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Evidence for a necrosis-inducing factor in tobacco mosaic virus-infected Nicotiana tabacum cv. Xanthi-nc grown at 22 °C but not at 32 °C
Physiological
Plant Pathology
(1984)
25,83-91
Evidence for a necrosis-inducing factor in tobacco mosaic virus-infected Nicotiana tabacum cv. Xanthi-nc grown at 22°C but not at 32 “C EMMA Willie
A.
WESTSTEIJN
Commclin
(Acceptedfor
Scholten Phytopathological
PubliGation
April
Laboratory,
Javalaan
20, 3742 CP Baam,
The Netherlands
1984)
Leaves of Nicotiana tabacum cv. Xanthi-nc, which have been inoculated with tobacco mosaic virus (TMV), remain symptomless when the plants are grown at 32 “C, but develop local lesions in about 8 h after transfer to 22 “C. The hypothesis that a necrosis-inducing factor is present but not functional at 32 “C was tested. Infected leaves were treated with metabolic inhibitors at 32 “C 2 h before transferring them to 22 “C to allow lesion development. Both cycloheximide and DMDMP caused a marked delay in lesion development, whereas chloramphenicol was without effect. Treatment with actinomycin D caused lesions to develop a few hours earlier than in untreated control leaves. The effect ofcycloheximide was reduced as treatment was delayed relative to the temperature change from 32 to 22 “C. Protoplasts were not killed by extracts from TMVinfected Xanthi-nc tobacco plants, which had been incubated at 32°C for 3 days, but toxic extracts were obtained from plants which had been incubated for at least 3 h at 22 “C after the reduction in temperature. These results indicate that a necrosis-inducing factor is not present in TMV-infected Xanthi-nc plants incubated at 32 “C.
INTRODUCTION
Nicotiana tabacum cv. Xanthi-nc reacts hypersensitively to infection with TMV when grown at temperatures below 28 “C. Necrotic local lesions appear about 2 days after inoculation and increase in size for 6-8 days. When lesion growth stops, the virus remains localized. This reaction is characteristic of N-gene-containing tobacco varieties. A toxic fraction, which kills tobacco protoplasts, can be extracted from hypersensitively reacting Xanthi-nc tobacco plants 21 h before lesions appear [12] and this factor may be responsible for the induction of necrosis. Infection of Xanthi-nc tobacco plants with TMV at 32 “C does not result in necrosis, and necrosis only develops after transfer of the plants to 22 “C [4, 13, IS, 181. Necrosis becomes visible to the naked eye about 8 h after transfer to 22 “C, but it is irreversibly induced within 3-6 h [lo, 13,181. The amount of necrotic tissue produced depends on the length of the induction period at 22 “C. When Xanthi-nc tobacco plants with small lesions formed at 22 “C, Abbreviations used in text: L- and D-MDMP, 2-(4-methyl-l,6 DNP, dinitrophenol; TMV, tobacco mosaic virus. 00484059/84/040083+09
$03.00/O
0
dinitroanilino)-N-methyl 1984 Academic
Press Inc.
proprionamide (London)
Limited
;
84 E. A. Weststeijn are transferred to 32 “C for 4 days, and this incubation is interrupted for 6.5 h at 22 “C on the third day, necrotic spots appear at a distance of approximately 1.5 mm from the lesion edge, but an interruption of 8.5 h at 22 “C on the third day results in a uniform necrotic zone extending from the lesion edge to beyond the necrotic spots [28]. These observations suggest that a necrosis-inducing factor may be synthesized during virus multiplication at 32 “C, but that it does not function at that temperature due to, perhaps, a conformational change in the factor itself or in its target. The different amounts of necrosis induced by the different times (6.5 or 8.5 h) at 22 “C, would then reflect different amounts of the factor which accumulate at 32 “C, the cells in the ring that become necrotic after 6.5 h at 22 “C, having more of the factor than the adjacent cells. The aim of the experiments described in this report was to investigate whether a necrosis-inducing factor is present in TMV-infected Xanthi-nc tobacco plants at 32 “C.
MATERIAL
AND
METHODS
Plant and virus material Tobacco plants (Nicotiana tabacum cv. Xanthi-nc) were grown in a glasshouse at about 22 “C, with additional illumination to provide a 16-h day during the winter. Tobacco mosaic virus was purified from frozen, systemically-infected Nicotiana tabacum cv. Samsun leaves according to Gooding & Hebert [8]. Inoculations Inoculations were carried out by rubbing a drop of suitably diluted inoculum over the leaves with a finger, using carborundum as an abrasive. After inoculation the leaves were rinsed with tap water. Immediately after inoculating two to seven leaves per plant, the plants were placed at 32 “C for 22 to 40 h, or for 3 days, and then transferred to 22 “C. Incubations at about 22 “C were carried out in the glasshouse, under the same conditions as described above but when continuous light was needed a growth chamber was used. Incubations at 32 “C were carried out in a growth chamber at 80% r.h. with a 16-h photoperiod. Metabolic inhibitors For metabolic inhibitor treatments, leaves were detached from the plants and their petioles placed in small vials filled with aqueous solutions of the inhibitor. Actinomycin D was used to study the possible involvement of DNA-dependent RNA-synthesis, and cycloheximide, D-MDMP and chloramphenicol were used to study the involvement of protein synthesis on cytoplasmic and chloroplast ribosomes, respectively. In the case of D-MDMP, the inactive L-isomer was used as a control [I]. Sodium azide and DNP were used to inhibit respiration. After allowing the inhibitor solution to be taken up for 1 h at 32 “C, the leaves were transferred to flat containers containing tap water of 32 “C; plates of polystyrene with holes for the petioles were used as a support. After a further 1 h at 32°C the containers with water and leaves were transferred to the glasshouse (22 “C).
