VIROLOGY
63, 459~465(1975)
Retention Mosaic
and Dissociation Virus by Tobacco
of Tobacco Protoplasts’
STEPHEN D. WYATT2 AND JOHN G. SHAW Department
of Plant Pathology,
University
Accepted
of Kentucky,
September
Lexington,
Kentucky
40506
16, 1974
When tobacco protoplasts were inoculated with radioactive tobacco mosaic virus (TMV) at a concentration of 1 &ml, about 15% of the particles (approximately 8 x lOa particles per protoplast) were retained. When poly-L-ornithine was not included in the inoculum, the amount of virus retained was reduced 6- to 30-fold. Maximum retention was observed 20-30 min after the addition of virus to the protoplasts. Thirty minutes after mixing virus and protoplasts, about 5% of the protein of the retained TMV had been removed and some of the virus particles appeared to be partially dissociated. A considerable amount of the retained viral material in extracts of inoculated protoplasts was found in the pellets after sucrose gradient centrifugation. This abnormally rapid sedimentation was correlated with the presence of poly-L-ornithine in the inoculum. INTRODUCTION
MATERIALS
The infection of protoplasts prepared from tobacco mesophyll cells with tobacco mosaic virus (TMV) has been demonstrated by Takebe and Otsuki (1969). This system has subsequently been utilized for studies of the attachment of virus particles to the plasmalemma, the appearance of the particles in cytoplasmic vesicles, and the production of progeny virus in the infected protoplasts (Otsuki and Takebe, 1969; Coutts et al., 1972; Otsuki et al., 1972; Hibi and Yora, 1972; Burgess et al., 1973). In the studies reported here, we used preparations of radioactive TMV in order to measure the extent of virus-particle attaehment to or absorption by tobacco protoplasts. In addition, we prepared extracts of protoplasts inoculated with labeled TMV and examined the physical state of the radioactive material therein by sucrose gradient centrifugation. 1Kentucky Agricultural Experiment Station Journal Article No. 74-11-85. zPresent address: Department of Plant Pathology and Plant Genetics, University of Georgia, Athens, GA 30602. 459 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Preparation of labeled virus. Nicotiana tubacun L. cv. Samsun nn plants, grow-
ing in glass jars containing Hoagland’s solution from which sulfates and phosphates were omitted, were inoculated with TMV. H,S6S0, and H,92P0, (or, in some cases, only H136S0,) were added to the root medium at various times over a period of 8 days. The virus was then extracted from the plants and purified by a modification of the procedure of Boedtker and Simmons (1958). Preparation of protoplasts. Protoplasts were obtained from tobacco (cv. Samsun nn) leaves by procedures similar to those developed by Takebe et al. (1968), Aoki and Takebe (1969), and Otsuki et al. (1972). Inoculation of protoplasts. The protoplasts were washed immediately before inoculation (Otsuki et al., 1972) and inoculated with poly-L-ornithine-treated, labeled TMV according to the procedure- of Takebe and Otsuki (1969). After inoculation, the protoplasts were washed four times by centrifugation in the wash me-
460
WYATT
dium (10 ml/ml virus-protoplast mixture for each wash). The completeness of removal of excess virus was determined from the radioactivity in aliquots of used wash media. Inoculated protoplasts were incubated (Takebe et al., 1968) for various periods of time and were then quickly frozen. Other samples of protoplasts were inoculated with nonlabeled TMV, incubated for 24 hr, and stained with fluorescent anti-TMV globulin according to the procedure of Otsuki and Takebe (1969). Analysis of protoplasts inoculated with labeled TMV. Frozen, inoculated protoplasts were thawed and brought to 5-6 ml with 0.05 M potassium phosphate buffer (pH 7.5) containing 1 mM EDTA. lOctanol (10 ~1) was added, and each sample was ground at room temperature at low speed for 5 min in the microcontainer of a Virtis 45 Homogenizer. One-tenth volume of 10% deoxycholate (DOC) in water was added, and the extract was kept at room temperature for 5 min. One-tenth volume of 10% Triton X-100 (Sigma Chemical Co.) was then added and the extract was cooled to 4”. Omission of the detergent treatment resulted in the formation of a floating green pellicle, containing a considerable amount of radioactivity, during subsequent centrifugation. Poly-L-ornithine-treated TMV binds to glass surfaces, thus, polyallomer tubes and silicone-treated pipets were used for all manipulations involving extracts of inoculated protoplasts. Radioactivity in aliquots of the extract was determined by measuring Cerenkov radiation followed by liquid scintillation and by subtracting from the scintillation counting rates those counts contributed by 32P. Other aliquots of the detergent-treated extracts were layered on cold, IO-40% (w/v) linear sucrose gradients containing 1 mM EDTA (adjusted to pH 7.8 with NaOH) and 0.5% Triton X-100. The gradients were centrifuged at 4’ for 2 % hr at 24,000 rpm in a Spinco SW 25.1 rotor. One-milliliter fractions from the gradients were collected with an ISCO Fractionator. These fractions and the resuspended pellet from the
AND SHAW
bottom of the tube were analyzed for radioactivity. RESULTS
Fluorescent antibody staining of tobacco protoplasts incubated for 24 hr after inoculation with TMV (at a final concentration of 1 pglml) indicated that 50, 47, 39, and 53% of the protoplasts were infected in four separate experiments. These values are within the range of those reported in several studies (Takebe and Otsuki, 1969; Hibi and Yora, 1972; Coutts et al., 1972; Burgess et al., 1973) but lower than those observed by Otsuki et al. (1972). Retention of TMV by protoplasts. Protoplast preparations were inoculated with various concentrations of labeled TMV. The washed protoplasts were homogenized in buffer and the homogenates were made 1 N in NaOH. The amount of radioactivity in the alkaline digests was measured. As shown in Table 1, large amounts of virus were retained by the protoplasts. Approximately the same percentage of the particles in the inoculum was retained by protoplasts inoculated with TMV at a final concentration of either 1 or 10 pg/ml (i.e., about 8 x lo3 and 8 x 10’ particles per protoplast, respectively). At a virus concentration of 100 pg/ml, a smaller proportion of the particles was retained. The results at this concentration suggest that the maximum number of virus particles which can be retained by a protoplast is between 3 x lo5 and 5 x 105. When poly-L-ornithine was not included in the inoculum, much less TMV was retained by the protoplasts (Table 2). The amount of time required for maximum retention of TMV by protoplasts was determined. Inoculated protoplasts were washed at various times after the addition of virus. As early as 10 min after mixing virus with protoplasts, 10% or more of the TMV could not be removed by washing (Table 3). Maximum retention of virus appeared to occur within 20-30 min. Simi. lar results were obtained when the inocu. lum contained virus at a final concentra tion of 10 pug/ml. Physical state of retained TMV parti cles. Protoplasts were inoculated witl,
RETENTION
AND DISSOCIATION TABLE
OF TMV 1
EFFECT OF VIRUS CONCENTRATION ON RETENTION OF TMV
BY INOCULATED TOBACCO PROTOPLASTS’
Virus added
Expt. no.
