The relationship between soil-borne wheat mosaic virus and tobacco mosaic virus

VIROLOGY

7l,

453-462 (1976)

The Relationship

between Soil-Borne Tobacco Mosaic CHARLES

Department OfPlantPathology,

Wheat Mosaic Virus’

Virus and

A. POWELL’

Nebraska Agricultural Experiment Station, University ofNebraska, Nebraska 68503 Accepted January 21,1976

Lincoln,

The relationship between soil-borne wheat mosaic virus (SBWMV) and tobacco mosaic virus (TMV) was investigated by serology, gel electrophoresis, and mixed infection. The two viruses cross-reacted in microprecipitin, Ouchterlony agar double-diffusion, and sucrose density gradient centrifugation serological tests. The coat proteins of SBWMV and TMV migrated the same when treated with SDS and electrophoresed on 5.0, 7.5, or 10.0% polyacrylamide gels. The infectivity of TMV on Pinto bean was decreased by as much as 80% by the addition of SBWMV. From these results, it is concluded that SBWMV is related to TMV. INTRODUCTION

Soil-borne wheat mosaic virus (SBWMV) (McKinney, 1925) and tobacco mosaic virus (TMV) (Beijerinck, 1898) were both recognized as plant pathogens in the early 1900’s. The host range of SBWMV (Tsuchizaki et al., 1973) is relatively narrow compared to that of TMV. SBWMV is a problem only in wheat, while TMV causes economic losses in tobacco and tomato. United States’ strains of SBWMV will not infect tobacco or tomato, and TMV does not normally infect wheat. The only hosts common to these two viruses are local lesion hosts such as Chenopodium quinoa Willd. (Tsuchizaki et al., 1973). Despite the host range difference, the two viruses have some properties in common. TMV is a rigid rod 300~nm long (Caspar, 1963). SBWMV also has rigid rods, some of which are 300~nm long (Brakke et al., 1965; Brandes et al., 1964). However, SBWMV also has shorter rods whose ’ Published with the approval of the Director, as Paper No. 4028, Journal Series, Nebraska Agriculture Experiment Station. 2 Present address: Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin 53706.

lengths vary from 110 to 160 nm depending on the strain (Tsuchizaki et al., 1973). Finally, potato mop top virus is serologically related to both TMV (Kassanis et al., 1972) and SBWMV (Cooper and Harrison, 1972). These similarities suggest that SBWMV and TMV may be related. This hypothesis was tested by serology, electrophoresis of the viral coat proteins, and mixed infection studies. MATERIALS

Virus purification. The source of SBWMV used in this study was originally obtained from a naturally infected Nebraska wheat field and propagated in wheat in a growth chamber. Approximately 70 g of SBWMV-infected wheat leaves were cut into small pieces (about 2.5cm long), placed in 150 ml of 0.5 M sodium borate buffer, pH 9.0, and homogenized in a Waring Blendor. The resulting homogenate was strained through cheesecloth and centrifuged at 6000 rpm for 10 min in a Servall type SS 34 rotor. The supernatant fluid from the low speed centrifugation was collected and made 1% in Triton X-100. This solution was centrifuged at 28,000 rpm for 3 hr through a loml pad of 20% sucrose with 1% Triton X453

Copyright Q 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND METHODS

454

CHARLES

100 in a Spinco No. 30 rotor. The supernatant fluid was discarded, and the pellets were allowed to resuspend overnight in l5 ml of distilled water. This solution was filtered through Kleenex. The virus was concentrated by high speed centrifugation in a Spinco No. 30 or No. 40 rotor. For serological experiments, the virus was then centrifuged at 23,000 rpm in the SW 27 rotor for 1 hr on 75-300 mg sucrose/ml density gradients. The virus was removed from the gradients with a syringe and pelleted by centrifugation. The approximate yield of SBWMV was 0.3 mg of virus/g of leaf tissue. The type strain of TMV was purified in the same manner as SBWMV except that it was initially extracted from tobacco in 0.0075 M Na,HPO,. The yield of TMV was approx 1.5 mg of virus/g of leaf tissue. Serology. The serological relationships between SBWMV and TMV were determined with microprecipitin, Ouchterlony agar double-diffusion, and density gradient centrifugation tests. Microprecipitin tests were performed as described by Ball (1961). Antiserum endpoints were measured at a 1:2 antigen dilution, and antigen endpoints were measured at a 1:16 antiserum dilution. Unpurified virus in clarified plant extracts was used as the antigen source in these tests because it was less aggregated than purified virus. SBWMV, in particular, aggregates extensively when purified, and endpoints vary with the degree of aggregation. Reactions of TMV antigen (Ag) vs TMV antiserum (As), TMV Ag vs SBWMV As, TMV Ag vs normal serum, SBWMV Ag vs SBWMV As, SBWMV Ag vs TMV As, and SBWMV Ag vs normal serum, were tested in each of five buffers: phosphate buffered saline (PBS; 0.14 M NaCl, 0.01 M potassium phosphate, pH 7.0); one-tenth strength PBS; standard saline citrate @SC; 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0); KH2P04-Na2HP04, 0.2 M in POI, pH 7.0; and TPE [0.06 M Tris (Tris hydroxymethyl aminomethane), 0.06 M NaH,PO,, and 0.003 M EDTA (disodium ethylenediamine tetraacetic acid), pH 7.31. Control drops with only antigen, antiserum, or buffer were also prepared for each plate. Plastic petri plates, 9 cm in diameter,

