VIROLOGY
92,
1- 14 (1979)
Structural Differences between Pea Enation Mosaic Virus Strains Affecting Transmissibility by Acyrthosiphon pisum (Harris) G. ADAM,* EVAMARIE
SANDER,* AND ROBERT J. SHEPHERD?’
*Institut fiir Biologie II, Universitit Tiibingen, D 74 Tiibingen, Auf der Morgenstelle 28, BRD, and TDepartment of Plant Pathology, University of California, Davis, California 95616 Accepted September 8, 1978 Aphid-transmissible and non-aphid-transmissible strains of pea enation mosaic virus were compared in biological and physical properties. When separated by electrophoresis into 3.4% polyacrylamide gels both centrifugal components of aphid-transmissible strains gave rise to multiple-banding, whereas non-aphid-transmissible strains migrated as two components without separation into multiple bands. The multiple banding phenomenon during gel electrophoresis may be caused by the association of different amounts of an additional protein of 56,666 daltons with the vu-ions, so that they migrate as different size classes. The mean increment between the diameters of any two size classes was determined to be 0.54 nm. The origin of this second coat protein may be accounted for by the molecular weight of the largest viral RNA from a transmissible strain being 1.2 x l(r daltons larger than that of the non-aphid-transmissible strains. A non-aphid-transmissible strain could be selected by continuous mechanical passages of an aphid-transmissible strain and resembled other non-aphid-transmissible strains in all characteristics tested, including the lack of the second protein associated with vu-ions. Hence, it is postulated that the second protein, unique to aphid-borne strains, is a gene product responsible for the insect transmission of PEMV. For both aphid-transmissible and non-aphid-transmissible strains of PEMV, examination of the RNA content of the two nucleoproteins largely confirmed the recent work of others on the multipartite nature of the virus genome. Evidence is presented for RNA, arising as a breakage product of RNA, during isolation with phenol-SDS. INTRODUCTION
ferences between the two types of strains. These differences are described herein.
Pea enation mosaic (PEMV) is a virus with two centrifugal components which can MATERIALS AND METHODS be transmitted mechanically and by several aphid species in a persistent manner. The Most experiments were done with three virus has been characterized extensively strains of PEMV, viz., Wt (wild-type) dephysically and chemically and, although scribed by Hull and Lane (1973), P-3 (Gonnon-aphid-transmissible strains have been salves and Shepherd, 1972); and Tii deselected (Tsai and Bath, 1974), until re- scribed by Adam and Sander (1976); some cently (Clarke and Bath, 1977; Harris et al., experiments utilized strains CAT and CNT 1975; Hull, 1977) no differences had been of Harris et al. (1975). First to third instars found between the aphid-transmissible and of three biotypes of pea aphids [Acyrthonon-aphid-transmissible types. In this in- siphon pisum (Harris)] reared on broad vestigation we have made an extensive com- bean were used for transmission tests. parison between the properties of PEMV Aphids were allowed to acquire virus by strains which are and are not transmissible feeding for 4 hr on infected plants or virus by aphids and have found some major dif- was injected into the hemocoele of CO,anesthesized aphids with a capillary tube us1 Author to whom reprint requests should be ing hydrostatic pressure. Single aphids addressed. were put onto test plants for a 7-day in1
0042-6822/79/01ooo1-14$~.~/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproductionin any form reserved.
2
ADAM,SANDER,ANDSHEPHERD
fection feeding period. Pisum sativum L. cv. Progress No. 9 was used as a host for propagation of virus and for aphid transmission trials. Virus purification. The method of Mahmood and Peters (1973).was used with slight modifications. Sucrose (5%) was added to the 0.2 M acetate medium for homogenizing leaves. After chloroform clarification, the pH precipitation step (1 hr at pH 5.2) was used to remove additional material followed immediately by precipitation with 6% polyethylene glycol (PEG) 6000-o. 1 M NaCl at pH 6.0. After two cycles of differential centrifugation the virus was stored in 0.1 M acetate, pH 6.0 containing 5% sucrose (VB). Before RNA isolation, the virus was centrifuged through a cushion of 15% sucrose (RNase-free). RNA isolation. RNA was isolated by either phenol-SDS or urea-SDS treatments followed by deproteinization with chloroform-isoamyl alcohol and storage in buffer G (0.04 M Tris, 0.2 M sodium acetate, 0.002 M EDTA, pH 7.5). When RNA was isolated by the SDSurea method, purified virus in VB (2-5 mg/ ml) was mixed with 0.1 vol of 10% SDS, 0.1 vol of 5 M urea, and 0.1 vol of 3 M acetate buffer, pH 5.5, and incubated for 5 min at 37”. Then 2.2 vol of a mixture of CHCl, and isoamyl alcohol (24:l) was added and the preparation was incubated for 15 min at 4” after shaking for 2 min at 23”. The emulsion was broken by centrifugation (2,500 g, 10 min, 0’) and the aqueous phase was treated again with the organic solvent mixture. The RNA was precipitated from the aqueous phase with ethanol. Rate zonal gradient centrifugation. The two components of each virus strain were separated by repeated centrifugation into sucrose gradients [lo-40% (w/v> sucrose in 0.1 M acetate buffer, pH 61 as described by Gonsalves and Shepherd (19’72). The separated components were concentrated between all gradient cycles by precipitation (10% PEG 6000, 0.05 M NaCl). Gradient centrifugation took place in SW27 or SW40 rotors (26,000 rpm, 4 hr or 39,000 rpm, 2.5 hr, both at 4°C) in a Spinco ultracentrifuge. Electrophoresis of intact virus particles.
Gels with a monomer concentration of 2.8, 3.0, and 3.4% acrylamide (5% crosslinked) were cast at pH 4.4 into glass tubes (10 cm long x 0.6 cm i.d.). Gradient gel slabs with a linear increase from 2.5 to 10% monomer concentration were made from two acrylamide solutions, viz., (a) 2.5% acrylamide with 3% sucrose and (b) 10% acrylamide with 10% sucrose. All monomer solutions were made with the electrophoresis buffer described by Hull and Lane (1973), 30 mM Tris, 3 mM Ca(OH), adjusted to pH 4.4 with lactic acid. The gels were stored at least 12 hr at 4” prior to use. Electrophoresis was carried out at 4” with 16 V/cm per gel at constant voltage, using 16 hr for 3.4% gels and 24 to 78 hr for gradient gels, both at pH 4.4. During electrophoresis the buffer was circulated between the buffer vessels. Virus bands in gels were located by scanning at 254 nm in a Gilford spectrophotometer or by staining with Coomassie blue (Weber and Osborn, 1969). To determine the serological properties of the virus bands, the gels were treated with antiserum against PEMV-Tii (titer l/512) at a dilution of 1:64 made with phosphate-buffered saline, pH 7.2. Excised virus bands were homogenized in VB containing 0.2% BSA and eluted by gentle shaking for 12 hr at 4”. After removal of acrylamide residue by lowspeed centrifugation, virus was precipitated with 10% PEG. Gel electrophoresis of RNA. Electrophoresis of RNA was done on 2.4% acrylamide gels (Gonsalves and Shepherd, 1972). Prior to electrophoresis the isolated RNA in buffer G was mixed 1:l with dissociation buffer (0.178 M Tris, 0.178 M boric acid, 2 mM EDTA, pH 8.3 containing 2% SDS, 2 M urea, 10% sucrose, 0.1 M mercaptoethanol) and heated for 10 min at 60”. Alternatively, purified virus in VB was mixed 1:l with dissociation buffer and heated for 10 min at 60” (Hull and Lane, 1973). Gels were loaded with 5 to 20 pg of RNA, subjected to electrophoresis for 2.5 hr with 80 V and then scanned at 260 nm or stained with toluidine blue 0 (Gonsalves and Shepherd, 1972). RNA was eluted from gel slices by shaking with buffer G containing 0.1% SDS for 12 hr at 4” (Gonsalves and Shepherd,
PEMV APHID TRANSMISSION
3
fied Tii or P-3 was injected into aphids 44 to 90% transmission was obtained but no transmission with Wt was obtained. Tii and Wt were selected as representatives of each type for more extensive investigation as the yield of purified virus was much higher (54 and 10 mg per 100 g of starting material) than the P-3 strain (1.4 mg per 100 g of material). No marked differences were observed between aphid-transmissible and non-aphidtransmissible isolates in sedimentation behavior during analytical or sucrose density gradient centrifugation. However, when purified virus was subjected to gel electrophoresis remarkably different types of electrophoretic patterns were obtained (Fig. 1). Wt separated into two homogeneous components as described by Hull and Lane (1973), a slower migrating prominent component and a faster migrating lesser component (Fig. la). In contrast, Tii (Fig. lb) and P-3 (Fig. lc), two aphid-transmissible strains, showed at least nine bands migrating slower than the major Wt component plus a minor band which migrated at the same rate as Wt lesser component. In addition, both aphid-transmissible strains showed several minor bands in between the two major components. Since the multiplicity of components during gel electrophoresis appeared to be related to aphid transmissibility and might provide a clue as to the mechanism of aphid transmission, the properties of Tii and Wt were investigated further. To determine whether the multiple banding in gels was caused by pH differences during isolation or gel electrophoresis, the effect of various pH modifications was evaluated. PEMV-Tu fractionated at pH 4.4 instead of pH 6.0 showed no significant differences in electrophoretic behavior. Gel RESULTS electrophoresis at pH values of 3.8,4.4, and In the initial phases of the investigation, 5.0 with gels cast at pH 4.4 showed the involving only three strains of PEMV, Tii, same type of multiplicity banding in the gels and P-3 were found to be aphid-transmis- although the resolution between bands was sible with about equal efficiency whereas most distinct at pH 3.8. The results indiWt was not transmissible by aphids. In ex- cated that the multiplicity phenomenon was periments repeated four times, Tii and P-3 a characteristic of the virus strains rather were transmitted by pea aphids to 27 to than an artifact of preparation or electro54% of the plants; Wt was never trans- phoresis. mitted in any of the four trials. When puriBoth Tii and Wt consisted of two centrif-
1972) followed by removal of acryhunide residue by filtration and centrifugation and precipitation of RNA with ethanol. Molecular weight estimations of RNA, as described by Loening (1969) were done using Esc~tichia coli ribosomal RNA (1.07 and 0.56 x lo6 daltons) and turnip yellow mosaic virus RNA (2 x lo6 daltons) and slab gel electrophoresis. Some experiments were done with formamide as solvent (Grierson and Hemleben, 1977). In this case RNA, precipitated with ethanol and dried in air, was dissolved in 95% buffered formamide (pH 9.0) containing 10% sucrose and heated at 60” for 2 min before loading 5 to 10 pg on each gel. Gel electrophoresis of PEMV coat protein. The coat protein was prepared by heating purified PEMV for 10 min in dissociation buffer at 60” (Hull and Lane, 1973). The resulting mixture was separated on 10% polyacrylamide gels (5% crosslinked) in Plexiglas tubes (12 cm, 6 mm diameter) at 20” with 20 V/cm using the buffer system of Peacock and Dingman (1967) at double strength. The gels, loaded with 10 to 30 pg of protein, were stained with Coomassie blue after electrophoresis. The molecular weight of the coat protein components was determined after simultaneous separation on slab gels of PEMV coat protein and marker proteins of bovine serum albumin (BSA), ovalbumin (OVA), chymotrypsinogen (CHY), and cytochrome c (CYT) with molecular weights of 68, 43, 25, and 12.5 x 103, respectively. Electrophoresis was terminated when the bromphenol blue front reached the gel bottom. The molecular weights were calculated according to Weber and Osborn (1969).