Necrosis-inducing
factor in TMV-infected
Nicotiana
85
The effect of metabolic inhibitors on lesion development was determined from counts of lesion number and measurements of lesion size. The number of lesions developing on each leaf after the transfer from 32 to 22 “C were counted at intervals until no further lesions developed. The final number was designated the lOOo/ovalue l‘or that leaf. From a graph relating the percentage of lesions present in the leaf to I.he time after transfer from 32 to 22 “C, the period at 22 “C needed for 50% of the lesions to develop (t& was determined. Lesion size was determined by measuring two diameters at right angles, per lesion, with the aid of a magnifier with a micrometer. Isolation of protophts
Protoplasts were isolated from fully-grown leaves of Xanthi-nc tobacco. Leaves from which the lower epidermis had been stripped were floated on 0.7 M mannitol (pH 5.8) in Petri dishes. After O-5-1.5 h the mannitol was replaced by a solution of 0.5% cellulase (Onozuka RlO, Yakult Hansha Co.) and 0.025% pectolyase (Y-23, Seishin Pharmaceutical Co.) in O-7 M mannitol (pH 5.8) and the incubation continued at 30 “C for 3-4 h. Protoplasts were then collected by centrifuging for 3 min at 100 g, washed twice with O-7 M mannitol (pH 5.8) and resuspended in 0.7 M mannitol !pH 5.8). Extraction
and assay of the necrosis-inducing
faGtor
To test for the presence of a necrosis-inducing factor, leaves with their mid-vein removed were ground in 0.7 M mannitol (pH 5.8) (1 ml g-l fresh weight) with a pestle and mortar. The slurry was centrifuged at 8000g for 25 min and the supernatant was used immediately or stored at -20 “C until use. Leaf extracts and protoplast suspensions were mixed in suitable proportions in small Petri dishes and incubated at about 22 “C under continuous light. The toxic effect of the extracts was determined after 1620 h by counting the percentage of dead or damaged protoplasts using a haemocytometer. A protoplast was considered dead or damaged when coloured brown after staining with phenosafranine (final concentration about 0.05%) [29]. Each extract was tested in two or three replications; in each replication two samples, each of 200 protoplasts were counted. RESULTS Rate of appearance of local lesions after transfer of the plants from 32 to 22°C
When TMV-infected Xanthi-nc tobacco plants were incubated at 32 “C for 2240 h and then transferred to 22 “C, local lesions appeared in the inoculated leaves. The first lesions were visible about 10 h after transfer and the maximum number attained about 7 h later. A time course of the development of the local lesions is shown in Fig. 1. Lesions started to appear about 10 h after transfer, and their number increased linearly over the period between 11 and 12 h. Lesion development on all plants followed a similar pattern. Although the rate of appearance varied between experiments, there was little difference within experiments. The time required for .5Oo/o of the lesions to appear (t& was determined in each experiment with an accuracy of 15 min; the tso in Fig. 1 was 11.5 h.
E. A. Weststeijn
86
o-•;lo ’ 12 14 16 Time after transfer from 32 to 22 “c ( h)
FIG. 1. Rate of appearance of local lesions in TMV-infected Xanthi-nc tobacco leaves incubated at 32 “C for 32 h after inoculation before transfer to 22 “C. The results are from one plant. Each point is the mean percentage number of lesions on six successive leaves. Final lesion number (100%) was determined 4 days after transfer.
Effect of metabolic inhibitors on the rate of appearanceof local lesions When metabolic inhibitors were applied 2 h before the transfer from 32 to 22 “C, the rate of appearance of local lesions in TMV-infected Xanthi-nc tobacco plants was clearly affected. Treatment with cycloheximide resulted in a concentration-dependent delay (Fig. 2) with lesions starting to appear later and increasing in number at a slower rate than in the water-treated control leaves, although the final number was approximately the same in both cases. Cloramphenicol, when applied together with cycloheximide did not increase the delay, indicating that it was due entirely to an inhibition of protein synthesis on cytoplasmic ribosomes. This conclusion was corroborated by the results of treatments with the L- and n-isomers of MDMP. At concentrations ranging from 10 to 30 pg ml-r, the inactive L-isomer did not affect the rate of lesion appearance, but D-MDMP caused a marked, concentration-dependent delay. For instance, in an experiment with a t3,, of about 12 h for water- or L-MDMP-treated plants, plants treated with D-MDMP at 10, 20 and 30 pg ml-’ showed, 36 h after the temperature shift, 85, 13 and 7%, respectively, of the final lesion number. In contrast, treatments with actinomycin D, an inhibitor of DNA-dependent RNA-synthesis decreased the time required for lesions to appear by up to 4 h, depending on the experiment and the concentration used (Table 1). At concentrations up to 1O-3 M, neither DNP nor sodium azide altered the t,, to a significant extent and only on leaves treated with 1O-2 M sodium azide was lesion development delayed. However, such leaves were damaged by the treatment and the t,, could not be determined. When cycloheximide was administered at times ranging from 2 h before until 3 h after transfer from 32 to 22 “C, lesion development was markedly delayed (Fig. 3) but there was no correlation between the time of treatment and the degree of
Necrosis-inducing
factor
in TMV-infected
Nicotiana
87
cloheximide loromphenlcol Concentrotlon
+g
ml-‘)
FIG. 2. Effect of cycloheximide (0) alone or with chloramphenicol (0) on the rate of appearance (t5,,) of local lessons in TMV-infected Xanthi-nc tobacco leaves after transfer from 32 to 22°C. Treatments with the inhibitors were carried out as described for actinomycin D in Table 1. Each point is the tsO for seven leaves from one plant. TABLE
Effect of actinomycin
1
D on the rate of appearance of local lesions tobacco plants=
(tso) in
TMV-infected
Xanthi-nc
tm (h) Actinomycin (wg ml-‘) 0 10 20 30 40
D Expt 14.50 11.75* 11.50* 11.50* 10.50*
lb
Expt 14.50 12.50* 11.75* 11.50* 11.00*
12.50 11.75* 11.00* 11.25* 11,50*
2’
Expt
3d
17.00 17,25* 15.75* 15.75* 13.00*
“Infected plants were incubated at 32 “C for 28 h. The infected leaves were then detached and placed in vials containing aqueous solutions of actinomycin D. After 1 h the leaves were transferred to tap water and after a further hour transferred to 22 “C. bSix leaves per plant were inoculated. CFour leaves per plant were inoculated; the plants were from the same batch as used in Expt 1. dThree leaves per plant were inoculated; the plants were from a different batch from those used in Expt 1. *Significantly different from water-treated control plant (trend test P = 0.05).
