The extraction buffer, modified from that described by Staples and Stahmann (1964) as reported by van Loon and van Kammen (1968), contained 0.1 M Tris-HCl, pH 8.0 (room temperature), 0.5 M sucrose, % ascorbic acid, and 75 mM 2-mercaptoethanol. The original buffer contained 1% cysteine-HCl, but it was found that substituting 2-mercaptoethanol at this concentration prevented browning during cell homogenization. The buffer was not kept more than 48 hours. The electrophoresis solutions were prepared as described by Davis (1964), except for the small-pore gel buffer (solution B); the solution 33 was brought to pH 6.9 with phosphoric acid (van Loon and van Kammen, 1968) instead of pH 6.7 with hydrochloric acid. Soluble protein extraction and electrophoresis Cell samples were harvested at 24 hour intervals, weighed and cooled to 4°. The cells were ground in a conical glass homogenizer in buffer 1:5 (v:w). The homogenate was immediately squeezed through 4 layers of cheesecloth (delay at this step resulted in extensive brown- ing of the homogenate), centrifuged at 10,000 g for 30 minutes, and the supernatant was subjected to 144,000 g for 150 minutes in the SW 39L rotor (Spinco Model L, 35,000 rpm). The final supernatant was removed and made 5% with respect to sucrose. Bromophenol-blue was added to serve as a marker. The procedure was designed to extract the soluble cell proteins, and to remove bound proteins by centrifugation. By this method, intact TMV would not be disrupted, and would be removed by cen- trifugation. Seven per cent and 10% polyacrylamide disc gels were prepared according to Davis (1964) in glass tubes 6 mm (inside diameter) x 8 cm which were acid-washed, and rinsed with Photoflo (Eastman Kodak Co.). The gels were placed in the electrophoresis apparatus and 500 ml of reservoir buffer (Davis, 1964) was added to the upper and lower reser- voirs. After placing 0.1 m1 of the protein extract on the gel surface, electrophoresis was performed at 4° with a Spinco Duostat constant cur- rent/constant voltage power source at 2 mA per gel (constant current) for 20 minutes, and 3 mA per gel until the marker dye reached the end of the gel (75-90 minutes). Gels were removed from the tubes, stained overnight in a solution of 0.5% amido black (van Loon and van Kammen, 1968) in 7% acetic acid, and electrophoretically destained at 6 mA per 34 gel (constant voltage) with 7% acetic acid in both reservoirs. The gels were scanned at 660 nm in a Gilford 2400 spectrophotometer with attached gel transporter. Electropherograms were traced directly from the originals. Immunodiffusion For immunodiffusion experiments 7% gels containing the extracted proteins were sliced lengthwise, and placed flat side down in a petri plate. The antiserum solution, consisting of rabbit anti-TMV serum 1:4 in a solution of 0.9 M NaCl, and 0.9% agar at 37-40°, was poured around the sliced gels, allowed to harden, and left at room temperature for several days. RESULTS AND DISCUSSION Burley NN and Xanthi-nc callus cultures were tested; both produced local lesions after infection with TMV. In repeated experiments the Xanthi-nc callus, a cell line with green pigments, consistently pro- duced more local lesions following inoculation, and was less likely to become brown after homogenization, than was Burley NN callus. Browning of the cell extract is caused by the oxidation of phenolic compounds (Loomis and Battalie, 1966) which are abundant in tobacco callus (Fritig and Hirth, 1971). Electrophoresis and subsequent staining of such ex- tracts resulted in poor protein separation and diffuse staining through- out the gels. This confirms the observations of van Loon and van Kammen (1968). The Xanthi-nc callus was used in all further eXperiments. In early experiments, extracts of healthy and TMV infected callus were separated on 7% and 10% gels to make more complete comparisons of protein components (van Loon and van Kammen, 1968). Essentially the 35 same results were obtained from both gels, but protein bands were sep- arated better on the 10% gels (Figure 10). There appeared to be better resolution of both the fast and slow migrating proteins on 10% gels, which were used routinely for all other experiments. In the first two experiments in which the cells were grown on the usual MS-W medium, no distinct changes in soluble proteins were detected from 24-144 hours after inoculation. Since it had been previously shown that Xanthi-nc callus grew rapidly on this medium (more than a 3-fold increase in fresh weight by 144 hours, Figure 19, Appendix), it