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Preliminary studies on the RNA components of three strains of cucumber mosaic virus
Physiological
Plant Patholqy
(1975)
;I, 139-145
Preliminary studies on the RNA components strains of cucumber mosaic virus K.
R. WOOD
and
Department of Microbiology, University of Birmingham, (Acceptedfor
publication
R. H. A. Birmingham April
of three
COUTTS 815
XT,
U.K.
1975)
In investigations of the causal relationships between disease symptoms and virus infection of plants, three strains of cucumber mosaic virus (viz. JV, Price’s No. 6 and W) which caused different symptoms in several plant species were studied. The ribonucleic acid components of the three strains were resolved by electrophoresis in 2.4% polyacrylamide/0.5% agarose gels and their sizes relative to those of the two ribosomal RNAs of Escherichia coli estimated. All three strains contained the four major RNA species.
INTRODUCTION There is presently very little information on the means by which virus infection leads to the development of disease symptoms, or why the same plant species reacts with different symptoms when infected with different strains of the same virus. It is possible that such differences reflect differences in virus-coded proteins. It was proposed, for example [S], that the yellowing of tobacco leaves and the concomitant reduced synthesis of ribosomal RNAs, particularly of chloroplast RNAs, after infection with certain strains of tobacco mosaic virus was associated with the repression of chloroplast RNA synthesis by a virus-coded protein. It is conceivable that with virus strains which invoke a less severe reaction such a protein may either not be produced, be produced in a reduced quantity or be produced in an ineffective form. However, other explanations are clearly possible. The relationship between virus strain and symptom expression is of particular interest, especially when the virus has a multicomponent genome. In such instances, the determinants of symptom expression may be coded for by one (or more) of the RNA species, which may or may not be the same as that which determines virus coat protein. Raspberry ringspot virus, for example, has two RNAs, RNA-1 and RNA-2. Recent studies by Harrison and his colleagues [5] in which the behaviour of homologous and heterologous mixtures of the two RNA species from different strains were investigated has revealed, inter &a, that RNA-l determines severity of symptoms on Chenopodium guinea while RNA-2 determines serological specificity. There are also many strains of cucumber mosaic virus (CMV; R/ 1; 1/I 8; S/S; S/Ap) causing symptoms of different degrees of severity on several plant species, while recent re-examinations of the RNA of some of the strains by improved techniques of polyacrylamide gel electrophoresis [4, 9, 10, 14, 15, 191 has revealed the multicomponent nature of the virus, as suggested by earlier studies [3, 8, 171. The
K. R. Wood
140
and
R. H. A. Coutts
strains investigated (D, Qand S) had four major RNA species (Nos. 1 to 4) together with two other lower molecular weight species (Nos. 5 and 6) occurring with some strains. The evidence suggested that these strains required the presence of the three largest RNAs (Nos. 1 to 3) for infectivity, and that No. 3 might be responsible for determining symptom expression in cowpea [16]. Though there is some evidence from ultracentrifuge studies that CMV preparations may be heterogeneous with respect to density [I3, 141 p ossibly indicating a heterogeneous distribution of RNA within the capsid, it is not yet clear how the RNA is distributed in the nucleocapsids [4, 141. Similarities with respect to some members of the bromovirus group are apparent [e.g. 1-j. The present work describes the species of RNA of three strains of CMV (w, fl and Price’s No. 6 or P6) and the disease symptoms caused by these three strains in various plant species, as a preliminary contribution to a study of the relationship translation products and symptom between the individual RNA species, their expression.