A.

POWELL

for the Ouchterlony agar double-diffusion tests contained 0.75% Ionagar No. 25 in 0.25% sodium aside, with 4-mm diameter wells at the corners and center of a regular hexagon of 6-cm circumference. TMV and SBWMV dilutions were prepared in dissociation buffer, 0.02 Tris-HCl, 0.1 M NaCl, 0.01 M EDTA, 1.0% sodium dodecyl sulfate (SDS), and 100 pg of bentonite/ml, pH 9.0. Antisera were diluted with PBS. One drop of each virus or antiserum dilution was placed in the appropriate well. The plates were sealed with parafilm to prevent the agar from drying. After 48 hr at room temperature, the plates were examined for precipitin lines. Density gradient centrifugation (DGC) serological tests were performed using the method of Ball and Brakke (1969). Purified TMV was mixed with a given antiserum to obtain the desired concentration of virus and antiserum in 0.01 M neutral potassium phosphate buffer, pH 7.0. The test tubes containing these mixtures were shaken at room temperature for 1 hr and placed at 2” for 24 hr. After this incubation period, the samples were centrifuged in an SW 27 or SW 50.1 rotor on 75-300 mg sucrose/ml density gradients. For SW 27 gradients, 1.5 ml of 200 Fg of virus/ml were centrifuged at 23,000 rpm for 1.75 hr, and for SW 50.1 gradients 0.2 ml of 200 pug of virus/ml were centrifuged at 45,000 rpm for 20 min. The absorbance at 254 nm was plotted vs depth for each gradient with an ISCO fractionator. Peak areas were measured with a planimeter. SBWMV-specific antibody (Ab) was prepared by adding 0.1 ml of antiserum prepared against SBWMV to 0.9 ml of 3.2 mg SBWMV/ml in 0.01 M potassium phosphate buffer, pH 7.0. The solution was shaken at room temperature for 1 hr, placed at 2” overnight, and centrifuged at 8000 rpm for 20 min in a Servall type SS 34 rotor to pellet the precipitate. The pellet was resuspended in 0.01 M neutral phosphate buffer and recentrifuged. The washed pellet was dissolved in 1 ml of 0.1 M glycine-HCl buffer, pH 2.5, to dissociate the antigen-antibody complex. The virus was then removed by centrifugation for 2 hr at 28,000 rpm in a Spinco No. 30 rotor. The supernatant fluid now contained only

