VIROLOGY 65, 565-573 (1975)
Synthesis Systemically
of Tobacco
Mosaic
Inoculated
W. 0. DAWSON,
Virus in intact Tobacco
by Differential D. E. SCHLEGEL,
Temperature AND
Leaves Treatment
M. C. Y. LUNG
Department of Plant Pathology and Cell Interaction Group, Unioersity of California, Riuerside, California 92502, and Department of Plant Pathology, University of California, Berkeley, California 94720 Accepted
February
1, 1975
A previously reported procedure which manipulated the systemic virus infection of intact plants using differential temperatures was developed into a model system that may provide synchronous virus replication in whole leaves. Cells of young leaves maintained at 5” (DTI-5” leaves) or 12” (DTI-12” leaves) became infected with tobacco mosaic virus (TMV) from mechanically inoculated lower leaves that were maintained at an optimum temperature for virus multiplication (DTI, differential-temperature inoculated.) The DTI leaves were then moved to a permissive temperature that allowed replication to begin in all infected cells simultaneously. The different, nonpermissive low temperatures inhibited early steps of TMV replication differently. The lag periods between the shift to the permissive temperature and the logarithmic and linear infectivity increases were shorter in DTI-12” leaves than DTI-5” leaves. In DTI-5” leaves, TMV infectivity began increasing logarithmically at 8 hr after the transfer to 25”, and a rapid linear increase began at 24 hr. In DTI-12” leaves, the logarithmic increase of TMV infectivity began between 0 and 6 hr, and the linear increase began 14 hr after transfer to 25”. No infectivity could be detected in DTI-5” leaves at the time of the shift from the low temperature, and only minute amounts could be detected in DTI-12” leaves. Infectivity increased at the same rate in DTI leaves detached before the transfer from the low temperature as in DTI leaves left on the intact plant. TMV synthesis in the DTI leaves was similar to that in other synchronous systems of virus replication and almost identical to those reported for TMV in synchronously infected tobacco protoplasts. INTRODUCTION
The lack of a suitable experimental systern for the study of virus replication in plants has been a major obstacle to plant virology. A system is needed in which cells can be infected simultaneously and in which the subsequent replicative events can be monitored sequentially. Plant protoplasts (Takebe et al., 1968) offer promise for such studies because they can be inoculated with TMV (Takebe and Otsuki, 1969) or TMV-RNA (Aoki and Takebe, 1969) with the resultant synthesis of TMV. This system appears to provide synchronous virus synthesis; however, protoplasts may be quite different from cells in intact leaves. The absence of necrosis in protoplasts that are hypersensitive to TMV is 565 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
evidence for physiological differences (Otsuki et al., 1972). Protoplasts cultured under conditions that favor virus synthesis survive only 2-3 days and, after the early periods, may be in various stages of physiological decline. Other difficulties associated with the use of protoplasts as an experimental system for the study of virus replication are the unpredictability of the procedure, the fragility of the cells, and the relatively limited number of cells with which to work. There is a need for a system that utilizes cells as they exist in the intact plant. Previously, we reported that, by manipulation of the systemic infection with differential temperatures, rapid rates of virus multiplication could be produced in intact leaves resulting in curves which suggested
566
DAWSON,
SCHLEGEL
that viral synthesis might be occurring synchronously in these leaves (Dawson and Schlegel, 1973). The present paper reports a detailed examination of the differential temperature inoculation system and demonstrates that different, nonpermissive low temperatures inhibit early steps of TMV synthesis differently. METHODS
As described previously (Dawson and Schlegel, 1973), the lower 4-6 leaves of greenhouse-grown tobacco plants (Nicotiana tabacum L. var. Xanthi) 20-30 cm tall were mechanically inoculated with TMV, strain Ul. Noninoculated leaves longer than 5-7 cm were removed, but young leaves less than 5-7 cm long were left intact. The plants were immediately put into a polyStyrofoam chamber with the young, noninoculated leaves extending out of the chamber, and the entire polyStyrofoam chamber was then placed in a plant growth chamber at 5 or 12”. The lower leaves (inside the polyStyrofoam chamber) were maintained at 28-30” with two 60 W light bulbs controlled by a rheostat. The plants were subjected to differential temperatures for 10 days, after which the intact plants were removed from the polyStyrofoam chamber and transferred to a plant growth chamber at 25” with a 14-hr photoperiod of 20,000 lx. All succeeding experiments were conducted on the upper, differential-temperature inoculated (DTI) leaves. Infectivity assays:. TMV infectivity was assayed by the half-leaf method on pinto beans (Phaseolus vulgaris L.) with 6-12 replications of each inoculum in a randomized block design. Samples to be assayed were removed from leaves with a cork borer and stored at -20” until ail samples could be assayed together. Frozen samples were ground in G-P buffer (0.05 M glycine, 0.03 M K2HP04, pH 9.2, 100 pg/ml of bentonite, and 1% Celite) and were further diluted in G-P buffer to produce lo-150 lesions per half-leaf (l/100-l/100,000 dilutions, w/v). RNA extraction. Frozen samples were ground in five volumes of 1% sodium pyrophosphate plus five volumes of water-
AND LUNG
saturated phenol with a mortar and pestle which had previously been stored at -20”. The resulting slurry was allowed to melt and was stirred at room temperature for 5 min. The phases were separated by centrifugation at 10,000 g for 10 min at 2”. The aqueous phase was removed and the RNA was precipitated with two volumes of ethanol. The precipitate was dissolved in G-P buffer and assayed as described above. RESULTS
Inhibition of TMV Multiplication Temperatures
by Low
Because low temperatures were used to inhibit TMV synthesis in the differentialtemperature inoculation procedure, the effectiveness of low-temperature inhibition of virus synthesis was examined. Tobacco plants were mechanically inoculated with TMV and maintained in a controlled-environment chamber at 12 or 25”. Figure 1A shows the amount of extractable infectivity produced in the mechanically inoculated 140
I
IZO-
I
I
I
I
A
I 25”
I
A--fi /
IOO-
:I
80 A
60: 5
/
40-
c20-
/
800
60o---o
40 o---o
zo/o-.6/ 0 2
4 DAYS
/I
/
I I 6 8 IO 12 AFTER INOCULATION
I 14
16
FIG. 1. (A) TMV infectivity in mechanically inoculated tobacco leaves maintained at 25 or 12”. (B) TMV infectivity in mechanically inoculated tobacco leaves which were incubated alternately at 12” (dashed lines) and 25” (solid lines).
