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Effects of ultraviolet radiation on infection by intact and phenol-disrupted red clover mottle virus
VIROLOGY
14,
19~296
Effects
(1961)
of Ultraviolet
Radiation
Phenol-disrupted
Red
F. C. BAWDEN Rothamsted
on Infection
Experimental
AND
Station, Accepted
Clover
Mottle
and
Virus
R. C. SINHAl
Harpenden,
February
by Intact
Hertfordshire,
England
fi?O, 196i
Exposing French bean leaves to ultraviolet radiation after inoculation with red clover mottle virus decreased numbers of infections more than did exposure before inoculation. The decrease produced by a given dose of radiation differed with plants in different physiological states. Treatments that increased susceptibility to infection also increased the effects of the radiation. Irradiation before inoculation affected lesion numbers similarly whether the inoculum was intact or phenol-disrupted virus provided the leaves were in the dark after irradiation, but when leaves were in the light effects were much greater with disrupted virus. Intact virus is apparently better able than disrupted virus to survive while light restores the capacity of leaves to support infection. The effects of photoreactivation depended on the radiation dose; leaves given small doses before inoculation and then exposed to visible light produced more lesions than the controls when the inoculum was intact virus, but usually fewer when it was disrupted virus. Irradiation immediately after inoculation decreased lesions more with disrupted virus than with intact virus, but infection centers initiated by disrupted virus increased their resistance sooner and more rapidly. Infection centers initiated by intact virus retained their original susceptibility for about 2 hours at 24-28” and 6-8 hours at 16”. At 32” resistance did not increase even after 22 hours; plants placed at 24” after 22 hours at 32” produced about half as many lesions as those kept at 24" continuously after inoculation. At 32”, infection centers initiated by disrupted virus also failed to increase their resistance to inactivation by the radiation, and few survived 4 hours, even in unirradiated leaves. Hence, whatever may happen at lower temperatures, intact virus seems not to dissociate into protein and nucleic acid in leaves at 32”.
of irradiation experiments are often difficult’ to interpret in terms of direct effects on virus or host; in attempts to gain more information about these different effects we have also studied how changing the physiological condition of leaves influences the response to irradiation.
INTRODUCTION
leaves to ultraviolet radiation has given information about changes in leaves infected with tobacco mosaic and tobacco necrosis viruses and has shown considerable differences between the initial behavior of intact virus particles and of infective nucleic acid preparations, Whether the results obtained with these two viruses apply at all generally can be determined only by experiments with other viruses, and we have therefore made similar experiments with red clover mottle virus. The irradiation not only inactivates virus particles in leaves, but also affects the capacity of leaf cells to support infection. Hence, the results Exposing
’ Present address : Botany Department, versity of Illinois, Urbana, Illinois.
METHODS
Red clover mottle virus (RCMV) (Sinha, 1960a) was propagated in inoculated leaves of French bean (Phaseolus vulgaris L. var. Prince), from which sap was extracted 4-6 days after inoculation and stored at -20” until used. The primary leaves of French bean were also used for infectivity assays by the half-leaf method already described (Sinha, 1960b).
Uni198
INFECTION
OF
IRRADIATED
In experiments with intact virus, the inoculum was usually sap clarified by centrifuging at 8000 rpm and diluted in water so that it produced about 50 lesions per halfleaf. Treating clarified sap with phenol (Gierer and Schramm, 1956) destroyed infectivity, but treating partially purified RCMV (Sinha, 1960a) in M/90 pH 7 phosphate buffer once with phenol disrupted the virus and left about one-tenth of the initial infectivity. Solutions of the phenol-disrupted material in water showed no virus particles when examined in the electron microscope. They lost their infectivity in a day at 18”, and in 10 minutes when incubated with pancreatic ribonuclease at concentrations harmless to intact virus. NO chemical tests were made on the phenoltreated material, but, by analogy with other viruses, the infectivity was probably conferred by nucleic acid. Ultraviolet radiation, mostly of wavelength 254 rnp, was provided by a Hanovia low-pressure, mercury-discharge lamp fitted with a filter to remove wavelengths shorter than 240 mp. At 20 cm from the lamp, the distance at which exposed leaves were placed, the intensity of radiation was about 1500 pW/cm2. Unless otherwise stated, after irradiation leaves were kept in daylight from a north window at about 20”. They were detached from plants immediately before irradiation and afterward were put on a moist cloth in trays covered with polyethylene sheets, where they were left until lesions were counted. Half of each leaf was exposed to the radiation while the other half was screened from it by cardboard and served as an unirradiated control. Each treatment was applied to at least eight halfleaves. The ratio, number of lesions formed on the irradiated half-leaves to number formed on the unirradiated halves, expressed as a percentage, will be called the “lesion survival.” Inocula of both intact and disrupted virus were used at dilutions that gave about 50 lesions per half-leaf. RESULTS
Experiments
with
Intact
V&s
Experiments in which leaves are in visible light after irradiation do not show the
199
LEAVES TABLE
EFFECT
Expt. no.
1 2 3
1
OF PHOTOREACTIVATION ON INFECTIONS IN LEAVES IRRADIATED BEFORE OR AFTER INOCULATION Irradiation dose (se4
30 30 60
Irradiated before inoculation”
Irradiated after inoculationa
Light
Dark
Light
Dark
91 77 63
94 80 37
9.0 6.6 1.1
3.7 4.0 0.8
a Values are the lesions produced on irradiated half leaves expressed as a percentage of those produced on the unirradisted halves. “Light” and “Dark” mean that leaves were in daylight or darkness for the day immediately after irradiation.
full effects of irradiation, because damage both to host cells and to some viruses can be partially counteracted by visible light. Table 1 shows results of experiments made to assess the amount of photoreactivation. Leaves irradiated for 30 seconds before inoculation were only slightly affected, and lesion survival was the same whether leaves were in the dark or light, but irradiation for 60 seconds did damage, some of which was counteracted by visible light. Irradiation after inoculation decreased lesion numberr much more than irradiation before inoculation, and there is evidence of photoreactivation with the 30 seconds’ exposure. Thiz probably indicates photoreactivation of the virus rather than the host, but unequivoca evidence that R,CMV can be photoreactivated could not be got; experiments wit1 virus irradiated in vitro and then inoculatec to plants, some of which were kept in the light and others in the dark, gave erratic and uninterpretable results because no re. liable dilution curve could be obtained wit1 control virus (Sinha, 1960a) from which tc compute the effects of irradiation. Bawden and Kleczkowski (1960) fount that the extent to which radiation affecter the capacity of Nicotiana glutinosa L. tc support infection by tobacco mosaic viru differed with different lots of leaves, am they noted that leaves that were most sus ceptible to infection were often those also most affected by irradiation. From the re
200
BAWDEN TABLE
AND
2
EFFECT OF PREINOCULATION TREATMENTS ON THE PERCENTAGE LESION SURVIVAL IN LEAVES IRRADIATED AFTER INOCULATION
Expt. IlO.