inhibition. Treatment applied in the period ranging from 3 to 8 h after transfer delayed lesion development which decreased linearly with time, and treatments given more than 8 h after transfer had no effect. Other experiments showed a similar pattern, although the t,, values varied. Administration of 20 pg ml-l cycloheximide
E. A. Weststeijn
I*,
’
-2
’
0
2
Time (h) before l-)01
3
3
4
6
’
‘:
8
IO HPC
after (+I tronsfer
from 32
iu22T FIG. 3. Effect of cycloheximide on the rate of appearance of local lesions (ts,) in TMVinfected Xanthi-nc tobacco leaves after transfer from 32 to 22 “C. Leaves were allowed to take up cycloheximide for 1 h, starting at the indicated time. Each point is the rs, for two leaves from one plant. 0, Treated with cycloheximide (20 pg ml-r) ; 0, water-treated controls,
50;i0 2 404 z0" 30h n 20$ D e-0
IO
0.25
0 Fmol
FIG. 4. Effect of extracts from incubated at 22 “C for 3 days, on of leaf extract were mixed with was determined after incubation dead cells stained brown. Each protoplasts being scored at each
concentration
.
050
0.75 of protoplasts
I-0 I IO5 ml-‘)
healthy (0) or TMV-infected (0) Xanthi-nc tobacco plants, the survival of Xanthi-nc tobacco protoplasts. Two millilitres 2 ml of protoplast suspension. Percentage of dead protoplasts for 18 h by observing their reaction to 0.05% phenosafranine; point is the mean of four counts from two replicates, 200 count.
2 h before the transfer from 32 to 22 “C resulted in a reduction in lesion size of about 40%, at a time when only about 50% of the final number of lesions were present. E$ects of leaf extracts on protoplast survival Extracts from TMV-infected Xanthi-nc tobacco plants with local lesions were toxic to Xanthi-nc tobacco protoplasts. This toxic effect was greatest when the protoplast concentration was low (Fig. 4).
Necrosis-inducing
factor in TMV-infected
Time
at 22’C
after
Nicofiana
lncubatian
89
at 32°C
for 3 days (hl
FIG. 5. Effect of extracts from TMV-infected Xanthi-nc tobacco plants, incubated at 32 “C for 3 days then at 22°C for further periods of from 0 to 8.75 h, on the survival of Xanthi-nc tobacco protoplasts. Three millilitres of extract were mixed with 0.5 ml of protoplast suspension (final concentration 0.25 x 10s protoplasts ml-‘). Percentage ofdead protoplasts was determined from their reaction to phenosafranine (see Fig. 4) after incubation for 18 h. Each point is the mean of six counts from three replicates, 200 protoplasts being scored at each count.
Extracts from TMV-infected Xanthi-nc tobacco plants, incubated continuously at 32 “C were not toxic to protoplasts, but extracts prepared from plants which had been transferred from 32 to 22 “C were toxic if the plants had been at the lower temperature for more than 3 h (Fig. 5). Lesions were not visible on these plants at the time that the extracts were made and in the plants used in the experiment they only became visible after 7.25 h. Extracts from healthy Xanthi-nc tobacco plants incubated at 32 “C for 3 days, with or without a further period at 22 “C for 9 h or from healthy plants grown continuously at 22 “C in the glasshouse were without effect. DISCUSSION
When cycloheximide was administered to detached infected leaves 2 h before transferring them from 32 to 22 “C it markedly delayed local lesion development (Fig. 2)) whereas actinomycin D, administered at a similar time advanced lesion development (Table 1). These results indicate that lesion formation does not require DNAdependent RNA-synthesis, but does involve at least one step which depends on cytoplasmic protein synthesis. If lesion formation is the direct result of the activity of a necrosis-inducing factor then the delay in lesion development after treatment with cycloheximide would tend to rule out the possibility that the factor is already present at 32 “C in a form which is simply activated at the lower temperature either by a single conformational change in itself or in its target. Both actinomycin D and cycloheximide can affect virus multiplication. Cycloheximide inhibits the synthesis of TMV independently of the time of administration [Z, S]. In contrast, actinomycin D only inhibits the multiplication of TMV when the treatment is given at a very early stage of infection [.5, 61, although an increase in virus production in response to actinomycin D treatment has also been reported [14].