RELATIONSHIP

BETWEEN

antibody molecules which had reacted with SBWMV. The pH of the specific antibody solution was raised to 5.5 with 0.1 M K,HPO,. Five-tenths milliliter, approx half of the original SBWMV-specific antibodies, was mixed with 0.5 ml of 400 pg of TMV/ml, and the reaction was analyzed by the sucrose density gradient centrifugation method. SDS polyacrylamide gel electrophoresis. TMV protein was purified by the method of Fraenkel-Conrat (195’7). SBWMV protein was prepared with a virus disruption buffer. Purified SBWMV was allowed to stand overnight at 2” in a buffer consisting of 0.02 M Tris, 0.001 M EDTA, 3.2 mg of bentonite/ml, 1% SDS, and enough HCl to lower the pH to 9.0. The protein was used for gel electrophoresis immediately after preparation. Occasionally, TMV protein was also prepared by this method. Prior to electrophoresis, all protein solutions were made 1% in SDS. One-tenth milliliter of 2-mercaptoethanol was then added to 0.5 ml of protein solution. These mixtures were heated in a water bath at 50” for 1.5 hr to dissociate protein aggregates. Next, 0.01 ml of 1% bromophenol blue and 0.39 ml of 600 mg sucrose/ml were added to each protein solution, and O.l0.2-ml aliquots were layered on top of the gels. Acrylamide gels with 5% of the total acrylamide as bis-acrylamide in a buffer of 0.1 M sodium phosphate, 0.1% SDS, and 0.1% sodium thioglycollate, pH 7.2, were polymerized in Plexiglas cylinders with the aid of ammonium persulfate and TEMED (N, N,N’, N’-Tetramethylethylenediamine). Both ends of the gels were cut with a razor blade so that their final lengths were 9 cm, and they were prexun at 5 mA/gel to remove the initiators. After the samples were added, electrophoresis was performed at 6.25 mA/gel for various time periods in the previously described gel buffer. After electrophoresis was completed, the gels were stained for 1 hr in a solution consisting of 0.25 g of Coomassie brilliant blue per 100 ml of solvent. The solvent was 45.5% methanol, 45.5% water, and 9% acetic acid by vol. The gels were then destained electrophoretically in a solution of

SBWMV

AND

TMV

455

50% methanol, 46.5% water, and 3.5% acetic acid by vol. A pattern was obtained for each gel by scanning it at 570 nm with a Gilford gel scanner. Infectivity. TMV-SBWMV mixtures of 0.01 mg of TMV/ml and 0.2 mg of SBWMV/ml, 0.005 mg of TMV/ml and 0.1 mg of SBWMV/ml, and 0.001 mg of TMV/ ml and 0.02 mg of SBWMV/ml; TMV solutions of 0.01 mg/ml, 0.005 mg/ml, and 0.001 mg/ml; and TMV-TMV (VV strain) mixtures of 0.01 mg of TMV/ml and 0.2 mg of TMV 1VV strainjlml, 0.005 mg of TMVlml and 0.1 mg of TMV (VV strain)/ml, and 0.001 mg of TMV/ml and 0.02 mg of TMV (VV strainUrn were prepared in inoculation buffer. The VV strain of TMV does not produce local lesions on Pinto bean (Veerisetty, unpublished results). The inoculation buffer was 0.01 M potassium phosphate buffer, pH 8.0, with 1% Celite, and 1 mg of bentonite/ml. Primary leaves of loday-old Pinto bean plants were inoculated by rubbing with cheesecloth which had been dipped into virus solution. The three different virus solutions with the same concentration of TMV were rubbed on three half-leaves of the same plant. The fourth half-leaf was rubbed with virus-free inoculation buffer. Twenty plants were inoculated with each virus dilution. After all the plants were inoculated, the leaves were rinsed with water, and the growing tips were removed. Five days later, the local lesions were counted. The experiment was repeated twice. Infectivity experiments with naked TMV-RNA and SBWMV-RNA were performed similarly. The RNA solutions consisted of 1 Fg of TMV-RNA/ml and 20 pug of SBWMV-RNA, 1 pg of TMV-RNA/ml and 20 pg of yeast-RNA/ml, and a 1 Fg/ml solution of TMV-RNA. RESULTS

Microprecipitin tests (Table 1) show that both TMV and SBWMV reacted with the heterologous antisera, and are therefore serologically cross-related. Neither antiserum reacted with healthy plant extracts and neither virus reacted with normal serum, indicating that the antisera were specific for the viral antigens. Purified SBWMV and TMV were com-

456

CHARLES TABLE MICROPRECIPITIN

Buffer

Ag

1

TABLE

TEST RESULTS”

Reciprocal of Ar endpointsb

0.1 x PBS ssc PO, TPE

TMV SBWMV TMV SBWMV TMV SBWMV TMV SBWMV TMV SBWMV

OUCHTERLONY

kciprocal of Ag endpoints’ I’MV As

PBS

A. POWELL

16 6 64d 16 16 12 64” 8 64” 8

Ag

2

DOUBLE-DIFFUSION

RESULTS

Lowest Ag concentration reacting

Reciprocal As dilution

of highest reacting

TMV As

TMV As

SBWMV As

SBWMV As 4 16 12 32 12 32 4 32 8 64

AGAR

TMV SBWMV

SBWMV As

20 200

200 2

256 16

64 256

(I Tested at concentrations of 2000, 200, 20, and 2 pg of purified virus/ml against undiluted antiserum. b Tested at fourfold dilutions against antigen at 2 mg of purified virus/ml.

