Helper components of two potyviruses are serologically distinct

VIROLOGY

125, 487-490

(19%)

omponents

W.

DAVID Department

of Plant

of Two

September

Are ~@r~lo~ic~~~y

AND THOMAS P.PIRONE'

THORNBURY

Pathology,

Received

Potyviruses

University

of Kentucky,

30, 1982; accepted

Lexington, December

Kentucky

&I546

1, 1982

The specificity of antisera to helper component (HC) from tobacco vein mottling virus (TVMV)or potato virus Y (PVY)-infected tobacco plants was tested in immunoprecipitation and immunoabsorption chromatography experiments. Treatment with the homologous antiserum abolished or drastically reduced the activity of either TVMV-HC or PVY-HC, as measured by their ability to effect aphid transmission of purified tobacco etch virus, while the heterologous antiserum had little or no effect on HG activity. Loss of TVMV-HC and PVY-HC activity in the immunoabsorption chromatography experiments was associated with the removal of a 53- and a 5%kDa polypeptide, respectively. The results indicate that serologically distinct HC proteins are produced in response to specific potyvirus infection and suggest that HC is virus coded.

Aphid transmission of purified preparations of viruses of the potyvirus group has been shown to be dependent on the presence of a protein, termed helper component (WC), which is present in extracts of potyvirus-infected but not healthy plants (1-3). Whether HC is a virus-specified protein or a protein produced as a plant response to potyvirus infection has been an unanswered question, although previous reports (3, J$)have shown that HC produced as the result of infection with a particular potyvirus is not necessarily effective in promoting the transmission of other potyviruses. In this report we present evidence, from two types of serological experiments, which indicates that HC is virus specific. The isolates of tobacco vein mottling virus (TVMV) and potato virus Y (PVY) have been described elsewhere (5, 2). Tobacco etch virus (TEV) was a highly aphidtransmissible, severe isolate obtained from field-infected tobacco plants. Purified virus and partially purified HC were prepared from systemically infected tobacco (Nicotiana tabacum L. “Burley 21”) that had been mechanically inoculated 2-4 weeks previously. All viruses were purified by method I of Moghal and Francki (6); ’ To whom

reprint

requests

should

virus concentrations were det~r~~~e~ spectrophotometrieally (A$;;l%n, = 2.4). Helper component for use as test material in all experiments, and for production of the antisera used in the immunoprecipitation experiments, was partially purified by sucrose gradient centrifugation as described (7’). Polyaerylamide electrophoresis indicated that the pre ration had numerous polypeptide components. Further purification, for ~r~dn~~~o~ of the antisera used in the immunoabsorption chromatography experiments, was achieved by high-performa~~~e liquid chromatography (HPLC). Sucrose gradient-purified WC was fra~tio~at~~ on three I-250 protein analysis columns (Waters Associates, 7.8 mm X 30 (0.5 ml, l-2 ABO) were run i HzS04, pH 7.2,0.02 M M,SQ4 plus 1% isopropanol. Colum assayed for NC activity by testing for aphid transmission of TEV (final con akion 80 pg/ml) after acquisition thr Parafilm as described (2). Antisera were prepared to gradient-purified and to HPLC-purified PVYTVMV-HC and to a similar g~a~ie~t-~~~rified fraction from uninoculated t~~~~~~~. Rabbits were injected three times at 2week intervals. For the first ~nje~~~~~, 1 ml of antigen (2 Azso units) was ern~~s~~~d

be addressed. 487

004%6822/83 Copyright All rights

$3.00

0 1983 by Acadenic Press, inc. of reproduction in any form resened.

