The influence of actinomycin D on cucumber mosaic virus (strain W) multiplication in cucumber cultivars

Physiological

Plant Pathology

(1974)

4, 45-50

The influence of actinomycin D on cucumber mosaic virus (strain W) multiplication in cucumber cultivars D. J. BARBARA? Department lJniversi@

and K. R. WOOD

of Microbiology, of Birmingham,

(Accepted for publication,

Birmingham,

3u&

U.K.

1973)

Actinomycin D was demonstrated, by its effect on the incorporation of PHJuridine into acidprecipitable material, to curtail RNA synthesis in cucumber cotyledons. Treatment of China cucumber plants, normally resistant to infection by cucumber mosaic virus (CMV), with actinomycin D allowed an enhanced accumulation of virus infectivity, measured 4 days after infection, compared to that in control plants, the maximum observed increase occurring following injection of an actinomycin solution containing 10 pg/ml 1h No significant influence on infectivity in susceptible Ashley plants was after infection. observed. While not ruling out alternative possibilities, the results are consistent with the hypothesis that the China cukivar responds to CMV infection with the synthesis of one or more antiviral compounds, a process restricted by actinomycin treatment. Although a different virus and cultivar were employed, these observations agree in principle with those reported by Nachman et al.

INTRODUCTION

Following inoculation with the W strain [S] of cucumber mosaic virus (CMV), the cucumber cultivar Ashley allows a high level of virus multiplication and responds with severe systemic symptoms, while the China cultivar supports virus multiplication at a low level, produces mild symptoms and can therefore be considered highly resistant to infection by this virus (though possibly not to some others, e.g. tobacco ringspot; Wood, unpublished information). Previous studies on the interaction between CMV- W and these two cultivars [l, 10, 111 had suggested that peroxidases and polyphenoloxidases were not involved in resistance to infection, but rather were associated with the susceptible response, their activity in infected tissue possibly increasing as a result of cell damage. This communication reports the results of studies designed to investigate an alternative possibility, namely that resistance of China plants is at least partially mediated through the rapid induction, following infection, of a compound or compounds capable of restricting virus multiplication, possibly in a manner analogous to the active responses which may contribute to localized or systemic induced resistance [e.g. reference 71. Actinomycin D has been used to curtail the induction of such hypothetical compounds, by suppressing de novo RNA synthesis, and the effect on subsequent accumulation of infective virus in inoculated cotyledons assessed. Such treatment had previously been reported by Loebenstein and his colleagues to reduce ham

t Present address: B15 ZTT, U.K.

Department

of Virology,

Medical

School,

University

of Birmingham,

Birming-

D. J. Barbara and K. R. Wood

46

the hypersensitive host responses which lead both to virus limitation [3] and to the development of localized acquired resistance [.?I (although a stimulation of the hypersensitive response has also been recorded [9]). MATERIALS Virus

AND

METHODS

The W strain of CMV described [I].

was purified

and used to inoculate

plants as previously

Plants

Plants were grown in glasshouses as previously reported, and experiments performed at 25 + 1 “C in a constant temperature room under illumination from mercury vapour lamps (Philips HPLR, 700 W) 14 h per day. Efect of actinomycin D on RNA

synthesis

Ashley (susceptible) and China (resistant) cucumber plants (eight of each cultivar) were cotyledon inoculated, when at the second leaf stage, with a purified suspension of CMV- W in 0.05 M-phosphate buffer, pH 7.5 (dilution end point 10m3 to 10-4). Actinomycin D (1 to 20 pg/ml in distilled water, containing 40 to 800 pg/ml mannitol), mannitol (40 to 800 pg/ml in water) or water (only) was injected into the mesophyll spaces of the cotyledons 1 h after virus inoculation with a 26G #-in hypodermic needle; uninfected plants were treated similarly. Since the actinomycin D used throughout these experiments (“Lyovac Cosmegen”; Merck, Sharp & Dohme Ltd) was originally intended for clinical use, it contained mannitol (1 : 40 w/w), so that in experiments involving administration of this drug, mannitol was administered to a control group of plants at the same concentration as in the actinomycin D solutions. After 24 h a disk (6.5 mm) was removed from each cotyledon, the combined disks vacuum infiltrated four times with a solution (1 ml) of rH]uridine (2 to 8 Ci/mmol, 10 @i/ml; The Radiochemical Centre, Amersham) and allowed to stand at 25 “C for 6 h. The disks were then rinsed three times with distilled water, homogenized in 0.05 M-phosphate buffer, pH 7.5 (3 ml) and the extracts brought to 1.0 M with ice-cold perchloric acid. The mixture was held at 0 “C for 10 mm and then centrifuged at 1000 g for 15 min. The precipitate was washed once with icecold 1.0 M-perchloric acid (3 ml), allowed to stand at 0 “C for 10 min and then recentrifuged. The pellet was then treated with 2.5 ml 0.1 M-sodium hydroxide for 2 h at 60 “C, 2 ml O-1 M-HCl added and l-ml aliquots used for scintillation counting of disintegrations (in 4 ml Triton X-100, together with 7 ml of a toluene solution containing 4 g 2,5-diphenyloxazole and O-12 g 1,4-bis(5..phenyloxazol-2-yl)benzene/l), using a Nuclear Chicago Series 720 system. Effect of actinomycin D on virus accumulation