was prob— able that changes which occurred following virus infection were ”diluted" by the new cell growth. Virus is localized in and around the lesion area of Xanthi-nc callus (Beachy and Murakishi, 1971) and the growth of uninfected cells would reduce the proportion of virus-infected cells. To reduce cell growth, mannitol was substituted for sucrose and dextrose to make the MS-W-mannitol medium described in Materials and Methods. Mannitol is an efficient osmoticum but is not metabolized by tobacco cells (Trip §t_§l, 1964). There was less than a 10% fresh weight in- crease in Xanthi-nc callus after 110 hours of incubation on mannitol medium. Callus kept under these conditions became greener under Gro- lux illumination than did other callus, and the cells appeared to re- main normal when observed by light microscopy. Xanthi-nc callus was inoculated with TMV, incubated on the manni- tol medium, and extracted for proteins at 24 hour intervals until 144 hours after infection for disc gel electrophoresis. There were 3 ex- periments of this type, and the same general observations were made in each experiment. The results of one experiment in which the greatest differences were observed are described. Since the gel lengths varied 36 IO! U" \ I or ./ du- 71" Figure 10. Soluble proteins from Xanthi-nc callus separated on 7% and 10% polyacrylamide disc gels. Gels were stained with amido black and scanned in a Gilford gel scanner. Migration is from the left. 37 slightly, and the rate of migration was not constant between gels, an Rf value was calculated by setting the migration of the marker dye equal to 1.00. Electropherograms from cells extracted 24 hours after inoculation are shown in Figure 11. The slight differences in proteins between the samples, such as those at Rf 0.25 and 0.28, were probably within the range of variability, and will not be considered in the discussion be- low. At 48 hours after inoculation, several changes in the stained pro- teins were observed (Figure 12). There was an increase in the 0.21 band in the inoculated sample and the 0.33 cells. The migration of other proteins were similar in both gels (for example, components 0.28 and 0.39). By 72 hours after inoculation a large peak, Rf=0.21, was prominent in the inoculated cells only (Figure 13). In addition, the 0.28 compo- nent was present in larger proportions in the inoculated cell extract. It appeared that the 0.21 and 0.28 peaks at 72 hours were similar to those with the same migration observed at 48 hours. Ninety-six hours after inoculation the 0.20 component remained prominent, and there were major changes at Rf 0.26-0.41 (Figure 14). The apparent increases in components 0.26 and 0.35 are substantiated by the similarity of the 0.41 and 0.54 bands in both cell extracts. This indicated that relative protein migration was consistent between the gels. The extracts made at 120 and 144 hours after inoculation (Figures 15, 16) showed the same kinds of differences between inoculated and control extracts as were seen at 96 hours. In addition, the component 0.043-0.05 was present in the inoculated sample as a distinct peak, but 38 II. 24 Im. In. Control '2 o 4. II". P.I. Figures 11, 12. Soluble proteins from infected and non-infected Xanthi- nc callus. Proteins were separated on 10% polyacrylamide disc gels and stained with amido black. The gels were monitored at 660 nm with a Gil- ford gel scanner. Migration is from the left. Rf was calculated from the migration on the marker dye. Extracts were taken at 24 and 48 hours post inoculation (hrs. P. 1.). ' 39 I3 . 12 Im. m. -h .I Control I4. 96 II". P.I. Figures l3, l4. Soluble proteins from infected and non-infected Xanthi- nc callus. See Figure 11 for details. Extracts were taken at 72 hours (Figure 13) and 96 hours (Figure 14) post inoculation (hrs. P. 1.). 4o '50 I20 Inn. P.l. 16. 144 Im. r.I. Control '9 Figures 15, 16. Soluble proteins from infected and non-infected Xanthi- nc callus. See Figure 11 for details. Extracts were taken at 120 hours (Figure 15) and 144 hours (Figure 16) post inoculation (hrs. P. 1.). 