90
E. A. Weststeijn
As the rate of appearance of local lesions is intimately linked with the rate of virus multiplication [3, 9, II], the most likely explanation for the effects of the inhibitors on lesion development is through their interference with the rate of virus multiplication. The fact that cycloheximide had less effect when given late after transfer (Fig. 3) is in accordance with this view. The presence of a necrosis-inducing factor in infected plants at 32 “C was further investigated by preparing extracts from infected plants grown under different temperature regimes and testing them on protoplasts at 22 “C. Extracts from infected plants grown at 32 “C were not toxic to protoplasts and such plants had to be incubated at 22 “C for at least 3 h before toxic extracts were obtainable (Fig. 5). This clearly suggests that the necrosis-inducing factor is only produced by plants after transfer to the lower temperature. However, production of the toxic factor preceded the development of visible necrosis on such plants by several hours and occurred at about the same time that an increase in the permeability of cell membranes occurs [17]. Such permeability changes are not found in virus-infected systemic hosts or in hypersensitive resistant tobacco plants grown at 32 “C [17], although the membranes of cells in which virus multiplication takes place, appear to be less stable than those of the cells where no virus multiplication occurs [7, 2.51. It is possible, therefore, that the more extensive membrane damage in the hypersensitive host allows for contact between different cell compartments, leading to the production of the necrosis-inducing factor. If this is the case, then it may be that membranes are less susceptible to damage during virus multiplication at 32 “C than at 22 “C, and so no toxic factor is produced. I thank Dr A. A. M. de Laat for a gift of n- and L-MDMP. REFERENCES R., KNELL, V. C., SOMERVILLE, H. J., SWAIN, H. M. & WEEKS, D. P. (1973). Effect of MDMP on protein synthesis in wheat and bacteria. Nature New Biology 243, 139-142. BEACHY, R. N. & MURAKISHI, H. H. (1973). Effect of cycloheximide on tobacco mosaic virus synthesis in callus from hypersensitive tobacco. Virology 55, 320-328. BEST, R. J. (1968). Tomato spotted wilt virus, Advances in Virus Research 13, 66-146. DA GRAYA, J. V. & MARTIN, M. M. (1976). An electron microscope study of hypersensitive tobacco infected with tobacco mosaic virus at 32 “C. Physiological Plan’t Pathology 8, 215-219. DAWSON, W. 0. (1978). Time course of actinomycin D inhibition of tobacco mosaic virus multiplication relative to the rate of spread of the infection. Intervirology 9, 304-309. DAWSON, W. 0. & SCHLEGEL, D. E. (1976). The sequence of inhibition of tobacco mosaic virus synthesis by actinomycin D, 2-thiouracil and cycloheximide in a synchronous infection. Phytopathology 66, 177-l 81. FOSTER, J. A. & Ross, A. F. (1975). The detection of symptomless virus infected tissue in inoculated tobacco leaves. Phytopathology 65, 600-610. GOODING, G. V. & HEBERT, T. T. (1976). A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology 57, 1285. GOTO, T. & TANIGUCHI, T. (1976). Local lesion formation and virus multiplication determined by intrinsic nature of strains of tobacco mosaic virus. Annals of the Phytopathological Society of Japan 42,
1. BAXTER,
2. 3. 4. 5. 6.
7. 8. 9.
549-555.
10. GUI.+AS, A. & FARKAS, G. L. (1978). Is cell to cell contact necessary for the expression of the N- gene in Nicotiana tabamm cv. Xanthi nc plants infected by TMV? Phytopathologische Zeitschrift 91, 182-187. 11. HIRTH, L. & LEBEURIER, G. (1964). Properties of tobacco mosaic virus developing at supraoptimal temperatures. In Host-pamite Relations in Plant Pathology, eds Z. Kiraly & G. Ubrizsy, pp. 3746. Symposium of the Hungarian Academy of Sciences, Budapest.
Necrosis-inducing
14.
15. 16.
17. 18.
19.
in TMV-infected
Nicotiana
91
R. & MCCARTHY, D. (1980). Extracts from virus infected hypersensitive tobacco leaves are detrimental to protoplast survival. Physiological Plant Pathology 16,25-38. MARTIN, C. (1966). Contribution a I’etude de phinomtne d’hypersensibilitt du virus de la mosaique du tabac. Bulletin de la Sociiti Franpise de Physiologic Vt!gLtale 12, 345-354. OHASHI, Y. & SHIMOMURA, T. (1972). Induction of localized necrotic lesions by actinomycin D on leaves systemically infected with tobacco mosaic virus. Virology 48, 601-603. OHASHI, Y. & SHIMOMURA, T. (1982). Modification of cell membranes of leaves systemically infected with tobacco mosaic virus. Physiological Plant Pathology 20, 125-128. TAKAHASHI, T. (1975). Studies on viral pathogenesis in plant hosts. VIII. Systemic virus invasion and localization of infection on “Samsun NW’ tobacco plants resulting from tobacco mosaic virus. Phytopathologische Zeitschrixt 84, 75-87. WESTSTEIJN, E. A. (1978). Permeability changes in the hypersensitive reaction of Nicotiana tabacum cv. Xanthi nc after infection with tobacco mosaic virus. Physiological Plant Pathology 13, 253-258. WESTSTEIJN, E. A. (1981). Lesion growth and virus localization in leaves of Nicotiana tabacum cv. Xanthi nc after inoculation with tobacco mosaic virus and incubation alternately at 22°C and 32°C. Physiological Plant Pathology 18, 357-368. WIDHOLM, .I. M. (1972). The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technology 47, 189-l 94.