MATERIALS AND Virus and plants
METHODS
The W strain of CMV (originally obtained through the courtesy of Dr J. A. Tomlinson, reference 24) was propagated in tobacco (Nicotiana tabacum L. cv. White Burley) ; Price’s No. 6 [ZO], a gift from Dr M. Hollings, was also cultured in tobacco; strain fl [25], kindly provided by Dr R. W. Fulton, was propagated in cucumber (Cucumis sativus L. cv. Ashley) or Nicotiana clevelandii and purified from cucumber. All three strains were purified by the method of Scott [22] with three cycles of low- and high-speed centrifugation followed by sucrose density gradient centrifugation (10 to 40% sucrose in 0.005 M-borate, pH 9.0) and dialysis against 0.005 M-borate buffer, pH 9.0. All plants used in this study were grown in J. Arthur Bowers’ loamless compost (Lindsey Kesteven Fertilizers, Saxilby, Lincoln, U.K.) and maintained in glasshouses at 23 + 2 “C with supplementary lighting from mercury (Philips 2F “Powerwhite”, 400 W) and sodium (GEC HPS/U “Solarcolour”, 360 W) lamps, with a 14 h photoperiod. For studies on symptom expression, plants were inoculated with partially purified virus (one cycle of low- and high-speed centrifugation), diluted in 0.05 M-sodium phosphate buffer, pH 7.5 or with freshly prepared inoculum from infected cucumber, in the same buffer. RNA
@reparation and electrophoresis
For studies involving RNA, virus (0.4 to O-7 mg/ml) was dissociated directly by heating at 50 “C for 10 min in electrophoresis buffer containing 1 y. (w/v) SDS, 1 M-urea, 5% (w/v) sucrose and 0.05 M-mercaptoethanol immediately before electrophoresis according to the procedure used by Lane & Kaesberg [12] for brome mosaic virus and by Hull for broad bean mottle virus [7] and CMV-TAV [14]. Ribosomal RNA was isolated from E. coli by the method of Bolton [2], stored at
RNA
components
of cucumber
mosaic
virus
strains
141
-20 “C in 95% (v/v) ethanol and dissolved in electrophoresis buffer containing 10% (w/v) sucrose immediately prior to electrophoresis. Electrophoresis was performed in glass tubes (75 x 5 mm) using 2.4% (w/v) acrylamide 0.12% bis-acrylamide (both re-crystallized before use) and O*5o/o agarose [18]. After filling, the tubes were stood at room temperature overnight. The gels were cut to c. 6.5 cm, and pre-run for 1 h (4 “C) at 5 mA per tube with 36 mM-Tris, 30 mM-NaH,P0,.2HsO and 1 m&r-Na EDTA, pH 7.2 containing 0.2% SDS as electrophoresis buffer. Viral RNA was applied to the gel with (25 ~1, 3 to 4 pg) or without (50 ~1, 6 to 8 pg) added E. coli RNA (2 ~1, 1 to 2 pg) as the internal marker. Following electrophoresis, for approximately I.5 h at 5 mA per gel, the gels were washed for 2 h in electrophoresis buffer (without SDS) and the position of RNA species located by staining for 3 min with 0.1% toluidine blue in 40% (v/v) 2-methoxyethanol. Destaining was achieved with 30% (v/v) 2 -methoxyethanol. Densitometer traces were obtained by scanning at 265 nm with an SP 1800 spectrophotometer (Pye U&am Ltd) equipped with a gel-scanning attachment.
RESULTS Symptoms
The symptoms of infection in tobacco, Nicotiana glutinosa, cucumber and cowpea following manual inoculation with infective sap are summarized in Table 1. Some of the more prominent symptom differences which occurred approximately 12 days after infection in .N. tabacum L. cv. White Burley and Xanthi nc, .hC glutinosa and in cucumber are shown in Plates 1 and 2. TABLE
I
Reactims of h W, N and P6 strainr
of CMV Reaction
Plant
species
W
Nicotiama tabacum (cm White Burley and Havana 425)
Broken mild
N. tabacum (cv. Xanthi
Inconspicuous
species
to strains P6
ringspots mosaic”
and
mosaics
nc.)