SYNTHESIS
OF TOBACCO
leaves maintained at each temperature. Less than 0.1% as much infectivity was produced after 15 days at 12” as at 25”. During shorter periods of time, even smaller amounts of TMV were produced at 12”. No TMV synthesis could be detected after 15 days at 5”. To determine whether all stages of TMV replication were inhibited by the low temperature or whether only an early, coldsensitive step was blocked, the accumulation of TMV infectivity was monitored in mechanically inoculated leaves incubated alternately at 25 and 12” (Fig. 1B). There was no measurable increase in infectivity at 12” at any stage of infection. The infectivity in young tobacco leaves that had been mechanically inoculated with TMV and maintained at 12” while the lower leaves of the plant were maintained at 30” (in the differential-temperature chamber) was similar to that in mechanically inoculated leaves where the whole plant was maintained at 12”. The infectivity curves of young leaves which were mechanically inoculated after the plants had been maintained in the differentialtemperature chamber for 10 days were similar to infectivity curves of mechanically inoculated young leaves in plants which were previously maintained at 25” before inoculation. Synthesis
of TMV
in DTI Leaves
The infectivity of TMV in DTI leaves, which had previously been maintained for 10 days at 5 or 12” as described above, was monitored beginning at the time when they were transferred to the permissive temperature (zero time). The infectivity of TMV in DTI leaves maintained at 5” (DTI-5” leaves) began increasing at 24 hr and continued increasing through the fourth day at 25” (Fig. 2). TMV infectivity began increasing in DTI leaves maintained at 12” (DTI-12” leaves) 14 hr after the transfer to 25” and continued increasing linearly for 3 days, at which time the maximum level of infectivity was approached. As a control, the lower four leaves of tobacco plants held continually at 25” were mechanically inoculated with TMV, and the infectivity increase in these leaves and
MOSAIC
I
567
VIRUS
2
3
4 DAYS
5 AT
6
7
8
9
IO
25’
FIG. 2. Infectivity of TMV in DTI-IZ”, DTI-5”, mechanically inoculated, and systemically infected leaves. Zero time for DTI-5” and DTI-12” curves was when the leaves were transferred to the 25” environment. Zero time for the mechanically inoculated curve was when the leaves were mechanically inoculated. The precise time the systemically infected leaves became infected could not be determined. The systemically infected curve is plotted with zero time being when the lower leaves of the plants were mechanically inoculated, although the younger leaves became infected about 2 days later. Infectivity is average number of lesions/half-leaf x dilution/g of tissue x 10e7.
in the young, systemically infected leaves (comparable in size and position on the plant to DTI leaves) was monitored. The lag period before rapid virus multiplication was much shorter in the DTI leaves than in the mechanically inoculated leaves, and the rate of multiplication was more rapid in the DTI leaves than in either the mechanically inoculated or systemically infected leaves (Fig. 2). Although rapid and linear increases in infectivity in DTI-5” leaves and DTI-12” leaves began after 24 and 14 hr at 25”, respectively, as demonstrated in Fig. 2, small increases in infectivity could be detected earlier. These small increases in infectivity can be more easily seen in a logarithmic plot (Fig. 3). There was no detectable infectivity in DTI-5” leaves during the first 8 hr. Virus became detectable after 8 hr and increased logarithmically until about 24 hr when the increase became linear. In the experiment shown in Fig. 3, TMV infectivity in DTI-12” leaves began increasing logarithmically after 6 hr at 25” and the linear increase began after 14 hr.
568
DAWSON,
SCHLEGEL
However, in 4 of 11 experiments, there was no lag before the ’ logarithmic increase (dashed line in Fig. 3). Although very little infectivity occurred at zero time, the logarithmic increase began concomitantly with the move to 25”. To demonstrate that TMV nucleoprotein increases were equivalent to the infectivity increases, TMV was purified from the DTI-12” leaves at intervals after transfer to 25o by polyethylene glycol precipitation (Gooding and Hebert, 1967) and two cycles of differential centrifugation. The basal 2/3 of DTI leaves 5-8 cm long was combined with all of the smaller leaves. The nucleoprotein accumulation curve (Fig.’ 4) was nearly identical to the infectivity curve for DTI-12” leaves shown in Fig. 2 and demonstrates the high concentration of virus nucleoprotein attained in DTI-12” leaves. The DTI leaves expanded during the incubation period at 25”, which means that samples measured by constant weight or area represent fewer cells as the leaves expand. Figure 5 shows infectivity curves corrected for leaf expansion. The similarity between Figs. 2 and 5 demonstrates that the rate of TMV synthesis was much greater than the rate of leaf expansion. Amount
of Infectivity
I
T
I
I
1
p,;&&z, B 048
2
I6
,
24
,
48 HOURS
FIG. 3. Semi-logarithmic increase in DTI-12” (A-A) leaves.
I /, cad0
0
l 2
4
( 6 DAYS
FIG. 4. Accumulation DTI-12’ leaves. I
”
l
l
l
8 IO AT 25“
,
I2
14
of TMV
nucleoprotein
’
I
in
II
at Zero Time
When DTI-5” leaves or DTI-12” leaves were detached and floated on water in petri dishes at 25” immediately before the whole plants were transferred to 25”, the infecI ’ I ’ I ’ I
AND LUNG
72 AT
,I 96
25”
plot of TMV and DTI-5”
infectivity (O---O)
FIG. 5. Infectivity of TMV in DTI-12” leaves corrected for leaf expansion.
and DTI-5”
tivity increased at the same rate in detached leaves as in intact leaves (Fig. 6). At the time the DTI-5” leaves were moved to the permissive temperature, no infectivity was detected in sap or in phenol-extracted RNA preparations. Although no infectivity could be detected in the detached DTI-5’ leaves immediately after they were detached, enough inoculum had moved into these leaves to produce the rapid increase in infectivity observed in them. In DTI-12’ leaves, small amounts of infectivity were occasionally detected at zero time (Table 1). Infectivity was detected in sap of DTI-12” leaves in 6 of 14 experiments. In only one experiment was infectivity detected in an RNA preparation
SYNTHESIS
I DAYS
2 AT 25”
3
OF TOBACCO
4
FIG. 6, Infectivity of TMV in DTI leaves detached immediately after the differential-temperature treatment and before they were transferred to 25” and in DTI leaves left on the intact tobacco plant which was moved to 25”: Detached DTI-12” (O---O), intact DTI-12” (A-A), detached DTI-5” (A---A), and intact DTI-5” leaves (O--Q).