12. HOOLEY, 13.
factor
Plant Pathology
(1984)
25,83-91
Evidence for a necrosis-inducing factor in tobacco mosaic virus-infected Nicotiana tabacum cv. Xanthi-nc grown at 22°C but not at 32 “C EMMA Willie
A.
WESTSTEIJN
Commclin
(Acceptedfor
Scholten Phytopathological
PubliGation
April
Laboratory,
Javalaan
20, 3742 CP Baam,
The Netherlands
1984)
Leaves of Nicotiana tabacum cv. Xanthi-nc, which have been inoculated with tobacco mosaic virus (TMV), remain symptomless when the plants are grown at 32 “C, but develop local lesions in about 8 h after transfer to 22 “C. The hypothesis that a necrosis-inducing factor is present but not functional at 32 “C was tested. Infected leaves were treated with metabolic inhibitors at 32 “C 2 h before transferring them to 22 “C to allow lesion development. Both cycloheximide and DMDMP caused a marked delay in lesion development, whereas chloramphenicol was without effect. Treatment with actinomycin D caused lesions to develop a few hours earlier than in untreated control leaves. The effect ofcycloheximide was reduced as treatment was delayed relative to the temperature change from 32 to 22 “C. Protoplasts were not killed by extracts from TMVinfected Xanthi-nc tobacco plants, which had been incubated at 32°C for 3 days, but toxic extracts were obtained from plants which had been incubated for at least 3 h at 22 “C after the reduction in temperature. These results indicate that a necrosis-inducing factor is not present in TMV-infected Xanthi-nc plants incubated at 32 “C.
INTRODUCTION
Nicotiana tabacum cv. Xanthi-nc reacts hypersensitively to infection with TMV when grown at temperatures below 28 “C. Necrotic local lesions appear about 2 days after inoculation and increase in size for 6-8 days. When lesion growth stops, the virus remains localized. This reaction is characteristic of N-gene-containing tobacco varieties. A toxic fraction, which kills tobacco protoplasts, can be extracted from hypersensitively reacting Xanthi-nc tobacco plants 21 h before lesions appear [12] and this factor may be responsible for the induction of necrosis. Infection of Xanthi-nc tobacco plants with TMV at 32 “C does not result in necrosis, and necrosis only develops after transfer of the plants to 22 “C [4, 13, IS, 181. Necrosis becomes visible to the naked eye about 8 h after transfer to 22 “C, but it is irreversibly induced within 3-6 h [lo, 13,181. The amount of necrotic tissue produced depends on the length of the induction period at 22 “C. When Xanthi-nc tobacco plants with small lesions formed at 22 “C, Abbreviations used in text: L- and D-MDMP, 2-(4-methyl-l,6 DNP, dinitrophenol; TMV, tobacco mosaic virus. 00484059/84/040083+09
$03.00/O
0
dinitroanilino)-N-methyl 1984 Academic
Press Inc.
proprionamide (London)
Limited
;
84 E. A. Weststeijn are transferred to 32 “C for 4 days, and this incubation is interrupted for 6.5 h at 22 “C on the third day, necrotic spots appear at a distance of approximately 1.5 mm from the lesion edge, but an interruption of 8.5 h at 22 “C on the third day results in a uniform necrotic zone extending from the lesion edge to beyond the necrotic spots [28]. These observations suggest that a necrosis-inducing factor may be synthesized during virus multiplication at 32 “C, but that it does not function at that temperature due to, perhaps, a conformational change in the factor itself or in its target. The different amounts of necrosis induced by the different times (6.5 or 8.5 h) at 22 “C, would then reflect different amounts of the factor which accumulate at 32 “C, the cells in the ring that become necrotic after 6.5 h at 22 “C, having more of the factor than the adjacent cells. The aim of the experiments described in this report was to investigate whether a necrosis-inducing factor is present in TMV-infected Xanthi-nc tobacco plants at 32 “C.
MATERIAL
AND
METHODS
Plant and virus material Tobacco plants (Nicotiana tabacum cv. Xanthi-nc) were grown in a glasshouse at about 22 “C, with additional illumination to provide a 16-h day during the winter. Tobacco mosaic virus was purified from frozen, systemically-infected Nicotiana tabacum cv. Samsun leaves according to Gooding & Hebert [8]. Inoculations Inoculations were carried out by rubbing a drop of suitably diluted inoculum over the leaves with a finger, using carborundum as an abrasive. After inoculation the leaves were rinsed with tap water. Immediately after inoculating two to seven leaves per plant, the plants were placed at 32 “C for 22 to 40 h, or for 3 days, and then transferred to 22 “C. Incubations at about 22 “C were carried out in the glasshouse, under the same conditions as described above but when continuous light was needed a growth chamber was used. Incubations at 32 “C were carried out in a growth chamber at 80% r.h. with a 16-h photoperiod. Metabolic inhibitors For metabolic inhibitor treatments, leaves were detached from the plants and their petioles placed in small vials filled with aqueous solutions of the inhibitor. Actinomycin D was used to study the possible involvement of DNA-dependent RNA-synthesis, and cycloheximide, D-MDMP and chloramphenicol were used to study the involvement of protein synthesis on cytoplasmic and chloroplast ribosomes, respectively. In the case of D-MDMP, the inactive L-isomer was used as a control [I]. Sodium azide and DNP were used to inhibit respiration. After allowing the inhibitor solution to be taken up for 1 h at 32 “C, the leaves were transferred to flat containers containing tap water of 32 “C; plates of polystyrene with holes for the petioles were used as a support. After a further 1 h at 32°C the containers with water and leaves were transferred to the glasshouse (22 “C).