Irradiation dose bed
Preinoculation Daylight at 20”
treatment9
Daylight at 36”
1
15 30 45 Go
12.4 5.3 2.1 1.3
8.8 0.7 0.5 0.5
2
15 30 45
27.6 8.2 1.9
10.7 1.8 0.3
3
15 30 45
29.1 11.3 2.0
-
Darkness at 20” 8.0 1.3 0.2 0 12.1 3.7 0
a Refers to the 24 hours before inoculation. Leaves were irradiated immediately after inoculation and then kept in daylight at 20” until lesions developed.
sults we have already given it will also be obvious that the same dose of radiation affected lesion survival differently in different lots of leaves inoculated with RCMV. To see whether these differences were correlated with differences in susceptibility to infection, experiments were made in which plants were treated before irradiation in ways known to affect susceptibility to infection. Keeping plants for the day before they are inoculated either at 36” (Sinha, 1960b), or in darkness instead of in daylight at 20”, approximately doubles the number of lesions produced by appropriately diluted inocula of RCMV. Table 2 shows that both these treatments also increased the effects of postinoculation irradiation on lesion survival. It is uncertain to what extent these increases reflect differences in the susceptibility of the leaves to damage by radiation or differences in the extent to which the virus is shielded from irradiation in leaves of different physiological states. That the treatment does affect the susceptibility of the leaves to damage was shown by irradiating leaves before they were inoculated with RCMV. In one such experiment, leaves from plants kept in day-
SINHA
light and 20” (normal glasshouse conditions) gave lesion survivals of 79, 40, and 20% when irradiated, respectively, for 30, 60, and 90 seconds, whereas comparable values for leaves from plants kept at 36” during the day before irradiation were 61, 20, and 9%. Some substance that absorbs ultraviolet radiation and helps to protect host-cell and infecting virus against damage may be decreased by keeping plants at 36” or in darkness. Both treatments will affect the amounts of many different substances, and there is no a priori reason to seek a common cause for the increased SUSceptibility to infection and to damage by the radiation. In these experiments the leaves were in daylight after irradiation, SO some effects of these treatments might have been on the mechanism responsible for photoreactivating the damaged leaves. The susceptibility of French bean leaves to infection by RCMV depends on their age; it increases as the leaves expand, reaches a maximum, and then decreases. The period of maximum susceptibility is reached sooner and is much briefer in summer than in winter. These changes in SUSceptibility to infection also coincide with changes in the effect of ultraviolet radiation on lesion survival. For example, in one experiment in summer, in which leaves were irradiated immediately after inoculation for 10,20,40, and 80 seconds,the lesion survivals in leaves from plants 12 days from sowing were, respectively, 42, 19, 6, and O%, whereas in leaves from plants 15 days from sowing they were 68, 32, 11, and 3%. When a constant dose of radiation is applied to leaves inoculated with tobacco mosaic or tobacco necrosis viruses, it has a constant effect on lesion survival for a few hours after inoculation, but its effect then decreases (Bawden and Harrison, 1955; Siegel and Wildman, 1956). The time when lesion survival starts to increase depends both on the identity of the virus used and on the temperature at which the inoculated plants are kept. Figure 1 shows how exposing leaves to radiation for 80 seconds at different intervals after inoculation with RCMV affected lesion survival in leave: kept at different temperatures between inoculation and irradiation. At 28”, lesion
INFECTION
I 0
I
I
2
4
OF IRRADIATED
I
1
6
8
Interval
between
I
I
IO
12
inoculation
and
LEAVES
I
14 irradiation
201
I
I
16
18
I
20
I
22
(hours]
FIG. 1. The effect on lesion survival of keeping leaves at different temperatures between the time they were inoculated with red clover mottle virus and exposed to ultraviolet radiation for 80 seconds. Note constant effect with leaves at 32”, a rapid increase in lesion survival after 2 hours at 28”, and a slow increase at 12”.
survival was constant after that it increased
for only rapidly.
2 hours, and At 12”, le-
sion survival did not obviously increase until at least 4 hours after inoculation, and t,hen the increase proceeded only slowly, taking 18 hours to reach the same percentage survival as that reached in 6 hours at 28”. At 32” there was no consistent increase even up to 22 hours. The virus is slowly inactivated in inoculated leaves kept at 32” and few lesions are produced in leaves left continuously at this temperature, but after 22 hours at 32” leaves will, when put at a lower temperature, still produce up to half as many lesions as leaves placed at the lower temperature immediately after inoculation. When, after 16 hours at 32”, plants were put at 28”, the lesion survival after exposure to ultraviolet radiation started to increase in 2-4 hours, as when plants were put at 28” immediately after inoculation. Some step in the infection process, which happens rapidly at 28” and slowly at 12”, is obviously prevented from happening at 32”. In this respect RCMV differs from the Rothamsted tobacco necrosis virus, which seems able to take this step at 32”, even though it also causesfewer lesions in plants at this than at lower temperatures. Figure 2 shows the behavior of RCMV at different temperatures as measured by
FIG. 2. The effect of temperature on the time required between inoculation and irradiation of leaves for half the infection centers initiated by red clover mottle and the Rothamsted tobacco necrosis virus to survive a standard dose (80 seconds) of ultraviolet radiation.
the time required for 50% of the lesions initially suppressible by a given dose of radiation to become insuppressible, and compares it with the behavior of the tobacco necrosis virus (Harrison, 1956). Between 8” and 28”, whatever change or changes at infection sites are measured by this method happen at much the same times with both
202
BAWDEN
AND
viruses. However, the two differ considerably in the times at which newly produced virus becomes detectable in inoculated leaves. At 24”, for example, with both viruses the interval between inoculation and the time when half the initially suppressible lesions survived irradiation for 80 seconds was 5-6 hours, but whereas sap from leaves inoculated with tobacco necrosis virus was infective within another 5 hours, sap from leaves inoculated with RCMV was not infective until another 13 hours. Presumably the tobacco necrosis virus either accumulates more rapidly than RCMV or fewer particles of the tobacco necrosis virus are needed to cause infections in French bean leaves. Experiments
with
Phenol-disrupted
Virus
Table 3 compares the effects of irradiating leaves before and after inoculation with intact and phenol-disrupted virus. In these experiments, in which the leaves were kept in daylight after irradiation, the two inocula behaved very differently. With each dose of irradiation, lesion survival was more affected in leaves inoculated with disrupted than with intact virus. The differences between the two inocula when irradiation followed inoculation could be partially explained by the disrupted virus being more sensitive than intact virus to inactivation by ultraviolet radiation, in the same way TABLE EFFECT ON PERCENTAGE IRRADIATING LEAVES AFTER INOCULATION DISRUPTED
-
Expt. no.
