Inhibition of cucumber mosaic virus infectivity by leaf extracts

VIROLOGY

24, 193-199 (1964)

Inhibition

of Cucumber

Mosaic

Virus

Infectivity

by Leaf Extracts

R. I. B. FRANCKI

When extracts of healthy cucumber or ~~~o~i~na glut~nosa leaves were added to partially purified preparations of cucumber mosaic virus (CMV), the infectivity of the virus was inhibited, sometimes by as much as a hundredfold. This inhibition is shown to be destroyed by phenol extraction of the sap. This inhibitor may account for the fact that phenol-buffer extracts of (XV-infected leaves are often more infectious than extracts made with buffer alone. The nature of the inhibitor and its mode of action are at present obscure but data preeented in this paper suggest that the inhibitor is of relatively high molecular weight and that its mode of a&ion involves someform of aggregationof C&W.

and hence confirm the results very recently It has been observed that when euculllber reported for SLAV-infected tobacco leaves mosaic virus (CMV)-infected tobacco Ieaves (Diener et al., 1964). However, it has been are extracted in a mixture of phenol and demonstrated that extracted saps from buffer, the preparation is as infectious, or healthy leaves of these plants reduce the inmore infectious, than when extracted in fectivity of partially purified CMV prepabuffer alone (Schlegel 1960a, b; Diener et al., rations by up to one-hundredfoId. This 1964). This is unexpected since phenol-ex- observation may account for the high intracted virus ribonucleic acids (RNA) fectivity of phenol-buffer extracts of CMVinfected leaves relative to those extracted in usually have only about 1 o/othe infectivity buffer alone. of the original virus. Schlegel (196Ob) suggested some possible explanations for his obMATERIALS AND ~ET~iODS servations, but his data did not support any single hypothesis to the exclusion of the CMV isolate. The same isolate of CMV others. One of the suggestions made was that was used throughout, one that was first isothere may be large alnounts of free virus lated from Cu~~~~~ sp. in Ql~ee~land and RXA in CXV-infected tissues which are de- maintained in the glasshouse in either N. graded by leaf ribonucleases on extraction glutinosu L. or Cwumis sutivus I,. var. Davis with buffer but are protected when the tissue Perfect. is treated with buffer and phenol. This sugPartial ~uri~cat~~ 0s CMli. Preparations gestion seemed particularly interesting in of CMV were partially purified from cucumthe light of recent work by Siegel et al. (1963) ber leaves by the method of Scott (1963). and Babos and Kassanis (1962), who have Plants were inoculated when at the cotydemonstrated the presence of proteinless ledonary stage, and Ieaves showing sympmutants of tobacco mosaic virus (TMV) and toms were harvested lo-14 days later. tobacco necrosis virus (TNV), respectively. Yields of 50-300 mg of virus per kilogram of Experi~~lents reported in this paper have leaf material were obtained and the virus failed to demonstrate the presence of de- was stored at 04°C for periods of up to 2 tectable amounts of free virus RNA in CXIV- months with retention of infectivity. infected leaves of cucumber and N. g~~~~~o~~ I~~ect~u~t~ assq~s.Relative infe~tivities of INTRODUCTION