yellow-green
Necrotic onlp
or chlorotic
lesions
Conspicuous mosaicb
yellow-green
Necrotic onlp
or chlorotic
lesions
yellow
No symptoms
Chlorotic lesions5 and conspicuous yellowgreen mosaicb
Conspicuous
Cucumis sativus (cv. Ashley)
Chlorotic mosaicb
Chlorotic spicuous
Vigna sine&s (cv. Blackeye)
Local lesions only,” smaller than with P6or N
lesions”
and
N
Conspicuous mosaicb
N. glutinosa
a Reactions b Reactions
in various plant
Local
of the inoculated leaves. of systemically infected leaves.
lesions” yellow
lesions
only0
mosaicb
and conmosaicb
Necrotic and chlorotic lesions,” severe necrosisb Local
lesions
onlya
K. R. Wood
142
and
R. H. A. Coutts
CMV RNA The RNA profiles of the three strains (Fig. 1, with peaks 1 to 6 labelled in increasing order of mobility) following polyacrylamide/agarose electrophoresis indicated that the four major species observed previously were present in all three strains. However; although six RNAs were invariably present in preparations from strain W (with species 5 making a significant contribution to the total amount of RNA present), only the four largest were normally present in preparations of the other two strains (N and P6). In addition to this difference in component number, the proportion of species 3 differed considerably between the three strains ( W> P6$>N).
Price’s
I
for
2 Distance
FIG. 1. Densitometer traces indicating the RNA species of three strains (W:
of
4 3 migration
5
No.6
6
(cm)
absorbance (265 nm) with distance 3v and Price’s No. 6) of CMV.
of migration
(cm)
(a)
Cd)
PLATE N
(a),
(d),
1. N. tabacum L. cv. Xanthi n.c. (a) to (c) and N. glutinosa Price’s No. 6 (b), (e) or W (c), (f) strain of CMV.
(d) to (f) inoculated
with
[ facing pact
142’
t
RNA
components
of cucumber
mosaic
virus
strains
143
Co-electrophoresis of viral RNA with E. coli ribosomal RNA (23 and 16s, I.07 and 0.55 x IO6 daltons respectively, reference 23) and measurement of mobilities enabled estimates to be made of the sizes of the various RNA species relative to those of E. coli RNAs (Table 2). TABLE Sizes
of CM V RNA species on co-electrophoresis CMV W Price’s N
strain
No. 6
1 1.24 1.17 1.16
2
with E. coli ribosomal
Size of RNA 2 3 1.10 1.05 1.07
0.73 0.76 0.74
species
R.NA ( I.07
and 0.55 da1t~n.s)
( x 10-s) 4
5
6
0.34 0.37 0.35
0.13 -
0.04 -
DISCUSSION The results of inoculating the three strains to the various hosts indicated (Plates 1 and 2 ; Table 1) that it was difficult to generalize about the type of symptom expected to be associated with a particular strain. Thus, whereas strain W gave mild symptoms in N. tabacum, the virus caused a severe systemic reaction in .N. glutinosa. Strain JV caused the formation of local lesions only, without systemic infection in fl. tabacum, a severe localized and systemic necrosis in cucumber, but no apparent infection in N. glutinosa. P6, however, caused systemic mosaic symptoms on these three species. Differences in the RNA components of the three strains were also apparent, but not such that would explain the symptom differences. Resolution on polyacrylamidelagarose gels indicated the presence of six components in the W strain (as found with S,, reference II), with four only in strains Jv and P6. The significance of species 5 and 6 is as yet unclear, though it is conceivable that they are host determined. Preparations of W also contained a much higher proportion of component 3 than strain fl, with P6 intermediate between the two. The apparent size of the four major species, estimated by co-electrophoresis with the two ribosomal RNAs of E. coli, were generally in close agreement with those estimated by Reijnders et al. [.?I] for the S strain using polyacrylamide gel electrophoresis under fully denaturing conditions (1.2 1, 1.06, 0.79 and 0.33 x 1O6 daltons). The two largest had apparent sizes rather higher than the “limiting” molecular weight for CMV RNA suggested by Kaper (0.97 to O-98; reference II), though smaller than values determined by Peden & Symons [19] for the Q strain, using polyacrylamide gel electrophoresis in the presence of formamide (1.35 and l-16 for species 1 and 2 respectively). Since aspects of the majority of procedures available for RNA molecular weight determinations are open to criticism, such data should be regarded with caution. It seems likely that differences in apparent size between corresponding species of the three strains studied here are attributable to small differences in secondary structure. The results obtained suggested, therefore, that it was unlikely that symptom expression was determined simply by the presence or absence of a particular RNA species, but rather by the function of their translation products. It is possible, however, that the replication of the virus in different hosts favours the enhancement of particular RNA species, so that the same strain purified from different hosts
K. R. Wood and R. H. A. Coutts
144
would differ in the proportions bility are in hand, together with mixtures of RNAs from the three tion of the relationship between expression.