where it was not detected in sap. When the differential temperature treatment was lengthened to 20 days, a detectable amount of infectivity was found in DTI-5” leaves. However, the amount of infectivity was only about 0.04% of the maximum level of infectivity attained after the leaves were moved to 25”. Slightly more infectivity occurred in DTI-12” leaves after 20 days of differential temperature treatment, but this amount was still less than 0.4% of the maximum level of infectivity after the leaves were moved to 25”. The length of time that the plants were maintained in the differential-temperature chamber (7-14 days) did not alter the lag periods in either DTI-5” or DTI-12” leaves. In DTI-12” leaves, the lag period did not become shorter as the plants were maintained for longer periods in the differential temperatures as would be expected if the difference between the lag periods in DTI5” leaves and DTI-12” leaves was due only to a less efficient inhibition of TMV synthesis by 12”. Free Viral RNA The relative amount of free viral RNA occurring during the early stages of infec-
MOSAIC
569
VIRUS
tion was estimated by comparing the infectivity of encapsidated TMV-RNA to that of total TMV-RNA. Total TMV-RNA infectivity was determined by assaying the total phenol-extracted RNA from infected leaves. Encapsidated TMV-RNA was determined by assaying phenol-extracted RNA from the sap of infected leaves kept at room temperature for 2 hr after homogenization in water. Free viral RNA represented the bulk of the infectivity in both DTI-5” and DTI-12” leaves during the initial stages of the infection when infectivity first became detectable (Fig. 7). However, there was no large excess of free viral RNA, nor was the lag period of the viral RNA infectivity curve appreciably shorter than that of intact virus. Distribution of the Initial Leaves
Infection
in DTI
It is important to know which leaves or leaf-areas are initially infected at the nonpermissive temperature by the DTI procedure and are usable for TMV replication experiments which require a synchronous infection. Nilsson-Tillgren et al. (1969) TABLE
AMOUNTOF TMV Expt no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1
INFECTIVITYIN DTI-12” ZEROTIME
Infectivity
of sap
Av no. of lesions
Dilution
7%of maximum infectivitp
0 1 0 0 0 0 26 0 2 1 0 0 13 8
l/l00 l/100 l/100 l/loo l/loo l/100 l/100 l/100 l/100 l/100 l/loo l/20 l/100 l/loo
0.02 0.065 0.002 0.01 0.018 0.17
LEAVESAT
Infectivity RNA Av no. of lesions
a Maximum infectivity was measured leaves at 4 days after transfer to 25”.
0 7 0 0 0 0 25 48 0 0 0 -
of
Dilution
l/25 l/5 l/l0 l/10 l/10 l/l l/10 l/10 l/20 l/20 l/10 -
in DTI-12”
570
DAWSON,
SCHLEGEL
1
AND LUNG
water. These experiments gave results similar to those shown in Table 2. DISCUSSION
5
DAYS AT 25’= FIG. 7. Infectivity and encapsidated leaves.
of total TMV-RNA TMV-RNA (O--O)
(A---A) in DTI
reported that during the systemic infection of young tobacco leaves, the base of some leaves became infected earlier than the rest of the leaf. To determine which areas of DTI leaves were infected initially, l-mm leaf discs collected from tip, middle, and base areas of detached leaves of different sizes were assayed. These samples were collected after the DTI leaves had incubated at 25’ for 24 hr, at the time the rapid increase in infectivity was beginning but before a substantial increase in infectivity could occur in cells not infected initially. Table 2 shows the results of representative experiments which suggest that all areas of leaves less than 4 cm long and the basal 213 of leaves 4-7 cm long became infected at approximately the same time. In other experiments, discs were removed from DTI leaves immediately before transfer to 25” and were then incubated at 25” floating on
A procedure that we (Dawson and Schlegel, 1973) previously reported to give enhanced virus multiplication rates in whole leaves by manipulation of the temperature of different parts of plants was further developed into a model system for studying virus replication and virus-cell interactions. The procedure allows leaf cells to become infected at a low temperature that is nonpermissive for virus replication. The cells of young leaves maintained at a low temperature become infected with inoculum from the lower leaves of the same plants that are maintained at a temperature permissive for virus multiplication. After sufficient inoculum has moved into the young leaves, they are moved to a permissive temperature that allows virus synthesis to begin simultaneously in all infected cells. We do not know in what form the inoculum moves from the lower leaves to the upper leaves. Enough inoculum moved into DTI-5” leaves to produce the rapid rates of synthesis observed, but infectivity could not be detected in sap or in phenolextracted RNA. This suggests that the differential-temperature inoculation is either very efficient, inoculating many cells with a small amount of inoculum which was below the level of detection, or that the inoculum was in some form which was not infectious when extracted and assayed on another host. TABLE
2
SIZE AND POSITION OF LEAVES INFECTED WITH TMV DIFFERENTIAL TEMPERATURE INOCULATION
Leaf no.
1 2 3 4
Size of leaves
BY
Infectivity after 24 hr at 25” (Av no. of lesions)
Max length (cm)
Max width (cm)
Tip
4 7 4 6
2.5 5 2.5 4.5
120 26 133 54
Middle
Base
108 90 113 125
124 91 127 125
SYNTHESIS
OF TOBACCO
The inoculum moved uniformly into young leaves smaller than 4 cm long and the basal portions of leaves 4-7 cm long. It moved sporadically or not at all into larger leaves. The inoculum appeared to move passively from photosynthesizing lower leaves to growing points, the direction metabolites in the phloem would be expected to move. The low temperatures apparently inhibited virus synthesis more than they inhibited host metabolism, in that the young leaves continued to expand at 12” and (very slowly) at 5”, thus serving as terminals of metabolites and virus inoculum. In both DTI-5” and DTI-12” leaves, TMV increased as rapidly when the leaves were detached as when they were left intact on the plants. This demonstrates that the rapid increase in infectivity occurring in DTI leaves resulted from TMV synthesis in these leaves and not from translocation of virus-specific products to DTI leaves from lower leaves of the plant. However, DTI leaves left on the plant have the same infectivity curve as detached leaves and are easier to maintain. Free viral RNA made up most of the infectivity in DTI leaves during the initial stages of synthesis (the beginning of the logarithmic infectivity increase) and was detectable during the first 24 hr at 25”. Because free TMV-RNA was measured by subtracting encapsidated RNA infectivity from total viral RNA infectivity, the amount of free TMV-RNA could not be accurately measured when it became less than about 10% of the total TMV-RNA infectivity. After 24 hr, the amount of encapsidated RNA was much greater than free viral RNA. These results are similar to those of Sarkar (1965), who examined free TMV-RNA after mechanical inoculation of tobacco leaves. This suggests that he observed free viral RNA only in initially infected cells, again because free viral RNA infectivity is difficult to measure in the presence of large excesses of encapsidated RNA which probably occurs in an asynchronous infection after the rapid linear increase of TMV in the initially infected cells and all stages of synthesis of subsequently infected cells.