4
ADAM, SANDER, AND SHEPHERD
a
FIG. 1. Electrophoretic patterns of virions of several PEMV strains after electrophoresis on 3.4% polyacrylamide gels: (a) Wt; (b) Tii; and (c) P-3. Thirty micrograms of purified virus was applied on each gel. Electrophoresis was carried out at 4”, pH 4.4, and 16 V/cm per gel for 16 hr.
ugal components as revealed by the sedimentation experiments. Hence, it was desirable to relate centrifugal to gel electrophoresis components. For these experiments Tii was subjected to three consecutive cycles of sucrose density gradient centrifugation and fractionation for-separation of its centrifugal components (Fig. 2). Thereafter the top component sedimented as a single band (Fig. 2e) whereas the bottom component was still slightly contaminated with top component and aggregation products (Fig. 2d). Analysis of such purified components in gels showed that the bottom centrifugal component was the slow component of gel electrophoresis and the top centrifugal component was the fast component in gels. To avoid confusion the gel components were called B and T to correspond to their identities during density gradient centrifugation. When the purified centrifugal components were subjected to electrophoretic separation on 3.4% gels and compared with unfractionated Tii and Wt (Fig. 3), the resulting scanning patterns revealed that the bottom component of Ti.i (Fig. 3b) was still contaminated with the top
component. Whether this contamination originated from a component mixture or aggregation products could not be determined since, when the aggregated product from the sucrose gradients was analyzed by gel electrophoresis, it contained all of the electrophoretically detectable bands. All of the latter bands, up to the one marked v in Fig. 3, which migrated as far as the major Wt bottom component, seem to belong to the Tti bottom component complex. The bottom centrifugal component of Ti.i consisted of 13 gel components, 9 forming a rather symmetrical series in the gels. The Tu top centrifugal component (Fig. 3d) was resolved into 10 to 11 gel components, of which the one marked V migrated as far as the Wt top centrifugal component. The electrophoretic patterns show that purified PEMV-Tti centrifugal components contain detectable amounts of the other heterologous component even though they sediment in a sucrose gradient as a single band. Biological activity of PEMV components. After separation on a sucrose density gradient, the infectivity of the centrifugal components of PEMV-Tii was deter-
PEMV APHID TRANSMISSION
5
d
I
bottom FIG. 2. Sedimentation patterns of PEMV-Tii components during three successive cycles of sucrose density gradient centrifugation. Virus or virus components were loaded onto lo-40% sucrose gradients and centrifuged in a Spinco SW40 rotor at 39,090 rpm for 2.5 hr at 4’. The centrifuged gradients were monitored with an ISCO scanner and fractionator. The arrows indicate the positions at which fractions were collected for further analysis. (a) Unfractionated virus. (b) Bottom component collected from ilrst cycle gradients. This consisted of the fraction indicated by the arrows in (a) subjected to a second cycle of gradient centrifugation. (c) Top component from (a) subjected to a second cycle of gradient centrifugation. (d) Bottom component collected from second cycle gradient (arrows in (b)) subjected to a third cycle of gradient centrifugation. (e) Top component from (c) (arrows) subjected to a third cycle of gradient centrifugation.
mined on a local lesion host. Each component was diluted to 2.5 pg/ml and compared with the other on eight halfleaves. The separated top component was 5 times more infectious than the bottom centrifugal component and 1.3 times more infectious than a mixture of 1.25 ,ug/ml bottom and 1.25 pg/ml top component (Table 1). The mixture was almost as infectious as the unfractionated virus material. Different results were obtained for the
infectivity of T and B components separated by gel electrophoresis. Wt components were cut out of gels as indicated in Fig. 3a and eluted for the assay. The results (Table 2) show that when the gel components were compared with unfractionated virus, the virus was 7.9-fold more infectious than the T component, 3.5-fold more infectious than the Wt B component, and 1.9-fold more infectious than the mixture of the two components. The Ti.i components were excised
ADAM, SANDER, AND SHEPHERD TABLE 1 INFECTIVITY OF THE PURIFIED CENTRIFUGAL COMPONENTS OF PEMV-TfY
Test pairsb
Local lesions/ half-leaf”
Ratio of lesions/test p&S
T:B T:T + B B:T + B U:T + B
37:8 38:29 327 46:33
5:l 1.3:1 1:9 1.21
a Tii-components were puriiled by three successive cycles of sucrose gradient centrifugation (Fig. 2) and adjusted to a concentration of 2.5 &ml. The infectivity of each component separately and as mixtures was determined on opposite half-leaves of Chenopodium quinoa as indicated. b T = top component; B = bottom component; U = unfractionated PEMV-Tii, (T + B) = mixture of 1.25 pg of T and 1.26 pg of B per milliliter. c With each test pair, eight leaves on four different plants were inoculated. The tabulated values are the mean number of local lesions per half-leaf.
FIG. 3. Scanning patterns after gel electrophoresis of purified PEMV strains: (a) Unfractionated PEMVWt; (b) PEMV-Tii bottom centrifugal component tier three cycles of density gradient centrifugation (Fig. 2d); (c) untiactionated PEMV-Tii, and (d) PEMV-Tii top centrifugal component after three cycles of density gradient centrifugation (see Fig. 2e). The electrophoretic separation was performed on 3.4% gels according to conditions described in Fig. 1. The gels were stained with Coomassie blue, destained, and
from the gels in two ways as indicated by the symbols TI’ and BI’ or TI and BI below the scanning pattern in Fig. 3c. At first Tii B component was excised as indicated by BI. Since infectivity tests (Table 3) showed that a mixture of the components was generally less infectious than each component alone (Table 3, Expts 1-3) the B component was then excised as indicated by BI’. This led to an increased infectivity of the component mixture as compared to that of single components (Table 3, Expt 4). The infectivity of each of the components isolated by gel electrophoresis, according to patterns TI’ and BI’ (Fig. 3c), wasalways less than that of unfractionated virus. However, the infectivity increased two to three times when the components were mixed. Properties of PEMV-RNA. The properscanned at 546 nm. The markers (v) and (V) show the locations of bands in the PEMV-Tii pattern which migrate as far as the PEMV-Wt B and T components, respectively. The arrows at the top of a and c .mark the locations of material cut out for subsequent bioassay. These sites for slicing were selected after scanning the unstained gels at 254 nm.