Virus retainedb
Total dpm
dml’
461
BY PROTOPLASTS
7c
dpm
No. particles
Particles/protoplast
1
100 10 1
760,000 76,000 7,600
37,250 11,590 1,070
4.9 15.3 14.1
2.9 x 10” 9.2 x 10’0 8.4 x 10’
2.9 x lo5 9.2 x 10’ 8.4x lOa
2
100 10 1
2,130,OOO 213,000 21,300
106,490 25,540 2,440
5.0 12.0 11.5
3.0 x 10” 7.2 x lOLo 6.9 x lo4
3.0 x 105 7.2 x 10’ 6.9 x lo3
3
100 10 1
1,320,OOO 132,000 13,200
104,270 19,840 2,240
7.9 15.0 17.0
4.7 x 10” 9.0 x 10’” 1.0 x 10’0
4.7 x 105 9.0 x 10’ 1.0 x 10’
a Protoplasts (1 x 108) in 2 ml were inoculated with 2 ml poly-L-ornithine-treated protoplasts were washed and frozen. b 35S not removed by repeated washing of the protoplasts. c Concentration of virus after mixing with protoplasts. TABLE
Poly-Lornithine bdml)
BY INOCULATED TOBACCO PROTOPLASTS’
Virus added rdml’
Total dw
after 1 hr the
2
EFFECT OF POLY-L-ORNITHINE ON RETENTION OF TMV Expt no.
[““S]TMV;
Virus retainedb dw
%
No. particles
Particles/ protoplast
1
1 0
1 1
512,000 512,000
149,660 5,150
29.2 1.0
1.8 x 10’0 6.0 x lOa
8.8 x 103 3.0 x 102
2
1 0
1 1
772,000 772,000
88,370 10,000
11.4 1.3
6.9 x lo9 7.8 x 10’
3.4 x 103 3.9 x 102
“Protoplasts (2 x 106) in 2 ml were inoculated with 2 ml [““SITMV 20 min the protoplasts were washed and frozen. b % not removed by repeated washing of protoplasts. c Concentration of virus after mixing with protoplasts.
[3”S]-[32P]TMV which had been incubated with poly-L-ornithine. The concentration of both virus and poly-L-ornithine, after mixing with the protoplasts, was 1 pg/ml. The inoculated protoplasts were washed 20 min after the addition of virus, incubated for various periods of time, and then ra.pidly frozen. The frozen samples were extracted and analyzed by sucrose gradient centrifugation. As a control, labeled virus was added to protoplasts which were then immediately extracted and analyzed. (The sedimentation of virus in control samples was identical to that of TMV which was not exposed to protoplasts.) The results of
(with or without
poly-L-ornithine);
after
one such experiment are shown in Table 4 and Fig. 1. Small amounts of radioactivity were detected near the tops of the gradients after centrifugation. After an infection period of 30 min, about 5% of the 35S and 3% of the szP from the retained virus were found in the top zone; these amounts did not change significantly as the infection period was prolonged. Somewhat more viral protein (3”S) than viral RNA (“‘P) appeared to be dissociated from the retained virus during the first 4 hr after inoculation. The small amounts of label detected at the tops of control gradients represent low
462
WYATT
AND SHAW
molecular-weight substances not completely removed during purification of the virus. (We have consistently found it more difficult to eliminate the last traces of nonviral 8sPthan nonviral Y3 from highly labeled preparations of TMV.) It is possible that these labeled contaminants in the virus preparations may have contributed slightly to the amount of label detected at the tops of gradients layered with extracts TABLE
of inoculated protoplasts. It should be pointed out, however, that such contaminants may well have been removed during the washing step prior to the analysis of inoculated protoplasts. In any case, most of the radioactivity detected at the tops of gradients layered with extracts of inoculated protoplasts probably represents viral protein and RNA dissociated from retained virus particles soon after inoculation. This observation is supported by the appearance of the virus zones in the gradients. In the case of extracts of inoculated protoplasts, the virus zone was always broader and contained labeled material which did not sediment quite as rapidly as intact TMV (Fig. 1). This region (fractions 11 to 17) probably contained partially dissociated virus particles. A considerable amount of the labeled material retained by protoplasts infected for short periods of time was found in the pellet after centrifugation through sucrose gradients. (The extent of this effect varied from one experiment to another-in one case over 80% of the label was found in the pellet of a gradient layered with an extract of protoplasts frozen 2 hr after inoculation.) Even when extracts of inoculated protoplasts were centrifuged at
3