VIRUS
Virus inoculum
-
I
3 LESION IMMEDIATELY
WITH
SURVIVAL BEFORE
INTACT
Timing of .rradiaTion relative to noculation
OF OR
OR PHENOL-
Irradiation (se4
dose
-
5
10
15
20
Intact Intact Disrupted Disrupted
Before After Before After
121 50 86 12
114 88 22 15 74 41 6 0
58 9 -
Intact Intact Disrupted Disrupted
Before After Before After
123 46 114 28
105 91 29 28 89 75 17 3
84 22 61 2 -
-
0
SINHA
as the nucleic acid of tobacco mosaic virus is more sensitive than the intact virus (McLaren and Takahashi, 1957). However, this cannot explain the differences between effects when irradiation preceded inoculation, when the two inocula often behaved so differently in leaves given small doses (for example, 5 and 10 seconds in experiment 1, Table 3) that lesion number was decreased with disrupted, and increased with intact, virus. This difference can be attributed to photoreactivation, because the two inocula behaved similarly when inoculated leaves were placed in darkness immediately after irradiation (Table 4). Bawden and Kleczkowski (1960) obtained similar results with tobacco mosaic virus and concluded that, whereas the intact virus could survive through the period during which visible light restored the damaged capacity of the leaves to support infection, the nucleic acid could not. Our results (Table 3) show that even brief exposures to the radiation can temporarily damage the ability to support infection, but this damage is so soon counteracted by visible light that it shows only with inocula of disrupted RCMV; indeed, with inocula of intact virus, photoreactivation more than restored the original capacity of the leaves and made conditions at some sites such that infection occurred although it would not have in unirradiated leaves. Table 4 shows that, when inoculation was delayed for some hours after leaves were irradiated, the two kinds of inocula gave similar results; both produced fewer lesions in leaves kept in darkness immediately after irradiation than in leaves exposed to light, and photoreactivation of leaves before inoculation increased lesion survival almost as much with disrupted as with intact RCMV. The main difference shows in the lesion survival in leaves inoculated immediately after irradiation and then kept in the light. With both inocula, exposure to light increased lesion survival, but neither produced as many lesions as in leaves kept in the light for 3 hours after irradiation before inoculation. In experiment 2 (Table 4) the average lesion survival in leaves placed in darkness after irradiation was 30%, whereas in leaves inoculated after 3 hours
INFECTION
OF IRRADIATED
in the light it was 76%. Hence, exposing irradiated leaves to light for 3 hours made 46% more lesion sites susceptible; disrupted virus inoculated to leaves immediately after irradiation and then kept in the light for 3 hours infected 15% of these sites (44 - 29) and intact virus 30% (61 - 31). Neither kind of inoculum, therefore, survived wholly through the 3 hours while photoreactivation was proceeding, and the disrupted virus seems to have inactivated in the leaves about twice as fast as did the inoculum of intact particles. Figure 3 compares the behavior of leaves inoculated with intact and disrupted RCMV and shows that, within the 2 hours during which lesion survival remained unchanged in leaves inoculated with intact virus, lesion survival increased greatly in leaves inoculated with disrupted virus. After lesion survival started to increase in leaves inoculated with intact virus, it did so more slowly than in leaves inoculated with disrupted virus, perhaps because the change that produced the increase happened not only later, but also over a more extended period, and so was less well synchronized than with disrupted virus. What change in state of the inoculum or host increases lesion survival is not known, but the association of the virus nucleic acid with, or its inclusion in, some cell component, such as the nucleus or ribosomes, could explain it. The different behavior of the two kinds of inocula is plausibly explained by postulating that any such association demands that the nucleic acid separates from the protein, and that the lag period with intact virus represents the time taken for the nucleic acid to become free in viva (Siegel et al., 1957). The fact that infection centers initiated by intact RCMV do not become increasingly resistant to inactivation by ultraviolet radiation when inoculated leaves are kept at 32” could then be explained by postulating that, at this temperature, the processes that normally lead to the separation of nucleic acid from protein do not operat,e. To test this possibility, leaves were inoculated with disrupted virus and then irradiated after the plants had spent different intervals at 32”. The lesion survival did not increase with increasing time, but with
203
LEAVES TABLE
EFFECT
ON
LIGHT
AND
ATION AND NOL-DISRUPTED
I
Expt. no.
PERCENTAGE OF
IlluminatioP I
INTERVAL
INOCULATION VIRUS
I
I
4 LESION
SURVIVAL
BETWEEN WITH
OF IRRADI-
INTACT
OR PHE-
Inoculum and interval (hr) between irradiation and inoculation Disrupted
virus
0
3
24
I
Intact
virus
0
3
24
-__~
1
Light
20
71
-
36
70
-
2
Light Dark
44 29
73 33
-
61 31
79 27
-
3
Light Dark
34 16
-
92 15
64 15
-
115 18
-
a “Light” indicates that leaves were kept in daylight between irradiation (80 seconds exposure) and inoculation, “Dark” that they were in darkness during this period. All leaves, except those in treatment “Light, 0 hr” which were kept in daylight throughout, were kept in darkness for 24 hours after inoculation.
0
I 2
I 4 Hours
after
I 6
I 8
inoculation
FIG. 3. The increase in percentage lesion survival with increasing time after French bean leaves were inoculated with phenol-disrupted (NA inoculum) and intact red clover mottle virus. Leaves were kept at 24” between inoculation and 20 seconds’ exposure to ultraviolet radiation, after which they were in daylight.
204
BAWDEN
increasing time at 32” the numbers of lesions produced on the unirradiated halfleaves fell sharply. To cite one experiment comparing the behavior of intact and disrupted virus, the unirradiated half-leaves inoculated with intact virus kept for 0, 2, 4, and 5 hours at 32” before being placed at 24” gave, respectively, 484, 515, 475, and 407 lesions, whereas those inoculated with disrupted virus gave 436, 173, 31, and 0. As stated earlier, even after 22 hours at 32” leaves inoculated with intact virus still produce many lesions when put at lower temperatures. These results give no direct information about whether infection by intact virus normally necessitates that the protein and nucleic acid separate from one another, but they do show that most of the virus particles do not liberate their nucleic acid within a few hours at 32”, for if they did so, it would be inactivated. The results also show that failure of intact virus to infect and produce lesions at 32” is not attributable solely to the nucleic acid failing to separate from the protein, because already disrupted virus also failed to multiply. Hence, if infection normally entails the nucleic acid becoming free, it is not only this step that is prevented in leaves at 32”, but also some other that follows this one. The slow inactivation of intact RCMV in inoculated leaves kept at 32” could be attributed to the processes that separate virus protein from nucleic acid being slowed but not eliminated, so that with increasing time more and more particles liberate their nucleic acid, which then becomes inactivated. However, there is no reason to postulate that nucleic acid must become free to be inactivated, because there are many treatments that inactivate intact virus particles in vitro. French bean leaves may contain a system able to do this, for they contain one that inactivates tobacco necrosis and some other viruses in vitro without destroying their serological specificity or affecting their gross morphology or physical properties (Bawden and Pirie, 1957). The general behavior of RCMV suggests that it is likely to be among the viruses inactivat>ed by these leaf systems.