of the various RNAs. Investigations of this possistudies on biological characteristics of appropriate strains, which are designed to assist in the clarificaRNA species and the determinants of symptom
The authors are grateful to Imperial Chemical Industries Limited for a Research Fellowship (to R. H. A. C.) and to Mr D. Lear for valuable assistance in the glasshouses. REFERENCES J. B., HIEBERT: E., REES, M. W. & MARKHAM, R. (1968). Properties of cowpea mottle virus, its protein and nucleic acid. Virolo~ 34, 224-239. BOLTON, E. T. (1966). The isolation and properties of the two high molecular weight fractions of Escherichia coli ribosomal RNA. In Procedures in Nucleic Acid Research, Ed. by G. L. Cantoni & D. R. Davies, pp. 437-443. Harper & Row, New York. DIENER, T. O., SCOTT, H. A. & KA~ER, J. M. (1964). Highly infectious nucleic acid from crude and purified preparations of cucumber mosaic virus (Y strain). Virology 22, 131-141. HABILI, N. & FRANCKI, R. I. B. (1974). Comparative studies on tomato aspermy and cucumber mosaic viruses. I. Physical and chemical properties. Virolo~ 57, 392-401. HARRISON, B. I)., MURANT, A. F., MAYO, M. A. & ROBERTS, I. M. (1974). Distribution of determinants for symptom production, host range and nematode transmissibility between the two RNA components of raspberry ringspot virus. 3ournal of General Virology 22, 233-247. HIRAI, A. & WILDMAN, S. G. (1969). Effect of TMV multiplication on RNA and protein synthesis in tobacco chloroplasts. Virology 38, 73-82. HULL, R. (1972). The multicomponent nature of broad bean mottle virus and its nucleic acid. 3ournal of General ViroloQ 17, 11 l-l 17. KAPER, J. M., DIENER, T. 0. & SCOTT, H. A. (1965). S ome physical and chemical properties of cucumber mosaic virus (strain Y) and of its isolated ribonucleic acid. Virology 27, 54-72. KAPER, J. M. & WEST, C. K. (1972). Polyacrylamide gel separation and molecular weight determination of the components of cucumber mosaic virus RNA. Preparative Biochemistry 2, 251-263. KAPER, J. M. & WATERWORTH, H. E. (1973). Comparison of molecular weights of single stranded viral RNAs by two empirical methods. Virology 51, 183-190. KAPER, J. M. & RE, G. G. (1974). Redetermination of the RNA content and the limiting RNA size of three strains of cucumber mosaic virus. ViroloQ 60, 308-3 11. LANE, L. C. & KAESBERG, P. (1971). Multiple genetic components in bromegrass mosaic virus.
1. BANCROFT,
chlorotic
2.
3. 4. 5.
6. 7. 8. 9.
10. 1 I. 12.
Jvature,
13. LOT, 14.
15.
16.
17.
18.
19.