193

194

FRANCKI

CMV preparations were compared by the local-lesion technique on half-leaves of cowpea (Vigna s~~~~~~sEndl. var. Black Eye) (Crowley, 1954). Inoculations were made with the forefinger after a single dip into the inoculum. With virus RNA or inocula suspected of containing ribon~~~lease-sensitive material a single drop from a l-ml pipette was placed on the surface of the half-leaf and was rubbed on with the forefinger. Unless otherwise stated the inoculunl was washed off the leaf surfaces with a jet of water from a wash-bottle 3-6 minutes after inoculation. Extmetion of healthy and CMV infected Eeaves. Buffer extracts were prepared by grinding leaf tissue in buffer with a pestle and mortar at room temperature. Unless otherwise stated 1 ml of 0.1 M phosphate buffer, pH 7.2, was used to extract 1 g of leaf tissue. The homogenates were strained through cheesecloth and centrifuged for 5 minutes at 3000 rpm in a R&SE Magnum centrifuge. Phe~~ol-buff er extracts were prepared by grinding 1 g leaf tissue in 1 ml buffer and 1 ml water-saturated phenol. Homogenates were centrifuged as above; the buffer layers were withdrawn with a hypodermic syringe and washed with about 10 volumes of ether. Analytical ultracent$fugation 0J CMV p~e~a~~~~o~s.Ultracentrifugal analyses were carried out in a Spinco model E machine using a plain and a wedge window cell in the An D rotor at 33,450 rpm. Relative concentrations of virus were determined by measuring areas under the peaks of schlieren diagrams; corrections for radial dilution were made. Preparation of radioactive CMV. Young cucumber plants about 5 days after infectio~l with CMV were supplied simultaneously with P32and S35for &6 days as described by Matthews (1960) for Chinese cabbage infect& with turnip yellow mosaic virus. R,adioactive CMV was purified by the method of Scott (1963), but the final preparations were dialyzed against 0.005 M borate buffer, pH 9, for a week, and the buffer was changed daily. Radioactivity measul’ements. Liquid samples were dried down on aluminum planchettes and counted with a thin end-~~indo~v

GX. tube. Estimates of material doubly labelled with both P32and S35were made by two n~easure~~lents, one without and one with an aluminum shield which absorbed 100 % of S35and 8;?% of P32radiation. RESULTS

Inectivity of Phenol Extracts from Cucumber Leaves InJected with CMV It has been found that homogenates of C~IV-infected cucu~~~ber leaves made in a mixture of 0.1 M phosphate buffer, pH 7.2, and water-saturated phenol are usually more infectious than those made in buffer alone. The ratio of infectivity of phenol-buffer extracts to those made with buffer alone varied from 11.6 to 0.37 but was usually greater than 1. This confirms earlier work on CMVinfected tobacco leaves (Schlegel 1960a, b), but unlike the results of Schlegel (1960b) the slopes of my dilution curves were not parallel. Those of the phenol-buffer extracts were usually steeper at t~he higher dilutions than those of the buffer homogenates. The results of a typical experiment are plotted in Fig. 1. Schlegel (196Ob) found that when R.NA was prepared from partially purified CMV by phenol extraction it had only O&0.7%

.m..buffer o-0

extract

phenol

extract

DILUTION

1. Dilution curve of phenol-buffer and buffer extracts of C~~~~-infected leaves from plants 7 days after infection. Tbirty leaves were cut into halves, one half of each being used for buffer-phenol extraction and the opposite half for buffer extraction. FIG.

INHIBITION

OF CMV

the infectivity of intact virus. In an experiment in which I prepared RNA from purified CMVinO.l M phosphate buffer, pH 7.2, by a single phenol extraction, the infectivity dropped by a factor of approximately 40 when calculated on an equal virus RNA basis. Thus it appears that CMV is like other viruses tested in that phenol prepared RNA has only very low infectivity compared to that of virus nucleoprotein. Extraction of Infectious Material from CMVInfected Leaves and from Healthy Leaves with Puri$ed Virus Added Schlegel (1960b) has suggested the possibility that in CMV-infected leaves there may be large amounts of free virus RNA or incomplete virus material sensitive to ribonuclease. To test this hypothesis the following experiments have been carried out using both cucumber and N. glutinosa. Samples of 24 leaf disks (each 1.5 cm in diameter) were taken from (XIV-infected and from healthy leaves. To a sample from healthy plants were added 0.25 ml of a purified virus preparation, 0.25 ml of water, and 0.50 ml of 0.2 M phosphate buffer, pH 7.2. To a sample from infected leaves 1 ml of 0.1 M phosphate buffer, pH 7.2, was added. One sample of healthy and one of infected leaf disks were each homogenized and centrifuged at 3000 rpm for 5 minutes; the supernatant solutions were inoculated to half-leaves of cowpeas. The opposite half-leaves were inoculated with a standard inoculum prepared from partially purified virus. Similar samples of healthy and infected leaf nlaterial were homogenized in the presence of 1 ml of watersaturated phenol. The homogenates were centrifuged; the buffer layer was washed with ether and inoculated to cowpeas as above. A further four samples, two containing infected and two healthy leaf disks plus purified virus as above were all homogenized and incubated at room temperature. After 30 minutes one sample from healthy disks and one from infected disks were centrifuged and tested for infectivity as above while the two remaining samples were extracted with phenol, washed with ether, and assayed for infectivity. The results of two such experiments are sum marized in Table I.