a Neither antisera reacted with healthy plant extracts. Neither virus reacted with normal serum. b The endpoints were based on the reaction with fourfold antiserum dilutions and are the highest dilution reacting. The antigens were in clarified extracts of infected plants diluted 1:2. The results presented are from one representative test. r The endpoints are reciprocals of the highest antigen dilution reacting with a 1:16 antiserum dilution. The antigens were in clarified extracts of infected plants. Twofold dilutions of antigen, based on volume of clarified sap, were tested. d Highest dilution tested.

pared by Ouchterlony agar double-diffusion tests. Since purified SBWMV aggregates and does not diffuse into agar, both viruses were disrupted with SDS before being placed in the wells. The agar, however, did not contain SDS. Serial dilutions of both viruses were tested against both antisera, and vice versa (Table 2). The Ouchterlony tests confirmed the relationship between SBWMV and TMV. There was no reaction of either virus with normal serum, healthy plant sap, or disruption buffer. When SBWMV and TMV in adjacent wells reacted with either SBWMV or TMV antiserum, spurs were formed, which shows the serological crossreaction is not one of identity. Additional serological experiments were performed with sucrose density gradients. Figure 1 shows the resulting DGC uv-absorbance patterns when 300 pg of TMV were treated with various concentrations of SBWMV As. The areas under the peaks are given in Table 3. These patterns again

0.2 0.1

0 12

IO

8 DEPTH

6

4

2

b-d

FIG. 1. DGC uv-absorbance patterns of 1.5 ml of 200 fig TMV/ml centrifuged at 23,000 rpm for 1.75 hr in an SW 27 rotor. (A) Untreated TMV; (B) TMV treated with a 1:16 dilution of SBWMV As; (C) TMV treated with a 1:8 dilution of SBWMV As; (D) TMV treated with a 1:4 dilution of SBWMV As; (E) TMV treated with a 1:2 dilution of normal serum.

RELATIONSHIP TABLE AREAS

OF PEAKS SHOWN

Figure

1A 1B 1c 1D 1E 2A 2B 2c 2D 3A 3B 3c 3D

IN FIGURES

Percentage of TMV removed

0.22 0.18 0.07 0.00 0.25

18 68 100 0

0.09 0.00 0.03 0.07 0.08 0.02

100 67 22 75

0.09

0

0.10

0

Untreated TMV TMV + 1:16 SBWMV As TMV + 1:8 SBWMV As TMV + 1:4 SBWMV As TMV + 1:2 normal serum Untreated TMV TMV + 1:lO TMV As TMV + 1:lOO TMV As TMV + 1:lOOO TMV As Untreated TMV TMV + SBWMV specific Ab TMV + SBWMV Ag-Ab complex TMV + glycine-HClPO, legends

for

l-3

Area (sq. in.)

complete

description

SBWMV

AND

457

TMV

but which would not react with SBWMV, were detected in a similar experiment. Ten milligrams of purified SBWMV were pelleted by centrifugation and resuspended in a small volume of antiserum prepared against TMV. The precipitate was removed by centrifugation, and the supernatant was used to resuspend another SBWMV pellet. The procedure was repeated until no further precipitate was visible. The SBWMV antibody-free supernatant fluid was then reacted with TMV, and the disappearance of virus was monitored by sucrose density gradient centrifugation. The TMV zone was completely removed, showing that antiserum prepared against TMV contained antibody molecules which reacted with TMV, but not SBWMV.

3

Treatment”

(1 See figure treatments.

BETWEEN

of

confirm that SBWMV As reacts with TMV. Normal serum broadened the TMV zone (Fig. lE), but did not remove any TMV. TMV was similarly tested with various dilutions of TMV As. Figure 2 shows the resulting DGC uv-absorbance patterns, and Table 3 shows the peak areas. The specificity of the heterologous reaction between TMV and SBWMV As was confirmed using antibody molecules which had reacted with SBWMV and then incubated with TMV (see Materials and Methods). Three controls were used. One sample of TMV was not treated, a second sample of TMV was treated with the undissociated antigen-antibody complex, and a third sample of TMV was treated with 0.1 M glycine-HCl-K,HPO, buffer, pH 5.5. The same antibody molecules which reacted with SBWMV also reacted with TMV (Fig. 3B, Table 3). TMV was not removed by treatment with the undissociated SBWMV Ag-SBWMV As complex (Fig. 3C, Table 31, nor by the partially neutralized glycine-HCl buffer (Fig. 3D, Table 3), although the zone was broadened in the latter case. Antibodies which were specific for TMV,