EFFECT OF LENGTH OF INOCULATION PERIOD ON RETENTION OF TMV BY TOBACCO PROTOPLASTS Per cent +I retainedb
E;y;rient
Inoculation
period (min)c
10
20
30
40
60
1
13.4
14.2
17.2
18.9
18.6
2
9.6
15.6
16.1
16.6
15.9
o Protoplasta (5 x IO&) in 1 ml were inoculated with I ml ~“SjTMV (2 rg) containing 2 ag poly+omithine. Specific radioactivity of virus in Expt. 1, 9.6 x 10’ dpm/pg; in Expt. 2, 9.5 x 10’ dpm/pg. b Percentage of the W in the inoculum not removed by washing of the protoplasts. c Interval between addition of virus to protoplasts and beginning of washing. TABLE
4
DISTRIBUTION OF RADIOACTWITV IN SUCROSEGRADIENTS AFTER CENTRIFUGATION OF EXTRACTS OF TOBACCO PROTOPLMTS INOWLATED WITH [3”S]-[3ZPjTMV Infection period@ (hr)
0.5 1 2 4 6 ControP
% of Label retained by protoplasts”
% of Retained label in top zonec
% of Retained label in pelletd
9
=P
%
=P
Y3
JZP
12.6 13.0 11.8 12.2 13.0 -
12.0 12.3 11.4 12.0 11.7 -
5.0 5.1 4.8 3.8 4.2 0.4
2.9 2.8 2.1 2.3 4.6 1.0
47 40 35 29
43 41 32 28
17
16
DProtoplasts (12 x 103 in 12 ml were inoculated with a mixture of 24 pg TMV (2.0 x 10” dpm a% and 9.2 x 10’ dpm r*P per rg) and 24 pg poly-L-omithine in 12 ml. After 20 min, the protoplasts were washed (which took 10 min), resuspended in 12 ml incubation medium, and divided into 2-ml samples. At various times thereafter, the samples were frozen. The infection period represents the interval between the addition of virus to the protoplasts and the freezing of the sample. Each sample was extracted as described in the text; the final volume of each extract was 6 ml. A 3-ml aliquot of each extract was layered on a sucrose gradient. b Percentage of label in the inoculum which was not removed from the protoplasts by washing. c Percentage of label layered on gradient which was in the upper six l-ml fractions after centrifugation. d Percentage of label layered on gradient which was in the pellet after centrifugation. ‘Labeled virus was added to uninoculated protoplasts immediately before extraction.
RETENTION
AND DISSOCIATION
OF TMV
463
BY PROTOPLASTS
Control
30 minutes
6 -.I ’ *:0 ; 8 v) A
FRACTION
FRACTION
1. Density-gradient centrifugation of extracts of control protoplasts (right) and protoplasts inoculated with [a*P]-[S5SJTMV (left). Data are from part of the experiment described in Table 4. The inoculated protoplast pattern shown is of the sample which was frozen 30 min after mixing virus with protoplasts. The patterns of samples incubated for longer periods of time are similar except for the proportions of label in the pellets and in the virus zones (see text). The left side of each diagram represents the top of the gradient. The numbers at the lo,ver right represent the 3”S (upper number) and 32P (lower number) in the pellets after centrifugation. FIG.
10,000 g for 10 min, instead of in sucrose gradients, considerable amounts of radioactivity could be sedimented. As the incubation period was prolonged, the amount of radioactive material in the pellet decreased and a corresponding increase in radioactivity appeared in the gradient at a position corresponding to the virus zone. However, the amount of label in the pellet was never found (even in the case of a sample which was incubated for 16 hr after inoculation and washing) to decrease to the levels found in the pellets in control gradients. In an effort to determine whether inclusion of poly-L-ornithine in the inoculum might be responsible for the unusually rapid sedimentation of some of the labeled material in extracts of inoculated protoplasts, samples were inoculated with virus which had not been preincubated with the polycation. As shown in Table 5, much less of the retained labeled material was found
TABLE
5
EFFECT OF POLY-L-ORNITHINE ON SEDIMENTATION THROUGH SUCROSE GRADIENTS OF TMV RETAINED BY INOCULATED PROTOPLASTS’
Poly-Lomithine
h/ml)
% of Label retained by protoplasts
% of Label in gradient pelletb
Ti
=P
Ti
32P
0
2.9
2.6
9.3
10.7
1
17.9
17.7
31.6
30.3
DProtoplasts (2 x 106) in 2 ml were inoculated with 4 rg TMV (1.7 x lo5 dpm 3sS and 4.1 x 10’ dpm 32P per pg) with or without poly-L-ornithine in 2 ml. After 20 min. the protoplasts were washed and frozen, then extracted and analyzed by sucrose gradient centrifugation. bPercentage of the labeled material layered on gradient which was in the pellet after centrifugation.