BY LEAF

195

EXTRACTS

It seems that there is no measurable amount of unstable infectious material in CMV-infected leaves. If there were, then we would expect the amount of phenol-extractable infectivity from virus-infected leaves, but not from mixtures of healthy leaves with added CMV, to drop subst,antially when homogenates are incubated prior to phenol treatment. Similar results from slightly differently designed experiments have very recently been reported by Diener et al. (1964). They concluded that the high infectivity of phenol-buffer extracts of CMVinfected tobacco leaves could not be due to the presence in the tissue of incomplete (or partially degraded) ribonuclease-sensitive material, to free virus RNA, or to entities similar to unstable variants of TNV described by Babos and Kassanis (1962). Inhibition

oj CMV

In,fectivity

by Extracts

from Healthy Leaves

To test the effect of host plant materials on the infectivity of CMV, extracts of cucumber or N. glutinosa leaves were prepared, mixed with partially purified CMV preparations, and inoculated to cowpeas about 10 minutes after mixing. Extracts were made by grinding samples of leaf tissue with 1 ml/g of 0.05 LIJ phosphate buffer, pH 7.2, 0.05 M boric acid or 0.05 M sodium borate. The effects of these extracts on infectivity of CMV were found to be very marked (Table 2). A subsequent experiment demonstrated that on dilution the inhibitory effect of sap on CMV infectivity is rapidly lost (Fig. 2). It has been observed that the magnitude of the inhibitory effect of leaf extract varies somewhat from preparation to preparation and may well be governed by the physiological condition of the plants. However, no attempt has yet been made to investigate this in detail. Preliminary tests have also shown that extracts from leaves of many plants inhibit CMV infection when mixed in the inoculum. Tomato (L ycopersicon esculentum Miller), lettuce (Lactuca sativa L.), Chinese cabbage (Brassica pekinensis Rupr. var. Wang Bok), lucerne (Medicago sativa L.), cauliflower (Brassica sativa 1,. var. botrytis), Chenopodium amaranticolo,~ Coste et Reyn., and C. quinoa Willdenow have all

FKANCKI

19G

TABLE INFECTIVITY

1

OF BUFFER EXTRACTS AND PHENOL-BUFFER EXTRACTS OF CM~-I~FECTE~ LEAVES AND HEALTHY LEAVES MIXED WITH PURIFIED CMV Experiment 1 (N. glufi?wsa)

Extract preparation

Lesions/half-IeaP By extract By standard inoculud inoculumC

Healthy leaf tissue plus purified CMV extracted in buffer only Healthy leaf tissue plus purified CMV extracted in buffer-phenol mixture Healthy leaf tissue plus purified CMV extracted in buffer and incubated 36 min Healthy leaf tissue plus purified CMV extracted in buffer, incubated 30 mm and extracted in phenol Infected leaf tissue extracted in buffer alone Infected leaf tissue extracted in bufferphenol mixture Infected leaf tissue extracted in buffer and incubated 30 min Infected leaf tissue extracted in buffer, incubated 30 min, and extracted in phenol

L&HIS

$$$a,6

Experiment 2 (cucumber) ______.-.Lesions/haIf-ieaP Lesions By extract By standard $82$;;c:fd inoculumb inoculunP

117

79

1.49

234

142

1.64

69

77

0.89

167

128

1.30

64

82

0.78

289

167

1.72

27

118

0.23

179

133

1.34

23

89

0.25

136

125

1.09

38

96

0.40

170

135

1.26

18

93

0.19

121

115

1.05

27

62

0.43

140

118

1.27

* For each assay 16 half-leaves were used. 6 Extract inoculum from healthy leaves corItai~~ed approximately 3.5 mg purified CM\: per m~llii~ter. c Standard inoculum conkned 0.1 rng-_purified CMV per milliliter in experiment 1 and 0.06 mg per milliliter in experiment 2.

shown strong inhibitory action, and no plant has yet been found whose leaf extract fails to inhibit CMV infection.