MOSAIC
VIRUS
571
The lag periods before free viral RNA accumulation in DTI leaves were not appreciably shorter than that of intact virus, and the linear increases did not occur measurably earlier, suggesting that assembly of particles closely follows viral RNA synthesis. Temperatures of 5 and 12” blocked TMV synthesis at different steps. Although TMV is synthesized more slowly at low temperatures (Lebeurier and Hirth, 1966), both 5 and 12” do more than slow virus synthesis. The infectivity curve of TMV in DTI-12” leaves was similar to that in DTI-5” leaves, except that the DTI-12” curve was about 6-10 hr earlier than the DTI-5” curve. Longer differential-temperature treatments did not alter the infectivity curves of TMV in DTI-5” or DTI-12” leaves. The similarity of TMV synthesis in DTI-5” leaves to that in protoplasts suggests that an initial event of virus replication was inhibited by 5”. The shorter lag period (and the occasional lack of a lag period) before the logarithmic infectivity increase in DTI12” leaves suggests that 12” blocked some step subsequent to infection but prior to viral RNA synthesis (that some steps of TMV synthesis occurred at 12’). Subsequent work (Dawson and Schlegel, in preparation) clearly demonstrates that 5 and 12’ inhibit different, specific steps of TMV replication in that 2-thiouracil totally inhibits TMV synthesis in DTI-5” leaves at the time of transfer to 25” but does not inhibit TMV synthesis in DTI-12” leaves when treatment begins at the same time. The rate of synthesis of TMV in DTI leaves is similar to that in other systems that provide synchronous virus synthesis. The infectivity curve of TMV in DTI leaves, consisting of logarithmic increase followed by a more rapid linear increase, is similar to synchronous RNA virus synthesis in animal (Baltimore et al., 1966) and bacterial systems (Godson, 1968) and to in vitro systems when below-saturating levels of viral RNA are added (Spiegelman et al., 1965). By infecting cells with poliovirus at various virus/cell multiplicities, Baltimore et a!. (1966) demonstrated that the logarithmic rate of viral RNA increase is due to the participation of progeny mole-
572
DAWSON,
SCHLEGEL
cules as templates and not to asynchronous entry into the replication process of parental RNA as the only templates for RNA replication. The infectivity curves of TMV in DTI leaves also are similar to those of TMV in protoplasts (Takebe et al., 1971; Otsuki et al., 1972). The logarithmic increase of TMV infectivity began at about 6 and 15 hr in protoplasts incubated at 28 and 22”, respectively. The logarithmic increase of TMV infectivity in DTI-5” leaves incubated at 25” began after 8 hr. The similarity between TMV synthesis in DTI leaves and protoplasts is particularly evident when the results of Takebe et al. (1971) are plotted on a linear scale (Fig. 8). In protoplasts, TMV infectivity began increasing linearly after 12 hr at 28 and 24 hr at 22”. At both temperatures, TMV infectivity continued to increase linearly until 72 hr of incubation (the duration of the experiment). In DTI-5” leaves, incubated at 25”, the linear increase began at 24 hr and continued until about 72 hr of incubation after which the rate of increase declined. The duration of synthesis of TMV is 3-5 days in DTI leaves. In the initial reports by Aoki and Takebe (1969) and Takebe and Otsuki (1969), the duration of TMV synthesis in isolated tobacco protoplasts was only 24 hr. However, these investigators
35 R 0 30 t
1ll.,~/f; HOURS
40 AFTER
60 72 INFECTION
FIG. 8. A linear plot of the time course of TMV multiplication in tobacco mesophyll protoplasts; adapted from Takehe et al. (1971, Fig. 2).
AND LUNG
recently demonstrated (Otsuki et al., 1972; Takebe et al., 1971) that TMV synthesis in protoplasts continued for 72 hr (the length of the experiment) and that the rate of synthesis at 72 hr was near the maximum rate (Fig. 8). TMV synthesis in separated tobacco cells isolated from leaves inoculated 3 days earlier was linear for 44 hr (the length of the experiment (Jackson et al., 1972)). Virus synthesis in some of the cells must have been longer than 44 hr. The results of Oxefelt (1970) using the conditions described by Nilsson-Tillgren et al. (1969) suggest that TMV synthesis in intact leaves continued for about 5 days after 90% of the cells were infected. The similarity of TMV infectivity curves in DTI leaves to that of TMV in protoplasts and to other synchronous virus systems suggests that TMV synthesis occurs synchronously in DTI leaves. However, a following paper (Dawson and Schlegel, in preparation) demonstrates that virus synthesis begins in DTI leaves at specific steps of replication and the subsequent steps occur sequentially and synchronously at the permissive temperature; a further argument for synchrony of TMV synthesis in DTI leaves will be given in that paper. The differential-temperature inoculation system should have many applications in plant virus research. It is simple, easy to perform, and highly reproducible. Large amounts of tissue with which to work are provided. The system should be applicable to many virus-host combinations and has previously been demonstrated applicable to cowpea chlorotic mottle virus in cowpea (Dawson and Schlegel, 1973). The only requirements should be a virus that is inhibited by low temperatures and a systemic host that is structurally suited to a differential-temperature chamber. The differential-temperature inoculation system provides a much needed complement to the protoplast system for studying plant virus replication. Protoplasts are more amenable for studies of the inoculation process, pulse-chase experiments, and the uptake of large molecules. However, they are subjected to a variety of relatively harsh treatments whose effects on the cell’s physiology are not known. Virus
SYNTHESIS
OF TOBACCO
synthesis in DTI leaves can be examined in cells in intact leaves that are not subjected to adverse treatment and therefore are more likely to retain their natural state. This system should prove especially useful in determining the effects of individual steps of virus replication upon host metabolism and the effect of viruses upon cellcell interactions. REFERENCES AOKI, S., and TAKEBE, I. (1969). Infection of tobacco mesophyll protoplasts by TMV-RNA. Virology 39, 439-448. BALTIMORE, D., GIRARD, M., and DARNELL, J. E. (1966). Aspects of the synthesis of poliovirus RNA and the formation of virus particles. Virology 29, 179-189. DAWSON, W. O., and SCHLEGEL,D. E. (1973). Differential temperature treatment of plants greatly enhances multiplication rates. Virology 53, 476-478. GODSON, G. N. (1968). Site of synthesis of viral ribonucleic acid and phage assembly in MS2infected Escherichia coli. J. Mol. Biol. 34,149-163. GOODING, G. V., and HEBERT, T. T. (1967). A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology 57, 1285. JACKSON, A. O., ZAITLIN, M., SEIGEL, A., and FRANCKI, R. I. B. (1972). Replication of tobacco mosaic virus. III. Viral RNA metabolism in separated leaf cells. Virology 48, 655-665. LEBEURIER, G., and HIRTH, L. (1966). Effect of tem-
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peratures on the development of two strains of tobacco mosaic virus. Virology 29, 385-395. NILSSON-TILLGREN, T., KOLEHMAINEN-SEVEUS, L., and VON WETTSTEIN, D. (1969). Studies on the biosynthesis of TMV. I. A system approaching a synchronized virus synthesis in a tobacco leaf. Molec. Gen. Genetics 104, 124-141. OXELFELT, P. (1970). Development of systemic tobacco mosaic virus infection. I. Initiation of infection and time course of virus multiplication. Phytopath. 2. 69, 202-211. OTSUKI, Y., SHIMOMURA, T., and TAKEBE, I. (1972). Tobacco mosaic virus multiplication and expression of the N gene in necrotic responding tobacco varieties. Virology 50, 45-50. SARKAR, S. (1965). The amount and nature of ribonuclease sensitive infectious material during biogenesis of tobacco mosaic virus. 2. Vererbunzsl. 97, 166-185. SPIEGELMAN, S., HARUNA, I., HOLLAND, I. B., BEUADREAU, G., and MILLS, D. (1965). The synthesis of a self-propagating and infectious nucleic acid with a purified enzyme. Proc. Nat. Acad. Sci. USA 54, 919-927. TAKEBE, I., and OTSUKI, Y. (1969). Infection of tobacco mesophyll protoplasts by TMV. 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. 9, 115-124. TAKEBE, I., OTSUKI, Y., and AOKI, S. (1971). Infection of isolated tobacco mesophyll protoplasts by tobacco mosaic virus. Colloq. Znt. Cent. Nat. Rech. Sci. 193.503-511.