New
Biology
232,
40-43.
H. & KAPER, J. M. (1973). Comparison of the buoyant density heterogeneity of cucumber mosaic virus and brome mosaic virus. Virology 54, 540-543. LOT, H., MARCHOUX, G., MARROU, J., KAPER, J. M., WEST, C. K., VAN VLOTEN-DOTING, L. 8r HULL, R. (1974). Evidence for three functional RNA species in several strains of cucumber mosaic virus. Journal of General Virology 22, 81-93. MARCHOUX, G., DOUINE, L., LOT, H. & ESVAN, C. (1973). Identification et estimation du poids moltculaire des acides ribonucleiques du virus de la mosa’ique du concombre (VMC souche D). Comf&s rendus hebdomadaires des stances de l’dcadkmie des Sciences, Paris 277D, 1409-1412. MARCHOUX, G., MARROU, J., DOUINE, L., LOT, H., QUIOT, J. B. & CLEMENT, M. (1974). ComplCmentation entre souches du virus de la mosa’ique du concombre. Localisation d’un gene sur I’ARN-3. Comptes rendus hebdomadaires des skances de l’Acad&nie des Sciences, Paris 278D, 889-892. MAY, J. T., GILLILAND, J. M. & SYMONS, R. H. (1969). Plant virus-induced RNA polymerase. Properties of the enzyme partly purified from cucumber cotyledons infected with cucumber mosaic virus. Virology 39, 54-65. PEACOCK, A. C. & DINGMAN, C. W. (1968). Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry 7, 668674. PEDEN, K. W. C. & SYMONS, R. H. (1973). Cucumber mosaic virus contains a functionally divided genome. Virology 53, 487-492.
RNA components 20.
of cucumber
PRICE, W. C. (1934).
Isolation
mosaic virus strains and study
of some
145 yellow
strains
of cucumber
mosaic
virus.
Phyto-
j~athology24, 742-76 1. 21.
L., AALBERS, A. M. J., VAN KAMMEN, A. & THURING, R. W. J. (1974). Molecular weights of plant viral RNAs determined by gel electrophoresis under denaturing conditions. ViroloD 60, 5 15-52 1. 22. SCOTT, H. A. (1963). Purification of cucumber mosaic virus. Virology 20, 103-106. 23. STANLEY, W. M. & BOCK, R. M. (1965). Isolation and physical properties of the ribosomal ribonucleic acid of Es&rich&a coli. Biochemistry 4, 1302-1311. 24. TOMLINSON, J. A., CARTER A. L., DALE, W. T. & SIMPSON, C. J. (1970). Weed plants as sources of cucumber mosaic virus. Annals of Applied Biology 66, 11-16. 25. TROUTMAN, J. L. & FULTON, R. W. (1958). Resistance in tobacco to cucumber mosaic virus. Virology 6, 303-3 16. REIJNDERS,
Plant Patholqy
(1975)
;I, 139-145
Preliminary studies on the RNA components strains of cucumber mosaic virus K.
R. WOOD
and
Department of Microbiology, University of Birmingham, (Acceptedfor
publication
R. H. A. Birmingham April
of three
COUTTS 815
XT,
U.K.
1975)
In investigations of the causal relationships between disease symptoms and virus infection of plants, three strains of cucumber mosaic virus (viz. JV, Price’s No. 6 and W) which caused different symptoms in several plant species were studied. The ribonucleic acid components of the three strains were resolved by electrophoresis in 2.4% polyacrylamide/0.5% agarose gels and their sizes relative to those of the two ribosomal RNAs of Escherichia coli estimated. All three strains contained the four major RNA species.