Although no attempt has been made to purify the substance in leaf extracts responsible for inhibitirlg CMV i~~fection, some tests were carried out to determine its stability and properties. From the data summarized in Table 3 it appears that the inhibitor can be removed from leaf extracts by phenol extraction. It was only partially destroyed by heating at 100°C and only partially sedimented by ~ent~ifugation at 144,000 g for 1 hour. It can be stored at 25” or 4” for a day without detectable loss of activity. It cannot pass through a dialysis membrane. These data indicate that the inhibitor may be a compound of fairly high i~~olecular weight,. The observation that phenol extraction removed all the inhibitory

effects of leaf extracts on CMV infectivity could explain why phenol-buffer extracts of infected leaves are so highly infectious. Sedimentation I3xtructs

of CMV

in Cucumber Leaf

In the analytical ult~eentrifuge partially purifi~ preparations of CMV showed a homogeneous schlieren peak with sedimentation coeflieient e-90 S. In some preparations there was also a very small peak -18 S. When purified virus preparations were mixed with ~ucuI~lber leaf extracts and analyzed in the ultracentrifuge the 90 S peak either disappeared completely or was considerably reduced in size. Aliquots of a virus preparation containing approximately 4 mg nucleoprotein per millilitre were mixed with equal volumes of healthy cucumber leaf extract at various dilutions. Results of this experiment (Fig. 3) indicate that the degree to which the area under the schlieren peak is reduced de-

INHIBITIVE

197

OF CMV BY LEAF EXTRACTS TABLE 2 HEALTHY WEAVES ON C&IV INFECTIVITY

THE EFFECT OF BUFFER EXTRACTS FROM

Experiment 1 (3’. gl&rsDsa leaf extract) Treatment of inoculum

6.1 9.1

/ 11

i

1 I 51

6.0

0.44

5.2

I 1 0.12 I

7.3

Experiment 2 (cucumberleaf extract) Lesions/haIf-leafa

Final pH of treated inoculum

Final of treated noculum PH

2 parts 0.025 M boric acid added to 1 part virus preparation 2 parts 0.025 M sodium borate added to I part virus preparation 2 parts leaf extract made with phosphate buffer, pH 7.2, added to 1 part of virus preparation 2 parts leaf extract made with boric acid added to 1 part of virus preparation 2 parts leaf extract made with sodium borate added to 1 pzlrt of virus preparation

/

! ji I

1.0

i

By ’ By std.h A’,$yudm inoculum

8.3

45 G3

I 0.81

66

0.007

22::

numbers treated/ std. -.--

0.26

1 6.1

I I

I 88

0.001

6.1

56

0.018

8.2

1 I I j I

-

i A.. a For each assay 16 half-leaves were used in experiment 1 and 20 in experiment 2. g Standard inoculum consisted of 2 parts 0.025 M phosphate buffer, pH 7.2, and 1 part of virus preparation.

J

. %zr,z

,‘b,,

b8

4

‘42

FIG. 2. Effect of cucumber leaf extract at various dilutions on inhibition of CM\’ infectivity. In this experiment every ino~u~um tested eontained 0.13 mg CMV per”milliliter.

pends on the concentration of sap added. In these experiments it could not be determined whether the di~ppearance of the sehlieren peaks was due to virus aggregation and pelleting or whether the virus disintegrate into materials with slow-sedimenting velocities. No such slow-sedimenting components could be observed, but they could easily have escaped detection owing to the presence of protein peaks aIways seen in leaf extracts. To investigate the effect of cueun~ber leaf extracts on the sedimentation properties of CMV protein and RNA siul~lltaneousIy, an experiment was carried out using doubly labelled virus with P3*in the RNA and S35in the protein. Aliquots of 207 pg C&IV were mixed with equal volumes of leaf extract and after 10 minutes were centrifuged at 44,000 y for 10 minutes. Radioactivity due to 1% and $335 remaining in the supernalant was determined. Results of this experiment (Table 4) show that C&IV is more headily sedirl~ented