41 remained as a shoulder in the control cells. This component was also extracted at 72 and 96 hours in lesser proportions. In summary, the 0.20-0.21 component increased by 48 hours after inoculation and was found through 144 hours. Other components with Rf values of 0.20-0.41 varied from day to day. An exception was the 0.26- 0.28 component which was extracted at 48-144 hours, but not always in sufficient proportions to be definitely correlated with virus infection. Another component, 0.043-0.05, was observed in only the inoculated cell extracts. Initially observed at 72 hours, the peak was also present at 96 and 120 hours and became intense by 144 hours. Since this band was most noticeable at 72 and 144 hours when the 0.20-0.21 component was also most intense, the two bands may be interrelated. It is possible that a quantitative aspect is also being observed, however, since the amount of protein applied to the gels at 72 and 144 hours was greater than the other times (as indicated by the height of the tracings above the baseline). The changes in protein bands other than virus antigen bands may represent changes in normal host proteins due to the infection. Curtis (1971) reported changes in isozymes of peroxidase in bean leaf extracts following the formation of tumors induced by Agrobacterium tumefaciens. The changes that he observed on 7% gels occurred at approximately Rf 0.4-0.55. The changes which were observed in stained proteins from to- bacco callus after virus infection were at Rf 0.3-0.4 on 10% gels; on 7% gels, their relative migration would have been 0.36 -0.48. The results suggest that changes in extractible proteins other than coat protein may represent increases or decreases in normal host proteins, perhaps isozymic changes. 42 van Loon and van Kammen (1970) were unable to demonstrate TMV antigen in cell extracts from infected Samsun NN, or N, glutinosa leaves, local lesion hosts of TMV. To determine whether or not soluble TMV-antigen was present in Xanthi-nc callus proteins were extracted from infected and non-infected cells at 96 hours after inoculation and subjected to electrophoresis on 7% gels. The gels were exposed to TMV antiserum as described in Materials and Methods. After 2-3 days of in- cubation, an antigen-antibody precipitin reaction was observed at the surface of the gel containing the extract from the infected cells (Figure 17). Figure 17. Identification of TMV protein by antigenic reaction. Sol- uble proteins from infected and non-infected Xanthi-nc callus were sep- arated on 7% polyacrylamide disc gels. Gels were sliced, and immersed in TMV antibody as described in the text. Migration was from the top. C = non-inoculated cells; I = inoculated cells. The arrows mark the 2 bands of antigen-antibody precipitation. 43 The non-infected cell extracts did not show this reaction. In other gels run at the same time and stained for proteins, two bands were ob- served in the infected cell extracts at positions identical to the two precipitin lines, and corresponded closely to the 0.20-0.21 and 0.26- 0.28 components reported above. van Loon and van Kammen (1970) reported that TMV coat protein in extracts from infected leaves of a systemic tobacco host had an Rf value of 0.22 in 10% gels. On these bases the 0.20-0.21 component from Xanthi-nc callus was thought to represent coat protein. The second precipitin band indicated that the 0.26-0.28 peak was also TMV protein, and may represent aggregated coat protein, per- haps X-protein (Takahashi and Ishii, 1953). Whether this band is an artifact of the isolation procedure, or is a true reflection of coat protein in the cells is not known. An interesting speculation is that the second band is a result of the hypersensitive response, and is some— how related to inhibition of virus synthesis in the host. van Loon and van Kammen (1970) reported finding 4 host proteins in larger amounts in leaves of Xanthi-nc 7 days after infection, with Rf values of 0.55-0.84 on 10% gels. Similar protein changes were not ob- served in the callus-TMV system, and several reasons are suggested: (1) synthesis of the proteins did not occur in the callus; or (2) the con- centration of the proteins was too low to be detected in the callus sys- tem. van Loon and van Kammen (1970) suggested that the new proteins were related to the acquired resistance phenomenon described by Ross (l96la, 1961b), but no further work on characterization of the proteins has yet been forthcoming. If their suggestions are correct, the failure of the infected callus to produce the proteins implies the absence of acquired resistance in TMV-infected Xanthi-nc callus. 