7
PEMV APHID TRANSMISSION TABLE 2 INFECTIVITY OF THE COMWNENTS OF PEMV-WT EXTRACTED FROMPOLYACRYLAMIDEGELS”
Ratio of lesions for each comparisonb to test pair
Expt
Virus concentration Ocghl)
T:B
T:(T + B)
B:(T + B)
(T + B):U
1” 2c 36
10 10 20
1:1.8 1:l 1:1.5
1:8. kg.8 k2.3
1:2.3 1:1.7 1:1.5
1:5.6 1:5.4 1:1.7
4d
10
T:U
B:U
(T + B):U
1:7.9
k3.5
1:1.9
a The top (T) and bottom (B) components were excised from 3.4% gel slices as indicated by the arrows and the T and B symbols in Fig. 3a. These gel slices were extracted with VB containing 0.2% BSA. Component concentration was calculated from the virus concentration loaded on the gels and the scanning pattern taken after electrophoresis (254 nm). The mixture (T + B) was prepared from the single components according to their concentrations calculated from the scanning pattern. U = unfractionated PEMV-Wt; the concentration was determined spectrophotometrically. b Eight half-leaves were used for each comparison except in Experiment 4, where six were used. c Chenepodiuvz quinoa was used as the local lesion host. d Chenopodium amuranticolor was used as the local lesion host.
ties of viral RNA were investigated to determine if any differences in the RNA genome were related to aphid transmissibility
and the gel multiplicity phenomenon. However, it was found that the method of preparation of the RNAs affected the relative
TABLE 3 INFECTIVITY OF THE COMPONENTS OF PEMV-TO EXTRACTED FROMPOLYACRYLAMIDEGELSO
Expt
vims concentration WmD
T:B
T$T + B)
B:(T + B)
(T + B):U
1= 2c 3’ 46
10 10 5 10
1:l l.l:l 1:1.4 1:1.5
1:2 1.3:1 1:l.l 1:3.3
k1.2 2.31 1.6:l lz2.7
1:4.1 1:6.6 1:2.2 1:4.6
5d 6d
10 10
Ratio of lesions for each comparisonb to test pairs
T:U
B:U
(T + B):U
1:14.6 k12.6
k10.6 1:14
k4.7 1:2.5
a The top (T) and bottom (B) components were excised from 3.4% gels after electrophoresis. Gel slices were eluted with VB containing 0.2% BSA. Component concentration was calculated from the virus concentration loaded on the gels and the scanning pattern after separation (254 nm). The mixture (T + B) was prepared from the single components according to their relation from the scanning pattern. U = unfractionated PEMV-Tii; the concentration was determined.photometrically. The local lesion host was CJlerconodium quinoa. b The infectivity is expressed as the ratio of the mean number of local lesions of each test pair. Eight halfleaves were used for each comparison except for Experiments 5 and 6, where. six leaves were used. e The components were excised from the gels as indicated in Fig. 3c by TI and BI. d The components were excised from the gels as indicated in Fig. 3d by TI’ and BI’.
8
ADAM, SANDER, AND SHEPHERD
0
-
0
FIG. 4. Scanning patterns after gel electrophoresis of viral RNA isolated by different methods from un-
fractionated PEMV-Tii. Electrophoresis was done on 2.4% gels at 20”, and 8 V/cm per gel, pH 7.5 for 2.5 hr. The direction of migration is shown by the arrow. The unstained gels were scanned at 254 nn. Numbers 1 to 3 refer to RNA,, RNA,, and RNA,, respectively. (a) RNA isolated with phenol-SDS. Ethanol-precipitated RNA was redissolved in dissociation buffer and heated for 10 min at 60”. (b) RNA isolated by the urea-SDS method and treated as described in a. (c) Purified virus was heated 10 min at 60” with dissociation buffer prior to loading the gel.
amounts of the two major species (RNA, and RNA,) and whether or not the smallest species (RNA,) was present. When the RNAs of Tii were liberated from density gradient-purified top and bottom by heating in dissociation buffer, top contained almost exclusively RNA, and bottom contained RNA,. RNA, was not detected. These re-
sults are similar to those of Hull and Lane (19’73)with PEMV-Wt. When phenol-SDS was used for preparation of the RNAs of PEMV-Tii, RNA3 was present and there was less RNA, than RNA, (Fig. 4a). However, when urea-SDS was used (dissociation buffer) for degrading Tti, more RNA, than RNA, was obtained and RNA, was
9
PEMV APHID TRANSMISSION
absent (Fig. 4~). Generally the amount of RNA, obtained with urea-SDS was much less than that obtained with phenol-SDS (Fig. 4b). With PEMV-Wt the amount of RNA, obtained was always greater than RNA%; RNA, was rarely present regardless of the method of preparation. RNA from purified Tii bottom component showed two different electrophoretic patterns, depending on the method for releasing the RNA (Fig. 5). When the RNA was extracted with phenol-SDS it contained almost equal amounts of RNA, and RNA, and a large amount of RNA, (Fig. 5a). In contrast, the RNA liberated with dissociation buffer contained considerably more
0 FIG. 5. Ultraviolet
RNA1 than RNA, and much less RNA, (Fig. 5b). Results with Tii support the suggestion of German and dezoeten (1975) that some RNA, molecules may be cleaved by phenolSDS treatment to give pieces of about the same size as RNA2 and RNA,. Such cleavage might account for the comparatively large amounts of RNA, and the heterogeneous low molecular weight RNAs between RNA, and RNA, (Figs. 4a and b). The fact that the molecular weights of RNA, and RNA, sum to that of RNA, (Gonsalves and Shepherd, 19’72) supports this notion. In three trials the molecular weights
w
0
scanning patterns after gel electrophoresis of RNA from PEMV-Tii bottom component. The RNA was isolated from the bottom component obtained by three cycles of rate zonal centrifugation in sucrose density gradients (see Fig. Zd). (a) RNA isolated with phenol-SDS. The RNA was precipitated with ethanol, redissolved in dissociation buffer, heated for 10 min at W, and subjected to electrophoresis as described for Fig. 6. (b) RNA was liberated from the purified bottom component by heating with dissociation buffer for 10 min at 60” and then subjected to gel electrophoresis as described in Fig. 6.
10
ADAM, SANDER, AND SHEPHERD
0-
servation by indicating that the difference arose in the virus strain rather than in the conformation of the RNAs. Nature of the multiple banding phenomenon of aphid-bovwe PEW-Tii. Two methods were used to examine whether charge or size differences were responsible for the separation of virion components during gel electrophoresis. In one case the effects of different pH values on relative mobilities of the different gel components were evaluated. When the relative mobilities of various Tti B components were measured relative to the fifth component as mobility standard, no significant differences were detected for seven of the nine gel components at pH values of 3.8, 4.4, and 5.0. According to Radloff and Kaesberg (1973) constant relative mobility at different pH values indicates similar charge shifts for the various components. Hence, size differences must account for the separation of the different components. This was examined using gradient gels which have a progressively decreasing pore size. When PEMV-Tii was electrophoresed FIG. 6. Stained components after electrophoresis of into gradient gels at constant pH, where PEMV- and E. coli ribosomal-RNAon3.596 formamide charge effects are nullified, the multiple gels (15% crosslinked). The isolated RNAs were pre- banding characteristic of Tti B component persisted. At the two longest periods of cipitated with ethanol and sedimented, and the pellets were dried with sterile air. Dry samples were dissolved electrophoresis (‘72 and 78 hr) the separain 95% buffered formamide containing 10% sucrose and tion patterns were almost the same (Fig. heated for 2 min at 60”. Five micrograms of PEMV7), demonstrating that the different comRNA and 5 pg of E. coli ribosomal-RNA were loaded ponents had reached their limiting pore size on each gel. Electrophoresis was carried out for 3.5 hr in the gradient. The equilibrium separation at 15” in 0.1 M N%HPOa, pH 9. The gels were stained pattern (Fig. 7) resembled that obtained with toluidine blue 0. (a) PEMV-Tii RNA + E. coli with Tii B component in 3.4% gels. When RNA (16 and 23 S); (b) PEMV-Wt RNA + E. coli Wt B component was electrophoresed into RNA (16 and 23 S); (c) RNA from a PEMV-strain selected from PEMV-Tti after several mechanical pas- gradient gels, it migrated to an intermedisages in Pisum sativum which rendered it nontrans- ate position between the T components and missible, plus 16 and 23-S RNA from E. coli. 1 and 2 the Tii B components, suggesting it is sigindicate PEMV-RNA, and PEMV-RNA,. nificantly smaller than the latter. From these results it was concluded that (~10~) for the three RNA species were: the multiple banding of PEMV-Tti (B comusing water as solvent, RNA,, 1.69 (Tii) ponent) is due to particles of at least nine and 1.52 (Wt), RNA,, 1.35 (Tii) and 1.32 different size classes. Since the distance be(Wt), RNAB, 0.15 (Tii) and 0.28 (Wt); using tween each of the nine bands appeared idenformamide as solvent, RNA,, 1.77 (Tii) and tical! it was concluded that the differences 1.65 (Wt), RNA2, 1.44 (Tii) and 1.48 (Wt). in migration are due to a uniform increment A consistent difference was shown between in particle diameter for each gel component. The magnitude of the increment, i.e., the Tii and Wt in the molecular weight of RNA,. Results with formamide validated this ob- differences in particle diameter between
1 2-
23s 16s
abc
0
PEMV APHID TRANSMISSION
78h
J
!2 h /
FIG. 7. Ultraviolet scanning pattern of PEMV-Wt and PEMV-Tii nucleoproteins after 72 and 78 hr of electrophoresis on gradient gels. A mixture of equal amounts PEMV-Wt and PEMV-Tii nucleoproteins was loaded on a polyacrylamide gradient gel (2.5-108) and electrophoresed for 72 and 78 hr at 4”, pH 4.4, and 240 V, with the buffer circulated between the electrode chambers. The gel was stained with Coomassie blue, destained, and scanned at 546 nm. v, T&B component; *, Wt-B component; Ir, Tii- and Wt-T components; 1,2, components believed to be dimer bands of Wt bottom and top components, respectively, as indicated by experiments with Wt components alone.