Necrosis-inducing
factor in TMV-infected
Nicotiana
85
The effect of metabolic inhibitors on lesion development was determined from counts of lesion number and measurements of lesion size. The number of lesions developing on each leaf after the transfer from 32 to 22 “C were counted at intervals until no further lesions developed. The final number was designated the lOOo/ovalue l‘or that leaf. From a graph relating the percentage of lesions present in the leaf to I.he time after transfer from 32 to 22 “C, the period at 22 “C needed for 50% of the lesions to develop (t& was determined. Lesion size was determined by measuring two diameters at right angles, per lesion, with the aid of a magnifier with a micrometer. Isolation of protophts
Protoplasts were isolated from fully-grown leaves of Xanthi-nc tobacco. Leaves from which the lower epidermis had been stripped were floated on 0.7 M mannitol (pH 5.8) in Petri dishes. After O-5-1.5 h the mannitol was replaced by a solution of 0.5% cellulase (Onozuka RlO, Yakult Hansha Co.) and 0.025% pectolyase (Y-23, Seishin Pharmaceutical Co.) in O-7 M mannitol (pH 5.8) and the incubation continued at 30 “C for 3-4 h. Protoplasts were then collected by centrifuging for 3 min at 100 g, washed twice with O-7 M mannitol (pH 5.8) and resuspended in 0.7 M mannitol !pH 5.8). Extraction
and assay of the necrosis-inducing
faGtor
To test for the presence of a necrosis-inducing factor, leaves with their mid-vein removed were ground in 0.7 M mannitol (pH 5.8) (1 ml g-l fresh weight) with a pestle and mortar. The slurry was centrifuged at 8000g for 25 min and the supernatant was used immediately or stored at -20 “C until use. Leaf extracts and protoplast suspensions were mixed in suitable proportions in small Petri dishes and incubated at about 22 “C under continuous light. The toxic effect of the extracts was determined after 1620 h by counting the percentage of dead or damaged protoplasts using a haemocytometer. A protoplast was considered dead or damaged when coloured brown after staining with phenosafranine (final concentration about 0.05%) [29]. Each extract was tested in two or three replications; in each replication two samples, each of 200 protoplasts were counted. RESULTS Rate of appearance of local lesions after transfer of the plants from 32 to 22°C
When TMV-infected Xanthi-nc tobacco plants were incubated at 32 “C for 2240 h and then transferred to 22 “C, local lesions appeared in the inoculated leaves. The first lesions were visible about 10 h after transfer and the maximum number attained about 7 h later. A time course of the development of the local lesions is shown in Fig. 1. Lesions started to appear about 10 h after transfer, and their number increased linearly over the period between 11 and 12 h. Lesion development on all plants followed a similar pattern. Although the rate of appearance varied between experiments, there was little difference within experiments. The time required for .5Oo/o of the lesions to appear (t& was determined in each experiment with an accuracy of 15 min; the tso in Fig. 1 was 11.5 h.
E. A. Weststeijn
86
o-•;lo ’ 12 14 16 Time after transfer from 32 to 22 “c ( h)
FIG. 1. Rate of appearance of local lesions in TMV-infected Xanthi-nc tobacco leaves incubated at 32 “C for 32 h after inoculation before transfer to 22 “C. The results are from one plant. Each point is the mean percentage number of lesions on six successive leaves. Final lesion number (100%) was determined 4 days after transfer.
Effect of metabolic inhibitors on the rate of appearanceof local lesions When metabolic inhibitors were applied 2 h before the transfer from 32 to 22 “C, the rate of appearance of local lesions in TMV-infected Xanthi-nc tobacco plants was clearly affected. Treatment with cycloheximide resulted in a concentration-dependent delay (Fig. 2) with lesions starting to appear later and increasing in number at a slower rate than in the water-treated control leaves, although the final number was approximately the same in both cases. Cloramphenicol, when applied together with cycloheximide did not increase the delay, indicating that it was due entirely to an inhibition of protein synthesis on cytoplasmic ribosomes. This conclusion was corroborated by the results of treatments with the L- and n-isomers of MDMP. At concentrations ranging from 10 to 30 pg ml-r, the inactive L-isomer did not affect the rate of lesion appearance, but D-MDMP caused a marked, concentration-dependent delay. For instance, in an experiment with a t3,, of about 12 h for water- or L-MDMP-treated plants, plants treated with D-MDMP at 10, 20 and 30 pg ml-’ showed, 36 h after the temperature shift, 85, 13 and 7%, respectively, of the final lesion number. In contrast, treatments with actinomycin D, an inhibitor of DNA-dependent RNA-synthesis decreased the time required for lesions to appear by up to 4 h, depending on the experiment and the concentration used (Table 1). At concentrations up to 1O-3 M, neither DNP nor sodium azide altered the t,, to a significant extent and only on leaves treated with 1O-2 M sodium azide was lesion development delayed. However, such leaves were damaged by the treatment and the t,, could not be determined. When cycloheximide was administered at times ranging from 2 h before until 3 h after transfer from 32 to 22 “C, lesion development was markedly delayed (Fig. 3) but there was no correlation between the time of treatment and the degree of
Necrosis-inducing
factor
in TMV-infected
Nicotiana
87
cloheximide loromphenlcol Concentrotlon
+g
ml-‘)
FIG. 2. Effect of cycloheximide (0) alone or with chloramphenicol (0) on the rate of appearance (t5,,) of local lessons in TMV-infected Xanthi-nc tobacco leaves after transfer from 32 to 22°C. Treatments with the inhibitors were carried out as described for actinomycin D in Table 1. Each point is the tsO for seven leaves from one plant. TABLE
Effect of actinomycin
1
D on the rate of appearance of local lesions tobacco plants=
(tso) in
TMV-infected
Xanthi-nc
tm (h) Actinomycin (wg ml-‘) 0 10 20 30 40
D Expt 14.50 11.75* 11.50* 11.50* 10.50*
lb
Expt 14.50 12.50* 11.75* 11.50* 11.00*
12.50 11.75* 11.00* 11.25* 11,50*
2’
Expt
3d
17.00 17,25* 15.75* 15.75* 13.00*
“Infected plants were incubated at 32 “C for 28 h. The infected leaves were then detached and placed in vials containing aqueous solutions of actinomycin D. After 1 h the leaves were transferred to tap water and after a further hour transferred to 22 “C. bSix leaves per plant were inoculated. CFour leaves per plant were inoculated; the plants were from the same batch as used in Expt 1. dThree leaves per plant were inoculated; the plants were from a different batch from those used in Expt 1. *Significantly different from water-treated control plant (trend test P = 0.05).
