Attachment of labeled TMV to tobacco mesophyll protoplasts

VIROLOGY

64, 43-48

(197.5)

Attachment

of Labeled TMV to Tobacco

Mesophyll

Protoplasts

YU. N. ZHURAVLEV, N. F. PISETSKAYA, L. A. SHUMILOVA, AND V. G. REIFMAN T. I. MUSOROK, Institute

of Biology and Pedology, Far East Science Centre, Academy of Sciences of the USSR, Vladiuostok-22, U.S.S.R. Accepted

September

23, 1974

“C-labeled TMV was used to examine virus attachment to tobacco protoplasts. About 600 virus particles per protoplast were attached under experimental conditions (1 rg/ml of TMV, 1 Kg/ml of poly-L-ornithine, 15 x lo5 cells/ml of protoplasts, 10 min at 25”). The number of virus particles associated with protoplasts increases during the course of inoculation, lo-15 min exposures being optimal. Approximately linear enhancement of attachment was observed at l-1,500 pg/ml TMV-concentration interval. Poly-L-ornithine (1 fig/ml) was shown to be a potent stimulator of virus retention at all TMV concentrations used. Increased poly-L-ornithine concentration results in greater virus retention. The authors conclude that virus attachment per se is stimulated by poly-L-ornithine and and penetration should be considered more maintain that the processes of attachmenl specifically. INTRODUCTION

pinocytosis in the early stages of infection is needed (Burgess et al., 1973a). The present report describes some results of studies on retention of “C-labeled TMV by tobacco protoplasts.

Tobacco mesophyll protoplasts appear to be a promising model for the study of the process of plant virus infection, since viruses were shown to penetrate through the plasmalemma rather easily (Cocking and MATERIALS AND METHODS Pojnar, 1969; Takebe and Otsuki, 1969). Plants. For protoplast preparation, toThe idea of pinocytotic uptake of TMV by tabacum L. cv. isolated protoplasts was put forward by bacco plants (Nicotiana laboratory White Burley) were grown in a greenhouse. experiments in Cocking’s (Cocking, 1966; Cocking and Pojnar, 1969) After the beginning of stem elongation, the leaves revealing no xeromorphic signs (usuand was supported later by the electron microscopic data of Otsuki et al. (1972) ally the third or fourth leaf) were harvested. who calculated that under experimental conditions one protoplast absorbs up to Preparation of tobacco protoplasts. Pro1,000 virus particles, this number being toplasts were isolated essentially according sufficient to induce infection in 80-90% of to the procedure of Takebe and collaborathe protoplasts. A marked decrease in tors (1968), using their latest modification infectivity, accompanied by the disappear(Aoki and Takebe, 1969) which we further modified by introducing 0.05 M potassium ance of visible particles in the cytoplasm, was observed during the initial 6-hr post- phosphate buffer and 1 x lo-’ M 2-merinto all of the solutions. A inoculation period (Cocking, 1966, 1970; captoethanol Aoki and Takebe, 1969; Takebe and Ot- mixture of 0.5% macerozyme R-10 (Yakult suki, 1969; Otsuki et al., 1972). Yet, nu- Biochemicals Co., Ltd., Japan) and 0.5% merous aspects of virus attachment and Shuchardt pectinase (Miinchen, Gerpenetration still remain obscure. Thus, many) in phosphate buffer, pH 5.8, was consideration of mechanisms other than used for tissue maceration. Cell walls were