2 E ?I N U W 0

0.3

-

0.2

-

0.1

-

I B

I

I

I

L

z 2 2

D

2

0.3

-

‘;I 5

0.2

-

$

0.1

*

0

DEPTH

I

I

4

2

(cm)

FIG. 2. DGC uv-absorbance patterns of 0.2 ml of 200 kg TMV/ml centrifuged at 45,000 rpm for 20 min in an SW 50.1 rotor. (A) Untreated TMV; (B) TMV treated with a 1:lO dilution of TMV As; (C) TMV treated with a 1:lOO dilution of TMV As; (D) TMV treated with a 1:lOOO dilution of TMV As.

458

CHARLES

I

I

I

I

4

2

4

2

DEPTH

(cm)

3. DGC uv-absorbance patterns of 0.2 ml of 200 pg TMV/ml centrifuged at 45,000 rpm for 20 min in an SW 50.1 rotor. (A) Untreated TMV; (B) TMV treated with SBWMV specific antibody; (C) TMV treated with the undissociated SBWMV AgSBWMV Ab complex; (D) TMV treated with 0.1 M glycine-HCl-K,HPO, buffer, pH 5.5. FIG.

The apparent molecular weight of the coat protein of SBWMV was determined by electrophoresis of SBWMV protein and TMV protein on the same 5, 7.5, and 10% polyacrylamide gels. The two proteins corn&rated at all three gel concentrations. The scans of the 5% gels are presented in Fig. 4. In addition, TMV protein, SBWMV protein, ovalbumin, and ribonuclease A were electrophoresed on separate gels at gel concentrations of 5, 7.5, and 10%. The relative mobility (RF) for each protein at each gel concentration was calculated by dividing the migration of the protein by the migration of bromophenol blue. A plot of log RF vs total gel concentration for each protein is presented in Fig. 5. The slope of each line is the retardation coefficient for that protein, and is proportional to the

A.

POWELL

molecular weight (Rodbard and Chrambath, 1971). The slopes of the lines for SBWMV and TMV are slightly different, indicating that the molecular weight of the coat protein of SBWMV may be slightly higher than the molecular weight of the coat protein of TMV. TMV coat protein prepared by the method of Fraenkel-Conrat (1957) had the same relative mobility as TMV coat protein prepared with the disruption buffer (see Materials and Methods). SBWMV has a low specific infectivity compared to TMV. One possible explanation for this low specific infectivity is that the 1 x 10” molecular weight RNA from the short particle is a defective RNA which suppresses the infectivity of the 2 x 10” molecular weight RNA. If this is true, and if TMV and SBWMV are indeed related, then the 1.0 x 10” RNA of SBWMV might also suppress the infectivity of TMV or its RNA. This proved to be the case. Three dilutions each of TMV, a TMV-SBWMV mixture, and a TMV-TMV (strain VV) mixture were rubbed on the half-leaves of Pinto bean plants. The ratio of SBWMV to TMV and TMV (strain VV) to TMV was 20 to 1. This ratio was chosen because the ratio of SBWMV short RNA to SBWMV long RNA is approx 20 to 1. Addition of 0.6

0

2

4

2 MIGRATION

4

2

4

(cm)

FIG. 4. SDS polyacrylamide gel electrophoresis scan of protein electrophoresed for 1.5 hr at 6.25 mA/ gel in 5% gels. (A) TMV protein; (B) SBWMV protein; (C) TMV protein and SBWMV protein.

RELATIONSHIP

BETWEEN

SBWMV

AND

459

TMV

decreased the local lesion production of TMV-RNA (Table 5). The control mixed infection with yeast-RNA increased TMVRNA local lesion production, although this increase was not statistically significant.

-0.6

DISCUSSION

By four criteria it is concluded that SBWMV is related to TMV. First,

-0.7

TABLE

IL

a

-0.8

INFECTIVITY OF TMV, AND TMV-TMV-VV” MEASURED IN

MIXTURES MIXTURES ON PINTO BEANS LOCAL LESIONS (LL)

Virus concentration

5 TOTAL

7.5 GEL

IO

CONCENTRATION

FIG. 5. Graph of log relative mobility vs total gel concentration for various proteins. The relative mobility is the mobility of a certain protein at a specific gel concentration compared to the mobility of bromophenol blue at the same gel concentration. The log relative mobilities are the averages from three separate experiments. (A) TMV coat protein; (B) SBWMV coat protein; (Cl ovalbumin; (D) ribonuclease A.