inhibition. Treatment applied in the period ranging from 3 to 8 h after transfer delayed lesion development which decreased linearly with time, and treatments given more than 8 h after transfer had no effect. Other experiments showed a similar pattern, although the t,, values varied. Administration of 20 pg ml-l cycloheximide
E. A. Weststeijn
I*,
’
-2
’
0
2
Time (h) before l-)01
3
3
4
6
’
‘:
8
IO HPC
after (+I tronsfer
from 32
iu22T FIG. 3. Effect of cycloheximide on the rate of appearance of local lesions (ts,) in TMVinfected Xanthi-nc tobacco leaves after transfer from 32 to 22 “C. Leaves were allowed to take up cycloheximide for 1 h, starting at the indicated time. Each point is the rs, for two leaves from one plant. 0, Treated with cycloheximide (20 pg ml-r) ; 0, water-treated controls,
50;i0 2 404 z0" 30h n 20$ D e-0
IO
0.25
0 Fmol
FIG. 4. Effect of extracts from incubated at 22 “C for 3 days, on of leaf extract were mixed with was determined after incubation dead cells stained brown. Each protoplasts being scored at each
concentration
.
050
0.75 of protoplasts
I-0 I IO5 ml-‘)
healthy (0) or TMV-infected (0) Xanthi-nc tobacco plants, the survival of Xanthi-nc tobacco protoplasts. Two millilitres 2 ml of protoplast suspension. Percentage of dead protoplasts for 18 h by observing their reaction to 0.05% phenosafranine; point is the mean of four counts from two replicates, 200 count.
2 h before the transfer from 32 to 22 “C resulted in a reduction in lesion size of about 40%, at a time when only about 50% of the final number of lesions were present. E$ects of leaf extracts on protoplast survival Extracts from TMV-infected Xanthi-nc tobacco plants with local lesions were toxic to Xanthi-nc tobacco protoplasts. This toxic effect was greatest when the protoplast concentration was low (Fig. 4).
Necrosis-inducing
factor in TMV-infected
Time
at 22’C
after
Nicofiana
lncubatian
89
at 32°C
for 3 days (hl
FIG. 5. Effect of extracts from TMV-infected Xanthi-nc tobacco plants, incubated at 32 “C for 3 days then at 22°C for further periods of from 0 to 8.75 h, on the survival of Xanthi-nc tobacco protoplasts. Three millilitres of extract were mixed with 0.5 ml of protoplast suspension (final concentration 0.25 x 10s protoplasts ml-‘). Percentage ofdead protoplasts was determined from their reaction to phenosafranine (see Fig. 4) after incubation for 18 h. Each point is the mean of six counts from three replicates, 200 protoplasts being scored at each count.
Extracts from TMV-infected Xanthi-nc tobacco plants, incubated continuously at 32 “C were not toxic to protoplasts, but extracts prepared from plants which had been transferred from 32 to 22 “C were toxic if the plants had been at the lower temperature for more than 3 h (Fig. 5). Lesions were not visible on these plants at the time that the extracts were made and in the plants used in the experiment they only became visible after 7.25 h. Extracts from healthy Xanthi-nc tobacco plants incubated at 32 “C for 3 days, with or without a further period at 22 “C for 9 h or from healthy plants grown continuously at 22 “C in the glasshouse were without effect. DISCUSSION
When cycloheximide was administered to detached infected leaves 2 h before transferring them from 32 to 22 “C it markedly delayed local lesion development (Fig. 2)) whereas actinomycin D, administered at a similar time advanced lesion development (Table 1). These results indicate that lesion formation does not require DNAdependent RNA-synthesis, but does involve at least one step which depends on cytoplasmic protein synthesis. If lesion formation is the direct result of the activity of a necrosis-inducing factor then the delay in lesion development after treatment with cycloheximide would tend to rule out the possibility that the factor is already present at 32 “C in a form which is simply activated at the lower temperature either by a single conformational change in itself or in its target. Both actinomycin D and cycloheximide can affect virus multiplication. Cycloheximide inhibits the synthesis of TMV independently of the time of administration [Z, S]. In contrast, actinomycin D only inhibits the multiplication of TMV when the treatment is given at a very early stage of infection [.5, 61, although an increase in virus production in response to actinomycin D treatment has also been reported [14].