INTRODUCTION There is presently very little information on the means by which virus infection leads to the development of disease symptoms, or why the same plant species reacts with different symptoms when infected with different strains of the same virus. It is possible that such differences reflect differences in virus-coded proteins. It was proposed, for example [S], that the yellowing of tobacco leaves and the concomitant reduced synthesis of ribosomal RNAs, particularly of chloroplast RNAs, after infection with certain strains of tobacco mosaic virus was associated with the repression of chloroplast RNA synthesis by a virus-coded protein. It is conceivable that with virus strains which invoke a less severe reaction such a protein may either not be produced, be produced in a reduced quantity or be produced in an ineffective form. However, other explanations are clearly possible. The relationship between virus strain and symptom expression is of particular interest, especially when the virus has a multicomponent genome. In such instances, the determinants of symptom expression may be coded for by one (or more) of the RNA species, which may or may not be the same as that which determines virus coat protein. Raspberry ringspot virus, for example, has two RNAs, RNA-1 and RNA-2. Recent studies by Harrison and his colleagues [5] in which the behaviour of homologous and heterologous mixtures of the two RNA species from different strains were investigated has revealed, inter &a, that RNA-l determines severity of symptoms on Chenopodium guinea while RNA-2 determines serological specificity. There are also many strains of cucumber mosaic virus (CMV; R/ 1; 1/I 8; S/S; S/Ap) causing symptoms of different degrees of severity on several plant species, while recent re-examinations of the RNA of some of the strains by improved techniques of polyacrylamide gel electrophoresis [4, 9, 10, 14, 15, 191 has revealed the multicomponent nature of the virus, as suggested by earlier studies [3, 8, 171. The
K. R. Wood
140
and
R. H. A. Coutts
strains investigated (D, Qand S) had four major RNA species (Nos. 1 to 4) together with two other lower molecular weight species (Nos. 5 and 6) occurring with some strains. The evidence suggested that these strains required the presence of the three largest RNAs (Nos. 1 to 3) for infectivity, and that No. 3 might be responsible for determining symptom expression in cowpea [16]. Though there is some evidence from ultracentrifuge studies that CMV preparations may be heterogeneous with respect to density [I3, 141 p ossibly indicating a heterogeneous distribution of RNA within the capsid, it is not yet clear how the RNA is distributed in the nucleocapsids [4, 141. Similarities with respect to some members of the bromovirus group are apparent [e.g. 1-j. The present work describes the species of RNA of three strains of CMV (w, fl and Price’s No. 6 or P6) and the disease symptoms caused by these three strains in various plant species, as a preliminary contribution to a study of the relationship translation products and symptom between the individual RNA species, their expression.
MATERIALS AND Virus and plants
METHODS
The W strain of CMV (originally obtained through the courtesy of Dr J. A. Tomlinson, reference 24) was propagated in tobacco (Nicotiana tabacum L. cv. White Burley) ; Price’s No. 6 [ZO], a gift from Dr M. Hollings, was also cultured in tobacco; strain fl [25], kindly provided by Dr R. W. Fulton, was propagated in cucumber (Cucumis sativus L. cv. Ashley) or Nicotiana clevelandii and purified from cucumber. All three strains were purified by the method of Scott [22] with three cycles of low- and high-speed centrifugation followed by sucrose density gradient centrifugation (10 to 40% sucrose in 0.005 M-borate, pH 9.0) and dialysis against 0.005 M-borate buffer, pH 9.0. All plants used in this study were grown in J. Arthur Bowers’ loamless compost (Lindsey Kesteven Fertilizers, Saxilby, Lincoln, U.K.) and maintained in glasshouses at 23 + 2 “C with supplementary lighting from mercury (Philips 2F “Powerwhite”, 400 W) and sodium (GEC HPS/U “Solarcolour”, 360 W) lamps, with a 14 h photoperiod. For studies on symptom expression, plants were inoculated with partially purified virus (one cycle of low- and high-speed centrifugation), diluted in 0.05 M-sodium phosphate buffer, pH 7.5 or with freshly prepared inoculum from infected cucumber, in the same buffer. RNA