two of the closely similar mobility species, eter of the fastest to be 29 nm. The mean was calculated from the diffusion coeffi- increment in diameter between particles of cients of the Wt monomer and dimer com- two neighboring gel components was calcuponents (Hull and Lane, 1973) and their dis- lated to be 0.54 nm. tance of migration. First-order regression If the incremental differences in particle analyses were calculated from which the dif- size of Tii B component were due to differfusion coefficients (D) for each of the nine ences in RNA content the particles should bands of Tii B component were obtained. show a difference in buoyant density. HowThe correlation coefficient ranged between ever, when unfractionated purified virus 0.96 and 0.99, indicating a close linear re- was centrifuged to equilibrium in CsCl, only lationship. By use of the equation 2r a single isopycnic component.of p = 1.43 gl = 4.258 x 10-13/D (Markham, 1962) the di- ml was obtained. Analysis of this component ameter of the slowest migrating component by gel electrophoresis revealed that it conwas estimated to be 34 nm and the diam- sisted of Tii B component only, with its char-
ADAM, SANDER, AND SHEPHERD
acteristic multiple banding behavior, T component having apparently been degraded by the CsCl. These experiments showed that differences in RNA content cannot account for the multiple banding phenomenon of Tti B component. PEMV-Wt showed a single density species in CsCl of p = 1.42. The analysis of the virus coat protein obtained by degradation of unfractionated virus with dissociation buffer as described for RNA extraction, carried out on 10% polyacrylamide gels, showed that the coat protein of Wt migrated as a single band with a molecular weight of about 17,000 daltons. In contrast, Tti protein showed two polypeptide bands, one with the same molecular weight as that of Wt coat protein and another of 56,000 daltons (Fig. 8). The second protein amounted to 3-5% of the main protein as calculated from the scanning patterns of the gels after staining with Coomassie blue. Characteristics of non-aphid-transmissible strains derived from aphid-transmissible ones. A non-aphid-transmissible variant of PEMV was sought by repeated sap transmissions of Tti. After seven mechanical passages of PEMV-Tti, an alteration of the electrophoretic pattern was observed. At the position of the Wt B component an additional electrophoretic component occurred which became more and more prominent during four further mechanical passages whereas the previously observed Ti.ispecific bands disappeared. After the last four mechanical transmissions, the virus was no longer aphid-transmissible. This newly selected virus strain was further analyzed with respect to the occurrence of the second coat protein and molecular weight of its RNA,. The results showed that the FIG. 8. Stained gels after electrophoresis of PEMV coat proteins, Puri6ed PEMV-Wt and PEMV-Tti (2 new strain lacked the second coat protein mg/ml) were mixed 1:l with dissociation buffer and and had an RNA, with a molecular weight heated for 10 min at 60’. Twenty micrograms of each 1.2 x lo5 daltons smaller than that of mixture was loaded on a 10% gel (5% crosslinked) and PEMV-Tti (Fig. 6). electrophoresed at 20”, 20 V/cm with 2x PeacockThe two PEMV strains of Harris et al. Dingman buffer containing 1% SDS until the front (19’75), the CAT strain being aphid-transreached the gel bottom. Gels were stained with Coomassie blue. (a) Wt coat protein. (b) Tii coat pro- missible and the CNT strain selected theretein. (c) Tii coat protein and the protein markers (10 from being non-aphid-transmissible, were pg each). BSA, bovine serum albumin; OVA, oval- compared by gel electrophoresis with the bumiq CHY, chymotrypsinogen A. (d) Wt coat protein virus strains we have investigated. The and the same marker proteins as in c. CAT strain coelectrophoresed with PEMV8
‘BSA OVA CHY
abed
e3
13
PEMV APHID TRANSMISSION
Tii whereas the CNT-strain behaved like our newly selected non-aphid-borne virus strain and PEMV-Wt. From these results it was concluded that the multiple banding phenomenon, and perhaps the second 56,000-dalton protein, is probably common to all aphid-transmissible PEMV strains. Hence, the second polypeptide is probably the gene product responsible for aphidtransmission of PEMV. DISCUSSION
The major differences observed in our study between aphid-transmissible and non-aphid-transmissible strains of PEMV consisted of a multiple banding in polyacrylamide gels of nucleoprotein components that were centrifugally homogeneous. This multiple banding occurred only with aphid - transmissible isolates; non - aphid transmissible isolates showed only the two electrophoretically homogeneous components described previously by Hull and Lane (1973). Our ability to isolate a non-aphid-transmissible strain of the virus by successive mechanical transmissions of an aphidborne strain and the lack of multiple electrophoretic zones in gels for this new isolate confirms the association of this phenomenon with aphid transmissibility. In a recent publication, Hull (1977) reported the same multiple banding for transmissible isolates of PEMV but reported no aphid transmission data, an essential element of such an investigation since the loss of aphid transmissibility has been reported to occur with some isolates after only three or four mechanical passages (Tsai and Bath, 1974). The main biochemical differences between strains of PEMV that appears to be associated with the multiplicity phenomenon are the occurrence of a second protein and a greater molecular weight of RNA, for virions of aphid-transmissible strains. RNA, of the Tii strain appears to be about 1.2 x lo5 daltons larger than that of RNA, of non-aphid-transmissible strains. This difference may be responsible for the slightly greater buoyant density of the bot-
tom component of Tti compared to that of wt. The second protein in bottom component virions of aphid-borne strains probably accounts for the size differences that lead to the multiple banding phenomenon. The addition of the 56,000-dalton protein probably occurs stepwise with distinct perturbations in the geometry of the virions leading to incremental increases in particle size. The loss of aphid transmissibility which occurs with successive mechanical transmissions of PEMV may be related to the loss of a particular segment of the virus genome which codes for the 54,000-dalton protein unique to aphid-borne strains. The reduced molecular weight for RNA, suggests that a deletion mutant has been selected from among native insect-borne strains during sequential mechanical transmissions of the virus. Although experiments have not been done to establish whether RNA, or RNA, contains the genetic information for aphid transmissibility, the apparent deletion of a portion of RNA, suggests that this portion of the genome specifies insect transmission. Polypeptides of 28,000 and 58,000 daltons have been reported by Hull (1977) to be associated with aphid-transmitted strains of PEMV. Although the larger of these two polypeptides was found in our investigation, we never obtained a protein of 23,000 daltons from either aphid-transmitted or nonaphid-transmitted strains that were used in this investigation. ACKNOWLEDGMENTS We wish to thank K. F. Harris, M. Hollings, and R. Hull for supplying us with virus strains. It is a pleasure for the senior author to acknowledge support from the Graduierten Forderung of the German Federal Republic during that portion of the investigation done at the University of California, Davis. REFERENCES ADAM, G., and SANDER, E. (1976). Isolation and culture of aphid cells for the assay of insect-transmitted plant viruses. Virology 70, 502-508. CLARKE, D. L. D., and BATH, J. E. (1977). Serological properties of aphid- and non aphid-transmissible pea
14
ADAM, SANDER, AND SHEPHERD
em&ion mosaic virus isolates. Phytopathology 67, 1935- 1946. GERMAN, T. L., and DEZOETEN, G. A. (1975). Purification and properties of the replicative intermediates of pea enation mosaic virus. Virology 66, 17% 184. GONSALVES,D., and SHEPHERD,R. J. (1972). Biological and physical properties of the two nucleoprotein components of pea enation mosaic virus and their associated nucleic acids. Virology 48, 709-722. GRIERSON,D., and HEMLEBEN, V. (1977). Ribonucleic acid from the higher plant Matthiola incano. Molecular weight measurements and DNA.RNA hybridization studies. Biochim. Biophys. Acta 475, 424-436. HARRIS, K. F., BATH, J. E., THOTTAPPILLY, G., and HOOPER,G. R. (1975). Fate of pea enation mosaic virus in PEMV-injected pea aphids. Virology 65, 148-162. HULL, R. (1977). Particle differences related to aphidtransmissibility of a plant virus. J. Gen. Virology 34, 183-187. HULL, R., and LANE, L. C. (1973). The unusual nature of the components of a strain of pea enation mosaic virus. Virology 55, 1-13.
LOENING, U. E-. (l&%9). The de&mination’oi the molecular weightofribonucleic acid by polyacrylamidegel electrophoresis. &o&m. J. 113, 131-138. MAHMOOD,K., and PETERS, D. (1973). Purification of pea enation mosaic virus and the infectivity of its components. NetL J. Plant. Pathol. 79, 138-147. MARKHAM, R. (1962). The analytic+. ultracentrifuge as a tool for the investigation of plant viruaes. Adv. Virus Ree. 9, 241-276. PEACOCK,R C., and DINGMAN, C. W. (1967). Resolution of multiple ribonucleic acid species by polyacrylamide gel electrophoresis. Bioehemietry 6, 1818-1827. RADLOFF, R. J., and KAESBERG, P. (1973). Electrophoretic and other properties of bacteriophage Q/3: The effect of a variable number of read-through proteins. J. Virology 11, 116-128. TSAI, J. H., and BATH, J. E. (1974). The loss of transmissibility of two pea enation mosaic virus isolates by the pea aphid, Amythoeiphon pisurn (Harris). Proc. Amer. Phytqathol. Sot. 1, 115-116. WEBER, K., and OSBORN,M. (1969). The reliability of molecular weight determinations by dodecyl-sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244,4496-4412.