Synthesis Systemically
of Tobacco
Mosaic
Inoculated
W. 0. DAWSON,
Virus in intact Tobacco
by Differential D. E. SCHLEGEL,
Temperature AND
Leaves Treatment
M. C. Y. LUNG
Department of Plant Pathology and Cell Interaction Group, Unioersity of California, Riuerside, California 92502, and Department of Plant Pathology, University of California, Berkeley, California 94720 Accepted
February
1, 1975
A previously reported procedure which manipulated the systemic virus infection of intact plants using differential temperatures was developed into a model system that may provide synchronous virus replication in whole leaves. Cells of young leaves maintained at 5” (DTI-5” leaves) or 12” (DTI-12” leaves) became infected with tobacco mosaic virus (TMV) from mechanically inoculated lower leaves that were maintained at an optimum temperature for virus multiplication (DTI, differential-temperature inoculated.) The DTI leaves were then moved to a permissive temperature that allowed replication to begin in all infected cells simultaneously. The different, nonpermissive low temperatures inhibited early steps of TMV replication differently. The lag periods between the shift to the permissive temperature and the logarithmic and linear infectivity increases were shorter in DTI-12” leaves than DTI-5” leaves. In DTI-5” leaves, TMV infectivity began increasing logarithmically at 8 hr after the transfer to 25”, and a rapid linear increase began at 24 hr. In DTI-12” leaves, the logarithmic increase of TMV infectivity began between 0 and 6 hr, and the linear increase began 14 hr after transfer to 25”. No infectivity could be detected in DTI-5” leaves at the time of the shift from the low temperature, and only minute amounts could be detected in DTI-12” leaves. Infectivity increased at the same rate in DTI leaves detached before the transfer from the low temperature as in DTI leaves left on the intact plant. TMV synthesis in the DTI leaves was similar to that in other synchronous systems of virus replication and almost identical to those reported for TMV in synchronously infected tobacco protoplasts. INTRODUCTION
The lack of a suitable experimental systern for the study of virus replication in plants has been a major obstacle to plant virology. A system is needed in which cells can be infected simultaneously and in which the subsequent replicative events can be monitored sequentially. Plant protoplasts (Takebe et al., 1968) offer promise for such studies because they can be inoculated with TMV (Takebe and Otsuki, 1969) or TMV-RNA (Aoki and Takebe, 1969) with the resultant synthesis of TMV. This system appears to provide synchronous virus synthesis; however, protoplasts may be quite different from cells in intact leaves. The absence of necrosis in protoplasts that are hypersensitive to TMV is 565 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
evidence for physiological differences (Otsuki et al., 1972). Protoplasts cultured under conditions that favor virus synthesis survive only 2-3 days and, after the early periods, may be in various stages of physiological decline. Other difficulties associated with the use of protoplasts as an experimental system for the study of virus replication are the unpredictability of the procedure, the fragility of the cells, and the relatively limited number of cells with which to work. There is a need for a system that utilizes cells as they exist in the intact plant. Previously, we reported that, by manipulation of the systemic infection with differential temperatures, rapid rates of virus multiplication could be produced in intact leaves resulting in curves which suggested
566
DAWSON,
SCHLEGEL
that viral synthesis might be occurring synchronously in these leaves (Dawson and Schlegel, 1973). The present paper reports a detailed examination of the differential temperature inoculation system and demonstrates that different, nonpermissive low temperatures inhibit early steps of TMV synthesis differently. METHODS
As described previously (Dawson and Schlegel, 1973), the lower 4-6 leaves of greenhouse-grown tobacco plants (Nicotiana tabacum L. var. Xanthi) 20-30 cm tall were mechanically inoculated with TMV, strain Ul. Noninoculated leaves longer than 5-7 cm were removed, but young leaves less than 5-7 cm long were left intact. The plants were immediately put into a polyStyrofoam chamber with the young, noninoculated leaves extending out of the chamber, and the entire polyStyrofoam chamber was then placed in a plant growth chamber at 5 or 12”. The lower leaves (inside the polyStyrofoam chamber) were maintained at 28-30” with two 60 W light bulbs controlled by a rheostat. The plants were subjected to differential temperatures for 10 days, after which the intact plants were removed from the polyStyrofoam chamber and transferred to a plant growth chamber at 25” with a 14-hr photoperiod of 20,000 lx. All succeeding experiments were conducted on the upper, differential-temperature inoculated (DTI) leaves. Infectivity assays:. TMV infectivity was assayed by the half-leaf method on pinto beans (Phaseolus vulgaris L.) with 6-12 replications of each inoculum in a randomized block design. Samples to be assayed were removed from leaves with a cork borer and stored at -20” until ail samples could be assayed together. Frozen samples were ground in G-P buffer (0.05 M glycine, 0.03 M K2HP04, pH 9.2, 100 pg/ml of bentonite, and 1% Celite) and were further diluted in G-P buffer to produce lo-150 lesions per half-leaf (l/100-l/100,000 dilutions, w/v). RNA extraction. Frozen samples were ground in five volumes of 1% sodium pyrophosphate plus five volumes of water-
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saturated phenol with a mortar and pestle which had previously been stored at -20”. The resulting slurry was allowed to melt and was stirred at room temperature for 5 min. The phases were separated by centrifugation at 10,000 g for 10 min at 2”. The aqueous phase was removed and the RNA was precipitated with two volumes of ethanol. The precipitate was dissolved in G-P buffer and assayed as described above. RESULTS
Inhibition of TMV Multiplication Temperatures
by Low
Because low temperatures were used to inhibit TMV synthesis in the differentialtemperature inoculation procedure, the effectiveness of low-temperature inhibition of virus synthesis was examined. Tobacco plants were mechanically inoculated with TMV and maintained in a controlled-environment chamber at 12 or 25”. Figure 1A shows the amount of extractable infectivity produced in the mechanically inoculated 140
I
IZO-
I
I
I
I
A
I 25”
I
A--fi /
IOO-
:I
80 A
60: 5
/
40-
c20-
/
800
60o---o
40 o---o
zo/o-.6/ 0 2
4 DAYS
/I
/
I I 6 8 IO 12 AFTER INOCULATION
I 14
16
FIG. 1. (A) TMV infectivity in mechanically inoculated tobacco leaves maintained at 25 or 12”. (B) TMV infectivity in mechanically inoculated tobacco leaves which were incubated alternately at 12” (dashed lines) and 25” (solid lines).
