Plant Cell Reports
Plant Cell Reports (1990) 9:224-228
9 Springer-Verlag1990
Beet necrotic yellow vein virus coat protein-mediated protection in sugarbeet (Beta vulgaris L.) protoplasts Jean Kallerhoff 1, Pascual Perez 1, Salah Bouzoubaa 2, Sofia Ben Tahar 1, and Joi~l Perret 1 1 BIOSEM Laboratory, Groupe Limagrain, 24, Avenue des Landais, F-63170 Aubiere, France 2 I.B.M.P., 12 rue du G6n6ral Zimmer, F-67000 Strasbourg, France Received March 29, 1990/Revised version received May 24, 1990 - Communicated by I. Potrykus
Abstract. Transformed Beta vulgaris L. suspension cultures were obtained after cocultivation of sugarbeet cells with Agrobacterium tumefaciens harbouring a binary vector containing the coat protein gene of beet necrotic yellow vein virus inserted between the kanamycin resistance gene and a l~-glucuronidase reporter gene. Protoplasts were isolated both from untransformed cells, and from transformed cells expressing the viral coat protein, and both were then infected with beet necrotic yellow vein virus. Comparison of the levels of infectivity shows that the expression of the coat protein gene in sugarbeet protoplasts mediates high levels of protection against infection by beet necrotic yellow vein virus.
: TMV : Tobacco Mosaic Virus, CP : Coat Protein, BNYVV : Beet Necrotic Yellow Vein Virus, l~-Glu : l~-glucuronidase, MS: Murashige and Skoog (1962), PEG : Polyethylene glycol, npt : neomycin phosphotransferase, nos: nopaline synthase, FITC : fluoresceine isothiocyanate, IAA : indole acetic acid, BAP : benzyl amino purine, MES : 2-[N-Morpholinolethane sulfonic acid, IgG : Immunoglobulin G, nt : nucleotide.
Abbrevations
Introduction
Powell Abel et al. (1986) first demonstrated that transgenic tobacco plants expressing Offprint requests to: J. Kallerhoff
TMV CP gene were protected against infection by TMV. This approach has since been extended to other viruses in tobacco, tomato and potato (Stark and Beachy 1989 ; Van Den Elzen et al. 1989). Sugarbeet is an agricultural crop of considerable economic importance and is severely damaged by rhizomania. This disease is characterized by massive lateral proliferation of rootlets on the main root resulting in severe stunting and consequent reduction in the sugar content. The causal agent is BNYVV, a well characterized multieomponent RNA virus (Bouzoubaa et al. 1989). There is to date no known source of natural resistance in the germplasm of breeding varieties of sugarbeet. As a first step towards obtaining sugarbeet resistant to BNYVV by means of nonconventional approaches, we have investigated whether engineered CP protection could be extended to sugarbeet. To test this hypothesis, we have expressed BNYVV CP gene in sugarbeet suspension cells and compared the ability of BNYW to infect protoplasts isolated from high CP expressing cells to the ability to infect protoplasts isolated from the non-expressing cells. M a t e r i a l s a n d methods Virus. BNYVV isolates (F13, $2) were gifts from Dr K. Riehards (IBMP, Strasbourg). Virus was purified as described by Putz and Kuszala (1978). Cell cultures. Sugarbeet suspension cultures were established from friable callus induced as described by Saunders and Doley (1986). This callus was cultured in MS liquid medium containing 1 rag/1 BAP and IAA for
225 one month and thereafter subcultured weeldy by 10 fold dilution with the same medium. Cultures were shaken at 200 rpm under continuous light at 25~ Protoplast isolation. Five days after subculture, cells were digested in 20 ml Caylase enzyme solution (0.25 % cellulase 345S ; 0.25 % hemi-cellulase T and 0.08 % pectolyase M2L) obtained from Soci6t6 CAYLA, 20 avenue de Larrieu, Cent~e Commercial de Gros, 31094 Toulouse Cedex, France. Enzymes were dissolved in 0.7 M mannitol containing 0.08 mM NaH2PO4, 0.03 mM MES, 0.68 mM CaC12 and adjusted to 760 mosm/kg at pH 5.8. After 18 h digestion at 24~ in the dark, protoplasts were filtered through 100 and 50 Izm sieves and sedimented at 50 g for 5 rain with an equal volume of iso-osmotic KC1 solution. Protoplasm were purified from debris by centrifuging twice in iso-osmotie sucrose. The protoplast band was removed and washed with mannitol. Viability was assessed by fluoresceine diacetate staining (Widholm 1972). Bacterial strains and plamids. E. Coll HBI01 was used for in vitro DNA transformation. The binary vector pGA 492 (An 1985) and derivatives were mobilized into A. tumefaciens LBA 4404 (Hoekema et al. 1983), EHA 101 (Hood et al. 1986) and C58'3 (Dale et al. 1989) by triparental mating (Ditta et al. 1980). Transformation of suspension cells. 