in the pellets of gradients layered with extracts of protoplasts when poly-L-orni-
464
WYATT
thine was omitted from the inoculum. Experiments with TMV at a final concentration of 10 fig/ml (and poly-L-ornithine at 1 pg/ml) produced results similar to those shown in Table 4. DISCUSSION
The amount of TMV attached to or absorbed by inoculated tobacco protoplasts after washing has been variously reported as from lo-lo3 particles per protoplast when the final concentration of virus was 1 kg/ml (Takebe and Otsuki, 1969; Otsuki et al., 1972; Hibi and Yora, 1972). These estimates were based on infectivity assays and electron microscopy. We have also made extracts of protoplasts which were washed and frozen 20-60 min after inoculation and found them to produce no, or very few, lesions when rubbed on leaves of a local-lesion host. Determination of attachment or absorption by means of radioactivity measurements, however, indicated that over 10% of the virus (from 3.4 x 10’ to 1 x 10’ particles per protoplast) was retained when the virus was inoculated at a concentration of 1 pglml (Tables 1, 2, and 3). (If only those protoplasts in which progeny virus could later be demonstrated were capable of retaining virus particles, for which we have no evidence, then these values would need to be approximately doubled.) Extracts of protoplasts which retained this many TMV particles should produce significant numbers of local lesions. One reason for the apparent low specific infectivity of the retained virus is suggested by the rapid sedimentation of large amounts of the labeled material in the extracts (Tables 4 and 5). Pretreatment of the virus with poly-L-ornithine, which under appropriate conditions, will coprecipitate with TMV, appears to cause either serious aggregation of the virus or binding of the virus to protoplast constituents. This condition does not seem to diminish until some time after the addition of virus to the protoplasts. Poly-L-ornithine has been shown to be required for the infection of tobacco protoplasts with TMV (Takebe and Otsuki, 1969), cucumber mosaic virus (Otsuki and
AND SHAW
Takebe, 1973), cowpea chlorotic mottle virus (Motoyoshi et al., 1973a), and potato virus X (Otsuki et al., 1974). Whether it is required for attachment to or penetration of the plasmalemma by the virus, for wounding of the membrane, or for some other event in the establishment of infection has not yet been shown conclusively. Our results indicate that omission of treatment of TMV with poly-L-ornithine results in a 6- to 30-fold decrease in retention of virus (Tables 2 and 5). The degree of dissociation of retained TMV particles (or uncoating of viral RNA) was surprisingly low, especially when compared with that which follows the inoculation of tobacco leaves with TMV (Shaw, 1967). With the protoplasts, dissociation appeared to occur and be completed by 30 min after the beginning of inoculation. About 5% of the total viral protein was released from virus particles; a smaller amount of viral RNA (2-3%) also appeared to be released though the conditions of extraction were such that uncoated viral RNA would very likely have been degraded prior to density gradient analysis. The position and shape of the virus zones (Fig. 1) suggest that much of the retained virus was not dissociated but that, within 30 min, enough protein had been removed from some of the other particles to render them significantly less sedimentable. In studies bearing on the same question, Otsuki et al. (1972) reported that the TMV particles had disappeared from cytoplasmic vesicles 30 min after the mixing of virus (at 1 pg/ml) and protoplasts and assumed that, by this time, the uncoating of the viral RNA was well under way. The reports of Hibi and Yora (1972) and Burgess et al. (1973), on the other hand, suggested that parental TMV particles (used at either 1 or 200 pg/ml) were visible for periods in excess of 30 min from the beginning of inoculation. Our results indicate that tobacco protoplasts are capable of absorbing or adsorbing very many TMV particles. However, only a small amount of this retained virus appears to undergo dissociation after inoculation. It appears, therefore, that only a small fraction of the TMV particles which
RETENTION
AND DISSOCIATION
OF TMV BY PROTOPLASTS
465
plasts with cowpea chlorotic mottle virus and its can be retained by the protoplasts is necesRNA. J. Gen. Viral. 20, 177-193. sary for and takes part in the establishment of infection. This view is consistent MOTOYOSHI,F., BANCROF~,J. B., and WATTS, J. W. (1973b). A direct estimate of the number of cowpea with those of Otsuki and Takebe (1973), chlorotic mottle virus particles absorbed by tobacco who obtained maximum infection with protoplasts that become infected. J. Gen. Viral. 21, TMV at 0.1 pg/ml, and Motoyoshi et al. 159-161. (1973b), who reported that as few as 760 OTSUKI,Y., and TAKEBE,I. (1969). Fluorescent anticowpea chlorotic mottle virus particles will body staining of tobacco mosaic virus antigen in infect a tobacco protoplast. tobacco mesophyll protoplasts. Virology 38, REFERENCES AOKI, S., and TAKEBE,I. (1969). Infection of tobacco mesophyll protoplasts by tobacco mosaic virus ribonucleic acid. Virology 39, 439-448. BOEDTKER,H., and SIMMONS,N. S. (1958). The preparation and characterization of essentially uniform tobacco mosaic virus particles. J. Amer. Chem. Sot. 80, 2550-2557.
BURGESS,F., MOTOYOSHI,F., and FLEMING, E. N. (1973). The mechanism of infection of plant protoplasts by viruses. Planta (Berlin) 112, 323-332. COV?TS,R. H. A., COCKING,E. C., and KASSANIS,B. (1972). Infection of tobacco mesophyll protoplasts with tobacco mosaic virus. J. Gem Viral. 17, 289-294.
HIBI, I., and YORA,K. (1972). Electron microscopy of tobacco mosaic virus infection in tobacco mesophyll protoplasts. Ann. Phytopathol. Sot. Jap. 38, 350-356.
MOTOYOSHI,F., BANCROFT,J. B., WATTS,J. W., and BURGESS, J. (1973a). The infection of tobacco proto-
497-499.
OTSUKI, Y., TAKEBE, I., MATSUI, C., and HONDA, Y. (1972). Ultrastructure of infection of tobacco mesophyll protoplasts by tobacco mosaic virus. Virology 49, 188-194. OTSUKI,T., and TAKEBE,I. (1973). Infection of tobacco mesophyll protoplasts by cucumber mosaic virus. Virology
52, 433-438.
OTSUKI, Y., TAKEBE, I., HONDA, Y., KAJITA, S., and MATSUI, C. (1974). Infection of tobacco mesophyll protoplasts by potato virus X. J. Gen. Viral. 22, 375-385.
SHAW,J. G. (1967). In vivo removal of protein from tobacco mosaic virus after inoculation of tobacco leaves. Virology 31, 665-675. TAKEBE,I., OTSUKI,Y., and AOKI, S. (1968). Isolation of tobacco mesophyll cells in intact and active state. Plant Cell Physiol. 9, 115-124. TAKEBE,I., and OTSUKI,Y. (1969). Infection of tobacco mesophyll protoplasts by tobacco mosaic virus. Proc. Nat. Acad. Sci. USA 64, 843-848.