SYNTHESIS
OF TOBACCO
leaves maintained at each temperature. Less than 0.1% as much infectivity was produced after 15 days at 12” as at 25”. During shorter periods of time, even smaller amounts of TMV were produced at 12”. No TMV synthesis could be detected after 15 days at 5”. To determine whether all stages of TMV replication were inhibited by the low temperature or whether only an early, coldsensitive step was blocked, the accumulation of TMV infectivity was monitored in mechanically inoculated leaves incubated alternately at 25 and 12” (Fig. 1B). There was no measurable increase in infectivity at 12” at any stage of infection. The infectivity in young tobacco leaves that had been mechanically inoculated with TMV and maintained at 12” while the lower leaves of the plant were maintained at 30” (in the differential-temperature chamber) was similar to that in mechanically inoculated leaves where the whole plant was maintained at 12”. The infectivity curves of young leaves which were mechanically inoculated after the plants had been maintained in the differentialtemperature chamber for 10 days were similar to infectivity curves of mechanically inoculated young leaves in plants which were previously maintained at 25” before inoculation. Synthesis
of TMV
in DTI Leaves
The infectivity of TMV in DTI leaves, which had previously been maintained for 10 days at 5 or 12” as described above, was monitored beginning at the time when they were transferred to the permissive temperature (zero time). The infectivity of TMV in DTI leaves maintained at 5” (DTI-5” leaves) began increasing at 24 hr and continued increasing through the fourth day at 25” (Fig. 2). TMV infectivity began increasing in DTI leaves maintained at 12” (DTI-12” leaves) 14 hr after the transfer to 25” and continued increasing linearly for 3 days, at which time the maximum level of infectivity was approached. As a control, the lower four leaves of tobacco plants held continually at 25” were mechanically inoculated with TMV, and the infectivity increase in these leaves and
MOSAIC
I
567
VIRUS
2
3
4 DAYS
5 AT
6
7
8
9
IO
25’
FIG. 2. Infectivity of TMV in DTI-IZ”, DTI-5”, mechanically inoculated, and systemically infected leaves. Zero time for DTI-5” and DTI-12” curves was when the leaves were transferred to the 25” environment. Zero time for the mechanically inoculated curve was when the leaves were mechanically inoculated. The precise time the systemically infected leaves became infected could not be determined. The systemically infected curve is plotted with zero time being when the lower leaves of the plants were mechanically inoculated, although the younger leaves became infected about 2 days later. Infectivity is average number of lesions/half-leaf x dilution/g of tissue x 10e7.
in the young, systemically infected leaves (comparable in size and position on the plant to DTI leaves) was monitored. The lag period before rapid virus multiplication was much shorter in the DTI leaves than in the mechanically inoculated leaves, and the rate of multiplication was more rapid in the DTI leaves than in either the mechanically inoculated or systemically infected leaves (Fig. 2). Although rapid and linear increases in infectivity in DTI-5” leaves and DTI-12” leaves began after 24 and 14 hr at 25”, respectively, as demonstrated in Fig. 2, small increases in infectivity could be detected earlier. These small increases in infectivity can be more easily seen in a logarithmic plot (Fig. 3). There was no detectable infectivity in DTI-5” leaves during the first 8 hr. Virus became detectable after 8 hr and increased logarithmically until about 24 hr when the increase became linear. In the experiment shown in Fig. 3, TMV infectivity in DTI-12” leaves began increasing logarithmically after 6 hr at 25” and the linear increase began after 14 hr.
568
DAWSON,
SCHLEGEL
However, in 4 of 11 experiments, there was no lag before the ’ logarithmic increase (dashed line in Fig. 3). Although very little infectivity occurred at zero time, the logarithmic increase began concomitantly with the move to 25”. To demonstrate that TMV nucleoprotein increases were equivalent to the infectivity increases, TMV was purified from the DTI-12” leaves at intervals after transfer to 25o by polyethylene glycol precipitation (Gooding and Hebert, 1967) and two cycles of differential centrifugation. The basal 2/3 of DTI leaves 5-8 cm long was combined with all of the smaller leaves. The nucleoprotein accumulation curve (Fig.’ 4) was nearly identical to the infectivity curve for DTI-12” leaves shown in Fig. 2 and demonstrates the high concentration of virus nucleoprotein attained in DTI-12” leaves. The DTI leaves expanded during the incubation period at 25”, which means that samples measured by constant weight or area represent fewer cells as the leaves expand. Figure 5 shows infectivity curves corrected for leaf expansion. The similarity between Figs. 2 and 5 demonstrates that the rate of TMV synthesis was much greater than the rate of leaf expansion. Amount
of Infectivity
I
T
I
I
1
p,;&&z, B 048
2
I6
,
24
,
48 HOURS
FIG. 3. Semi-logarithmic increase in DTI-12” (A-A) leaves.
I /, cad0
0
l 2
4
( 6 DAYS
FIG. 4. Accumulation DTI-12’ leaves. I
”
l
l
l
8 IO AT 25“
,
I2
14
of TMV
nucleoprotein
’
I
in
II
at Zero Time
When DTI-5” leaves or DTI-12” leaves were detached and floated on water in petri dishes at 25” immediately before the whole plants were transferred to 25”, the infecI ’ I ’ I ’ I
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72 AT
,I 96
25”
plot of TMV and DTI-5”
infectivity (O---O)
FIG. 5. Infectivity of TMV in DTI-12” leaves corrected for leaf expansion.
and DTI-5”
tivity increased at the same rate in detached leaves as in intact leaves (Fig. 6). At the time the DTI-5” leaves were moved to the permissive temperature, no infectivity was detected in sap or in phenol-extracted RNA preparations. Although no infectivity could be detected in the detached DTI-5’ leaves immediately after they were detached, enough inoculum had moved into these leaves to produce the rapid increase in infectivity observed in them. In DTI-12’ leaves, small amounts of infectivity were occasionally detected at zero time (Table 1). Infectivity was detected in sap of DTI-12” leaves in 6 of 14 experiments. In only one experiment was infectivity detected in an RNA preparation
SYNTHESIS
I DAYS
2 AT 25”
3
OF TOBACCO
4
FIG. 6, Infectivity of TMV in DTI leaves detached immediately after the differential-temperature treatment and before they were transferred to 25” and in DTI leaves left on the intact tobacco plant which was moved to 25”: Detached DTI-12” (O---O), intact DTI-12” (A-A), detached DTI-5” (A---A), and intact DTI-5” leaves (O--Q).