AND
SINHA DISCUSSION
There are two differences between the behavior of RCMV and tobacco necrosis virus. Kassanis (1960) found that infection centers initiated by intact and disrupted tobacco necrosis virus were equally susceptible to inactivation when leaves were irradiated immediately after they were inoculated, whereas with RCMV, as with tobacco mosaic virus, the infection centers initiated by disrupted virus are more readily inactivated than those initiated by intact virus. This could mean that the protein of RCMV protects its nucleic acid from irradiation damage, as does the protein of tobacco mosaic virus, whereas the protein of tobacco necrosis virus does not, but whether this interpretation is correct will remain uncertain until intact and disrupted virus preparations are irradiated in vitro and the relative rates of inactivation are determined. The second difference is in leaves at 32”, in which RCMV retains its initial susceptibility to inactivation by radiation for many hours, whereas the susceptibility of tobacco necrosis virus seems to decrease (Harrison, 1956). Perhaps the most significant feature in common with tobacco mosaic and tobacco necrosis viruses is that inoculation with disrupted virus shortens the period required for infection centers to gain resistance to ultraviolet radiation. This difference between the behavior of the two inocula is probably the major evidence in favor of the hypothesis that infection by intact virus requires, as a first step, that the protein and nucleic acid separate. Other phenomena from the irradiation experiments also fit this hypothesis. For instance the fact that, when leaves newly inoculated with intact RCMV are irradiated, lesion survival is greater in leaves kept in light than in darkness but photoreactivation has little effect on lesion survival in leaves inoculated with disrupted virus, receives a ready explanation by postulating that the free nucleic acid inactivates during the time when the capacity of the leaves to support infection is being photoreactivated, whereas virus particles are still intact during this period and
INFECTION
OF
IRRADIATED
disrobe to infect later. However, this explanation is not wholly satisfactory, for with tobacco mosaic virus there is equal photoreactivation whether leaves are irradiated immediat.ely after inoculation or after the lag period (Bawden and Kleczkowski, 1960), when according to the hypothesis the nucleic acid should already be free. Irradiation experiments show that changes proceed in infected cells and that changes become detectable sooner with inocula of nucleic acid than with intact virus. In addition to the effect on the lag period already mentioned, irradiated nucleic acid of tobacco mosaic virus becomes photoreactivable immediately after inoculation to leaves (Bawden and Kleczkowski, 1959) whereas few irradiated potato virus X particles become photoreactivable until half an hour after leaves are inoculated (Bawden and Kleczkowski, 1955). Unfortunately, although the irradiation experiments establish phenomena, they provide no information that allows the phenomena to be interpreted with certainty. The only experiments with RCMV that give unequivocal evidence about the behavior of intact virus are those with inoculated leaves kept at 32”, and these show clearly that the nucleic acid does not become free from the protein. However, the fact that nucleic acid does not become free in leaves at 32” is not evidence about what may happen at lower temperatures, at which infection becomes established. Indeed, it is plausibly argued that nothing much happens to virus particles in leaves at 32” simply because the mechanism that normally separates the protein and nucleic acid is inactive at this temperature. Inocula of disrupted and intact virus both lead to the same end results, virus multiplication and lesions. Hence, at some stage between inoculation and the production of new virus, effects from the two inocula must coincide, but it is well to stress that where this happens is by no means certain. Because infection centers initiated by both kinds of inocula in time increase their resistance to inactivation by ultraviolet radiation, it is plausible to assume that their
205
LEAVES
activities coincide at this point. However, this is still an assumption, and the increased resistance quickly acquired by infection centers initiated by disrupted virus may come from a process quite different from the one by which infection centers initiated by intact virus slowly acquire resistance. In establishing infection both host cell and virus are concerned, and effects of radiation on one are not readily disentangled from effects on the other. Differences between effects obtained by irradiating immediately before and after inoculation are helpful only in part, because our results show that changes in the physiological state of leaves greatly affect their susceptibility to radiation damage and the physiological changes caused in leaf cells by infection are likely to be greater than those caused by a few days’ aging or by a few hours in darkness or at 36”. Also, the practice of putting irradiated leaves in the light, which most workers have adopted with the idea that photoreactivation would compensate for damage to the leaves, is confusing rather than otherwise, particularly when the behavior of intact and disrupted virus is being compared. With intact virus, photoreactivation always increases lesion survival, whereas with disrupted virus, its effects depend on the time interval between inoculation and irradiation. Ideally all experiments should be done in duplicate, with half the irradiated leaves put in darkness and half in the light, to show the effects of photoreactivation. We did not do this, and so are less well able to interpret Fig. 3 than if we knew what would have happened had a comparable set of leaves been kept in darkness. The different behavior of the two inocula may be at least partially explicable by photoreactivation increasing lesion survival in leaves irradiated soon after inoculation more with intact than with disrupted virus. REFERENCES BAWDEN, F. C., and HARRISON, B. D. (1955). on the multiplication of a tobacco virus in inoculated leaves of French-bean J. Gen. Microbial. 13, 494-508.
Studies necrosis plants.
206
BAWDEN
BAWDEN, F. C., and KLECZKOWSXI, A. (1955). Studies on the ability of light to counteract the inactivating action of ultraviolet radiation on plant viruses. J. Gem Microbial. 13, 370-382. BAWDEN, F. C., and KLECZKOWSKI, A. (1959). Photoreactivation of nucleic acid from tobacco mosaic virus. Nature 183, 50%504. BAWDEN, F. C., and KLECZKOWSKI, A. (1960). Some effects of ultraviolet radiation on the infection of Nicotiana glutinosa leaves by tobacco mosaic virus. ViroZog?J 10, 163-181. BAWDEN, F. C., and PIRIE, N. W. (1957). A virusinactivating system from tobacco leaves. J. Gen. Microbial. 16, 696-710. GIERER, A., and SCHRAMM, G. (1956). Infectivity of ribonucleic acid from tobacco mosaic virus. Nature 177, 702-703. HARRISON, B. D. (1956). Studies on the effect of temperature on virus multiplication in inoculated leaves. Ann. Appl. Biol. 44, 215-226.
AND
SINHA
KASSANIS, B. (1960). Comparison of the early stages of infection by intact and phenol-disrupted tobacco necrosis virus. Virology 10, 353-369. MCLAREN, A. D., and TAKAHASHI, W. N. (1957). Inactivation of infectious nucleic acid from tobacco mosaic virus by ultraviolet light (2537 A). Radiation Research 6, 532-542. SIEGEL, A., and WILDMAN, S. G. (1956). The inactivation of the infectious centers of tobacco mosaic virus by ultraviolet light. Virology 2, 69-
82. SIEGEL, A., GINOZA, W., and WILDMAN, S. G. (1957). The early events of infection with tobacco mosaic virus nucleic acid. Virology 3, 554-559. SINHA, R. C. (1960a). Red clover mottle virus. Ann. AppZ. BioZ. 48, 742-748. SINHA, R. C., (196Ob). Some effects of temperature and of virus inhibitors on infection of French-bean leaves by red clover mottle virus. Ann. AppZ. Biol. 48,749-753.