488

SHORT

COMMUNICATIONS TABLE

EFFECT

OF ANTISERA

1

ON THE ABILITY OF TVMV-HC AND PVY-HC THE APHID TRANSMISSION OF TEV” Percentage Antiserum

TVMV Expt

l/5

HC

PVY-HC

l/10

TVMV-HC

1 2 3 4 5

0 -d 0

PVY-HC

1 2 3 4 5

90 60

aphid transmission and dilution used

TO PROMOTE

of TEVb to treat HC

Healthy

tobacco

l/5

l/10

l/5

30 0 0 40 30

90 90

100 90 100 100

100 100 90

100 50 100 -

0 -

0 0 0 0 10

50

60 90 100 80

40 100 100 -

-

ControlC

40 80

“Helper component (HC) was incubated with the indicated antiserum; following centrifugation, TEV added to the supernatant and aphids were tested for their ability to transmit TEV. b Percentage of infected plants; 10 aphids were placed on each of 10 test plants for each treatment. ‘Helper component not treated with antiserum. d Not tested.

TABLE

2

EFFECT OF I~~IMUNQAB~~RPTION CnRohiIAToGRAPHy ON THE ABILITY OF TVMV-HC AND PVY-HC TO PROMOTE THE APHID TRANSMISSION OF TEV” Percentage

aphid transmission of TEVb Antiserum bound to protein A agarose

Source of HC

Expt

TVMV-HC

PVY-HC

Healthy

TVMV

1 2 3

0 0 0

20 50 60

50 60 30

PVY

1 2 3

100 100 80

30 20 0

90 60 85

a Helper component (HC) was allowed to react with the indicated immunoglobulins bound to protein A agarose. The unbound uv-absorbing material was collected and tested for HC activity with TEV. bPercentage of infected plants; 10 aphids were placed on each of 10 test plants for each treatment.

was

with 1 ml of complete Freund’s adjuvant (Difco) and injected subcutaneously. For subsequent injections, 0.5 ml of antigen was emulsified with an equal volume of incomplete Freund’s adjuvant. Rabbits were bled 1 week after the last injection. When tested in double-diffusion tests in agar containing sodium dodecyl sulfate (SDS) (8), antisera against gradient-purified TVMV-HC and PVY-HC, and uninoculated tobacco preparations produced similar patterns against all three of the corresponding antigens, with antiuninoculated tobacco producing slightly fewer zones than the other antisera. Still fewer zones were seen with anti-HPLC-purified HC, but this also reacted with the uninoculated tobacco preparation. Thus, the preparations appeared to be complex mixtures, all containing plant proteins, by both gel electrophoresis and immunological analyses. For the immunoprecipitation experiments a HC dilution was used that could

SHORT

COMMUNICATIONS

ected to result in approximately 80100% transmission of TEV. Tobacco etch virus was used because, although TVMVHC and PVY-HC are able to mediate the transmission of either TVMV or PVY, there is a bias toward preferential transmission of the homologous virus (3). The use of TEV reduced the likelihood of such bias and simplified the experimental design. The effect of the antisera on HC activity was measured by adding a series of dilutions of antiserum to HC of predetermined activity, incubating for 2 hr at 4”, and centrifuging the samples for 5 min at 8400 g in order to pellet the immunoprecipitates. The supernatant was then mixed with TEV and assayed for HC activity by aphid transmission tests. The data in Table 1 show that the aetivity of TVMV-HC was abolished when HC preparations were treated with the homologous antiserum at a l/5 dilution and was redueed to O-40% of the control when treated with homologous antiserum at a 1/10 dilution. Similarly, treatment of PVY-

<&_1

HC with the homologous antiserum also abolished or drastically reduced the activity of PVY-HC. In contrast, treatment of either TVMV-HC or PVYWith heterologous antiserum bad le or no feet on MC activity. For immunoabsor~t~on ~hrorna~~~r~phy each antiserum was passed thr s-ml column of protein A agarose in l/zX phosphate-buffered saline pH 7.2, following the method of God to bind the IgG. The columns were then washed with 0.1 &t?Tris-H&I4 remove phosphate, and then e with TSM buffer. For immuno gradient-prepared HC (0.5 Am units) was incubated on the columns for 2 hr at 4”; the unbound uv-absorbing material was collected and assayed for HC activity. The data in Table 2 skow that Xg@ from antisera to TVMV-WC and PVY-HC remove HC activity from the bornol~~o~~ HC preparations while there is no re~~~t~o~ in activity of either WC passed protein A-bound heterolo~o~s Ig

VMV HC ST ASH ASP HC

3k

BIG. I. SDS-polyacrylamide gel electrophoresis of TVMV-HC (left) and PVY-HC (right) preparations immunoabsorbed by protein A agarose-bound anti-TVMV-RC antiserum (AsT), anti-realtor plant extract antiserum (ASH), and anti-PVY-HC antiserum (ASP). Profiles of helper component (HC), and uninoculated healthy plant extract (Hty), prepared by sucrose gradient cent~~f~~~t~~~, are shown for comparison. Proteins were stained with Coomassie brilliant blue R-250.