where it was not detected in sap. When the differential temperature treatment was lengthened to 20 days, a detectable amount of infectivity was found in DTI-5” leaves. However, the amount of infectivity was only about 0.04% of the maximum level of infectivity attained after the leaves were moved to 25”. Slightly more infectivity occurred in DTI-12” leaves after 20 days of differential temperature treatment, but this amount was still less than 0.4% of the maximum level of infectivity after the leaves were moved to 25”. The length of time that the plants were maintained in the differential-temperature chamber (7-14 days) did not alter the lag periods in either DTI-5” or DTI-12” leaves. In DTI-12” leaves, the lag period did not become shorter as the plants were maintained for longer periods in the differential temperatures as would be expected if the difference between the lag periods in DTI5” leaves and DTI-12” leaves was due only to a less efficient inhibition of TMV synthesis by 12”. Free Viral RNA The relative amount of free viral RNA occurring during the early stages of infec-
MOSAIC
569
VIRUS
tion was estimated by comparing the infectivity of encapsidated TMV-RNA to that of total TMV-RNA. Total TMV-RNA infectivity was determined by assaying the total phenol-extracted RNA from infected leaves. Encapsidated TMV-RNA was determined by assaying phenol-extracted RNA from the sap of infected leaves kept at room temperature for 2 hr after homogenization in water. Free viral RNA represented the bulk of the infectivity in both DTI-5” and DTI-12” leaves during the initial stages of the infection when infectivity first became detectable (Fig. 7). However, there was no large excess of free viral RNA, nor was the lag period of the viral RNA infectivity curve appreciably shorter than that of intact virus. Distribution of the Initial Leaves
Infection
in DTI
It is important to know which leaves or leaf-areas are initially infected at the nonpermissive temperature by the DTI procedure and are usable for TMV replication experiments which require a synchronous infection. Nilsson-Tillgren et al. (1969) TABLE
AMOUNTOF TMV Expt no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1
INFECTIVITYIN DTI-12” ZEROTIME
Infectivity
of sap
Av no. of lesions
Dilution
7%of maximum infectivitp
0 1 0 0 0 0 26 0 2 1 0 0 13 8
l/l00 l/100 l/100 l/loo l/loo l/100 l/100 l/100 l/100 l/100 l/loo l/20 l/100 l/loo
0.02 0.065 0.002 0.01 0.018 0.17
LEAVESAT
Infectivity RNA Av no. of lesions
a Maximum infectivity was measured leaves at 4 days after transfer to 25”.
0 7 0 0 0 0 25 48 0 0 0 -
of
Dilution
l/25 l/5 l/l0 l/10 l/10 l/l l/10 l/10 l/20 l/20 l/10 -
in DTI-12”
570
DAWSON,
SCHLEGEL
1
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water. These experiments gave results similar to those shown in Table 2. DISCUSSION
5
DAYS AT 25’= FIG. 7. Infectivity and encapsidated leaves.
of total TMV-RNA TMV-RNA (O--O)
(A---A) in DTI
reported that during the systemic infection of young tobacco leaves, the base of some leaves became infected earlier than the rest of the leaf. To determine which areas of DTI leaves were infected initially, l-mm leaf discs collected from tip, middle, and base areas of detached leaves of different sizes were assayed. These samples were collected after the DTI leaves had incubated at 25’ for 24 hr, at the time the rapid increase in infectivity was beginning but before a substantial increase in infectivity could occur in cells not infected initially. Table 2 shows the results of representative experiments which suggest that all areas of leaves less than 4 cm long and the basal 213 of leaves 4-7 cm long became infected at approximately the same time. In other experiments, discs were removed from DTI leaves immediately before transfer to 25” and were then incubated at 25” floating on
A procedure that we (Dawson and Schlegel, 1973) previously reported to give enhanced virus multiplication rates in whole leaves by manipulation of the temperature of different parts of plants was further developed into a model system for studying virus replication and virus-cell interactions. The procedure allows leaf cells to become infected at a low temperature that is nonpermissive for virus replication. The cells of young leaves maintained at a low temperature become infected with inoculum from the lower leaves of the same plants that are maintained at a temperature permissive for virus multiplication. After sufficient inoculum has moved into the young leaves, they are moved to a permissive temperature that allows virus synthesis to begin simultaneously in all infected cells. We do not know in what form the inoculum moves from the lower leaves to the upper leaves. Enough inoculum moved into DTI-5” leaves to produce the rapid rates of synthesis observed, but infectivity could not be detected in sap or in phenolextracted RNA. This suggests that the differential-temperature inoculation is either very efficient, inoculating many cells with a small amount of inoculum which was below the level of detection, or that the inoculum was in some form which was not infectious when extracted and assayed on another host. TABLE
2
SIZE AND POSITION OF LEAVES INFECTED WITH TMV DIFFERENTIAL TEMPERATURE INOCULATION
Leaf no.
1 2 3 4
Size of leaves
BY
Infectivity after 24 hr at 25” (Av no. of lesions)
Max length (cm)
Max width (cm)
Tip
4 7 4 6
2.5 5 2.5 4.5
120 26 133 54
Middle
Base
108 90 113 125
124 91 127 125
SYNTHESIS
OF TOBACCO
The inoculum moved uniformly into young leaves smaller than 4 cm long and the basal portions of leaves 4-7 cm long. It moved sporadically or not at all into larger leaves. The inoculum appeared to move passively from photosynthesizing lower leaves to growing points, the direction metabolites in the phloem would be expected to move. The low temperatures apparently inhibited virus synthesis more than they inhibited host metabolism, in that the young leaves continued to expand at 12” and (very slowly) at 5”, thus serving as terminals of metabolites and virus inoculum. In both DTI-5” and DTI-12” leaves, TMV increased as rapidly when the leaves were detached as when they were left intact on the plants. This demonstrates that the rapid increase in infectivity occurring in DTI leaves resulted from TMV synthesis in these leaves and not from translocation of virus-specific products to DTI leaves from lower leaves of the plant. However, DTI leaves left on the plant have the same infectivity curve as detached leaves and are easier to maintain. Free viral RNA made up most of the infectivity in DTI leaves during the initial stages of synthesis (the beginning of the logarithmic infectivity increase) and was detectable during the first 24 hr at 25”. Because free TMV-RNA was measured by subtracting encapsidated RNA infectivity from total viral RNA infectivity, the amount of free TMV-RNA could not be accurately measured when it became less than about 10% of the total TMV-RNA infectivity. After 24 hr, the amount of encapsidated RNA was much greater than free viral RNA. These results are similar to those of Sarkar (1965), who examined free TMV-RNA after mechanical inoculation of tobacco leaves. This suggests that he observed free viral RNA only in initially infected cells, again because free viral RNA infectivity is difficult to measure in the presence of large excesses of encapsidated RNA which probably occurs in an asynchronous infection after the rapid linear increase of TMV in the initially infected cells and all stages of synthesis of subsequently infected cells.