92,
1- 14 (1979)
Structural Differences between Pea Enation Mosaic Virus Strains Affecting Transmissibility by Acyrthosiphon pisum (Harris) G. ADAM,* EVAMARIE
SANDER,* AND ROBERT J. SHEPHERD?’
*Institut fiir Biologie II, Universitit Tiibingen, D 74 Tiibingen, Auf der Morgenstelle 28, BRD, and TDepartment of Plant Pathology, University of California, Davis, California 95616 Accepted September 8, 1978 Aphid-transmissible and non-aphid-transmissible strains of pea enation mosaic virus were compared in biological and physical properties. When separated by electrophoresis into 3.4% polyacrylamide gels both centrifugal components of aphid-transmissible strains gave rise to multiple-banding, whereas non-aphid-transmissible strains migrated as two components without separation into multiple bands. The multiple banding phenomenon during gel electrophoresis may be caused by the association of different amounts of an additional protein of 56,666 daltons with the vu-ions, so that they migrate as different size classes. The mean increment between the diameters of any two size classes was determined to be 0.54 nm. The origin of this second coat protein may be accounted for by the molecular weight of the largest viral RNA from a transmissible strain being 1.2 x l(r daltons larger than that of the non-aphid-transmissible strains. A non-aphid-transmissible strain could be selected by continuous mechanical passages of an aphid-transmissible strain and resembled other non-aphid-transmissible strains in all characteristics tested, including the lack of the second protein associated with vu-ions. Hence, it is postulated that the second protein, unique to aphid-borne strains, is a gene product responsible for the insect transmission of PEMV. For both aphid-transmissible and non-aphid-transmissible strains of PEMV, examination of the RNA content of the two nucleoproteins largely confirmed the recent work of others on the multipartite nature of the virus genome. Evidence is presented for RNA, arising as a breakage product of RNA, during isolation with phenol-SDS. INTRODUCTION
ferences between the two types of strains. These differences are described herein.
Pea enation mosaic (PEMV) is a virus with two centrifugal components which can MATERIALS AND METHODS be transmitted mechanically and by several aphid species in a persistent manner. The Most experiments were done with three virus has been characterized extensively strains of PEMV, viz., Wt (wild-type) dephysically and chemically and, although scribed by Hull and Lane (1973), P-3 (Gonnon-aphid-transmissible strains have been salves and Shepherd, 1972); and Tii deselected (Tsai and Bath, 1974), until re- scribed by Adam and Sander (1976); some cently (Clarke and Bath, 1977; Harris et al., experiments utilized strains CAT and CNT 1975; Hull, 1977) no differences had been of Harris et al. (1975). First to third instars found between the aphid-transmissible and of three biotypes of pea aphids [Acyrthonon-aphid-transmissible types. In this in- siphon pisum (Harris)] reared on broad vestigation we have made an extensive com- bean were used for transmission tests. parison between the properties of PEMV Aphids were allowed to acquire virus by strains which are and are not transmissible feeding for 4 hr on infected plants or virus by aphids and have found some major dif- was injected into the hemocoele of CO,anesthesized aphids with a capillary tube us1 Author to whom reprint requests should be ing hydrostatic pressure. Single aphids addressed. were put onto test plants for a 7-day in1
0042-6822/79/01ooo1-14$~.~/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproductionin any form reserved.
2
ADAM,SANDER,ANDSHEPHERD
fection feeding period. Pisum sativum L. cv. Progress No. 9 was used as a host for propagation of virus and for aphid transmission trials. Virus purification. The method of Mahmood and Peters (1973).was used with slight modifications. Sucrose (5%) was added to the 0.2 M acetate medium for homogenizing leaves. After chloroform clarification, the pH precipitation step (1 hr at pH 5.2) was used to remove additional material followed immediately by precipitation with 6% polyethylene glycol (PEG) 6000-o. 1 M NaCl at pH 6.0. After two cycles of differential centrifugation the virus was stored in 0.1 M acetate, pH 6.0 containing 5% sucrose (VB). Before RNA isolation, the virus was centrifuged through a cushion of 15% sucrose (RNase-free). RNA isolation. RNA was isolated by either phenol-SDS or urea-SDS treatments followed by deproteinization with chloroform-isoamyl alcohol and storage in buffer G (0.04 M Tris, 0.2 M sodium acetate, 0.002 M EDTA, pH 7.5). When RNA was isolated by the SDSurea method, purified virus in VB (2-5 mg/ ml) was mixed with 0.1 vol of 10% SDS, 0.1 vol of 5 M urea, and 0.1 vol of 3 M acetate buffer, pH 5.5, and incubated for 5 min at 37”. Then 2.2 vol of a mixture of CHCl, and isoamyl alcohol (24:l) was added and the preparation was incubated for 15 min at 4” after shaking for 2 min at 23”. The emulsion was broken by centrifugation (2,500 g, 10 min, 0’) and the aqueous phase was treated again with the organic solvent mixture. The RNA was precipitated from the aqueous phase with ethanol. Rate zonal gradient centrifugation. The two components of each virus strain were separated by repeated centrifugation into sucrose gradients [lo-40% (w/v> sucrose in 0.1 M acetate buffer, pH 61 as described by Gonsalves and Shepherd (19’72). The separated components were concentrated between all gradient cycles by precipitation (10% PEG 6000, 0.05 M NaCl). Gradient centrifugation took place in SW27 or SW40 rotors (26,000 rpm, 4 hr or 39,000 rpm, 2.5 hr, both at 4°C) in a Spinco ultracentrifuge. Electrophoresis of intact virus particles.
Gels with a monomer concentration of 2.8, 3.0, and 3.4% acrylamide (5% crosslinked) were cast at pH 4.4 into glass tubes (10 cm long x 0.6 cm i.d.). Gradient gel slabs with a linear increase from 2.5 to 10% monomer concentration were made from two acrylamide solutions, viz., (a) 2.5% acrylamide with 3% sucrose and (b) 10% acrylamide with 10% sucrose. All monomer solutions were made with the electrophoresis buffer described by Hull and Lane (1973), 30 mM Tris, 3 mM Ca(OH), adjusted to pH 4.4 with lactic acid. The gels were stored at least 12 hr at 4” prior to use. Electrophoresis was carried out at 4” with 16 V/cm per gel at constant voltage, using 16 hr for 3.4% gels and 24 to 78 hr for gradient gels, both at pH 4.4. During electrophoresis the buffer was circulated between the buffer vessels. Virus bands in gels were located by scanning at 254 nm in a Gilford spectrophotometer or by staining with Coomassie blue (Weber and Osborn, 1969). To determine the serological properties of the virus bands, the gels were treated with antiserum against PEMV-Tii (titer l/512) at a dilution of 1:64 made with phosphate-buffered saline, pH 7.2. Excised virus bands were homogenized in VB containing 0.2% BSA and eluted by gentle shaking for 12 hr at 4”. After removal of acrylamide residue by lowspeed centrifugation, virus was precipitated with 10% PEG. Gel electrophoresis of RNA. Electrophoresis of RNA was done on 2.4% acrylamide gels (Gonsalves and Shepherd, 1972). Prior to electrophoresis the isolated RNA in buffer G was mixed 1:l with dissociation buffer (0.178 M Tris, 0.178 M boric acid, 2 mM EDTA, pH 8.3 containing 2% SDS, 2 M urea, 10% sucrose, 0.1 M mercaptoethanol) and heated for 10 min at 60”. Alternatively, purified virus in VB was mixed 1:l with dissociation buffer and heated for 10 min at 60” (Hull and Lane, 1973). Gels were loaded with 5 to 20 pg of RNA, subjected to electrophoresis for 2.5 hr with 80 V and then scanned at 260 nm or stained with toluidine blue 0 (Gonsalves and Shepherd, 1972). RNA was eluted from gel slices by shaking with buffer G containing 0.1% SDS for 12 hr at 4” (Gonsalves and Shepherd,
PEMV APHID TRANSMISSION
3
fied Tii or P-3 was injected into aphids 44 to 90% transmission was obtained but no transmission with Wt was obtained. Tii and Wt were selected as representatives of each type for more extensive investigation as the yield of purified virus was much higher (54 and 10 mg per 100 g of starting material) than the P-3 strain (1.4 mg per 100 g of material). No marked differences were observed between aphid-transmissible and non-aphidtransmissible isolates in sedimentation behavior during analytical or sucrose density gradient centrifugation. However, when purified virus was subjected to gel electrophoresis remarkably different types of electrophoretic patterns were obtained (Fig. 1). Wt separated into two homogeneous components as described by Hull and Lane (1973), a slower migrating prominent component and a faster migrating lesser component (Fig. la). In contrast, Tii (Fig. lb) and P-3 (Fig. lc), two aphid-transmissible strains, showed at least nine bands migrating slower than the major Wt component plus a minor band which migrated at the same rate as Wt lesser component. In addition, both aphid-transmissible strains showed several minor bands in between the two major components. Since the multiplicity of components during gel electrophoresis appeared to be related to aphid transmissibility and might provide a clue as to the mechanism of aphid transmission, the properties of Tii and Wt were investigated further. To determine whether the multiple banding in gels was caused by pH differences during isolation or gel electrophoresis, the effect of various pH modifications was evaluated. PEMV-Tu fractionated at pH 4.4 instead of pH 6.0 showed no significant differences in electrophoretic behavior. Gel RESULTS electrophoresis at pH values of 3.8,4.4, and In the initial phases of the investigation, 5.0 with gels cast at pH 4.4 showed the involving only three strains of PEMV, Tii, same type of multiplicity banding in the gels and P-3 were found to be aphid-transmis- although the resolution between bands was sible with about equal efficiency whereas most distinct at pH 3.8. The results indiWt was not transmissible by aphids. In ex- cated that the multiplicity phenomenon was periments repeated four times, Tii and P-3 a characteristic of the virus strains rather were transmitted by pea aphids to 27 to than an artifact of preparation or electro54% of the plants; Wt was never trans- phoresis. mitted in any of the four trials. When puriBoth Tii and Wt consisted of two centrif-
1972) followed by removal of acryhunide residue by filtration and centrifugation and precipitation of RNA with ethanol. Molecular weight estimations of RNA, as described by Loening (1969) were done using Esc~tichia coli ribosomal RNA (1.07 and 0.56 x lo6 daltons) and turnip yellow mosaic virus RNA (2 x lo6 daltons) and slab gel electrophoresis. Some experiments were done with formamide as solvent (Grierson and Hemleben, 1977). In this case RNA, precipitated with ethanol and dried in air, was dissolved in 95% buffered formamide (pH 9.0) containing 10% sucrose and heated at 60” for 2 min before loading 5 to 10 pg on each gel. Gel electrophoresis of PEMV coat protein. The coat protein was prepared by heating purified PEMV for 10 min in dissociation buffer at 60” (Hull and Lane, 1973). The resulting mixture was separated on 10% polyacrylamide gels (5% crosslinked) in Plexiglas tubes (12 cm, 6 mm diameter) at 20” with 20 V/cm using the buffer system of Peacock and Dingman (1967) at double strength. The gels, loaded with 10 to 30 pg of protein, were stained with Coomassie blue after electrophoresis. The molecular weight of the coat protein components was determined after simultaneous separation on slab gels of PEMV coat protein and marker proteins of bovine serum albumin (BSA), ovalbumin (OVA), chymotrypsinogen (CHY), and cytochrome c (CYT) with molecular weights of 68, 43, 25, and 12.5 x 103, respectively. Electrophoresis was terminated when the bromphenol blue front reached the gel bottom. The molecular weights were calculated according to Weber and Osborn (1969).