14,
19~296
Effects
(1961)
of Ultraviolet
Radiation
Phenol-disrupted
Red
F. C. BAWDEN Rothamsted
on Infection
Experimental
AND
Station, Accepted
Clover
Mottle
and
Virus
R. C. SINHAl
Harpenden,
February
by Intact
Hertfordshire,
England
fi?O, 196i
Exposing French bean leaves to ultraviolet radiation after inoculation with red clover mottle virus decreased numbers of infections more than did exposure before inoculation. The decrease produced by a given dose of radiation differed with plants in different physiological states. Treatments that increased susceptibility to infection also increased the effects of the radiation. Irradiation before inoculation affected lesion numbers similarly whether the inoculum was intact or phenol-disrupted virus provided the leaves were in the dark after irradiation, but when leaves were in the light effects were much greater with disrupted virus. Intact virus is apparently better able than disrupted virus to survive while light restores the capacity of leaves to support infection. The effects of photoreactivation depended on the radiation dose; leaves given small doses before inoculation and then exposed to visible light produced more lesions than the controls when the inoculum was intact virus, but usually fewer when it was disrupted virus. Irradiation immediately after inoculation decreased lesions more with disrupted virus than with intact virus, but infection centers initiated by disrupted virus increased their resistance sooner and more rapidly. Infection centers initiated by intact virus retained their original susceptibility for about 2 hours at 24-28” and 6-8 hours at 16”. At 32” resistance did not increase even after 22 hours; plants placed at 24” after 22 hours at 32” produced about half as many lesions as those kept at 24" continuously after inoculation. At 32”, infection centers initiated by disrupted virus also failed to increase their resistance to inactivation by the radiation, and few survived 4 hours, even in unirradiated leaves. Hence, whatever may happen at lower temperatures, intact virus seems not to dissociate into protein and nucleic acid in leaves at 32”.
of irradiation experiments are often difficult’ to interpret in terms of direct effects on virus or host; in attempts to gain more information about these different effects we have also studied how changing the physiological condition of leaves influences the response to irradiation.
INTRODUCTION
leaves to ultraviolet radiation has given information about changes in leaves infected with tobacco mosaic and tobacco necrosis viruses and has shown considerable differences between the initial behavior of intact virus particles and of infective nucleic acid preparations, Whether the results obtained with these two viruses apply at all generally can be determined only by experiments with other viruses, and we have therefore made similar experiments with red clover mottle virus. The irradiation not only inactivates virus particles in leaves, but also affects the capacity of leaf cells to support infection. Hence, the results Exposing
’ Present address : Botany Department, versity of Illinois, Urbana, Illinois.
METHODS
Red clover mottle virus (RCMV) (Sinha, 1960a) was propagated in inoculated leaves of French bean (Phaseolus vulgaris L. var. Prince), from which sap was extracted 4-6 days after inoculation and stored at -20” until used. The primary leaves of French bean were also used for infectivity assays by the half-leaf method already described (Sinha, 1960b).
Uni198
INFECTION
OF
IRRADIATED
In experiments with intact virus, the inoculum was usually sap clarified by centrifuging at 8000 rpm and diluted in water so that it produced about 50 lesions per halfleaf. Treating clarified sap with phenol (Gierer and Schramm, 1956) destroyed infectivity, but treating partially purified RCMV (Sinha, 1960a) in M/90 pH 7 phosphate buffer once with phenol disrupted the virus and left about one-tenth of the initial infectivity. Solutions of the phenol-disrupted material in water showed no virus particles when examined in the electron microscope. They lost their infectivity in a day at 18”, and in 10 minutes when incubated with pancreatic ribonuclease at concentrations harmless to intact virus. NO chemical tests were made on the phenoltreated material, but, by analogy with other viruses, the infectivity was probably conferred by nucleic acid. Ultraviolet radiation, mostly of wavelength 254 rnp, was provided by a Hanovia low-pressure, mercury-discharge lamp fitted with a filter to remove wavelengths shorter than 240 mp. At 20 cm from the lamp, the distance at which exposed leaves were placed, the intensity of radiation was about 1500 pW/cm2. Unless otherwise stated, after irradiation leaves were kept in daylight from a north window at about 20”. They were detached from plants immediately before irradiation and afterward were put on a moist cloth in trays covered with polyethylene sheets, where they were left until lesions were counted. Half of each leaf was exposed to the radiation while the other half was screened from it by cardboard and served as an unirradiated control. Each treatment was applied to at least eight halfleaves. The ratio, number of lesions formed on the irradiated half-leaves to number formed on the unirradiated halves, expressed as a percentage, will be called the “lesion survival.” Inocula of both intact and disrupted virus were used at dilutions that gave about 50 lesions per half-leaf. RESULTS
Experiments
with
Intact
V&s
Experiments in which leaves are in visible light after irradiation do not show the
199
LEAVES TABLE
EFFECT
Expt. no.
1 2 3
1
OF PHOTOREACTIVATION ON INFECTIONS IN LEAVES IRRADIATED BEFORE OR AFTER INOCULATION Irradiation dose (se4
30 30 60
Irradiated before inoculation”
Irradiated after inoculationa
Light
Dark
Light
Dark
91 77 63
94 80 37
9.0 6.6 1.1
3.7 4.0 0.8
a Values are the lesions produced on irradiated half leaves expressed as a percentage of those produced on the unirradisted halves. “Light” and “Dark” mean that leaves were in daylight or darkness for the day immediately after irradiation.
full effects of irradiation, because damage both to host cells and to some viruses can be partially counteracted by visible light. Table 1 shows results of experiments made to assess the amount of photoreactivation. Leaves irradiated for 30 seconds before inoculation were only slightly affected, and lesion survival was the same whether leaves were in the dark or light, but irradiation for 60 seconds did damage, some of which was counteracted by visible light. Irradiation after inoculation decreased lesion numberr much more than irradiation before inoculation, and there is evidence of photoreactivation with the 30 seconds’ exposure. Thiz probably indicates photoreactivation of the virus rather than the host, but unequivoca evidence that R,CMV can be photoreactivated could not be got; experiments wit1 virus irradiated in vitro and then inoculatec to plants, some of which were kept in the light and others in the dark, gave erratic and uninterpretable results because no re. liable dilution curve could be obtained wit1 control virus (Sinha, 1960a) from which tc compute the effects of irradiation. Bawden and Kleczkowski (1960) fount that the extent to which radiation affecter the capacity of Nicotiana glutinosa L. tc support infection by tobacco mosaic viru differed with different lots of leaves, am they noted that leaves that were most sus ceptible to infection were often those also most affected by irradiation. From the re
200
BAWDEN TABLE
AND
2
EFFECT OF PREINOCULATION TREATMENTS ON THE PERCENTAGE LESION SURVIVAL IN LEAVES IRRADIATED AFTER INOCULATION
Expt. IlO.