490

SHORT

COMMUNICATIONS

Protein patterns of HC fractions from the immunoabsorption columns, concentrated by vacuum dialysis, were compared with gradient-purified HC and extract of uninoculated tobacco on 10% polyacrylamide gels using the SDS-buffer system of Laemmli (10). Active fractions of TVMVWC contained a 53,000-Da polypeptide which was removed by the protein A-bound anti-TVMV-HC IgG. A E&,000-Da polypeptide, present in active PVY-HC fractions, was removed by the anti-PVY-HC IgG. Neither protein was detected in the extract of uninoculated tobacco (Fig. 1). Previous studies (3, .4) have shown that HC from certain potyviruses is ineffective in promoting the transmission of other potyviruses, suggesting a functional dissimilarity. Although there is a bias toward preferential transmission of the homologous virus, in the case of PVY and TVMV, the respective HCs are functionally interchangeable (3). The demonstration that antisera to TVMV-HC and PVY-HC inhibit the activity of homologous, but not heterologous, HC preparations in both immunoprecipitation and immunoabsorption experiments shows that the WC proteins are serologically distinct. This, and the identification of HC-specific protein bands in the active fractions of TVMV-HC and PVY-HC, in the immunoabsorption experiments, suggests that HC is virus specified. Other virus-specified proteins such as coat (11) and cytoplasmic inclusion (1%) of different potyviruses are also serologically distinct (6, 13). Since the HC preparations used in our study contain proteins which are of plant origin, the possibility that different viruses cause a differential induction of a host-specific HC protein cannot be ruled

out on the basis of these data alone. However, taken together with the fact that these respective HC antisera specifically precipitate a unique subset of products of the cell-free translation of TVMV RNA (7) or PVY RNA (Thornbury et al., unpublished) they provide very strong evidence that HC is a product of the virus genome. ACKNOWLEDGMENTS This work is Kentucky Agricultural Experiment Station Journal Series Paper 82-11-230. This work was supported in part by U. S.-Israel Agricultural Research and Development Fund (BARD) Grant l27-79. Teresa Perrone provided excellent technical assistance. REFERENCES 1. GOVIER,

D. A., and KASSANIS, B., virology 57,285286 (1974). 2. GOVIER, D. A., KASSANIS, B., and PIRONE, T. P., virology 78, 306-314 (1977). 3. PIRONE, T. P., Ph@q&hology 71,911-924 (1981). 4 SAKO, N., and OGATA, K., Erology 112, 762-765 (1981). 5. PIRONE, T. P., GOODING, G. V., and SMILEY, J. H., Plant Dis. Rep. 57, 841-844 (1973). 6. MOGHAL, S. M., and FRANCKI, R. I. B., virology 73, 350-362 (1976). z HELLMAN, G. M., THORNBURY, D. W., HIEBERT, E., SHAW, J. G., PIRONE, T. P., and RHOADS, R. E., Virology 124, 434-444. 8. GOODING, G. V., and BING, W. W., Phytopathology 60, 1293 (1970). 9. GODING, J. W., ImmunoL Methods 20, 24-253 (1978). 10. LAEMMLI, U. K., Nature (London) 227, 680-685 (1970). 12. DOUGHERTY, W. G., and HIEBERT, E., ‘virologv 104, 174-182 (1980). 12. DOUGHERTY, W. G., and HIEBERT, E., virology 104, 183-194 (1980). 13. PURCIFULL, D. E., HIEBERT, E., and MCDONALD, J. G., ViroZogy 55, 275-279 (1973).