44

ZHURAVLEV

digested by treatment with 2% cellulase (Onozuka R-10, Kinki Yakult, Japan); for details see Pisetskaya et al. (1975). Labeled uirus. To prepare “C-labeled TMV, 10 plants of N. tabacum cv. Samsun (5-6 weeks old) were used. Upper and lower leaves were removed and discarded. The remaining three leaves were dusted with Carborundum and inoculated with TMV (0.1 mg/ml). The leaves were carefully washed with tap water before the plants were transferred to a Plexiglas box under conditions of continuous illumination at a temperature of 25”. Twenty millicuries of ‘“CTMV (5-25) pCi/mg specific activity, ing Ba”C0, with lactic acid. After 10 days the plants were harvested for purification of TMV by common differential centrifugation. After three cycles of centrifugation, it was possible to obtain 50-100 mg of [14C]TMV (5-25 &i/mg specific activity, Azso:Azeo equal 1.2-1.3. of protoplasts with virus. Inoculation The isolated protoplasts were washed twice with a wash solution ca. 100 g for 1 min (Takebe et al., 1968) and suspended at a concentration of l-3 x lo8 cells/ml in 0.02 M potassium citrate buffer, pH 5.2, containing 0.7 M mannitol. They were allowed to sediment slowly in a tall glass vessel for lo-20 min. Immediately before adding the protoplasts to the virus suspension, the supernatant fluid was decanted and replaced with a corresponding volume of fresh buffer solution. Labeled TMV (for concentration, see Results) was suspended in 0.02 M potassium citrate buffer, pH 5.2, containing 0.7 M mannitol and poly-L-ornithine (MW, 165,000; Sigma, USA) at 2 pg/ml. After standing for 10 min at 25”, the virus and protoplast suspensions were mixed (1: 1, v/v). The final medium contained 1 pg/ml of both virus and poly-r.-ornithine and 0.5-1.5 x 10’ protoplasts/ml. The length of time of inoculation varied in different experiments. To calculate the number of virus particles attached per protoplast, the experiment was run in strict accordance with Otsuki et al. (1972). The final concentrations were 1 pg/ml of virus, 1 pg/ml of poly-L-ornithine and 1.5 x 10’ protoplasts/ ml.

ET AL.

Incubation of inoculated protoplasts. The inoculation mixture was centrifuged (ca. 100 g, 1 min) and the radioactive supernatant liquid was discarded. The protoplasts were washed three times with a wash medium (Takebe et al., 1968) and divided into several samples. One of them was harvested immediately, while the others were incubated at 25” with occasional shaking under continuous illumination with fluorescent lamps (ca. 3,000 lux). Before harvesting, each sample was washed once with diluted (I/;0 of stock solution) wash medium and centrifuged (ca. 1000 g, 5 min) to remove excess mannitol. Because of shortterm experiments, sterility during isolation and incubation was not necessary, even though some solutions were prepared under aseptic conditions. attachof 14C TMV Determination ment. After having been inoculated and washed, the protoplasts were collected, again washed with diluted medium, and dried on an aluminium planchet. An aliquot of the incubation medium and part of the wash solution were dried on planchets to assay radioactivity. The radioactivity of the protoplasts was also measured with a thin-end window tube (T-25-BFL) and correction for selfabsorption, etc. was carried out according to Vosnesensky et al. (1965). After these corrections, the percentage of virus attached to the protoplasts and that of the virus “free” (nonattached) in the protoplasts fraction were calculated. RESULTS

Virus Retention About 6% of the TMV present in the inoculum is retained by protoplasts (Table l), and less than 1% of the amount retained appears to be free (nonadsorbed) virus as indicated by the low radioactivity of the wash solution. The radioactivity retained by the protoplasts corresponds to attachment of about 600 virus particles per protoplast . At a concentration of 1.5 x IO6 protoplasts/ml, the protoplasts (approximate diam, 30 pm; vol, 1.0 x 10’ pm”) occupy about 1.5 x 10” pms/ml of the suspension, corresponding to 1.5% of the total volume.

ATTACHMENT TABLE AVACHMENT

OF TMV

45

TO PROTOPLASTS

1

OF [I’C]TMV TO ISOLATED TOBACCO PROTOPLASTS

Fractions

Radioactivity %

cpm Inoculation mixture Protoplasts after lo-min inoculation Wash solution of last washing” “Free” virus in protoplasts*

15,756 938

100 6

160

1

8

0.05’ 0

0 Only traces of radioactivity were detected in the subsequent wash solution. D“Free” virus content in protoplasts was determined as radioactivity of the wash solution remaining in the protoplast fraction (about !40 of the entire volume) c This value corresponds to 1% of the radioactivity of the protoplast fraction.