@reparation and electrophoresis
For studies involving RNA, virus (0.4 to O-7 mg/ml) was dissociated directly by heating at 50 “C for 10 min in electrophoresis buffer containing 1 y. (w/v) SDS, 1 M-urea, 5% (w/v) sucrose and 0.05 M-mercaptoethanol immediately before electrophoresis according to the procedure used by Lane & Kaesberg [12] for brome mosaic virus and by Hull for broad bean mottle virus [7] and CMV-TAV [14]. Ribosomal RNA was isolated from E. coli by the method of Bolton [2], stored at
RNA
components
of cucumber
mosaic
virus
strains
141
-20 “C in 95% (v/v) ethanol and dissolved in electrophoresis buffer containing 10% (w/v) sucrose immediately prior to electrophoresis. Electrophoresis was performed in glass tubes (75 x 5 mm) using 2.4% (w/v) acrylamide 0.12% bis-acrylamide (both re-crystallized before use) and O*5o/o agarose [18]. After filling, the tubes were stood at room temperature overnight. The gels were cut to c. 6.5 cm, and pre-run for 1 h (4 “C) at 5 mA per tube with 36 mM-Tris, 30 mM-NaH,P0,.2HsO and 1 m&r-Na EDTA, pH 7.2 containing 0.2% SDS as electrophoresis buffer. Viral RNA was applied to the gel with (25 ~1, 3 to 4 pg) or without (50 ~1, 6 to 8 pg) added E. coli RNA (2 ~1, 1 to 2 pg) as the internal marker. Following electrophoresis, for approximately I.5 h at 5 mA per gel, the gels were washed for 2 h in electrophoresis buffer (without SDS) and the position of RNA species located by staining for 3 min with 0.1% toluidine blue in 40% (v/v) 2-methoxyethanol. Destaining was achieved with 30% (v/v) 2 -methoxyethanol. Densitometer traces were obtained by scanning at 265 nm with an SP 1800 spectrophotometer (Pye U&am Ltd) equipped with a gel-scanning attachment.
RESULTS Symptoms
The symptoms of infection in tobacco, Nicotiana glutinosa, cucumber and cowpea following manual inoculation with infective sap are summarized in Table 1. Some of the more prominent symptom differences which occurred approximately 12 days after infection in .N. tabacum L. cv. White Burley and Xanthi nc, .hC glutinosa and in cucumber are shown in Plates 1 and 2. TABLE
I
Reactims of h W, N and P6 strainr
of CMV Reaction
Plant
species
W
Nicotiama tabacum (cm White Burley and Havana 425)
Broken mild
N. tabacum (cv. Xanthi
Inconspicuous
species
to strains P6
ringspots mosaic”
and
mosaics
nc.)
yellow-green
Necrotic onlp
or chlorotic
lesions
Conspicuous mosaicb
yellow-green
Necrotic onlp
or chlorotic
lesions
yellow
No symptoms
Chlorotic lesions5 and conspicuous yellowgreen mosaicb
Conspicuous
Cucumis sativus (cv. Ashley)
Chlorotic mosaicb
Chlorotic spicuous
Vigna sine&s (cv. Blackeye)
Local lesions only,” smaller than with P6or N
lesions”
and
N
Conspicuous mosaicb
N. glutinosa
a Reactions b Reactions
in various plant
Local
of the inoculated leaves. of systemically infected leaves.
lesions” yellow
lesions
only0
mosaicb
and conmosaicb
Necrotic and chlorotic lesions,” severe necrosisb Local
lesions
onlya
K. R. Wood
142
and
R. H. A. Coutts
CMV RNA The RNA profiles of the three strains (Fig. 1, with peaks 1 to 6 labelled in increasing order of mobility) following polyacrylamide/agarose electrophoresis indicated that the four major species observed previously were present in all three strains. However; although six RNAs were invariably present in preparations from strain W (with species 5 making a significant contribution to the total amount of RNA present), only the four largest were normally present in preparations of the other two strains (N and P6). In addition to this difference in component number, the proportion of species 3 differed considerably between the three strains ( W> P6$>N).