SBWMV significantly decreased the number of local lesions produced by TMV on Pinto bean (Table 4). Addition of TMV (strain VV) to TMV also decreased the number of local lesions, although this reduction was not as great as with SBWMV. Inhibition was greatest at the higher virus concentrations. The ability of SBWMV-RNA to decrease the infectivity of TMV-RNA was also examined. TMV-RNA, a mixture of TMVRNA and short SBWMV-RNA, and a mixture of TMV-RNA and Torula yeast-RNA were rubbed on the half-leaves of Pinto bean plants. The ratio of SBWMV-RNA to TMV-RNA and yeast-RNA to TMV-RNA was 20 to 1. SBWMV-RNA significantly

4

TMV-SBWMV

Average LL/halfIear

10 pg TMV/ml 5 pg TMV/ml 1 pg TMV/ml 10 Fg TMV + 200 pg SB WMV/ml 5 pg TMV + 100 pg SBWMV/ml 1 fig TMV + 20 Fg SBWMV/ ml 10 pg TMV + 200 pg TMVVV/ml 5 fig TMV + 100 Fg TMVVV/ml 1 /*g TMV + 20 fig TMVVV/ml

Percentage reduction in infectivity

105 60 10 20

80

19

68

8

20

49

53

39

35

8

20

(1 A strain of TMV which gives no LL on Pinto bean. b Average was based on the counts from 40 different half-leaves. TABLE

5

INFECTIVITY OF TMV RNA, TMV RNA-SBWMV RNA” MIXTURES AND TMV RNA-YEAST RNA MIXTURES

ON PINTO BEANS MEASURED LESIONS (LL)

RNA preparation

1 pg TMV RNA/ml 1 fig TMV RNA SBWMV RNA”/ml 1 PcLg TMV RNA yeast RNA/ml

Average LL/halfleafb

IN

LOCAL

Percentage infectivity compared to TMV RNA

+

20 fig

13 3

100 23

+

20 pg

17

130

’ Only RNA from the short SBWMV component was used. b Average was based on the counts from 40 different half-leaves.

460

CHARLES

SBWMV is serologically related to TMV. This was confirmed by microprecipitin, Ouchterlony agar double-diffusion, and sucrose density gradient centrifugation tests. In the microprecipitin tests, the homologous endpoints were from 2 to 32 times higher than the heterologous endpoints. In the Ouchterlony tests, the homologous antigen endpoints were between 10 and 100 times higher than the heterologous endpoints, whereas the difference in antisera endpoints was 18fold. In sucrose density gradient tests, approx 12 times more SBWMV As than TMV As was required to remove two-thirds of the TMV zone (Table 31, assuming the two antisera had equal titers (Table 2). The reciprocal density gradient tests were not feasible because SBWMV aggregates too much to band in sucrose density gradients. Crossreactions could not have resulted from cross-contamination of the antisera because the specific antibody test showed that the same antibody molecules which reacted with SBWMV could also react with TMV (Fig. 3). The serological relationship between SBWMV and TMV is not as close as for some TMV strains, but it is closer than the relationships reported for others. For example, the Ni 118 strain is serologically identical to the type strain of TMV (von Sengbusch and Wittmann, 19651, the Rosette strain is about as closely related to the type strain of TMV (Ball, 1966) as SBWMV is related to TMV, and the relationship of the cowpea strain of TMV to the type strain (Bawden, 1958) is not as close as between SBWMV and TMV. Under certain conditions, two unrelated viruses can apparently cross-react in serological tests. For example, antiserum prepared against TMV, a rod-shaped virus, will cross-react with cocksfoot mild mosaic virus, an icosahedral virus, and vice versa (Bercks and Querfurth, 1971). However, the homologous endpoints are approx 200 times higher than the heterologous endpoints, suggesting a much more distant relationship than that of SBWMV to TMV. A second criterion is particle morphology. Both SBWMV and TMV are rigid