This amount is one-fourth the percentage of radioactivity retained by the protoplasts (6%). Time of Inoculation

Time

4 of

2;

inoculation

(minutes)

FIG. 1. Timing of TMV absorption by isolated tobacco protoplasts. The virus (2 rg/ml) was preincubated with poly-L-ornithine (2 fig/ml) for 10 min, and an equal volume of protoplast suspension (1 x lOYm1) was added at the end of preincubation. After the inoculation times indicated, the protoplasts were washed three times with wash medium and once with dilute wash medium. After centrifugation the pellet was transferred onto an aluminum planchet to measure radioactivity.

with TMV

A suspension containing 5 x lo6 protoplasts/ml was mixed with 14C! TMV (1 pg/ml) and poly-L-ornithine (1 &ml), and samples were taken at different time intervals. As shown in Fig. 1, the radioactivity retained by protoplasts rises in the course of inoculation. However, the rate of increase declines after 9 min. According to our observations, the time when the increase begins to decline depends on the rate at which the incubation solution is agitated. TMV Concentration

i

SOl-isi!

E 400”

in Inoculum

[“C ]TMV was preincubated at different concentrations with poly-L-ornithine (2 pg/ml) for 10 min, and then protoplast suspensions were added. In this experiment, protoplast inoculation lasted 15 min. The amount of radioactivity retained by protoplasts as a function of inoculum concentration is shown in Fig. 2. Higher concentrations of virus result in greater retention of virus by protoplasts. Departure of the curve in Fig. 2 from a straight line is slight. Hence, in this case, the relation of retention to TMV concentration is linear.

0

0.5

Concentration

1.0

40 of

TYV

(mg/ml)

2. Relation between TMV absorption by protoplasts and TMV concentration in inoculation medium. TMV was preincubated with poly-L-ornithine (2 pglml) for 10 min and mixed with a protoplast suspension (1:l). After 15-min inoculation, the protoplasts were prepared for radioactivity assay. The abscissa represents TMV concentration in the final mixture.

46

ZHURAVLEV

ET AL.

Effect of Poly-L-Ornithine The effect of poly-L-ornithine on attachment is illustrated in Table 2 and Fig. 3. At a final poly-L-ornithine concentration of 1 pg/ml, a potent stimulation of attachment is observed at each of the TMV concentrations used, even though the absence of poly-L-ornithine in the inoculation medium cannot fully prevent attachment. An extremely high level of attachment (about 7,500 particles per protoplast) was observed in the absence of poly-L-ornithine at a TMV concentration of 1.5 mg/ml. An increase in the concentration of polyL-ornithine results in more virus attachment (Fig. 3). However, protoplasts become damaged at concentrations higher than 3 pglml. DISCUSSION

Whether or not TMV is actually attached to the surface of protoplasts cannot be determined from our studies. Some or all of the radioactivity associated with the protoplasts could be due to virus particles trapped in vesicles of the plasmalemma. Nevertheless, we use the term attachment in the sense of its representing the early events in the virus-protoplast relationship. It is hard to compare virus retention by isolated protoplasts with leaf cells, since, in TABLE

2

EFFECT OF POLY-L-ORNITHINE ON TMV ATTACHMENT TO ISOLATED TOBACCO PROTOPLASTS

Virus concentration

TMV attachment (particles/protoplast)” Poly-L-ornithine

k/ml) 0.5 5 50 150 500 1,500

+ 360 640 1,560 2,940 6,000 17,660

140 200 620 1,240 2,480 7,560

“Protoplasta (2.4 x 106/ml) were inoculated with [“CITMV (either preincubated (+) or nonpreincubated (-) with poly-L-omithine at a concentration of 2.0 &ml). After washing, protoplast radioactivity was measured, and the number of virus particles attached was calculated.