4
ADAM, SANDER, AND SHEPHERD
a
FIG. 1. Electrophoretic patterns of virions of several PEMV strains after electrophoresis on 3.4% polyacrylamide gels: (a) Wt; (b) Tii; and (c) P-3. Thirty micrograms of purified virus was applied on each gel. Electrophoresis was carried out at 4”, pH 4.4, and 16 V/cm per gel for 16 hr.
ugal components as revealed by the sedimentation experiments. Hence, it was desirable to relate centrifugal to gel electrophoresis components. For these experiments Tii was subjected to three consecutive cycles of sucrose density gradient centrifugation and fractionation for-separation of its centrifugal components (Fig. 2). Thereafter the top component sedimented as a single band (Fig. 2e) whereas the bottom component was still slightly contaminated with top component and aggregation products (Fig. 2d). Analysis of such purified components in gels showed that the bottom centrifugal component was the slow component of gel electrophoresis and the top centrifugal component was the fast component in gels. To avoid confusion the gel components were called B and T to correspond to their identities during density gradient centrifugation. When the purified centrifugal components were subjected to electrophoretic separation on 3.4% gels and compared with unfractionated Tii and Wt (Fig. 3), the resulting scanning patterns revealed that the bottom component of Ti.i (Fig. 3b) was still contaminated with the top
component. Whether this contamination originated from a component mixture or aggregation products could not be determined since, when the aggregated product from the sucrose gradients was analyzed by gel electrophoresis, it contained all of the electrophoretically detectable bands. All of the latter bands, up to the one marked v in Fig. 3, which migrated as far as the major Wt bottom component, seem to belong to the Tti bottom component complex. The bottom centrifugal component of Ti.i consisted of 13 gel components, 9 forming a rather symmetrical series in the gels. The Tu top centrifugal component (Fig. 3d) was resolved into 10 to 11 gel components, of which the one marked V migrated as far as the Wt top centrifugal component. The electrophoretic patterns show that purified PEMV-Tti centrifugal components contain detectable amounts of the other heterologous component even though they sediment in a sucrose gradient as a single band. Biological activity of PEMV components. After separation on a sucrose density gradient, the infectivity of the centrifugal components of PEMV-Tii was deter-
PEMV APHID TRANSMISSION
5
d
I
bottom FIG. 2. Sedimentation patterns of PEMV-Tii components during three successive cycles of sucrose density gradient centrifugation. Virus or virus components were loaded onto lo-40% sucrose gradients and centrifuged in a Spinco SW40 rotor at 39,090 rpm for 2.5 hr at 4’. The centrifuged gradients were monitored with an ISCO scanner and fractionator. The arrows indicate the positions at which fractions were collected for further analysis. (a) Unfractionated virus. (b) Bottom component collected from ilrst cycle gradients. This consisted of the fraction indicated by the arrows in (a) subjected to a second cycle of gradient centrifugation. (c) Top component from (a) subjected to a second cycle of gradient centrifugation. (d) Bottom component collected from second cycle gradient (arrows in (b)) subjected to a third cycle of gradient centrifugation. (e) Top component from (c) (arrows) subjected to a third cycle of gradient centrifugation.
mined on a local lesion host. Each component was diluted to 2.5 pg/ml and compared with the other on eight halfleaves. The separated top component was 5 times more infectious than the bottom centrifugal component and 1.3 times more infectious than a mixture of 1.25 ,ug/ml bottom and 1.25 pg/ml top component (Table 1). The mixture was almost as infectious as the unfractionated virus material. Different results were obtained for the
infectivity of T and B components separated by gel electrophoresis. Wt components were cut out of gels as indicated in Fig. 3a and eluted for the assay. The results (Table 2) show that when the gel components were compared with unfractionated virus, the virus was 7.9-fold more infectious than the T component, 3.5-fold more infectious than the Wt B component, and 1.9-fold more infectious than the mixture of the two components. The Ti.i components were excised
ADAM, SANDER, AND SHEPHERD TABLE 1 INFECTIVITY OF THE PURIFIED CENTRIFUGAL COMPONENTS OF PEMV-TfY
Test pairsb
Local lesions/ half-leaf”
Ratio of lesions/test p&S
T:B T:T + B B:T + B U:T + B
37:8 38:29 327 46:33
5:l 1.3:1 1:9 1.21
a Tii-components were puriiled by three successive cycles of sucrose gradient centrifugation (Fig. 2) and adjusted to a concentration of 2.5 &ml. The infectivity of each component separately and as mixtures was determined on opposite half-leaves of Chenopodium quinoa as indicated. b T = top component; B = bottom component; U = unfractionated PEMV-Tii, (T + B) = mixture of 1.25 pg of T and 1.26 pg of B per milliliter. c With each test pair, eight leaves on four different plants were inoculated. The tabulated values are the mean number of local lesions per half-leaf.
FIG. 3. Scanning patterns after gel electrophoresis of purified PEMV strains: (a) Unfractionated PEMVWt; (b) PEMV-Tii bottom centrifugal component tier three cycles of density gradient centrifugation (Fig. 2d); (c) untiactionated PEMV-Tii, and (d) PEMV-Tii top centrifugal component after three cycles of density gradient centrifugation (see Fig. 2e). The electrophoretic separation was performed on 3.4% gels according to conditions described in Fig. 1. The gels were stained with Coomassie blue, destained, and
from the gels in two ways as indicated by the symbols TI’ and BI’ or TI and BI below the scanning pattern in Fig. 3c. At first Tii B component was excised as indicated by BI. Since infectivity tests (Table 3) showed that a mixture of the components was generally less infectious than each component alone (Table 3, Expts 1-3) the B component was then excised as indicated by BI’. This led to an increased infectivity of the component mixture as compared to that of single components (Table 3, Expt 4). The infectivity of each of the components isolated by gel electrophoresis, according to patterns TI’ and BI’ (Fig. 3c), wasalways less than that of unfractionated virus. However, the infectivity increased two to three times when the components were mixed. Properties of PEMV-RNA. The properscanned at 546 nm. The markers (v) and (V) show the locations of bands in the PEMV-Tii pattern which migrate as far as the PEMV-Wt B and T components, respectively. The arrows at the top of a and c .mark the locations of material cut out for subsequent bioassay. These sites for slicing were selected after scanning the unstained gels at 254 nm.