Irradiation dose bed
Preinoculation Daylight at 20”
treatment9
Daylight at 36”
1
15 30 45 Go
12.4 5.3 2.1 1.3
8.8 0.7 0.5 0.5
2
15 30 45
27.6 8.2 1.9
10.7 1.8 0.3
3
15 30 45
29.1 11.3 2.0
-
Darkness at 20” 8.0 1.3 0.2 0 12.1 3.7 0
a Refers to the 24 hours before inoculation. Leaves were irradiated immediately after inoculation and then kept in daylight at 20” until lesions developed.
sults we have already given it will also be obvious that the same dose of radiation affected lesion survival differently in different lots of leaves inoculated with RCMV. To see whether these differences were correlated with differences in susceptibility to infection, experiments were made in which plants were treated before irradiation in ways known to affect susceptibility to infection. Keeping plants for the day before they are inoculated either at 36” (Sinha, 1960b), or in darkness instead of in daylight at 20”, approximately doubles the number of lesions produced by appropriately diluted inocula of RCMV. Table 2 shows that both these treatments also increased the effects of postinoculation irradiation on lesion survival. It is uncertain to what extent these increases reflect differences in the susceptibility of the leaves to damage by radiation or differences in the extent to which the virus is shielded from irradiation in leaves of different physiological states. That the treatment does affect the susceptibility of the leaves to damage was shown by irradiating leaves before they were inoculated with RCMV. In one such experiment, leaves from plants kept in day-
SINHA
light and 20” (normal glasshouse conditions) gave lesion survivals of 79, 40, and 20% when irradiated, respectively, for 30, 60, and 90 seconds, whereas comparable values for leaves from plants kept at 36” during the day before irradiation were 61, 20, and 9%. Some substance that absorbs ultraviolet radiation and helps to protect host-cell and infecting virus against damage may be decreased by keeping plants at 36” or in darkness. Both treatments will affect the amounts of many different substances, and there is no a priori reason to seek a common cause for the increased SUSceptibility to infection and to damage by the radiation. In these experiments the leaves were in daylight after irradiation, SO some effects of these treatments might have been on the mechanism responsible for photoreactivating the damaged leaves. The susceptibility of French bean leaves to infection by RCMV depends on their age; it increases as the leaves expand, reaches a maximum, and then decreases. The period of maximum susceptibility is reached sooner and is much briefer in summer than in winter. These changes in SUSceptibility to infection also coincide with changes in the effect of ultraviolet radiation on lesion survival. For example, in one experiment in summer, in which leaves were irradiated immediately after inoculation for 10,20,40, and 80 seconds,the lesion survivals in leaves from plants 12 days from sowing were, respectively, 42, 19, 6, and O%, whereas in leaves from plants 15 days from sowing they were 68, 32, 11, and 3%. When a constant dose of radiation is applied to leaves inoculated with tobacco mosaic or tobacco necrosis viruses, it has a constant effect on lesion survival for a few hours after inoculation, but its effect then decreases (Bawden and Harrison, 1955; Siegel and Wildman, 1956). The time when lesion survival starts to increase depends both on the identity of the virus used and on the temperature at which the inoculated plants are kept. Figure 1 shows how exposing leaves to radiation for 80 seconds at different intervals after inoculation with RCMV affected lesion survival in leave: kept at different temperatures between inoculation and irradiation. At 28”, lesion
INFECTION
I 0
I
I
2
4
OF IRRADIATED
I
1
6
8
Interval
between
I
I
IO
12
inoculation
and
LEAVES
I
14 irradiation
201
I
I
16
18
I
20
I
22
(hours]
FIG. 1. The effect on lesion survival of keeping leaves at different temperatures between the time they were inoculated with red clover mottle virus and exposed to ultraviolet radiation for 80 seconds. Note constant effect with leaves at 32”, a rapid increase in lesion survival after 2 hours at 28”, and a slow increase at 12”.
survival was constant after that it increased
for only rapidly.
2 hours, and At 12”, le-
sion survival did not obviously increase until at least 4 hours after inoculation, and t,hen the increase proceeded only slowly, taking 18 hours to reach the same percentage survival as that reached in 6 hours at 28”. At 32” there was no consistent increase even up to 22 hours. The virus is slowly inactivated in inoculated leaves kept at 32” and few lesions are produced in leaves left continuously at this temperature, but after 22 hours at 32” leaves will, when put at a lower temperature, still produce up to half as many lesions as leaves placed at the lower temperature immediately after inoculation. When, after 16 hours at 32”, plants were put at 28”, the lesion survival after exposure to ultraviolet radiation started to increase in 2-4 hours, as when plants were put at 28” immediately after inoculation. Some step in the infection process, which happens rapidly at 28” and slowly at 12”, is obviously prevented from happening at 32”. In this respect RCMV differs from the Rothamsted tobacco necrosis virus, which seems able to take this step at 32”, even though it also causesfewer lesions in plants at this than at lower temperatures. Figure 2 shows the behavior of RCMV at different temperatures as measured by
FIG. 2. The effect of temperature on the time required between inoculation and irradiation of leaves for half the infection centers initiated by red clover mottle and the Rothamsted tobacco necrosis virus to survive a standard dose (80 seconds) of ultraviolet radiation.
the time required for 50% of the lesions initially suppressible by a given dose of radiation to become insuppressible, and compares it with the behavior of the tobacco necrosis virus (Harrison, 1956). Between 8” and 28”, whatever change or changes at infection sites are measured by this method happen at much the same times with both
202
BAWDEN
AND
viruses. However, the two differ considerably in the times at which newly produced virus becomes detectable in inoculated leaves. At 24”, for example, with both viruses the interval between inoculation and the time when half the initially suppressible lesions survived irradiation for 80 seconds was 5-6 hours, but whereas sap from leaves inoculated with tobacco necrosis virus was infective within another 5 hours, sap from leaves inoculated with RCMV was not infective until another 13 hours. Presumably the tobacco necrosis virus either accumulates more rapidly than RCMV or fewer particles of the tobacco necrosis virus are needed to cause infections in French bean leaves. Experiments
with
Phenol-disrupted
Virus
Table 3 compares the effects of irradiating leaves before and after inoculation with intact and phenol-disrupted virus. In these experiments, in which the leaves were kept in daylight after irradiation, the two inocula behaved very differently. With each dose of irradiation, lesion survival was more affected in leaves inoculated with disrupted than with intact virus. The differences between the two inocula when irradiation followed inoculation could be partially explained by the disrupted virus being more sensitive than intact virus to inactivation by ultraviolet radiation, in the same way TABLE EFFECT ON PERCENTAGE IRRADIATING LEAVES AFTER INOCULATION DISRUPTED
-
Expt. no.