To ensure that mannitol, contained in the actinomycin solutions, was not itself having a drastic effect on virus infectivity, groups of eight plants of each cultivar were inoculated and then either left without further treatment or injected 1 h after

Influence

of actinomycin

D on cucumber

47

inoculation with mannitol (usually 400 pg/ml in water) or water only. Cotyledons were collected 4 days after infection, homogenized in 0.05 M-phosphate, pH 7.5 and virus infectivity assessedon 20 primary cowpea leaves, comparing the infectivity of extracts from treated plants with that from untreated plants on opposite leaves. To test the effect of actinomycin D on the accumulation of infective virus in cotyledons, eight plants of each cultivar were inoculated with CMV, 1 h later injected either with actinomycin D/mannitol or mannitol alone (c. 5 ml) and 4 days later the cotyledons were collected and infectivity assessed as described above. RESULTS

Efect on RNA synthesis The incorporation of [3H]uridine into the acid-insoluble fraction of cotyledons of China plants following injection with mannitol solutions (400 yg/ml, point 0) and with various concentrations of actinomycin is indicated in Fig. 1(a), together with the corresponding data for infected plants [Fig. l(b)] and for plants which had been infected, but received no further treatment (point I). Incorporation following injection with water only or mannitol at 800 pg/rnl was not significantly different from that following injection with manmtol at 400 pg/ml. Although there was a considerable variation between experiments, the evidence suggests that a reduction in RNA synthesis followed treatment of plants of this cultivar with actinomycin D. Comparable experiments involving the cultivar Ashley gave essentially similar results.

Actinomycin

FIG. 1. Effect

of

D concentration

(pg/mi)

actinomycin D on the incorporation of

fraction of cucumber leaf tissue, expressed and infected (b) ; I, infected but otherwise experiments, with standard error.

[?SJuridine into the acid-insoluble as yO untreated control: China plants uninfected (a) untreated. Each point represents the results of three

Effect on virus accumulation In 18 out of a total of 29 experiments designed to test the effect of mannitol on infectivity extractable from China cotyledons, the infectivity from mannitol-treated leaves fell within the range 60 to 100% of that from untreated leaves; in 24 of the 29 experiments, the corresponding range was 50 to 140%. A similar pattern was observed for Ashley plants, and when water alone was injected.

48

D. J. Barbara

and

K. R. Wood

The infectivity from resistant China plants treated with various concentrations of actinomycin compared with that from control plants is indicated in Fig. 2, where it is evident that following administration of a solution containing 10 Q.g/ml actinomycin, the increase in extractable infectivity was approximately sevenfold, the effect being

Actinomycin

FIG. 2. Effect extracts error.

of actinomycin D, injected from China plants. The data represent * Increase significant; P = 0.05.