1 0

0.5

Concentration

1.5

4.5 of

poly-L-ornithtne(yg/ml)

FIG. 3. Effect of poly-L-ornithine on TMV absorption by isolated tobacco protoplasts. TMV (2 fig/ml) was preincubated with different concentrations of poly-L-ornithine for 10 min and mixed with equal volumes of protoplast suspensions. The protoplasts were inoculated and washed to measure radioactivity. The abscissa represents poly-L-ornithine concentration in the final mixture.

the case of a leaf, the number of cells in contact with the inoculum is uncertain. The difference between the relative inoculum volumes and inoculation times presents another difficulty in such a comparison. Hence, the 6% retention observed in our experiments may be a rather high attachment level, even though the experiments of other authors show that as much as 30-40% of the inoculum may be retained by the leaves (Reddy, 1966; Shaw, 1972). The retention level may be considerably increased by using denser protoplast suspensions. Virus attachment is retarded 9 min after the beginning of incubation. At this time there is four times more virus in the protoplasts than in the inoculation medium. This indicates that virus absorption by the protoplast is not simple diffusion into the protoplast but a process involving certain specific forces whose nature is still obscure. In their experiments Takebe and collaborators reported that about lOO-1,000 virus particles were absorbed per protoplast (Takebe and Otsuki, 1969; Otsuki et

ATTACHMENT

OF TMV

showed an al., 1972). Our calculations association of 600 particles per protoplast, which is close to Takebe’s values. The number of virus particles absorbed is rather large. Hence, it is not clear why every protoplast does not become infected under these conditions (Takebe and Otsuki, 1969). Furumoto and Wildman (1963) reported that at least one of ten TMV particles is infectious in purified preparations, while Schramm and Engler (1958) reported that about half of the tobacco plants inoculated with ten virus particles became infected. Thus, the excess virus in the protoplasts seems to be rather high. Furthermore, in our experiments, increased TMV concentration in the inoculum leads to greater virus attachment. Yet, the largest number of infected protoplasts was obtained with virus concentrations of 1 pg/ml (Otsuki et al., 1972; Hibi and Yora, 1972). Virus retention by the protoplast is ostensibly only the first step in a more complex process leading to penetration of virus into the protoplast. The possible interdependence of actual attachment and penetration per se needs more detailed study. The fact that poly-L-ornithine stimulates virus attachment with varied TMV concentrations appears to be significant. Shaw (1972) demonstrated that only poly-L-ornithine concentrations exceeding 1 mg/ml were sufficient to promote virus absorption by tobacco leaves. However, no stimulating effect on the part of poly-L-ornithine was observed in experiments with tissue culture (Murakishi et al., 1971). Different concentrations of TMV and poly-L-ornithine were used in Shaw’s and Murakishi’s investigations. Hence, one would expect the need for a specific virus-polycation relationship for stimulation. Inasmuch as poly-L-ornithine is effective with any TMV concentration, it is likely that simple stoichiometry will not be observed in the uptake of these two components. On the other hand, the difference in susceptibility to poly-L-ornithine may indicate that different plasmalemma states are characteristic of protoplasts and intact cells. This possibility is all the more probable, since