7
PEMV APHID TRANSMISSION TABLE 2 INFECTIVITY OF THE COMWNENTS OF PEMV-WT EXTRACTED FROMPOLYACRYLAMIDEGELS”
Ratio of lesions for each comparisonb to test pair
Expt
Virus concentration Ocghl)
T:B
T:(T + B)
B:(T + B)
(T + B):U
1” 2c 36
10 10 20
1:1.8 1:l 1:1.5
1:8. kg.8 k2.3
1:2.3 1:1.7 1:1.5
1:5.6 1:5.4 1:1.7
4d
10
T:U
B:U
(T + B):U
1:7.9
k3.5
1:1.9
a The top (T) and bottom (B) components were excised from 3.4% gel slices as indicated by the arrows and the T and B symbols in Fig. 3a. These gel slices were extracted with VB containing 0.2% BSA. Component concentration was calculated from the virus concentration loaded on the gels and the scanning pattern taken after electrophoresis (254 nm). The mixture (T + B) was prepared from the single components according to their concentrations calculated from the scanning pattern. U = unfractionated PEMV-Wt; the concentration was determined spectrophotometrically. b Eight half-leaves were used for each comparison except in Experiment 4, where six were used. c Chenepodiuvz quinoa was used as the local lesion host. d Chenopodium amuranticolor was used as the local lesion host.
ties of viral RNA were investigated to determine if any differences in the RNA genome were related to aphid transmissibility
and the gel multiplicity phenomenon. However, it was found that the method of preparation of the RNAs affected the relative
TABLE 3 INFECTIVITY OF THE COMPONENTS OF PEMV-TO EXTRACTED FROMPOLYACRYLAMIDEGELSO
Expt
vims concentration WmD
T:B
T$T + B)
B:(T + B)
(T + B):U
1= 2c 3’ 46
10 10 5 10
1:l l.l:l 1:1.4 1:1.5
1:2 1.3:1 1:l.l 1:3.3
k1.2 2.31 1.6:l lz2.7
1:4.1 1:6.6 1:2.2 1:4.6
5d 6d
10 10
Ratio of lesions for each comparisonb to test pairs
T:U
B:U
(T + B):U
1:14.6 k12.6
k10.6 1:14
k4.7 1:2.5
a The top (T) and bottom (B) components were excised from 3.4% gels after electrophoresis. Gel slices were eluted with VB containing 0.2% BSA. Component concentration was calculated from the virus concentration loaded on the gels and the scanning pattern after separation (254 nm). The mixture (T + B) was prepared from the single components according to their relation from the scanning pattern. U = unfractionated PEMV-Tii; the concentration was determined.photometrically. The local lesion host was CJlerconodium quinoa. b The infectivity is expressed as the ratio of the mean number of local lesions of each test pair. Eight halfleaves were used for each comparison except for Experiments 5 and 6, where. six leaves were used. e The components were excised from the gels as indicated in Fig. 3c by TI and BI. d The components were excised from the gels as indicated in Fig. 3d by TI’ and BI’.
8
ADAM, SANDER, AND SHEPHERD
0
-
0
FIG. 4. Scanning patterns after gel electrophoresis of viral RNA isolated by different methods from un-
fractionated PEMV-Tii. Electrophoresis was done on 2.4% gels at 20”, and 8 V/cm per gel, pH 7.5 for 2.5 hr. The direction of migration is shown by the arrow. The unstained gels were scanned at 254 nn. Numbers 1 to 3 refer to RNA,, RNA,, and RNA,, respectively. (a) RNA isolated with phenol-SDS. Ethanol-precipitated RNA was redissolved in dissociation buffer and heated for 10 min at 60”. (b) RNA isolated by the urea-SDS method and treated as described in a. (c) Purified virus was heated 10 min at 60” with dissociation buffer prior to loading the gel.
amounts of the two major species (RNA, and RNA,) and whether or not the smallest species (RNA,) was present. When the RNAs of Tii were liberated from density gradient-purified top and bottom by heating in dissociation buffer, top contained almost exclusively RNA, and bottom contained RNA,. RNA, was not detected. These re-
sults are similar to those of Hull and Lane (19’73)with PEMV-Wt. When phenol-SDS was used for preparation of the RNAs of PEMV-Tii, RNA3 was present and there was less RNA, than RNA, (Fig. 4a). However, when urea-SDS was used (dissociation buffer) for degrading Tti, more RNA, than RNA, was obtained and RNA, was
9
PEMV APHID TRANSMISSION
absent (Fig. 4~). Generally the amount of RNA, obtained with urea-SDS was much less than that obtained with phenol-SDS (Fig. 4b). With PEMV-Wt the amount of RNA, obtained was always greater than RNA%; RNA, was rarely present regardless of the method of preparation. RNA from purified Tii bottom component showed two different electrophoretic patterns, depending on the method for releasing the RNA (Fig. 5). When the RNA was extracted with phenol-SDS it contained almost equal amounts of RNA, and RNA, and a large amount of RNA, (Fig. 5a). In contrast, the RNA liberated with dissociation buffer contained considerably more
0 FIG. 5. Ultraviolet
RNA1 than RNA, and much less RNA, (Fig. 5b). Results with Tii support the suggestion of German and dezoeten (1975) that some RNA, molecules may be cleaved by phenolSDS treatment to give pieces of about the same size as RNA2 and RNA,. Such cleavage might account for the comparatively large amounts of RNA, and the heterogeneous low molecular weight RNAs between RNA, and RNA, (Figs. 4a and b). The fact that the molecular weights of RNA, and RNA, sum to that of RNA, (Gonsalves and Shepherd, 19’72) supports this notion. In three trials the molecular weights
w
0
scanning patterns after gel electrophoresis of RNA from PEMV-Tii bottom component. The RNA was isolated from the bottom component obtained by three cycles of rate zonal centrifugation in sucrose density gradients (see Fig. Zd). (a) RNA isolated with phenol-SDS. The RNA was precipitated with ethanol, redissolved in dissociation buffer, heated for 10 min at W, and subjected to electrophoresis as described for Fig. 6. (b) RNA was liberated from the purified bottom component by heating with dissociation buffer for 10 min at 60” and then subjected to gel electrophoresis as described in Fig. 6.
10
ADAM, SANDER, AND SHEPHERD
0-
servation by indicating that the difference arose in the virus strain rather than in the conformation of the RNAs. Nature of the multiple banding phenomenon of aphid-bovwe PEW-Tii. Two methods were used to examine whether charge or size differences were responsible for the separation of virion components during gel electrophoresis. In one case the effects of different pH values on relative mobilities of the different gel components were evaluated. When the relative mobilities of various Tti B components were measured relative to the fifth component as mobility standard, no significant differences were detected for seven of the nine gel components at pH values of 3.8, 4.4, and 5.0. According to Radloff and Kaesberg (1973) constant relative mobility at different pH values indicates similar charge shifts for the various components. Hence, size differences must account for the separation of the different components. This was examined using gradient gels which have a progressively decreasing pore size. When PEMV-Tii was electrophoresed FIG. 6. Stained components after electrophoresis of into gradient gels at constant pH, where PEMV- and E. coli ribosomal-RNAon3.596 formamide charge effects are nullified, the multiple gels (15% crosslinked). The isolated RNAs were pre- banding characteristic of Tti B component persisted. At the two longest periods of cipitated with ethanol and sedimented, and the pellets were dried with sterile air. Dry samples were dissolved electrophoresis (‘72 and 78 hr) the separain 95% buffered formamide containing 10% sucrose and tion patterns were almost the same (Fig. heated for 2 min at 60”. Five micrograms of PEMV7), demonstrating that the different comRNA and 5 pg of E. coli ribosomal-RNA were loaded ponents had reached their limiting pore size on each gel. Electrophoresis was carried out for 3.5 hr in the gradient. The equilibrium separation at 15” in 0.1 M N%HPOa, pH 9. The gels were stained pattern (Fig. 7) resembled that obtained with toluidine blue 0. (a) PEMV-Tii RNA + E. coli with Tii B component in 3.4% gels. When RNA (16 and 23 S); (b) PEMV-Wt RNA + E. coli Wt B component was electrophoresed into RNA (16 and 23 S); (c) RNA from a PEMV-strain selected from PEMV-Tti after several mechanical pas- gradient gels, it migrated to an intermedisages in Pisum sativum which rendered it nontrans- ate position between the T components and missible, plus 16 and 23-S RNA from E. coli. 1 and 2 the Tii B components, suggesting it is sigindicate PEMV-RNA, and PEMV-RNA,. nificantly smaller than the latter. From these results it was concluded that (~10~) for the three RNA species were: the multiple banding of PEMV-Tti (B comusing water as solvent, RNA,, 1.69 (Tii) ponent) is due to particles of at least nine and 1.52 (Wt), RNA,, 1.35 (Tii) and 1.32 different size classes. Since the distance be(Wt), RNAB, 0.15 (Tii) and 0.28 (Wt); using tween each of the nine bands appeared idenformamide as solvent, RNA,, 1.77 (Tii) and tical! it was concluded that the differences 1.65 (Wt), RNA2, 1.44 (Tii) and 1.48 (Wt). in migration are due to a uniform increment A consistent difference was shown between in particle diameter for each gel component. The magnitude of the increment, i.e., the Tii and Wt in the molecular weight of RNA,. Results with formamide validated this ob- differences in particle diameter between
1 2-
23s 16s
abc
0
PEMV APHID TRANSMISSION
78h
J
!2 h /
FIG. 7. Ultraviolet scanning pattern of PEMV-Wt and PEMV-Tii nucleoproteins after 72 and 78 hr of electrophoresis on gradient gels. A mixture of equal amounts PEMV-Wt and PEMV-Tii nucleoproteins was loaded on a polyacrylamide gradient gel (2.5-108) and electrophoresed for 72 and 78 hr at 4”, pH 4.4, and 240 V, with the buffer circulated between the electrode chambers. The gel was stained with Coomassie blue, destained, and scanned at 546 nm. v, T&B component; *, Wt-B component; Ir, Tii- and Wt-T components; 1,2, components believed to be dimer bands of Wt bottom and top components, respectively, as indicated by experiments with Wt components alone.