Price’s
I
for
2 Distance
FIG. 1. Densitometer traces indicating the RNA species of three strains (W:
of
4 3 migration
5
No.6
6
(cm)
absorbance (265 nm) with distance 3v and Price’s No. 6) of CMV.
of migration
(cm)
(a)
Cd)
PLATE N
(a),
(d),
1. N. tabacum L. cv. Xanthi n.c. (a) to (c) and N. glutinosa Price’s No. 6 (b), (e) or W (c), (f) strain of CMV.
(d) to (f) inoculated
with
[ facing pact
142’
t
RNA
components
of cucumber
mosaic
virus
strains
143
Co-electrophoresis of viral RNA with E. coli ribosomal RNA (23 and 16s, I.07 and 0.55 x IO6 daltons respectively, reference 23) and measurement of mobilities enabled estimates to be made of the sizes of the various RNA species relative to those of E. coli RNAs (Table 2). TABLE Sizes
of CM V RNA species on co-electrophoresis CMV W Price’s N
strain
No. 6
1 1.24 1.17 1.16
2
with E. coli ribosomal
Size of RNA 2 3 1.10 1.05 1.07
0.73 0.76 0.74
species
R.NA ( I.07
and 0.55 da1t~n.s)
( x 10-s) 4
5
6
0.34 0.37 0.35
0.13 -
0.04 -
DISCUSSION The results of inoculating the three strains to the various hosts indicated (Plates 1 and 2 ; Table 1) that it was difficult to generalize about the type of symptom expected to be associated with a particular strain. Thus, whereas strain W gave mild symptoms in N. tabacum, the virus caused a severe systemic reaction in .N. glutinosa. Strain JV caused the formation of local lesions only, without systemic infection in fl. tabacum, a severe localized and systemic necrosis in cucumber, but no apparent infection in N. glutinosa. P6, however, caused systemic mosaic symptoms on these three species. Differences in the RNA components of the three strains were also apparent, but not such that would explain the symptom differences. Resolution on polyacrylamidelagarose gels indicated the presence of six components in the W strain (as found with S,, reference II), with four only in strains Jv and P6. The significance of species 5 and 6 is as yet unclear, though it is conceivable that they are host determined. Preparations of W also contained a much higher proportion of component 3 than strain fl, with P6 intermediate between the two. The apparent size of the four major species, estimated by co-electrophoresis with the two ribosomal RNAs of E. coli, were generally in close agreement with those estimated by Reijnders et al. [.?I] for the S strain using polyacrylamide gel electrophoresis under fully denaturing conditions (1.2 1, 1.06, 0.79 and 0.33 x 1O6 daltons). The two largest had apparent sizes rather higher than the “limiting” molecular weight for CMV RNA suggested by Kaper (0.97 to O-98; reference II), though smaller than values determined by Peden & Symons [19] for the Q strain, using polyacrylamide gel electrophoresis in the presence of formamide (1.35 and l-16 for species 1 and 2 respectively). Since aspects of the majority of procedures available for RNA molecular weight determinations are open to criticism, such data should be regarded with caution. It seems likely that differences in apparent size between corresponding species of the three strains studied here are attributable to small differences in secondary structure. The results obtained suggested, therefore, that it was unlikely that symptom expression was determined simply by the presence or absence of a particular RNA species, but rather by the function of their translation products. It is possible, however, that the replication of the virus in different hosts favours the enhancement of particular RNA species, so that the same strain purified from different hosts
K. R. Wood and R. H. A. Coutts
144
would differ in the proportions bility are in hand, together with mixtures of RNAs from the three tion of the relationship between expression.
of the various RNAs. Investigations of this possistudies on biological characteristics of appropriate strains, which are designed to assist in the clarificaRNA species and the determinants of symptom
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