198

FItANCXI

TABLE 3 EFFECTS OF SOME TREATMENTS ON THE INHIBITORY Accnoru OF CUCUMBER LEAF EXTRACTS ON CMV INFECTIVITY ExperimentI ---.--_----l--l--__ll_---------_-

Experiment2

~sions~half-leafa Lesions/hdf-leafa Ratio ~._I__~---. ----~ Treatment of extract from hedthy cucumberleaves lesions with By By BY BY extract/ inaculum inocuium inoculu~ inoculum without with without with without extract extract extract CXtraCt extract - ._ ~ ~___ -__- -___- __-._- -_ l-l-_---

Extract untreated Extract t,reated with phenol and washed with ether Extract heated 1OOO”C for 10 min Supernatant of extract after centrifugation for 1 hr at X44,000g Extract stored for 22 hrs at 25°C Extract stored for 22 hrs at 4°C Extract dialyzed for 22 hr against 2 volumes of water at 4°C Dialysis water from above

,Ratio lWOliJ~c~t without extract .I ,-_._--.

0.2 9.8

12 7.1

0.02 1.38

47

36 45

0.00 1.05

5.3 4.0

6.4 I8

0.83 0.22

49 39

88 59

0.50 0.66

1.9 0.3 1.1

9.5 7.0

0.20 0.04

0.0 0.0

31 73

20

0.06

1.2

99

0.00 0.00 0.01

15

0.93

87

1.03

14

2.1

89

a For each assay I6 half-leaves were used. TABLE 4 S~QIME~~ATION OF CMV DOUBLP LABELLED WITH Pa2 AND 6% IN BCFFER AND IN HEALTHY

CTKXIMBER LEAF EXTBACT

Treatment of purified C&IV labelled with P32and S=

To Radioactivity remaining in supernatant ~_______I_ pa SS

___ I_-._____-- -. - __l-l-Virus mixed with equal volume of healthy cucumber leaf ext~ract) Virus mixed with equal volume of 0.05 IM phosphate buffer pB 7.2

Ratio Pa2:Sssin supernatant

_ ._..- - -__.._.-...--.. “- - ...-l.---~-.

.-._-

45.8 4, 0.88”

50>1 Zk 3.99

0.96 f

8u.s =I? 1.05

94.8 f

0.86 It 0.047

3.78

0.075

a Values indicate t,he standard errors of means.

when mixed with leaf extract and that both virus protein and RNA undergo parallel changes in their sedimentation behaviour. This suggests that the virus particles do not undergo any significant fragmentation on exposure to leaf extract.

3 100 $ 2 4 3 0

E 25 IS&ct of Cwumbw Leaf Exkzcl on ~ow~eas s I~~~~~~~~~~ w&z C&T s I have failed to demonstrate any inhibi~o~y effect of eucumber extracts on lesion 2 1o 5: production by CMV on cowpea leaves by applying the extracts to the surface of halfleaves after inoculation with virus. Experi,“! 6 m&s were carried out in whieh cowpea I?~LUTION OF SAP leaves were inoculated with C%IV aud cucumber leaf extract was appli& with a catnelFro. 3. .kXeect of cucumber leaf extract at varihair brush to half-leaves while the opposite ous dilutions as determined by measuring aress half-leaves were sinlilarly treat& with 0.05 under schlieren diagrams,

INHIBITION

OF

CMV BY LEAF EXTRACTS

Al phosphate buffer. In a typical experiment when leaf extract was incorporated into the ir~oculunl the lesion number was reduced from 27 to 5 per half-leaf. When the same extract was applied to the leaf surface imIl~edi~tely after inoculation the lesion numbers were 23 and 39 on control and treated half-leaves, respectively.