A. POWELL

rods which look identical in the electron microscope except that most SBWMV rods are shorter than TMV rods. However, some SBWMV rods contain RNA whose molecular weight is 1.84 x 10” as determined by sedimentation of formaldehydetreated RNA (Gumpf, 1971), or 2.10 x 10” as determined by gel electrophoresis of formaldehyde-treated RNA (Brakke, personal communication). These molecular weights are close to the molecular weight of TMV-RNA and suggest that SBWMV has some rods whose monomeric lengths are approx 300 nm. A third criterion for concluding that SBWMV is related to TMV is that the molecular weights of their coat proteins are similar. This should be true for related viruses because most mutations resulting in nonlethal alterations in the coat protein will not appreciably change the molecular weight. A fourth criterion indicating that SBWMV is related to TMV is competition during infection. The addition of SBWMV or its RNA decreased the infectivity of TMV (Tables 4 and 5). This viewpoint is strengthened by the observation that TMV (strain VV), a strain of TMV that does not infect Pinto bean, also decreased TMV local lesion production; and yeast RNA, which is unrelated to TMV-RNA, did not decrease local lesion production by TMVRNA. This phenomenon of competition between related viruses in a local lesion host may prove to be a useful tool for plant virus classification. One of the most interesting differences between SBWMV and TMV is that SBWMV has two types of particles, but TMV has only one. There are several possible explanations. First, SBWMV could have a defective particle; second, SBWMV could be a multicomponent variant of TMV; and third, the short SBWMV particle could be a pseudovirion. There are two types of defective particles that could explain the short particle of SBWMV. First, the short particle could be a satellite virus which possesses its own coat protein, but cannot replicate without the help of the long SBWMV particle. The

RELATIONSHIP

BETWEEN

SDS polyacrylamide gel electrophoresis results are evidence against this type of defective particle because SBWMV protein only gave one band in polyacrylamide gels. This means that the short and long particles of SBWMV both have the same coat protein, unless they have different coat proteins with the same molecular weight. A second type of defective particle is one which depends on another particle for both its replication and coat protein and often interferes with the propagation of the virus. There are many examples of such defective particles in animal virology (Bellett and Cooper, 1959; Von Magnus, 1954). The defective particle explanation would explain several observations concerning SBWMV. The relative amount of the two RNAs recovered from disruption of SBWMV suggests that the concentration of the long particle is much less than that of the short particle. This is unusual because the relative concentrations of the particles of most multiparticulate viruses are close to equal. This may mean that the short SBWMV particles are suppressing the synthesis of long particles, as is the case with vesicular stomatitis virus (Bellett and Cooper, 1959). The defective particle hypothesis would also explain the low specific infectivity of SBWMV and the decrease in TMV-RNA infectivity when it is mixed with short SBWMV-RNA. However, the defective particle hypothesis would not explain the enhancement of infectivity observed by Tsuchizaki et al. (1975) when short and long particles of SBWMV were mixed. SBWMV has been referred to as a multicomponent virus. A multicomponent virus is a virus with a divided genome in which at least two segments of the genome are encapsidated into separate particles. The concept that each particle of SBWMV contains distinct genetic information is favored by the recent work of Tsuchizaki et al. (1975) who showed enhanced infectivity by mixing preparations of short and long rods. However, interpretations of their results is somewhat uncertain because they did not test their preparations of long rods

SBWMV

AND

TMV

461

for aggregates of short rods. Such contamination is possible because SBWMV (Nebraska isolates) aggregates readily and relatively irreversibly, as can be shown by analysis of the RNA from preparations of short and long rods. It is possible that the short particle of SBWMV is a messenger RNA which has been encapsidated. This idea has already been proposed to explain the short particle associated with the cowpea strain of TMV (Morris, 1974). The production of a short RNA by specific cleavage or partial replication should be a distinct advantage to the virus because it could increase translation efficiency as well as provide a mechanism for separating the synthesis of early and late proteins. The messenger RNA could easily be encapsidated if it contained a recognition site for coat protein. The encapsidation would not be a disadvantage unless it interfered with entry into the cell, replication, or control mechanisms; or competed for a limiting supply of coat protein. A final explanation for the two particle nature of SBWMV is that the short particle is a pseudovirion; a host RNA encapsidated in viral protein. It would be unusual for a plant virus to encapsidate such a high concentration of host nucleic acid. The evidence concerning the role of the short SBWMV particle is still incomplete. Nucleic acid or virus preparations which are known to contain predominantly long RNA (2.0 x 10” daltons) without considerable contamination with short RNA (1.0 x 10” daltons) have not yet been obtained. Further research in this area is needed before positive conclusions can be drawn. SBWMV is the second soil-borne virus with a fungal vector shown to be related to TMV, potato mop top virus being the first (Kassanis et al., 1972). This is significant since TMV is also soil-borne, although it has no known soil-borne vector. Perhaps TMV also had a fungal vector, but this method of transmission was lost or became insignificant because of the virus’ stability and the advent of human cultural practices. The new findings concerning SBWMV