VIRUS
Virus inoculum
-
I
3 LESION IMMEDIATELY
WITH
SURVIVAL BEFORE
INTACT
Timing of .rradiaTion relative to noculation
OF OR
OR PHENOL-
Irradiation (se4
dose
-
5
10
15
20
Intact Intact Disrupted Disrupted
Before After Before After
121 50 86 12
114 88 22 15 74 41 6 0
58 9 -
Intact Intact Disrupted Disrupted
Before After Before After
123 46 114 28
105 91 29 28 89 75 17 3
84 22 61 2 -
-
0
SINHA
as the nucleic acid of tobacco mosaic virus is more sensitive than the intact virus (McLaren and Takahashi, 1957). However, this cannot explain the differences between effects when irradiation preceded inoculation, when the two inocula often behaved so differently in leaves given small doses (for example, 5 and 10 seconds in experiment 1, Table 3) that lesion number was decreased with disrupted, and increased with intact, virus. This difference can be attributed to photoreactivation, because the two inocula behaved similarly when inoculated leaves were placed in darkness immediately after irradiation (Table 4). Bawden and Kleczkowski (1960) obtained similar results with tobacco mosaic virus and concluded that, whereas the intact virus could survive through the period during which visible light restored the damaged capacity of the leaves to support infection, the nucleic acid could not. Our results (Table 3) show that even brief exposures to the radiation can temporarily damage the ability to support infection, but this damage is so soon counteracted by visible light that it shows only with inocula of disrupted RCMV; indeed, with inocula of intact virus, photoreactivation more than restored the original capacity of the leaves and made conditions at some sites such that infection occurred although it would not have in unirradiated leaves. Table 4 shows that, when inoculation was delayed for some hours after leaves were irradiated, the two kinds of inocula gave similar results; both produced fewer lesions in leaves kept in darkness immediately after irradiation than in leaves exposed to light, and photoreactivation of leaves before inoculation increased lesion survival almost as much with disrupted as with intact RCMV. The main difference shows in the lesion survival in leaves inoculated immediately after irradiation and then kept in the light. With both inocula, exposure to light increased lesion survival, but neither produced as many lesions as in leaves kept in the light for 3 hours after irradiation before inoculation. In experiment 2 (Table 4) the average lesion survival in leaves placed in darkness after irradiation was 30%, whereas in leaves inoculated after 3 hours
INFECTION
OF IRRADIATED
in the light it was 76%. Hence, exposing irradiated leaves to light for 3 hours made 46% more lesion sites susceptible; disrupted virus inoculated to leaves immediately after irradiation and then kept in the light for 3 hours infected 15% of these sites (44 - 29) and intact virus 30% (61 - 31). Neither kind of inoculum, therefore, survived wholly through the 3 hours while photoreactivation was proceeding, and the disrupted virus seems to have inactivated in the leaves about twice as fast as did the inoculum of intact particles. Figure 3 compares the behavior of leaves inoculated with intact and disrupted RCMV and shows that, within the 2 hours during which lesion survival remained unchanged in leaves inoculated with intact virus, lesion survival increased greatly in leaves inoculated with disrupted virus. After lesion survival started to increase in leaves inoculated with intact virus, it did so more slowly than in leaves inoculated with disrupted virus, perhaps because the change that produced the increase happened not only later, but also over a more extended period, and so was less well synchronized than with disrupted virus. What change in state of the inoculum or host increases lesion survival is not known, but the association of the virus nucleic acid with, or its inclusion in, some cell component, such as the nucleus or ribosomes, could explain it. The different behavior of the two kinds of inocula is plausibly explained by postulating that any such association demands that the nucleic acid separates from the protein, and that the lag period with intact virus represents the time taken for the nucleic acid to become free in viva (Siegel et al., 1957). The fact that infection centers initiated by intact RCMV do not become increasingly resistant to inactivation by ultraviolet radiation when inoculated leaves are kept at 32” could then be explained by postulating that, at this temperature, the processes that normally lead to the separation of nucleic acid from protein do not operat,e. To test this possibility, leaves were inoculated with disrupted virus and then irradiated after the plants had spent different intervals at 32”. The lesion survival did not increase with increasing time, but with
203
LEAVES TABLE
EFFECT
ON
LIGHT
AND
ATION AND NOL-DISRUPTED
I
Expt. no.
PERCENTAGE OF
IlluminatioP I
INTERVAL
INOCULATION VIRUS
I
I
4 LESION
SURVIVAL
BETWEEN WITH
OF IRRADI-
INTACT
OR PHE-
Inoculum and interval (hr) between irradiation and inoculation Disrupted
virus
0
3
24
I
Intact
virus
0
3
24
-__~
1
Light
20
71
-
36
70
-
2
Light Dark
44 29
73 33
-
61 31
79 27
-
3
Light Dark
34 16
-
92 15
64 15
-
115 18
-
a “Light” indicates that leaves were kept in daylight between irradiation (80 seconds exposure) and inoculation, “Dark” that they were in darkness during this period. All leaves, except those in treatment “Light, 0 hr” which were kept in daylight throughout, were kept in darkness for 24 hours after inoculation.
0
I 2
I 4 Hours
after
I 6
I 8
inoculation
FIG. 3. The increase in percentage lesion survival with increasing time after French bean leaves were inoculated with phenol-disrupted (NA inoculum) and intact red clover mottle virus. Leaves were kept at 24” between inoculation and 20 seconds’ exposure to ultraviolet radiation, after which they were in daylight.
204
BAWDEN
increasing time at 32” the numbers of lesions produced on the unirradiated halfleaves fell sharply. To cite one experiment comparing the behavior of intact and disrupted virus, the unirradiated half-leaves inoculated with intact virus kept for 0, 2, 4, and 5 hours at 32” before being placed at 24” gave, respectively, 484, 515, 475, and 407 lesions, whereas those inoculated with disrupted virus gave 436, 173, 31, and 0. As stated earlier, even after 22 hours at 32” leaves inoculated with intact virus still produce many lesions when put at lower temperatures. These results give no direct information about whether infection by intact virus normally necessitates that the protein and nucleic acid separate from one another, but they do show that most of the virus particles do not liberate their nucleic acid within a few hours at 32”, for if they did so, it would be inactivated. The results also show that failure of intact virus to infect and produce lesions at 32” is not attributable solely to the nucleic acid failing to separate from the protein, because already disrupted virus also failed to multiply. Hence, if infection normally entails the nucleic acid becoming free, it is not only this step that is prevented in leaves at 32”, but also some other that follows this one. The slow inactivation of intact RCMV in inoculated leaves kept at 32” could be attributed to the processes that separate virus protein from nucleic acid being slowed but not eliminated, so that with increasing time more and more particles liberate their nucleic acid, which then becomes inactivated. However, there is no reason to postulate that nucleic acid must become free to be inactivated, because there are many treatments that inactivate intact virus particles in vitro. French bean leaves may contain a system able to do this, for they contain one that inactivates tobacco necrosis and some other viruses in vitro without destroying their serological specificity or affecting their gross morphology or physical properties (Bawden and Pirie, 1957). The general behavior of RCMV suggests that it is likely to be among the viruses inactivat>ed by these leaf systems.