50 pl of an overnight culture of each Agrobacterium swain harbouring binary vectors was shaken for 3 days at 24~ with 2 ml of a 7 day old cell culture diluted 2-fold. Following cocultivation, cells were washed twice in culture medium containing 600 and 300 rag/1 cefotaxim respectively and were plated on two 7 cm Whatman filter paper discs on solid MS medium containing 300 mg/1 cefotaxim and 200 mg/1 kanamycin. Transformed colonies appearing on solid selective medium were cultured in kanamyein containing liquid medium. They were tested for l~-glu activity according to Jefferson et al. (1987) and for the expression of fgglu and BNYVV CP by standard western blotting techniques using purified rabbit polyclonal antibodies and alkaline phosphatase conjugated antirabbit IgC made in goats. Inoculation of protoplasts. Protoplasts were inoculated by the PEG method essentially as described by Samac et al. (1983) except that 5 p g virus was used as inoeulum for a pellet of 1 x 1 0 v protoplasts. Inoculation of protoplasm by electroporation was done according to Watts et al. (1987) using a capacitor discharge apparatus and chamber already described (Guerche et al. 1987). Two pulses were delivered at 15 s interval from 100 nF to 200 pF capacitors charged at different voltages. Assay of infected protoplasts. The percentage of infected protoplasts was quantified by using an indirect fluorescent antibody staining technique (Maule et al. 1980). BNYVV polyclonal antibodies produced in rabbits were used as primary antibodies and FITC conjugated
anti-rabbit IgG made in goats, were used as secondary antibodies to reveal BNYVV infected protoplasts. Replication of viral RNA was followed by isolating total RNA from protoplasts lysed in 0.2 M borate buffer pH 9, containing 1 % (w/v) SDS followed by phenol extraction and selective precipitation by 2 M LIE1. RNA was spotted on nitrocellulose and hybridised with 32p-labelled cDNA reversed transcribed from total viral RNA using standard techniques.
Results and discussion
Binary vectors containing BNYVV CP pGA.3.1.b
30 kd) was treated with f~-glu antibodies and the lower part with BNYW antibodies. Numbers 1 to 17 correspond to different transformed cell lines. 2 and 20 ng of purified BNYVV were loaded as controls. WT is a non transformed line.
Optimisation of the method was performed on protoplasts isolated from non transformed cells.
0
Approximately 2 % of the co-cuttivamd cells were transformed and could grow on 200 mg/l kanamycin containing media. Transformed colonies showed different levels of g-Glu activity ranging from 10 to 50 fold induction when compared to levels obtained with non transformed colonies. The latter did not grow on selective media. Western blotting analysis of transformed cells demonstrated the expression of g-Glu as well as the BNYVV CP gene (Fig. 2). In addition to the 22 kd CP band, transformed cells also express a 29 kd protein which immuno-reacts with specific BNYVV antibodies. The BNYVV coat protein cistron ends with an amber termination codon (UAG) at nt. 709 which sometimes undergoes translational suppression to produce a longer protein of 85 kd apparent molecular weight having the coat protein aminoacid sequence at its N-terminus (Ziegler et a'l. 1985, Bouzoubaa et al. 1986). The BNYVV coat protein sequence in plasmid pGA g.3.1.b, also contains the first 163 residues of the readthrough domain following this amber termination codon (Fig. 2). Suppression of the UAG codon in transcripts of this region would produce a polypeptide with a calculated molecular weight of 29 kd containing the coat protein plus this truncated readthrough domain and ending at a termination codon in the nopaline synthase termination signal. We suggest that the immunoreactive 29 kd polypeptide observed in the transformed cell lines arises from such
25 50 (7~ l i m e in culture
Fig. 3. Time course of infection of sugarbeet protoplasts with F13 ( H and V ) and $2 ( O ) isolates of BNYVV by the PEG method. 9 represents results obtained with inactivated F13 isolate.