MOSAIC
VIRUS
571
The lag periods before free viral RNA accumulation in DTI leaves were not appreciably shorter than that of intact virus, and the linear increases did not occur measurably earlier, suggesting that assembly of particles closely follows viral RNA synthesis. Temperatures of 5 and 12” blocked TMV synthesis at different steps. Although TMV is synthesized more slowly at low temperatures (Lebeurier and Hirth, 1966), both 5 and 12” do more than slow virus synthesis. The infectivity curve of TMV in DTI-12” leaves was similar to that in DTI-5” leaves, except that the DTI-12” curve was about 6-10 hr earlier than the DTI-5” curve. Longer differential-temperature treatments did not alter the infectivity curves of TMV in DTI-5” or DTI-12” leaves. The similarity of TMV synthesis in DTI-5” leaves to that in protoplasts suggests that an initial event of virus replication was inhibited by 5”. The shorter lag period (and the occasional lack of a lag period) before the logarithmic infectivity increase in DTI12” leaves suggests that 12” blocked some step subsequent to infection but prior to viral RNA synthesis (that some steps of TMV synthesis occurred at 12’). Subsequent work (Dawson and Schlegel, in preparation) clearly demonstrates that 5 and 12’ inhibit different, specific steps of TMV replication in that 2-thiouracil totally inhibits TMV synthesis in DTI-5” leaves at the time of transfer to 25” but does not inhibit TMV synthesis in DTI-12” leaves when treatment begins at the same time. The rate of synthesis of TMV in DTI leaves is similar to that in other systems that provide synchronous virus synthesis. The infectivity curve of TMV in DTI leaves, consisting of logarithmic increase followed by a more rapid linear increase, is similar to synchronous RNA virus synthesis in animal (Baltimore et al., 1966) and bacterial systems (Godson, 1968) and to in vitro systems when below-saturating levels of viral RNA are added (Spiegelman et al., 1965). By infecting cells with poliovirus at various virus/cell multiplicities, Baltimore et a!. (1966) demonstrated that the logarithmic rate of viral RNA increase is due to the participation of progeny mole-
572
DAWSON,
SCHLEGEL
cules as templates and not to asynchronous entry into the replication process of parental RNA as the only templates for RNA replication. The infectivity curves of TMV in DTI leaves also are similar to those of TMV in protoplasts (Takebe et al., 1971; Otsuki et al., 1972). The logarithmic increase of TMV infectivity began at about 6 and 15 hr in protoplasts incubated at 28 and 22”, respectively. The logarithmic increase of TMV infectivity in DTI-5” leaves incubated at 25” began after 8 hr. The similarity between TMV synthesis in DTI leaves and protoplasts is particularly evident when the results of Takebe et al. (1971) are plotted on a linear scale (Fig. 8). In protoplasts, TMV infectivity began increasing linearly after 12 hr at 28 and 24 hr at 22”. At both temperatures, TMV infectivity continued to increase linearly until 72 hr of incubation (the duration of the experiment). In DTI-5” leaves, incubated at 25”, the linear increase began at 24 hr and continued until about 72 hr of incubation after which the rate of increase declined. The duration of synthesis of TMV is 3-5 days in DTI leaves. In the initial reports by Aoki and Takebe (1969) and Takebe and Otsuki (1969), the duration of TMV synthesis in isolated tobacco protoplasts was only 24 hr. However, these investigators
35 R 0 30 t
1ll.,~/f; HOURS
40 AFTER
60 72 INFECTION
FIG. 8. A linear plot of the time course of TMV multiplication in tobacco mesophyll protoplasts; adapted from Takehe et al. (1971, Fig. 2).
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recently demonstrated (Otsuki et al., 1972; Takebe et al., 1971) that TMV synthesis in protoplasts continued for 72 hr (the length of the experiment) and that the rate of synthesis at 72 hr was near the maximum rate (Fig. 8). TMV synthesis in separated tobacco cells isolated from leaves inoculated 3 days earlier was linear for 44 hr (the length of the experiment (Jackson et al., 1972)). Virus synthesis in some of the cells must have been longer than 44 hr. The results of Oxefelt (1970) using the conditions described by Nilsson-Tillgren et al. (1969) suggest that TMV synthesis in intact leaves continued for about 5 days after 90% of the cells were infected. The similarity of TMV infectivity curves in DTI leaves to that of TMV in protoplasts and to other synchronous virus systems suggests that TMV synthesis occurs synchronously in DTI leaves. However, a following paper (Dawson and Schlegel, in preparation) demonstrates that virus synthesis begins in DTI leaves at specific steps of replication and the subsequent steps occur sequentially and synchronously at the permissive temperature; a further argument for synchrony of TMV synthesis in DTI leaves will be given in that paper. The differential-temperature inoculation system should have many applications in plant virus research. It is simple, easy to perform, and highly reproducible. Large amounts of tissue with which to work are provided. The system should be applicable to many virus-host combinations and has previously been demonstrated applicable to cowpea chlorotic mottle virus in cowpea (Dawson and Schlegel, 1973). The only requirements should be a virus that is inhibited by low temperatures and a systemic host that is structurally suited to a differential-temperature chamber. The differential-temperature inoculation system provides a much needed complement to the protoplast system for studying plant virus replication. Protoplasts are more amenable for studies of the inoculation process, pulse-chase experiments, and the uptake of large molecules. However, they are subjected to a variety of relatively harsh treatments whose effects on the cell’s physiology are not known. Virus
SYNTHESIS
OF TOBACCO
synthesis in DTI leaves can be examined in cells in intact leaves that are not subjected to adverse treatment and therefore are more likely to retain their natural state. This system should prove especially useful in determining the effects of individual steps of virus replication upon host metabolism and the effect of viruses upon cellcell interactions. REFERENCES AOKI, S., and TAKEBE, I. (1969). Infection of tobacco mesophyll protoplasts by TMV-RNA. Virology 39, 439-448. BALTIMORE, D., GIRARD, M., and DARNELL, J. E. (1966). Aspects of the synthesis of poliovirus RNA and the formation of virus particles. Virology 29, 179-189. DAWSON, W. O., and SCHLEGEL,D. E. (1973). Differential temperature treatment of plants greatly enhances multiplication rates. Virology 53, 476-478. GODSON, G. N. (1968). Site of synthesis of viral ribonucleic acid and phage assembly in MS2infected Escherichia coli. J. Mol. Biol. 34,149-163. GOODING, G. V., and HEBERT, T. T. (1967). A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology 57, 1285. JACKSON, A. O., ZAITLIN, M., SEIGEL, A., and FRANCKI, R. I. B. (1972). Replication of tobacco mosaic virus. III. Viral RNA metabolism in separated leaf cells. Virology 48, 655-665. LEBEURIER, G., and HIRTH, L. (1966). Effect of tem-
MOSAIC
VIRUS
573
peratures on the development of two strains of tobacco mosaic virus. Virology 29, 385-395. NILSSON-TILLGREN, T., KOLEHMAINEN-SEVEUS, L., and VON WETTSTEIN, D. (1969). Studies on the biosynthesis of TMV. I. A system approaching a synchronized virus synthesis in a tobacco leaf. Molec. Gen. Genetics 104, 124-141. OXELFELT, P. (1970). Development of systemic tobacco mosaic virus infection. I. Initiation of infection and time course of virus multiplication. Phytopath. 2. 69, 202-211. OTSUKI, Y., SHIMOMURA, T., and TAKEBE, I. (1972). Tobacco mosaic virus multiplication and expression of the N gene in necrotic responding tobacco varieties. Virology 50, 45-50. SARKAR, S. (1965). The amount and nature of ribonuclease sensitive infectious material during biogenesis of tobacco mosaic virus. 2. Vererbunzsl. 97, 166-185. SPIEGELMAN, S., HARUNA, I., HOLLAND, I. B., BEUADREAU, G., and MILLS, D. (1965). The synthesis of a self-propagating and infectious nucleic acid with a purified enzyme. Proc. Nat. Acad. Sci. USA 54, 919-927. TAKEBE, I., and OTSUKI, Y. (1969). Infection of tobacco mesophyll protoplasts by TMV. 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. 9, 115-124. TAKEBE, I., OTSUKI, Y., and AOKI, S. (1971). Infection of isolated tobacco mesophyll protoplasts by tobacco mosaic virus. Colloq. Znt. Cent. Nat. Rech. Sci. 193.503-511.