63, 459~465(1975)
Retention Mosaic
and Dissociation Virus by Tobacco
of Tobacco Protoplasts’
STEPHEN D. WYATT2 AND JOHN G. SHAW Department
of Plant Pathology,
University
Accepted
of Kentucky,
September
Lexington,
Kentucky
40506
16, 1974
When tobacco protoplasts were inoculated with radioactive tobacco mosaic virus (TMV) at a concentration of 1 &ml, about 15% of the particles (approximately 8 x lOa particles per protoplast) were retained. When poly-L-ornithine was not included in the inoculum, the amount of virus retained was reduced 6- to 30-fold. Maximum retention was observed 20-30 min after the addition of virus to the protoplasts. Thirty minutes after mixing virus and protoplasts, about 5% of the protein of the retained TMV had been removed and some of the virus particles appeared to be partially dissociated. A considerable amount of the retained viral material in extracts of inoculated protoplasts was found in the pellets after sucrose gradient centrifugation. This abnormally rapid sedimentation was correlated with the presence of poly-L-ornithine in the inoculum. INTRODUCTION
MATERIALS
The infection of protoplasts prepared from tobacco mesophyll cells with tobacco mosaic virus (TMV) has been demonstrated by Takebe and Otsuki (1969). This system has subsequently been utilized for studies of the attachment of virus particles to the plasmalemma, the appearance of the particles in cytoplasmic vesicles, and the production of progeny virus in the infected protoplasts (Otsuki and Takebe, 1969; Coutts et al., 1972; Otsuki et al., 1972; Hibi and Yora, 1972; Burgess et al., 1973). In the studies reported here, we used preparations of radioactive TMV in order to measure the extent of virus-particle attaehment to or absorption by tobacco protoplasts. In addition, we prepared extracts of protoplasts inoculated with labeled TMV and examined the physical state of the radioactive material therein by sucrose gradient centrifugation. 1Kentucky Agricultural Experiment Station Journal Article No. 74-11-85. zPresent address: Department of Plant Pathology and Plant Genetics, University of Georgia, Athens, GA 30602. 459 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Preparation of labeled virus. Nicotiana tubacun L. cv. Samsun nn plants, grow-
ing in glass jars containing Hoagland’s solution from which sulfates and phosphates were omitted, were inoculated with TMV. H,S6S0, and H,92P0, (or, in some cases, only H136S0,) were added to the root medium at various times over a period of 8 days. The virus was then extracted from the plants and purified by a modification of the procedure of Boedtker and Simmons (1958). Preparation of protoplasts. Protoplasts were obtained from tobacco (cv. Samsun nn) leaves by procedures similar to those developed by Takebe et al. (1968), Aoki and Takebe (1969), and Otsuki et al. (1972). Inoculation of protoplasts. The protoplasts were washed immediately before inoculation (Otsuki et al., 1972) and inoculated with poly-L-ornithine-treated, labeled TMV according to the procedure- of Takebe and Otsuki (1969). After inoculation, the protoplasts were washed four times by centrifugation in the wash me-
460
WYATT
dium (10 ml/ml virus-protoplast mixture for each wash). The completeness of removal of excess virus was determined from the radioactivity in aliquots of used wash media. Inoculated protoplasts were incubated (Takebe et al., 1968) for various periods of time and were then quickly frozen. Other samples of protoplasts were inoculated with nonlabeled TMV, incubated for 24 hr, and stained with fluorescent anti-TMV globulin according to the procedure of Otsuki and Takebe (1969). Analysis of protoplasts inoculated with labeled TMV. Frozen, inoculated protoplasts were thawed and brought to 5-6 ml with 0.05 M potassium phosphate buffer (pH 7.5) containing 1 mM EDTA. lOctanol (10 ~1) was added, and each sample was ground at room temperature at low speed for 5 min in the microcontainer of a Virtis 45 Homogenizer. One-tenth volume of 10% deoxycholate (DOC) in water was added, and the extract was kept at room temperature for 5 min. One-tenth volume of 10% Triton X-100 (Sigma Chemical Co.) was then added and the extract was cooled to 4”. Omission of the detergent treatment resulted in the formation of a floating green pellicle, containing a considerable amount of radioactivity, during subsequent centrifugation. Poly-L-ornithine-treated TMV binds to glass surfaces, thus, polyallomer tubes and silicone-treated pipets were used for all manipulations involving extracts of inoculated protoplasts. Radioactivity in aliquots of the extract was determined by measuring Cerenkov radiation followed by liquid scintillation and by subtracting from the scintillation counting rates those counts contributed by 32P. Other aliquots of the detergent-treated extracts were layered on cold, IO-40% (w/v) linear sucrose gradients containing 1 mM EDTA (adjusted to pH 7.8 with NaOH) and 0.5% Triton X-100. The gradients were centrifuged at 4’ for 2 % hr at 24,000 rpm in a Spinco SW 25.1 rotor. One-milliliter fractions from the gradients were collected with an ISCO Fractionator. These fractions and the resuspended pellet from the
AND SHAW
bottom of the tube were analyzed for radioactivity. RESULTS
Fluorescent antibody staining of tobacco protoplasts incubated for 24 hr after inoculation with TMV (at a final concentration of 1 pglml) indicated that 50, 47, 39, and 53% of the protoplasts were infected in four separate experiments. These values are within the range of those reported in several studies (Takebe and Otsuki, 1969; Hibi and Yora, 1972; Coutts et al., 1972; Burgess et al., 1973) but lower than those observed by Otsuki et al. (1972). Retention of TMV by protoplasts. Protoplast preparations were inoculated with various concentrations of labeled TMV. The washed protoplasts were homogenized in buffer and the homogenates were made 1 N in NaOH. The amount of radioactivity in the alkaline digests was measured. As shown in Table 1, large amounts of virus were retained by the protoplasts. Approximately the same percentage of the particles in the inoculum was retained by protoplasts inoculated with TMV at a final concentration of either 1 or 10 pg/ml (i.e., about 8 x lo3 and 8 x 10’ particles per protoplast, respectively). At a virus concentration of 100 pg/ml, a smaller proportion of the particles was retained. The results at this concentration suggest that the maximum number of virus particles which can be retained by a protoplast is between 3 x lo5 and 5 x 105. When poly-L-ornithine was not included in the inoculum, much less TMV was retained by the protoplasts (Table 2). The amount of time required for maximum retention of TMV by protoplasts was determined. Inoculated protoplasts were washed at various times after the addition of virus. As early as 10 min after mixing virus with protoplasts, 10% or more of the TMV could not be removed by washing (Table 3). Maximum retention of virus appeared to occur within 20-30 min. Simi. lar results were obtained when the inocu. lum contained virus at a final concentra tion of 10 pug/ml. Physical state of retained TMV parti cles. Protoplasts were inoculated witl,
RETENTION
AND DISSOCIATION TABLE
OF TMV 1
EFFECT OF VIRUS CONCENTRATION ON RETENTION OF TMV
BY INOCULATED TOBACCO PROTOPLASTS’
Virus added
Expt. no.