~xperil~le~lts described in this paper indicate that phenol buffer is a more efficient medium than buffer alone for extracting infectious material from C~IV-infected leaves of cucumber, X. g~~~ino~a,and probabIy also many other plants because of the presence in sap of a phenol-labile inhibitor of CR/IV infection. Evidence for the presence of a CMV il~hibitor in ~ncul~~ber has been reported previously by Sill (1951) and Sill and Walker (1952). The presence of this inhibitor in leaf extracts and its removal by phenol would account for the shapes of dilution curves of buffer and buffer-phenol extracts of CMVinfected leaves (Fig. 1) and also the dilution curve for CMV-infected sap reported by Crowley (1954). ~i~culties experienced in the past of purifying CMV in reasonable yields may also be due, at Ieast in part, to the presence of this inhibitor (Sill et al., 1952; Tomlinson et af., 1959; Schlegel 1960b). The exact nature of the CMV inhibitor in plant leaves is obscure, but it seems likely that it is a substance of relatively high molecular weight. The mechanism by which C&IV infectivity is inhibited is unknown, but is unlikely to involve the disintegration of the virus particle as shown by the results of experi~nents with double-labelled C&IV (Table 4). The sedimentation behaviour of CRIV following exposure to leaf extracts suggests that some form of aggregation takes place. Very recent work of Diener et al. (1964) strongly supports the suggestion that CXIV in sap tends to aggregate. There is also some evidence (van Regenmortel, 1961) that host leaf ~laterials readily adsorb to the surfaces of CMV particles and are difhcult to remove. It seems not unreasonable to suggest that the loss of infectivity of CMV on exposure to leaf extracts could be due to the aggregation of virus particles with themselves, or to the association of some host

199

plant materials with the particles, thus preventing them from starting an infection.

I wish to thank Mr. R. Greber for praviding the CMV isolate, Dr. S. B. Wilson for carrying out the ultra~entrifugal analyses, and Miss Geraldine Norman for able technical assistance. I am also grateful to Dr. H. Scott for sending me his manuscript before publication. This work was supported in part by a grant from the Rockefeller FoundatioI~. REFERENCES BABOS, P., and KASSANIS, B. (1962). Unstable variants of tobacco necrosis virus. Virology 18, 206-211. CRO~LEY, N. C. (1954). Some variables affecting the use of cowpea as an assay host for cucumber mosaic virus. ~~~s~~ff.1~~~ f. l3ioZ. Sci. 7, 141-150. DIENER, I’. O., SCOTT, H. A., and KAPER, J. M. (1964). Highly infectious nucleic acid from crude and purified preparations of cucumber mosaic virus (Y strain). ~~~~Zo~~22, U--141. MATTHEWS, R. E. F. (1960). Properties of nucleoprotein fractions isolated from turnip yellow mosaic virus preparations. Virology 12,521-539. SCHLEGEL,D. E. (1960a). Transmission of several plant viruses by phenol-water extracts of diseased tissues. ~~~~0~~~~~~0~~50, 156-158. SCHLEGEL, D. E. (1960b). Highfy infectious phenol extracts from tobacco leaves infected with cucumber mosaic virus. Virology 11, 329338. SCOTT,H. (lQ63). Purification of cucumber mosaic virus. Virology 20, 103-106. SIEGEL, A., ZAITLIN, M., and SEHGAL, 0. P. (1963). The isolation of defective tobacco mosaic virus strains. proc. i\icatf. Bcud. Sei. V. S. 48, 1845-1851. SILL, W. H., JR. (1951). Some characteristics of a virus inhibitor in cucumber. (Abstract.) P&topathology

41, 32.

SILL, W. II., JR., and WALKER, J. C. (1952). A virus inhibitor in cucumber in relation to mosaic resistance. Phytopathology 42,349-352. SILL, W. II., JR., BURGER, W. C., S~A~MANN, M. A., and WALKER, J. C. (1952). Electron microscopy of cucumber mosaic virus 1. Phytopathology

42, 420-422.

TOMLINSON, J. A., SHEPHERD,R. J., and WALKER, J. C. (1959). Purification, properties and serology of cucumber mosaic virus. Ph,ytopathology

49, 293-299.

VAN REGENMORTEL,ivI. H. V. (1961). Zone electrophoresis and particle size of cucumber mosaic virus. ~~ro~o~~~15, 221-223.