44 The results of this study suggest that Xanthi-nc callus inocu- lated with TMV and incubated on mannitol-containing medium would be a good system in which to study viral proteins or virus-induced host-pro- teins: (1) local lesions (indicative of virus infection) are produced under these conditions; (2) electrophoresis of extracts of cells grown on mannitol-containing medium demonstrated that changes in cell proteins could be observed by this technique; (3) since the weight of cells does not increase under these conditions, it is possible to study the changes which occur only in those cells present at the time of inocula- tion; (4) soluble protein extracts of tobacco callus resolved into more, distinct bands upon electrophoresis than extracts from infected tobacco leaves (see van Loon and van Kammen, 1968, 1970). In other preliminary experiments it was determined that mannitol treatment did not greatly affect the incorporation of 14C-amino acid into an acid insoluble frac- tion of the cell homogenate. The system would, therefore, be amenable to the type of double labeling eXperiments such as those of Singer (1971, 1972) and Zaitlin and Hariharasubramanian (1970, 1972). This procedure would more accurately detect changes in extracted proteins due to virus infection. REFERENCES Beachy, R. N., and Murakishi, H. H. (1971). Local lesion formation in tobacco tissue culture. Phytopathology_§4, 877-878. Baltimore, D. (1969). The replication of picornaviruses. 32_"The Bio- chemistry of Viruses,” (H. B. Levy, ed.) Marcel Dekker, New York. Bradley, D. W., and Zaitlin, M. (1971). Replication of tobacco mosaic virus. 11. The 32_vitro synthesis of high molecular weight virus- specific RNA. Virology 43, 192-199. Brishammar, S. (1970). Identification and characterization of an RNA replicase from TMV-infected tobacco leaves. Biochem. Biophys. Res. Comm. 41, 506-511. Crick, F. H. C. (1962). The genetic code. Scientific American 207, 66-74. Curtis, C. R. (1971). Disc electrophoretic comparisons of proteins and peroxidases from Phaseolus vulgaris leaves infected with Agrobacter- ium tumefaciens. Can. 3, Bot. 42, 333-337. Davis, B. J. (1964). Disc electrophoresis - 11 Method and application to human serum proteins. Ann. N, I, Acad. Sci. 121, 404-427. Fraenkel-Conrat, H., and Williams, R. C. (1955). Reconstitution of active TMV from its inactive protein and nucleic acid components. Proc. Natl. Acad. Sci. U, 3, 44, 690-698. Fraenkel-Conrat, H. (1965). Structure and function of virus proteins and viral nucleic acid. 32 "The Proteins” (H. Neurath, ed.), 3, 99-151. Academic Press, New York. Fritig, B., and Hirth, L. (1971). Biosynthesis of phenylpropanoids and coumarins in TMV-infected tobacco leaves and tobacco tissue culture. Acta. Phytopath. Acad. Sci. Hupg, 3, 21-29. Hart, R. G. (1955). Electron-microscopic evidence for the localization of ribonucleic acid in particles of tobacco mosaic virus. Proc. Natl. Acad. Sci. U, 3, 43, 261-264. Kiho, Y. (1968). Isolation of polyribosome from tobacco plants infected with tobacco mosaic virus. Japan. 3, Microbiol. 32, 211-217. Kozak, M., and Nathans, D. (1972). Translation of the genome of a ribo- nucleic acid bacteriophage. Bacteriol. Rev. 33, 109-134. Loomis, W. D., and Battalie, J. (1966). Plant phenolic compounds and the isolation of plant enzymes. Phytochem. 3, 423-438. Maizel, J. V. (1965). Acrylamide-gel electrophorograms by mechanical fractionation: radioactive adenovirus proteins. Science 151, 988- 990. 4S 46 Matthews, R. E. F. (1970). Plant Virology. 165-171. Academic Press, New York. Nirenberg, M. W., and Matthaei, J. H. (1961). The dependence of cell free protein synthesis in E, coli upon naturally occurring or syn- thetic polyribonucleotides. Proc. Natl. Acad. Sci. U, 3, 42, 1588- 1602. Murakishi, H. H., Hartmann, J. X., Pelcher, L. E., and Beachy, R. N. (1970). Improved inoculation of cultured plant cells resulting in high virus titer and crystal formation. Virology 43, 365-367. Ralph, R. K., and Wojcik, S. J. (1969). Double-stranded tobacco mosaic virus RNA. Virology 32, 276-282. Reichmann, M. E., and Clark, J. M. (1968). Current status in 32_ vitro synthesis of plant viruses. Ann. Rev. Phytopathology_§, 295-316. Ross, A. F. (1961 a). Localized acquired resistance to plant virus infection in hypersensitive hosts. Virology 34, 329-339. Ross, A. F. (1961 b). Systemic acquired resistance induced by local- ized virus infection in plants. Virology 34, 340-358. Singer, B. (1971). Protein synthesis in virus-infected plants I. The number and nature of TMV-directed-proteins detected on poly- acrylamide gels. Virology 43, 247-255. Singer, B. (1972). Protein synthesis in virus-infected plants 11. The synthesis and accumulation of TMV and TMV coat protein in subcellular fractions of TMV-infected tobacco. Virology 41, 397- 404. Staples, R. C., and Stahmann, M. A. (1964). Changes in proteins and several enzymes in susceptible bean leaves after infection by the bean rust fungus. Phytopathology_34, 760—764. Takahashi, W. N., and Ishii, N. (1953). An abnormal protein asso- ciated with tobacco mosaic virus infection. Nature 169, 419-420. Trip, P., Krotkov, G., and Nelson, C. D. (1964). Metabolism of mannitol in higher plants- Amer. Jour. Bot. 31, 828-835. van Loon, L. C., and van Kammen, A. (1968). Polyacrylamide disc electrOphoresis of the soluble leaf proteins from Nicotiana tabacum var. "Samsun NN" and "Samsun" - I. Phytochem. Z, 1727- 1735. van Loon, L. C., and van Kammen, A. (1970). Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var. "Samsun" and "Samsun NN" 11. Changes in protein constitution after infection with tobacco mosaic virus. Virology 40, 199-211. 47 Weber, K., and Osborn, M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrOphoresis. 3, Biol. Chem. 244, 4406-4412. Zaitlin, M., and Hariharasubramanian, V. (1971). Proteins in tobacco mosaic virus-infected tobacco plants. Biochem. BioPhys. Res. Comm. 32, 1031-1036. Zaitlin, M., and Hariharasubramanian, V. (1972). A gel electrOphoretic analysis of proteins from plants infected with tobacco mosaic and potato spindle tuber viruses. Virology 41, 296-305. SUMMARY AND RECOMMENDATIONS SUMMARY AND RECOMMENDATIONS Cultured cells of 4 hypersensitive tobacco cultivars produced necrotic local lesions following infection with TMV. Specific cultural conditions are required for high lesion numbers: (1) the callus cul- ture must be composed of cell aggregates with a ”grainy" texture at the time of inoculation; and (2) the growth medium must contain Murashige and Skoog mineral and vitamin stocks, coconut water, 2,4-D, and NAA. An important advance towards characterization of the necrotic response in cultured cells would be the elimination of coconut water which re- mains largely undefined. If the growth, virus infectibility, and le- sion expression of the cultured cells could be controlled by altering specified components in a defined medium, the steps involved in these processes could be closely examined. Whether or not lesions can be produced in a defined medium, clearly warrants further study. More than 90% of the TMV synthesized in Xanthi-nc callus occurred after necrotic lesions were formed. Microscopic studies of TMV-infected leaves of hypersensitive bean indicated that materials deposited between the cells blocked the plasmodesmatal connections, and thereby prevented virus spread (Wu and Dimitman, 1970). This occurs, however, several cells distant from the point of infection, and 5-7 days after necrosis is first observed. All of the data raise questions regarding the effect, if any, of the necrotic response on the synthesis and/or pro- cessing of viral protein(s), and of viral RNA. A comparison of the kinetics of viral RNA and protein synthesis in callus from Xanthi-nc 48 49 and Xanthi (a systemic host) before, during, and after lesion develop- ment on the hypersensitive callus may elucidate the effects of the ne- crotic reaction, and the nature of the disease resistance conferred by the necrotic (N) gene (Holmes, 1939). Within 1 hour of applying cycloheximide (CHX) to infected Xanthi-nc callus, the formation of complete TMV was prevented, but the synthesis of TMV-RNA was unaffected even after 4 hours of treatment. Further studies may answer questions related to the nature of TMV synthesis and assembly such as the following: (1) How long does the synthesis of TMV- RNA continue in the absence of protein synthesis (in the presence of CHX)? Answering this question will help to characterize the lifetime of the enzyme (replicase) responsible for the synthesis of viral RNA. (2) Does CHX prevent the formation of complete virus by blocking the move- ment of TMV-RNA from its ”site of synthesis" to its "site of assembly”? (3) Are pre-existing pools of coat protein available for encapsulating TMV-RNA synthesized in the presence of CHX, or (4) are proteins, other than virus coat protein, required for virus assembly? Questions 2, 3, and 4 were raised when TMV-RNA synthesized in the presence of CHX was not incorporated into complete virus, even though a pool of coat protein had been identified in Xanthi-nc callus. Further studies are needed to clarify the nature of TMV synthesis and assembly. Changes in soluble proteins following TMV infection of Xanthi-nc callus were detected by polyacrylamide disc gel electrophoresis. The TMV coat protein was detected by the antibody-antigen reaction. An additional protein component was detected (by staining) which may be related to virus infection. The staining technique, however, is