TO PROTOPLASTS

47

the fermentative activity of protoplasts isolated enzymatically was shown to differ from that of protoplasts isolated mechanically. We (Musorok and Zhuravlev, 1975) also established that part of the cell proteins, particularly the urease moiety, can pass into the surrounding solution in the course of protoplast isolation. Loss of protein during isolation may be highly significant, since under certain conditions, e.g., after osmotic shock of Phaseolus vulgaris leaf strips, the loss of even a small amount (5%) of cell protein is accompanied by a sharp decline in the ability of cells to perform membrane transport (Amar and Reinhold, 1973). It is known that poly-L-ornithine is essential for infecting tobacco protoplasts with TMV (Takebe and Otsuki, 1969; Takebe and Nagata, 1973); yet, quite large amounts of the virus are also attached to the protoplasts in the absence of poly-Lornithine (Table 2). This is particularly apparent at high TMV concentrations. Nevertheless, we still do not know of SUCcessful attempts to infect tobacco protoplasts with TMV in the absence of poly-Lornithine. Burgess et al. (1973a) arrived at the conclusion that polyornithine affects the initial stages of virus retention but does not induce pinocytosis. In accord with this, our observations show that poly-L-ornithine affects the attachment process. However, our results do not exclude the simultaneous action of the compound on subsequent steps in the process of TMV penetration into the cell, e.g., on pinocytosis. ACKNOWLEDGMENT Translation of the paper from the Russian Joseph C. Shapiro is hereby acknowledged.

by

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BURGESS, J., MOTOYOSHI, F., and FLEMING, E. N. (1973b). The mechanism of infection of plant protoplasts by viruses. Planta 112,323-332. COCKING, E. C. (1966). An electron microscopic study of the initial stages of infection of isolated tomato fruit protoplasts by tobacco mosaic virus. Planta 68,206-214.

COCKING, E. C. (1970). Virus uptake, cell wall regeneration, and virus multiplication in isolated plant protoplasts. Int. Rev. Cytol. 28,899124. COCKING, E. C., and POJNAR, E. (1969). An electron microscopic study of infection of isolated tomato fruit protoplasts by tobacco mosaic virus. J. Gen.

Viral. 4, 305-312. FURUMOTO, W. A., and WILDMAN, S. G. (1963). The specific infectivity of tobacco mosaic virus. Virology 20, 53-61.

HIBI, T., and YORA, K. (1972). Electron microscopy of tobacco mosaic virus infection on tobacco mesophyll protoplasts. Ann. Phytopathol. Sot. Japan 38, 350-356.

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. MUSOROK, T. I., and ZHURAVLEV,Yu. N. (1975). Urease activity of isolated tobacco protoplasts. Plant Physiol. USSR (in press). OTSUKI, Y., TAKEBE, I., HONDA, Y., and MATSUI, C. (1972). Ultrastructure of infection of tobacco mesophyll protoplasts by tobacco mosaic virus. Virology

ET AL. 49, 188-194. PISETSKAYA, N. F., MUSOROK, T. I., REIFMAN, V. G., and ZHURAVLEV, Yu. N. (1975). Takebe’s method: two modifications for rapid tobacco mesophyll protoplasts preparation. Plant Physiol. USSR (in press). REDDY, K. K. (1966). Studies on the formation of tobacco mosaic virus ribonucleic acid. III. Fate of tobacco mosaic virus after entering the host cell.

Proc. Nat. Acad. Sci. USA 55, 593-598. SCHRAMM, G., and ENGLER, R. (1958). The latent period after infection with tobacco mosaic virus and virus nucleic acid. Nature (London) 181, 916. SHAW, J. G. (1972). Effect of poly-L-ornithine on the attachment of tobacco mosaic virus to tobacco leaves and on the uncoating of viral RNA. Virology 48, 380-385.

TAKEBE, I., and NAGATA, T. (1973). Culture of isolated tobacco mesophyll protoplasts. In “Protoplastes et Fusion de Cellules Somatiques Vegetales,” pp. 175-178, CNRS, Paris. TAKEBE, I., and OTSUKI, Y. (1969). Infection of tobacco mesophyll protoplasts by tobacco mosaic virus.

Proc. Nat. Acad. Sci. USA 64, 843-848. TAKEBE, I., OTSUKI, Y., and AOKI, S. (1968). Isolation of tobacco mesophyll cells in intact and active state. Plant Cell Physiol. (Tokyo) 9, 115-124. VOSNESENSKY, V. L., ZALENSKY, 0. V., and SEMICHATOVA, 0. A. (1965). “Methods in Plant Photosynthesis and Respiration,” Nauka M.-L. U.S.S.R.