D~concentrotion~(~g/ml)

1 h after infection, the results of three

on infectivity experiments,

of cotyledon with standard

rather less at other concentrations. When the time of administration relative to infection was varied, the effect on infectivity extractable 4 days later also varied (Fig. 3). Of the combinations tested, a solution containing 10 pg/m.l actinomycin injected 1 h after virus inoculation had the most significant effect on China plants, while either varying actinomycin concentration or increasing time after infection diminished the effect. In no case, however, was a significant increase in infectivity extractable from susceptible Ashley plants stimulated by treatment with this drug. DISCUSSION

Although there was evidence that actinomycin D does restrict de nova host RNA synthesis in some situations [e.g. references 5, 61, it was clearly desirable to ensure that this was indeed the effect on the cucumber plants used in these experiments involving changes in virus accumulation. To this end, the effect of actinomycin D on the incorporation of [sH]uridine into the acid-insoluble fraction of extracts from both infected and control plants was evaluated, as an indication of the extent of RNA synthesis. The results indicated that RNA synthesis was indeed restricted, though not completely, and in some instances, injection of water or mannitol solutions also affected RNA synthesis. By curtailing de novo host RNA synthesis with actinomycin D, accumulation of infective virus in normally resistant China plants was allowed to proceed to a significantly greater extent than in untreated control plants, though this accumulation was still considerably less than that achieved in susceptible Ashley plants [I].

Influence of actinomycin

49

D on cucumber

8 t

*

I-

* ID*

!4

Actinomycin D Concentration (pg/ml)

5

IO

48

:

1

24

15

FIG. 3. Effect on infectivity of cotyledon extracts from China plants (5 to 15 pg/ml) at various times after infection. The data represent ments, with standard error. * Increase significant; P = 0.05.

of injecting the results

actinomycin D of three experi-

While not ruling out the possibility of contributions to resistance from unrelated passive factors, these results are consistent with the hypothesis that China plants respond to infection by CMV with the induction of a compound or compounds which are able to restrict virus multiplication, a process which is curtailed by administration of actinomycin and which probably does not occur, or is much less important, in Ashley plants. While these studies were in progress, Nachman et al. [4] reported a phenomenon essentially similar, though differing in detail, in resistant Elem plants; a maximum increase in infectivity was induced when actinomycin D was administered rather later (24 h) than reported here. Actinomycin D has also been reported [9] to stimulate the hypersensitive response, an observation at variance with the conclusions of Loebenstein et al. [Z, 31 suggesting that factors other than induced antiviral compounds (e.g. peroxidases) may be involved in virus limitation in this type of response. The observation that, even in the presence of actinomycin, virus multiplication was considerably less than in untreated, susceptible Ashley plants suggests either that host RNA synthesis was not fully inhibited under the present conditions, allowing 4

50

D. J. Barbara and K. R. Wood

limited synthesis of such compounds, or additional factors are involved in conferring resistance to CMV in the cultivar China. However, the biochemical effects of actinomycin D on plants cells are not well understood and other processes which may or may not influence virus multiplication could be affected, so that further experiments are in hand to clarify the role, if any, of antiviral compounds in the resistance of China cucumber plants to CMV. The authors are indebted to Dr J. A. Tomlinson of the National Vegetable Research Station, Wellesbourne, Warwickshire, for his continued interest and provision of materials, and to the Royal Society and the Science Research Council for financial assistance (to K. R. W. and D. J. B. respectively). REFERENCES 1. BARBARA, D. J. & WOOD, K. R. (1972). V’ nus multiplication, peroxidase and polyphenoloxidase activity in leaves of two cucumber (Cucamis sulivus L.) cultivars inoculated with cucumber mosaic virus. Physiological Plant Pathology 2, 167-173. 2. LOEBENSTEIN, G., RABINA, S. & VAN PRAAGH, T. (1968). Sensitivity of induced localized acquired resistance to actinomycin D. Virology 34, 264268. 3. LOEBENSTEIN, G., SELA, B. & VAN PFUAGH, T. (1969). Increase of tobacco mosaic local lesion size and virus multiplication in hypersensitive hosts in the presence of actinomycin D. Virology 37, 42-48. 4. NACHMAN, I., LOEBENSTEIN, G., VAN PRAAGH, T. & ZELCER, A. (1971). Increased multiplication of cucumber mosaic virus in a resistant cucumber cultivar caused by actinomycin D. Physiological Plant Pathology 1, 67-72. 5. %NGER, H. L. & KNIGHT, C. A. (1963). Action of actinomycin D or RNA synthesis in healthy and virus infected tobacco leaves. B&hem&al and BiophysiGal Research Communications 13,455-461. 6. SEMAL, J. & KUMMERT, J. (1969). Effects of actinomycin D on the incorporation of uridine into virus infected leaf fragments. Phytopathologische