not accurate for detecting small changes in protein synthesis. Tissue culture cells take up radioactive amino acids readily, and double 50 labeling experiments similar to those reported by Zaitlin and Harihara- subramanian (1970) and Singer (1971) would more accurately detect the effects of virus infection on host cell protein synthesis, and may lead to the identification of additional viral proteins other than coat pro- tein. APPENDIX APPENDIX The three chapters of the thesis were prepared primarily for pub- lication. Additional observations and results of experiments not re- corded in the thesis proper are present in the Appendix. It gives in- formation related to tissue culture cell lines used, the value of the growth medium, and the inoculation procedures used. It is hoped that the information given here will serve as a background for further work on the hypersensitive callus - TMV system. 51 52 *Local lesion and viral crystal formation in tissue culture cells of different species of Nicotiana Callus derived from cultivars of Nicotiana tabacum L. and other Nicotiana species was studied to determine which cell lines would pro- duce large amounts of virus and would therefore be amenable to further studies of virus infection. Most of the cultures were developed by H. H. Murakishi during 1965-1968 and were maintained on agar-solidified White's medium (1954). Nicotiana tabacum cvs Samsun NN (SNN), Burley NN (BNN), and U, glutinosa were developed by the author. Two cultivars of U, tabacum, an albino mutant, and an habituated cell line, were ob- tained from S. Venketeswaran (University of Houston). Agar-grown callus (300-400 mg) were placed into 40 m1 test tubes containing 3 ml liquid medium and 36 pg TMV, and the tubes were vi- brated with a Vortex mixer for 15 seconds. Contents of each tube were poured into a funnel lined with coarse filter paper and allowed to drain for 5 minutes. After washing with 15 m1 fresh medium, the cells were dispersed on solid medium and placed under Gro-lux lamps. A small sample of cells was harvested at 0-time and frozen. After 7 days the inoculated cells were harvested, frozen, and later homogenized with 0.01 M potassium phosphate buffer, pH 7.3, and appropriately diluted to give an easily countable number of local lesions on the assay host. Homogenates of the O-time and 7-day harvests were applied with a glass spatula to opposite half-leaves of U, tabacum Xanthi-nc previously kept in the dark for 24 hours, and dusted with carborundum. Virus increase *This material, presented at the Sixty-Second Annual Meeting of the American Phytopathological Society, Hot Springs, Arkansas, October 4-8, 1970, was published as an abstract (Phytopathology 33, 1283, 1970). S3 was determined by comparing the number of lesions produced by the two homogenates. The data are given as the number of local lesions produced by the homogenates of 4 samples, each rubbed on 6 half leaves of Xanthi-nc (total of 24 half leaves). The cell lines H-38 (Havana-38), White Burley, Maryland Mammoth, "Habituated", and U, sylvestris produced large amounts of virus (Figure 18). Only the H-38 callus produced high numbers of TMV crystalline inclusions, with 79% of 500 cells examined containing crystals. Only 3-5% of the cells of other cultivars con- tained inclusions. The lack of virus development in U, ambalema was expected because this cultivar was reported to be a "tolerant" host of TMV. Callus from U, tabacum cvs Samsun NN, and Burley NN, and U, glutinosa produced ne- crotic spots after TMV inoculation; crystalline inclusions were found only around the local lesions. The data indicate that the H-38 callus produced more virus in 7 days than did the other cultures, under the conditions of these experi- ments. It is clear, however, that several cell lines produce virus following TMV infection, and could be used in further virus studies. Cultural procedures to increase the efficiengy of virus infection The study reported above indicated that callus from U, glutinosa and U, tabacum cvs Samsun NN, and Burley NN responded to TMV infection by producing the necrotic response, similar to the reaction known to occur on the leaves of these hosts (Holmes, 1929; Holmes, 1938; Valleau, 1952). Nicotiana tabacum cv Xanthi-nc (Takahashi, 1956) callus re- sponded in a similar manner. 54 Figure 18. Increase of TMV in callus tissue from cultivars of Nicotiana tabacum, and other Nicotiana species. Virus increase was calculated by comparing the infectivity associated with homogenates of O-time and 7-day harvests. 55 «2036.. ._