Virus retainedb
Total dpm
dml’
461
BY PROTOPLASTS
7c
dpm
No. particles
Particles/protoplast
1
100 10 1
760,000 76,000 7,600
37,250 11,590 1,070
4.9 15.3 14.1
2.9 x 10” 9.2 x 10’0 8.4 x 10’
2.9 x lo5 9.2 x 10’ 8.4x lOa
2
100 10 1
2,130,OOO 213,000 21,300
106,490 25,540 2,440
5.0 12.0 11.5
3.0 x 10” 7.2 x lOLo 6.9 x lo4
3.0 x 105 7.2 x 10’ 6.9 x lo3
3
100 10 1
1,320,OOO 132,000 13,200
104,270 19,840 2,240
7.9 15.0 17.0
4.7 x 10” 9.0 x 10’” 1.0 x 10’0
4.7 x 105 9.0 x 10’ 1.0 x 10’
a Protoplasts (1 x 108) in 2 ml were inoculated with 2 ml poly-L-ornithine-treated protoplasts were washed and frozen. b 35S not removed by repeated washing of the protoplasts. c Concentration of virus after mixing with protoplasts. TABLE
Poly-Lornithine bdml)
BY INOCULATED TOBACCO PROTOPLASTS’
Virus added rdml’
Total dw
after 1 hr the
2
EFFECT OF POLY-L-ORNITHINE ON RETENTION OF TMV Expt no.
[““S]TMV;
Virus retainedb dw
%
No. particles
Particles/ protoplast
1
1 0
1 1
512,000 512,000
149,660 5,150
29.2 1.0
1.8 x 10’0 6.0 x lOa
8.8 x 103 3.0 x 102
2
1 0
1 1
772,000 772,000
88,370 10,000
11.4 1.3
6.9 x lo9 7.8 x 10’
3.4 x 103 3.9 x 102
“Protoplasts (2 x 106) in 2 ml were inoculated with 2 ml [““SITMV 20 min the protoplasts were washed and frozen. b % not removed by repeated washing of protoplasts. c Concentration of virus after mixing with protoplasts.
[3”S]-[32P]TMV which had been incubated with poly-L-ornithine. The concentration of both virus and poly-L-ornithine, after mixing with the protoplasts, was 1 pg/ml. The inoculated protoplasts were washed 20 min after the addition of virus, incubated for various periods of time, and then ra.pidly frozen. The frozen samples were extracted and analyzed by sucrose gradient centrifugation. As a control, labeled virus was added to protoplasts which were then immediately extracted and analyzed. (The sedimentation of virus in control samples was identical to that of TMV which was not exposed to protoplasts.) The results of
(with or without
poly-L-ornithine);
after
one such experiment are shown in Table 4 and Fig. 1. Small amounts of radioactivity were detected near the tops of the gradients after centrifugation. After an infection period of 30 min, about 5% of the 35S and 3% of the szP from the retained virus were found in the top zone; these amounts did not change significantly as the infection period was prolonged. Somewhat more viral protein (3”S) than viral RNA (“‘P) appeared to be dissociated from the retained virus during the first 4 hr after inoculation. The small amounts of label detected at the tops of control gradients represent low
462
WYATT
AND SHAW
molecular-weight substances not completely removed during purification of the virus. (We have consistently found it more difficult to eliminate the last traces of nonviral 8sPthan nonviral Y3 from highly labeled preparations of TMV.) It is possible that these labeled contaminants in the virus preparations may have contributed slightly to the amount of label detected at the tops of gradients layered with extracts TABLE
of inoculated protoplasts. It should be pointed out, however, that such contaminants may well have been removed during the washing step prior to the analysis of inoculated protoplasts. In any case, most of the radioactivity detected at the tops of gradients layered with extracts of inoculated protoplasts probably represents viral protein and RNA dissociated from retained virus particles soon after inoculation. This observation is supported by the appearance of the virus zones in the gradients. In the case of extracts of inoculated protoplasts, the virus zone was always broader and contained labeled material which did not sediment quite as rapidly as intact TMV (Fig. 1). This region (fractions 11 to 17) probably contained partially dissociated virus particles. A considerable amount of the labeled material retained by protoplasts infected for short periods of time was found in the pellet after centrifugation through sucrose gradients. (The extent of this effect varied from one experiment to another-in one case over 80% of the label was found in the pellet of a gradient layered with an extract of protoplasts frozen 2 hr after inoculation.) Even when extracts of inoculated protoplasts were centrifuged at
3