two of the closely similar mobility species, eter of the fastest to be 29 nm. The mean was calculated from the diffusion coeffi- increment in diameter between particles of cients of the Wt monomer and dimer com- two neighboring gel components was calcuponents (Hull and Lane, 1973) and their dis- lated to be 0.54 nm. tance of migration. First-order regression If the incremental differences in particle analyses were calculated from which the dif- size of Tii B component were due to differfusion coefficients (D) for each of the nine ences in RNA content the particles should bands of Tii B component were obtained. show a difference in buoyant density. HowThe correlation coefficient ranged between ever, when unfractionated purified virus 0.96 and 0.99, indicating a close linear re- was centrifuged to equilibrium in CsCl, only lationship. By use of the equation 2r a single isopycnic component.of p = 1.43 gl = 4.258 x 10-13/D (Markham, 1962) the di- ml was obtained. Analysis of this component ameter of the slowest migrating component by gel electrophoresis revealed that it conwas estimated to be 34 nm and the diam- sisted of Tii B component only, with its char-
ADAM, SANDER, AND SHEPHERD
acteristic multiple banding behavior, T component having apparently been degraded by the CsCl. These experiments showed that differences in RNA content cannot account for the multiple banding phenomenon of Tti B component. PEMV-Wt showed a single density species in CsCl of p = 1.42. The analysis of the virus coat protein obtained by degradation of unfractionated virus with dissociation buffer as described for RNA extraction, carried out on 10% polyacrylamide gels, showed that the coat protein of Wt migrated as a single band with a molecular weight of about 17,000 daltons. In contrast, Tti protein showed two polypeptide bands, one with the same molecular weight as that of Wt coat protein and another of 56,000 daltons (Fig. 8). The second protein amounted to 3-5% of the main protein as calculated from the scanning patterns of the gels after staining with Coomassie blue. Characteristics of non-aphid-transmissible strains derived from aphid-transmissible ones. A non-aphid-transmissible variant of PEMV was sought by repeated sap transmissions of Tti. After seven mechanical passages of PEMV-Tti, an alteration of the electrophoretic pattern was observed. At the position of the Wt B component an additional electrophoretic component occurred which became more and more prominent during four further mechanical passages whereas the previously observed Ti.ispecific bands disappeared. After the last four mechanical transmissions, the virus was no longer aphid-transmissible. This newly selected virus strain was further analyzed with respect to the occurrence of the second coat protein and molecular weight of its RNA,. The results showed that the FIG. 8. Stained gels after electrophoresis of PEMV coat proteins, Puri6ed PEMV-Wt and PEMV-Tti (2 new strain lacked the second coat protein mg/ml) were mixed 1:l with dissociation buffer and and had an RNA, with a molecular weight heated for 10 min at 60’. Twenty micrograms of each 1.2 x lo5 daltons smaller than that of mixture was loaded on a 10% gel (5% crosslinked) and PEMV-Tti (Fig. 6). electrophoresed at 20”, 20 V/cm with 2x PeacockThe two PEMV strains of Harris et al. Dingman buffer containing 1% SDS until the front (19’75), the CAT strain being aphid-transreached the gel bottom. Gels were stained with Coomassie blue. (a) Wt coat protein. (b) Tii coat pro- missible and the CNT strain selected theretein. (c) Tii coat protein and the protein markers (10 from being non-aphid-transmissible, were pg each). BSA, bovine serum albumin; OVA, oval- compared by gel electrophoresis with the bumiq CHY, chymotrypsinogen A. (d) Wt coat protein virus strains we have investigated. The and the same marker proteins as in c. CAT strain coelectrophoresed with PEMV8
‘BSA OVA CHY
abed
e3
13
PEMV APHID TRANSMISSION
Tii whereas the CNT-strain behaved like our newly selected non-aphid-borne virus strain and PEMV-Wt. From these results it was concluded that the multiple banding phenomenon, and perhaps the second 56,000-dalton protein, is probably common to all aphid-transmissible PEMV strains. Hence, the second polypeptide is probably the gene product responsible for aphidtransmission of PEMV. DISCUSSION
The major differences observed in our study between aphid-transmissible and non-aphid-transmissible strains of PEMV consisted of a multiple banding in polyacrylamide gels of nucleoprotein components that were centrifugally homogeneous. This multiple banding occurred only with aphid - transmissible isolates; non - aphid transmissible isolates showed only the two electrophoretically homogeneous components described previously by Hull and Lane (1973). Our ability to isolate a non-aphid-transmissible strain of the virus by successive mechanical transmissions of an aphidborne strain and the lack of multiple electrophoretic zones in gels for this new isolate confirms the association of this phenomenon with aphid transmissibility. In a recent publication, Hull (1977) reported the same multiple banding for transmissible isolates of PEMV but reported no aphid transmission data, an essential element of such an investigation since the loss of aphid transmissibility has been reported to occur with some isolates after only three or four mechanical passages (Tsai and Bath, 1974). The main biochemical differences between strains of PEMV that appears to be associated with the multiplicity phenomenon are the occurrence of a second protein and a greater molecular weight of RNA, for virions of aphid-transmissible strains. RNA, of the Tii strain appears to be about 1.2 x lo5 daltons larger than that of RNA, of non-aphid-transmissible strains. This difference may be responsible for the slightly greater buoyant density of the bot-
tom component of Tti compared to that of wt. The second protein in bottom component virions of aphid-borne strains probably accounts for the size differences that lead to the multiple banding phenomenon. The addition of the 56,000-dalton protein probably occurs stepwise with distinct perturbations in the geometry of the virions leading to incremental increases in particle size. The loss of aphid transmissibility which occurs with successive mechanical transmissions of PEMV may be related to the loss of a particular segment of the virus genome which codes for the 54,000-dalton protein unique to aphid-borne strains. The reduced molecular weight for RNA, suggests that a deletion mutant has been selected from among native insect-borne strains during sequential mechanical transmissions of the virus. Although experiments have not been done to establish whether RNA, or RNA, contains the genetic information for aphid transmissibility, the apparent deletion of a portion of RNA, suggests that this portion of the genome specifies insect transmission. Polypeptides of 28,000 and 58,000 daltons have been reported by Hull (1977) to be associated with aphid-transmitted strains of PEMV. Although the larger of these two polypeptides was found in our investigation, we never obtained a protein of 23,000 daltons from either aphid-transmitted or nonaphid-transmitted strains that were used in this investigation. ACKNOWLEDGMENTS We wish to thank K. F. Harris, M. Hollings, and R. Hull for supplying us with virus strains. It is a pleasure for the senior author to acknowledge support from the Graduierten Forderung of the German Federal Republic during that portion of the investigation done at the University of California, Davis. REFERENCES ADAM, G., and SANDER, E. (1976). Isolation and culture of aphid cells for the assay of insect-transmitted plant viruses. Virology 70, 502-508. CLARKE, D. L. D., and BATH, J. E. (1977). Serological properties of aphid- and non aphid-transmissible pea
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ADAM, SANDER, AND SHEPHERD
em&ion mosaic virus isolates. Phytopathology 67, 1935- 1946. GERMAN, T. L., and DEZOETEN, G. A. (1975). Purification and properties of the replicative intermediates of pea enation mosaic virus. Virology 66, 17% 184. GONSALVES,D., and SHEPHERD,R. J. (1972). Biological and physical properties of the two nucleoprotein components of pea enation mosaic virus and their associated nucleic acids. Virology 48, 709-722. GRIERSON,D., and HEMLEBEN, V. (1977). Ribonucleic acid from the higher plant Matthiola incano. Molecular weight measurements and DNA.RNA hybridization studies. Biochim. Biophys. Acta 475, 424-436. HARRIS, K. F., BATH, J. E., THOTTAPPILLY, G., and HOOPER,G. R. (1975). Fate of pea enation mosaic virus in PEMV-injected pea aphids. Virology 65, 148-162. HULL, R. (1977). Particle differences related to aphidtransmissibility of a plant virus. J. Gen. Virology 34, 183-187. HULL, R., and LANE, L. C. (1973). The unusual nature of the components of a strain of pea enation mosaic virus. Virology 55, 1-13.
LOENING, U. E-. (l&%9). The de&mination’oi the molecular weightofribonucleic acid by polyacrylamidegel electrophoresis. &o&m. J. 113, 131-138. MAHMOOD,K., and PETERS, D. (1973). Purification of pea enation mosaic virus and the infectivity of its components. NetL J. Plant. Pathol. 79, 138-147. MARKHAM, R. (1962). The analytic+. ultracentrifuge as a tool for the investigation of plant viruaes. Adv. Virus Ree. 9, 241-276. PEACOCK,R C., and DINGMAN, C. W. (1967). Resolution of multiple ribonucleic acid species by polyacrylamide gel electrophoresis. Bioehemietry 6, 1818-1827. RADLOFF, R. J., and KAESBERG, P. (1973). Electrophoretic and other properties of bacteriophage Q/3: The effect of a variable number of read-through proteins. J. Virology 11, 116-128. TSAI, J. H., and BATH, J. E. (1974). The loss of transmissibility of two pea enation mosaic virus isolates by the pea aphid, Amythoeiphon pisurn (Harris). Proc. Amer. Phytqathol. Sot. 1, 115-116. WEBER, K., and OSBORN,M. (1969). The reliability of molecular weight determinations by dodecyl-sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244,4496-4412.