462

CHARLES

should cause no problems in nomenclature. As a member of the TMV group of viruses, SBWMV is decidedly atypical, and it is recommended that the name “soilborne wheat mosaic virus” be retained. ACKNOWLEDGMENTS I thank Dr. Myron K. Brakke for his advice and helpful suggestions. I thank Dr. Ellen Ball for providing the antisera used in this study. REFERENCES BALL, E. M. (1961). “Serological Tests for the Identification of Plant Viruses.” Published by the American Phytopathological Society. 16 pp. BALL, E. M. (1966). A technique for comparing the electrophoretic mobility rates of viruses or virus strains. Arch. Biochem. Biophys. 114, 547-556. BALL, E. M., and BRAKKE, M. K. (1969). Analysis of antigen-antibody reactions of two plant viruses by density-gradient centrifugation and electron microscopy. Virology 39, 746-758. BAWDEN, F. C. (1958). Reversible changes in strains of tobacco mosaic virus from leguminous plants. J. Gen. Microbial. 18, 751-766. BEIJERINCK, M. W. (1898). Over een contagium virum fluidum als oorzaak van de vlekziekte der tabaksbladen. Verh. Kon. Akad. Wetenschup.,Afdel. Wis-Natuurk 7, 229-235. BELLETT, A. J. D., and COOPER, P. D. (1959). Some properties of the transmissible interfering component of vesicular stomatitis virus preparations. J. Gen. Microbial. 21, 498-509. BERCKS, R., and QUERFURTH, G. (1971). Serologische Beziehungen zwischen einem gestreckten (tobacco mosaic) und einem isometrischen (cocksfoot mild mosaic) virus. Phytopathol. 2. 72,354-367. BRAKKE, M. K., ESTES, ALICE P., and SCHUSTER, M. L. (1965). Transmission of soil-borne wheat mosaic virus. Phytoputhology 55, 79-86. BRANDES, J., PHILLIPPE, M. R., and THORNBERRY, H. H. (1964). Electron microscopy of particles associ-

A. POWELL ated with soil-borne wheat mosaic. Phytopathol. 2. 50, 181-190. CASPAR, D. L. D. (1963). Assembly and stability of the tobacco mosaic virus particle. Aduan. Protein Chem. 18, 37-121. COOPER, J. I., and HARRISON, B. D. (1972). Potato mop-top virus. Rep. Scott. Hort. Res. Inst. 1971, 63. FRAENKEL-CONRAT, H. (1957). Degradation of tobacco mosaic virus with acetic acid. Virology 4, l4. GUMPF, D. J. (1971). Purification and properties of soil-borne wheat mosaic virus. Virology 43, 588596. KASSANIS, B., WOODS, R. D., and WHITE, R. F. (1972). Some properties of potato mop-top virus and its serological relationship to tobacco mosaic virus. J. Gen. Virol. 14, 123-132. MC KINNEY, H. H. (1925). A mosaic disease of winter wheat and rye. U. S. Dept. Agr. Bull. 1361, 10 pp. MORRIS, T. J. (1974). Two nucleoprotein components associated with the cowpea strain of TMV. Proc. Amer. Phytopathol. Sot. 1, 83. RODBARD, D., and CHRAMBACH, A. (1971). Estimation of molecular radius, free mobility, and valence using polyacrylamide gel electrophoresis. Anal. Biochem. 40, 95-134. TSUCHIZAKI, T., HIBINO, H., and SAITO, Y. (1973). Comparisons of soil-borne wheat mosaic virus isolates from Japan and the United States. Phytopathology 63, 634-639. TSUCHIZAKI, T., HIBINO, H., and SAITO, Y. (1975). The biological functions of short and long particles of soil-borne wheat mosaic virus. Phytopathology 65, 523-532. VON MAGNUS, P. (1954). Incomplete forms of influenza virus. Adv. Virus Res. 2, 59-79. VON SENGBUSCH, P., and WITTMANN, H. G. (1965). Serological and physicochemical properties of the wild strain and two mutants of tobacco mosaic virus with the same amino acid exchange in different positions of the protein chain. Biochem. Biophys. Res. Commun. 18, 780-787.