EFFECT OF LENGTH OF INOCULATION PERIOD ON RETENTION OF TMV BY TOBACCO PROTOPLASTS Per cent +I retainedb
E;y;rient
Inoculation
period (min)c
10
20
30
40
60
1
13.4
14.2
17.2
18.9
18.6
2
9.6
15.6
16.1
16.6
15.9
o Protoplasta (5 x IO&) in 1 ml were inoculated with I ml ~“SjTMV (2 rg) containing 2 ag poly+omithine. Specific radioactivity of virus in Expt. 1, 9.6 x 10’ dpm/pg; in Expt. 2, 9.5 x 10’ dpm/pg. b Percentage of the W in the inoculum not removed by washing of the protoplasts. c Interval between addition of virus to protoplasts and beginning of washing. TABLE
4
DISTRIBUTION OF RADIOACTWITV IN SUCROSEGRADIENTS AFTER CENTRIFUGATION OF EXTRACTS OF TOBACCO PROTOPLMTS INOWLATED WITH [3”S]-[3ZPjTMV Infection period@ (hr)
0.5 1 2 4 6 ControP
% of Label retained by protoplasts”
% of Retained label in top zonec
% of Retained label in pelletd
9
=P
%
=P
Y3
JZP
12.6 13.0 11.8 12.2 13.0 -
12.0 12.3 11.4 12.0 11.7 -
5.0 5.1 4.8 3.8 4.2 0.4
2.9 2.8 2.1 2.3 4.6 1.0
47 40 35 29
43 41 32 28
17
16
DProtoplasts (12 x 103 in 12 ml were inoculated with a mixture of 24 pg TMV (2.0 x 10” dpm a% and 9.2 x 10’ dpm r*P per rg) and 24 pg poly-L-omithine in 12 ml. After 20 min, the protoplasts were washed (which took 10 min), resuspended in 12 ml incubation medium, and divided into 2-ml samples. At various times thereafter, the samples were frozen. The infection period represents the interval between the addition of virus to the protoplasts and the freezing of the sample. Each sample was extracted as described in the text; the final volume of each extract was 6 ml. A 3-ml aliquot of each extract was layered on a sucrose gradient. b Percentage of label in the inoculum which was not removed from the protoplasts by washing. c Percentage of label layered on gradient which was in the upper six l-ml fractions after centrifugation. d Percentage of label layered on gradient which was in the pellet after centrifugation. ‘Labeled virus was added to uninoculated protoplasts immediately before extraction.
RETENTION
AND DISSOCIATION
OF TMV
463
BY PROTOPLASTS
Control
30 minutes
6 -.I ’ *:0 ; 8 v) A
FRACTION
FRACTION
1. Density-gradient centrifugation of extracts of control protoplasts (right) and protoplasts inoculated with [a*P]-[S5SJTMV (left). Data are from part of the experiment described in Table 4. The inoculated protoplast pattern shown is of the sample which was frozen 30 min after mixing virus with protoplasts. The patterns of samples incubated for longer periods of time are similar except for the proportions of label in the pellets and in the virus zones (see text). The left side of each diagram represents the top of the gradient. The numbers at the lo,ver right represent the 3”S (upper number) and 32P (lower number) in the pellets after centrifugation. FIG.
10,000 g for 10 min, instead of in sucrose gradients, considerable amounts of radioactivity could be sedimented. As the incubation period was prolonged, the amount of radioactive material in the pellet decreased and a corresponding increase in radioactivity appeared in the gradient at a position corresponding to the virus zone. However, the amount of label in the pellet was never found (even in the case of a sample which was incubated for 16 hr after inoculation and washing) to decrease to the levels found in the pellets in control gradients. In an effort to determine whether inclusion of poly-L-ornithine in the inoculum might be responsible for the unusually rapid sedimentation of some of the labeled material in extracts of inoculated protoplasts, samples were inoculated with virus which had not been preincubated with the polycation. As shown in Table 5, much less of the retained labeled material was found
TABLE
5
EFFECT OF POLY-L-ORNITHINE ON SEDIMENTATION THROUGH SUCROSE GRADIENTS OF TMV RETAINED BY INOCULATED PROTOPLASTS’
Poly-Lomithine
h/ml)
% of Label retained by protoplasts
% of Label in gradient pelletb
Ti
=P
Ti
32P
0
2.9
2.6
9.3
10.7
1
17.9
17.7
31.6
30.3
DProtoplasts (2 x 106) in 2 ml were inoculated with 4 rg TMV (1.7 x lo5 dpm 3sS and 4.1 x 10’ dpm 32P per pg) with or without poly-L-ornithine in 2 ml. After 20 min. the protoplasts were washed and frozen, then extracted and analyzed by sucrose gradient centrifugation. bPercentage of the labeled material layered on gradient which was in the pellet after centrifugation.