90
E. A. Weststeijn
As the rate of appearance of local lesions is intimately linked with the rate of virus multiplication [3, 9, II], the most likely explanation for the effects of the inhibitors on lesion development is through their interference with the rate of virus multiplication. The fact that cycloheximide had less effect when given late after transfer (Fig. 3) is in accordance with this view. The presence of a necrosis-inducing factor in infected plants at 32 “C was further investigated by preparing extracts from infected plants grown under different temperature regimes and testing them on protoplasts at 22 “C. Extracts from infected plants grown at 32 “C were not toxic to protoplasts and such plants had to be incubated at 22 “C for at least 3 h before toxic extracts were obtainable (Fig. 5). This clearly suggests that the necrosis-inducing factor is only produced by plants after transfer to the lower temperature. However, production of the toxic factor preceded the development of visible necrosis on such plants by several hours and occurred at about the same time that an increase in the permeability of cell membranes occurs [17]. Such permeability changes are not found in virus-infected systemic hosts or in hypersensitive resistant tobacco plants grown at 32 “C [17], although the membranes of cells in which virus multiplication takes place, appear to be less stable than those of the cells where no virus multiplication occurs [7, 2.51. It is possible, therefore, that the more extensive membrane damage in the hypersensitive host allows for contact between different cell compartments, leading to the production of the necrosis-inducing factor. If this is the case, then it may be that membranes are less susceptible to damage during virus multiplication at 32 “C than at 22 “C, and so no toxic factor is produced. I thank Dr A. A. M. de Laat for a gift of n- and L-MDMP. REFERENCES R., KNELL, V. C., SOMERVILLE, H. J., SWAIN, H. M. & WEEKS, D. P. (1973). Effect of MDMP on protein synthesis in wheat and bacteria. Nature New Biology 243, 139-142. BEACHY, R. N. & MURAKISHI, H. H. (1973). Effect of cycloheximide on tobacco mosaic virus synthesis in callus from hypersensitive tobacco. Virology 55, 320-328. BEST, R. J. (1968). Tomato spotted wilt virus, Advances in Virus Research 13, 66-146. DA GRAYA, J. V. & MARTIN, M. M. (1976). An electron microscope study of hypersensitive tobacco infected with tobacco mosaic virus at 32 “C. Physiological Plan’t Pathology 8, 215-219. DAWSON, W. 0. (1978). Time course of actinomycin D inhibition of tobacco mosaic virus multiplication relative to the rate of spread of the infection. Intervirology 9, 304-309. DAWSON, W. 0. & SCHLEGEL, D. E. (1976). The sequence of inhibition of tobacco mosaic virus synthesis by actinomycin D, 2-thiouracil and cycloheximide in a synchronous infection. Phytopathology 66, 177-l 81. FOSTER, J. A. & Ross, A. F. (1975). The detection of symptomless virus infected tissue in inoculated tobacco leaves. Phytopathology 65, 600-610. GOODING, G. V. & HEBERT, T. T. (1976). A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology 57, 1285. GOTO, T. & TANIGUCHI, T. (1976). Local lesion formation and virus multiplication determined by intrinsic nature of strains of tobacco mosaic virus. Annals of the Phytopathological Society of Japan 42,
1. BAXTER,
2. 3. 4. 5. 6.
7. 8. 9.
549-555.
10. GUI.+AS, A. & FARKAS, G. L. (1978). Is cell to cell contact necessary for the expression of the N- gene in Nicotiana tabamm cv. Xanthi nc plants infected by TMV? Phytopathologische Zeitschrift 91, 182-187. 11. HIRTH, L. & LEBEURIER, G. (1964). Properties of tobacco mosaic virus developing at supraoptimal temperatures. In Host-pamite Relations in Plant Pathology, eds Z. Kiraly & G. Ubrizsy, pp. 3746. Symposium of the Hungarian Academy of Sciences, Budapest.
Necrosis-inducing
14.
15. 16.
17. 18.
19.
in TMV-infected
Nicotiana
91
R. & MCCARTHY, D. (1980). Extracts from virus infected hypersensitive tobacco leaves are detrimental to protoplast survival. Physiological Plant Pathology 16,25-38. MARTIN, C. (1966). Contribution a I’etude de phinomtne d’hypersensibilitt du virus de la mosaique du tabac. Bulletin de la Sociiti Franpise de Physiologic Vt!gLtale 12, 345-354. OHASHI, Y. & SHIMOMURA, T. (1972). Induction of localized necrotic lesions by actinomycin D on leaves systemically infected with tobacco mosaic virus. Virology 48, 601-603. OHASHI, Y. & SHIMOMURA, T. (1982). Modification of cell membranes of leaves systemically infected with tobacco mosaic virus. Physiological Plant Pathology 20, 125-128. TAKAHASHI, T. (1975). Studies on viral pathogenesis in plant hosts. VIII. Systemic virus invasion and localization of infection on “Samsun NW’ tobacco plants resulting from tobacco mosaic virus. Phytopathologische Zeitschrixt 84, 75-87. WESTSTEIJN, E. A. (1978). Permeability changes in the hypersensitive reaction of Nicotiana tabacum cv. Xanthi nc after infection with tobacco mosaic virus. Physiological Plant Pathology 13, 253-258. WESTSTEIJN, E. A. (1981). Lesion growth and virus localization in leaves of Nicotiana tabacum cv. Xanthi nc after inoculation with tobacco mosaic virus and incubation alternately at 22°C and 32°C. Physiological Plant Pathology 18, 357-368. WIDHOLM, .I. M. (1972). The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technology 47, 189-l 94.
12. HOOLEY, 13.
factor