AND
SINHA DISCUSSION
There are two differences between the behavior of RCMV and tobacco necrosis virus. Kassanis (1960) found that infection centers initiated by intact and disrupted tobacco necrosis virus were equally susceptible to inactivation when leaves were irradiated immediately after they were inoculated, whereas with RCMV, as with tobacco mosaic virus, the infection centers initiated by disrupted virus are more readily inactivated than those initiated by intact virus. This could mean that the protein of RCMV protects its nucleic acid from irradiation damage, as does the protein of tobacco mosaic virus, whereas the protein of tobacco necrosis virus does not, but whether this interpretation is correct will remain uncertain until intact and disrupted virus preparations are irradiated in vitro and the relative rates of inactivation are determined. The second difference is in leaves at 32”, in which RCMV retains its initial susceptibility to inactivation by radiation for many hours, whereas the susceptibility of tobacco necrosis virus seems to decrease (Harrison, 1956). Perhaps the most significant feature in common with tobacco mosaic and tobacco necrosis viruses is that inoculation with disrupted virus shortens the period required for infection centers to gain resistance to ultraviolet radiation. This difference between the behavior of the two inocula is probably the major evidence in favor of the hypothesis that infection by intact virus requires, as a first step, that the protein and nucleic acid separate. Other phenomena from the irradiation experiments also fit this hypothesis. For instance the fact that, when leaves newly inoculated with intact RCMV are irradiated, lesion survival is greater in leaves kept in light than in darkness but photoreactivation has little effect on lesion survival in leaves inoculated with disrupted virus, receives a ready explanation by postulating that the free nucleic acid inactivates during the time when the capacity of the leaves to support infection is being photoreactivated, whereas virus particles are still intact during this period and
INFECTION
OF
IRRADIATED
disrobe to infect later. However, this explanation is not wholly satisfactory, for with tobacco mosaic virus there is equal photoreactivation whether leaves are irradiated immediat.ely after inoculation or after the lag period (Bawden and Kleczkowski, 1960), when according to the hypothesis the nucleic acid should already be free. Irradiation experiments show that changes proceed in infected cells and that changes become detectable sooner with inocula of nucleic acid than with intact virus. In addition to the effect on the lag period already mentioned, irradiated nucleic acid of tobacco mosaic virus becomes photoreactivable immediately after inoculation to leaves (Bawden and Kleczkowski, 1959) whereas few irradiated potato virus X particles become photoreactivable until half an hour after leaves are inoculated (Bawden and Kleczkowski, 1955). Unfortunately, although the irradiation experiments establish phenomena, they provide no information that allows the phenomena to be interpreted with certainty. The only experiments with RCMV that give unequivocal evidence about the behavior of intact virus are those with inoculated leaves kept at 32”, and these show clearly that the nucleic acid does not become free from the protein. However, the fact that nucleic acid does not become free in leaves at 32” is not evidence about what may happen at lower temperatures, at which infection becomes established. Indeed, it is plausibly argued that nothing much happens to virus particles in leaves at 32” simply because the mechanism that normally separates the protein and nucleic acid is inactive at this temperature. Inocula of disrupted and intact virus both lead to the same end results, virus multiplication and lesions. Hence, at some stage between inoculation and the production of new virus, effects from the two inocula must coincide, but it is well to stress that where this happens is by no means certain. Because infection centers initiated by both kinds of inocula in time increase their resistance to inactivation by ultraviolet radiation, it is plausible to assume that their
205
LEAVES
activities coincide at this point. However, this is still an assumption, and the increased resistance quickly acquired by infection centers initiated by disrupted virus may come from a process quite different from the one by which infection centers initiated by intact virus slowly acquire resistance. In establishing infection both host cell and virus are concerned, and effects of radiation on one are not readily disentangled from effects on the other. Differences between effects obtained by irradiating immediately before and after inoculation are helpful only in part, because our results show that changes in the physiological state of leaves greatly affect their susceptibility to radiation damage and the physiological changes caused in leaf cells by infection are likely to be greater than those caused by a few days’ aging or by a few hours in darkness or at 36”. Also, the practice of putting irradiated leaves in the light, which most workers have adopted with the idea that photoreactivation would compensate for damage to the leaves, is confusing rather than otherwise, particularly when the behavior of intact and disrupted virus is being compared. With intact virus, photoreactivation always increases lesion survival, whereas with disrupted virus, its effects depend on the time interval between inoculation and irradiation. Ideally all experiments should be done in duplicate, with half the irradiated leaves put in darkness and half in the light, to show the effects of photoreactivation. We did not do this, and so are less well able to interpret Fig. 3 than if we knew what would have happened had a comparable set of leaves been kept in darkness. The different behavior of the two inocula may be at least partially explicable by photoreactivation increasing lesion survival in leaves irradiated soon after inoculation more with intact than with disrupted virus. REFERENCES BAWDEN, F. C., and HARRISON, B. D. (1955). on the multiplication of a tobacco virus in inoculated leaves of French-bean J. Gen. Microbial. 13, 494-508.
Studies necrosis plants.
206
BAWDEN
BAWDEN, F. C., and KLECZKOWSXI, A. (1955). Studies on the ability of light to counteract the inactivating action of ultraviolet radiation on plant viruses. J. Gem Microbial. 13, 370-382. BAWDEN, F. C., and KLECZKOWSKI, A. (1959). Photoreactivation of nucleic acid from tobacco mosaic virus. Nature 183, 50%504. BAWDEN, F. C., and KLECZKOWSKI, A. (1960). Some effects of ultraviolet radiation on the infection of Nicotiana glutinosa leaves by tobacco mosaic virus. ViroZog?J 10, 163-181. BAWDEN, F. C., and PIRIE, N. W. (1957). A virusinactivating system from tobacco leaves. J. Gen. Microbial. 16, 696-710. GIERER, A., and SCHRAMM, G. (1956). Infectivity of ribonucleic acid from tobacco mosaic virus. Nature 177, 702-703. HARRISON, B. D. (1956). Studies on the effect of temperature on virus multiplication in inoculated leaves. Ann. Appl. Biol. 44, 215-226.
AND
SINHA
KASSANIS, B. (1960). Comparison of the early stages of infection by intact and phenol-disrupted tobacco necrosis virus. Virology 10, 353-369. MCLAREN, A. D., and TAKAHASHI, W. N. (1957). Inactivation of infectious nucleic acid from tobacco mosaic virus by ultraviolet light (2537 A). Radiation Research 6, 532-542. SIEGEL, A., and WILDMAN, S. G. (1956). The inactivation of the infectious centers of tobacco mosaic virus by ultraviolet light. Virology 2, 69-
82. SIEGEL, A., GINOZA, W., and WILDMAN, S. G. (1957). The early events of infection with tobacco mosaic virus nucleic acid. Virology 3, 554-559. SINHA, R. C. (1960a). Red clover mottle virus. Ann. AppZ. BioZ. 48, 742-748. SINHA, R. C., (196Ob). Some effects of temperature and of virus inhibitors on infection of French-bean leaves by red clover mottle virus. Ann. AppZ. Biol. 48,749-753.