in the pellets of gradients layered with extracts of protoplasts when poly-L-orni-
464
WYATT
thine was omitted from the inoculum. Experiments with TMV at a final concentration of 10 fig/ml (and poly-L-ornithine at 1 pg/ml) produced results similar to those shown in Table 4. DISCUSSION
The amount of TMV attached to or absorbed by inoculated tobacco protoplasts after washing has been variously reported as from lo-lo3 particles per protoplast when the final concentration of virus was 1 kg/ml (Takebe and Otsuki, 1969; Otsuki et al., 1972; Hibi and Yora, 1972). These estimates were based on infectivity assays and electron microscopy. We have also made extracts of protoplasts which were washed and frozen 20-60 min after inoculation and found them to produce no, or very few, lesions when rubbed on leaves of a local-lesion host. Determination of attachment or absorption by means of radioactivity measurements, however, indicated that over 10% of the virus (from 3.4 x 10’ to 1 x 10’ particles per protoplast) was retained when the virus was inoculated at a concentration of 1 pglml (Tables 1, 2, and 3). (If only those protoplasts in which progeny virus could later be demonstrated were capable of retaining virus particles, for which we have no evidence, then these values would need to be approximately doubled.) Extracts of protoplasts which retained this many TMV particles should produce significant numbers of local lesions. One reason for the apparent low specific infectivity of the retained virus is suggested by the rapid sedimentation of large amounts of the labeled material in the extracts (Tables 4 and 5). Pretreatment of the virus with poly-L-ornithine, which under appropriate conditions, will coprecipitate with TMV, appears to cause either serious aggregation of the virus or binding of the virus to protoplast constituents. This condition does not seem to diminish until some time after the addition of virus to the protoplasts. Poly-L-ornithine has been shown to be required for the infection of tobacco protoplasts with TMV (Takebe and Otsuki, 1969), cucumber mosaic virus (Otsuki and
AND SHAW
Takebe, 1973), cowpea chlorotic mottle virus (Motoyoshi et al., 1973a), and potato virus X (Otsuki et al., 1974). Whether it is required for attachment to or penetration of the plasmalemma by the virus, for wounding of the membrane, or for some other event in the establishment of infection has not yet been shown conclusively. Our results indicate that omission of treatment of TMV with poly-L-ornithine results in a 6- to 30-fold decrease in retention of virus (Tables 2 and 5). The degree of dissociation of retained TMV particles (or uncoating of viral RNA) was surprisingly low, especially when compared with that which follows the inoculation of tobacco leaves with TMV (Shaw, 1967). With the protoplasts, dissociation appeared to occur and be completed by 30 min after the beginning of inoculation. About 5% of the total viral protein was released from virus particles; a smaller amount of viral RNA (2-3%) also appeared to be released though the conditions of extraction were such that uncoated viral RNA would very likely have been degraded prior to density gradient analysis. The position and shape of the virus zones (Fig. 1) suggest that much of the retained virus was not dissociated but that, within 30 min, enough protein had been removed from some of the other particles to render them significantly less sedimentable. In studies bearing on the same question, Otsuki et al. (1972) reported that the TMV particles had disappeared from cytoplasmic vesicles 30 min after the mixing of virus (at 1 pg/ml) and protoplasts and assumed that, by this time, the uncoating of the viral RNA was well under way. The reports of Hibi and Yora (1972) and Burgess et al. (1973), on the other hand, suggested that parental TMV particles (used at either 1 or 200 pg/ml) were visible for periods in excess of 30 min from the beginning of inoculation. Our results indicate that tobacco protoplasts are capable of absorbing or adsorbing very many TMV particles. However, only a small amount of this retained virus appears to undergo dissociation after inoculation. It appears, therefore, that only a small fraction of the TMV particles which
RETENTION
AND DISSOCIATION
OF TMV BY PROTOPLASTS
465
plasts with cowpea chlorotic mottle virus and its can be retained by the protoplasts is necesRNA. J. Gen. Viral. 20, 177-193. sary for and takes part in the establishment of infection. This view is consistent MOTOYOSHI,F., BANCROF~,J. B., and WATTS, J. W. (1973b). A direct estimate of the number of cowpea with those of Otsuki and Takebe (1973), chlorotic mottle virus particles absorbed by tobacco who obtained maximum infection with protoplasts that become infected. J. Gen. Viral. 21, TMV at 0.1 pg/ml, and Motoyoshi et al. 159-161. (1973b), who reported that as few as 760 OTSUKI,Y., and TAKEBE,I. (1969). Fluorescent anticowpea chlorotic mottle virus particles will body staining of tobacco mosaic virus antigen in infect a tobacco protoplast. tobacco mesophyll protoplasts. Virology 38, REFERENCES AOKI, S., and TAKEBE,I. (1969). Infection of tobacco mesophyll protoplasts by tobacco mosaic virus ribonucleic acid. Virology 39, 439-448. BOEDTKER,H., and SIMMONS,N. S. (1958). The preparation and characterization of essentially uniform tobacco mosaic virus particles. J. Amer. Chem. Sot. 80, 2550-2557.
BURGESS,F., MOTOYOSHI,F., and FLEMING, E. N. (1973). The mechanism of infection of plant protoplasts by viruses. Planta (Berlin) 112, 323-332. COV?TS,R. H. A., COCKING,E. C., and KASSANIS,B. (1972). Infection of tobacco mesophyll protoplasts with tobacco mosaic virus. J. Gem Viral. 17, 289-294.
HIBI, I., and YORA,K. (1972). Electron microscopy of tobacco mosaic virus infection in tobacco mesophyll protoplasts. Ann. Phytopathol. Sot. Jap. 38, 350-356.
MOTOYOSHI,F., BANCROFT,J. B., WATTS,J. W., and BURGESS, J. (1973a). The infection of tobacco proto-
497-499.
OTSUKI, Y., TAKEBE, I., MATSUI, C., and HONDA, Y. (1972). Ultrastructure of infection of tobacco mesophyll protoplasts by tobacco mosaic virus. Virology 49, 188-194. OTSUKI,T., and TAKEBE,I. (1973). Infection of tobacco mesophyll protoplasts by cucumber mosaic virus. Virology
52, 433-438.
OTSUKI, Y., TAKEBE, I., HONDA, Y., KAJITA, S., and MATSUI, C. (1974). Infection of tobacco mesophyll protoplasts by potato virus X. J. Gen. Viral. 22, 375-385.
SHAW,J. G. (1967). In vivo removal of protein from tobacco mosaic virus after inoculation of tobacco leaves. Virology 31, 665-675. TAKEBE,I., OTSUKI,Y., and AOKI, S. (1968). Isolation of tobacco mesophyll cells in intact and active state. Plant Cell Physiol. 9, 115-124. TAKEBE,I., and OTSUKI,Y. (1969). Infection of tobacco mesophyll protoplasts by tobacco mosaic virus. Proc. Nat. Acad. Sci. USA 64, 843-848.