Fluorescent antibody staining was used to monitor the frequency of infection. BNYVV infected protoplasts display a characteristic bright green fluorescence dispersed through the cytoplasm ; non infected protoplasts are dull brown in appearance. Fig 3 shows results obtained in 4 experiments using the PEG method of inoculation. More than 50 % of the protoplasts were infected 24 h post-inoculation. Inactivated virus obtained after repeated freezing and thawing does not replicate in protoplasts. Weakly fluorescent protoplasts detected at early stages of infection may be due t o the inoculum retained by the protoplasts. This varied from one experiment to another and based on data from more than 50 successful experiments, we found no correlation between the amount of retained inoculum and the level of infectivity. On the average, 40 to 50 % of the protoptasts were infected in these experiments. Higher levels of infectivity occured rarely, probably in cases where protoplast preparations were exceptionally competent for infection and/or when the virus in the inoculum had very high specific infectivity. Heterologous antibodies and non-immune serum did not stain BNYVV infected protoplasts.
227 Eleetroporation also proved to be an efficient means of introducing BNYVV particles into sugarbeet protoplasts (data not shown). Highest levels of infectivity (55 ~ were obtained when using two 100 ms pulses delivered by a 63 ~F capacitor charged to 220 V to protoplasts suspended in mannitol containing 5 mM CaC12. Omission of CaCI~ led to a three fold decrease in infectivit~ levels. Under all other conditions tested, electroporation did not prove to be superior to the PEG inoculation method to achieve higher levels of infectivity. In order to determine whether BNYVV CP expressed in sugarbeet protoplasts could confer protection against infection by the virus, we inoculated protoplasts isolated from transformed and non transformed cells strictly in parallel and cultured them on nonselective media. Indeed, on medium without kanamycin, the plating efficiencies of protoplasts from transformed and nontransformed cells were not significantly different to those of protoplasts from transformed cells on medium containing 200 rag/1 kanamycin. Protoplasts from untransformed cells did not survive under selective conditions. Inoculation of protoplasts was performed using PEG and eleetroporation, and infection was monitored by fluorescent antibody staining 30 h postinoculation. No background immunofluorescence was detected in protoplasts isolated from the CP expressing lines prior to inoculation. Table 1 summarizes typical results obtained with the two highest CP expressing lines (f~7 and 1~14), with the non transformed line (Pll) and with a transformed line (f~D) not expressing BNYVV CP. The I~D line was obtained after transformation of sugarbeet cells with A. tumefaciens harbouring a binary vector containing the kanamycin and phleomycin resistance genes (Perez et al. 1989). In all cases and irrespective of virus isolate, or of the inoculation method, the percentage of protoplasts issued from transformed cells expressing CP and which were infected by BNYVV, was significantly lower than that from the non-transformed line. Experiments 7 and 8 reveal that transformed protoplasts not expressing CP also become infected by BNYVV. Thus, it can
be concluded that the observed protection is indeed due to the expression of viral coat protein and that the expression of foreign genes other than the BNYVV CP gene is not responsible for the protective effects.
Infected protoplasts
Protection
(%)
Protoplast source
nontransformed CPPl i
/~7
(%)
transformed
CP + 814
CPI~D
Exp 1
30
1
97
2
68
10
85
3
70
4
94
4
27
5
35
5
85
6
25
2
92
7
37
10
8
1
12
96
36
73
48
75
Table 1. Infection of wansformed (1~7, 1~14, I~D) and nontransformed ( P l l ) sugarbeet protoplasm by BNYVV. CP + and CP- are transformed lines expressing and not expressing BNYVV CP respectively. 1 x 105 protoplasts were inoculated by PEG (exp. 1, 2, 4, 6, 7) or electroporation (exp. 3, 5, 8) with 5 pg BNYW (F13 or $2) except for experiment 4 where inoeulum consisted of 30 lag BNYVV. Numbers represent the % of fluorescent protoplasm detected 30 h post-inoeulatlon.
The effect of using high amolants of inoculum (30 lag instead of 5 ~tg/10" protoplasts) is shown m expertment 4. Total RNA was extracted from infected protoplasts and approximately 500 ng of total RNA, determined spectrophotometrically, was spotted on nitrocellulose, followed by hybridization with a radioactive cDNA probe complementary to viral RNA.
228 References
Relative 3 30 ..50
optical density 'p
J
Fig 4. Densitometric profiles of RNA dot-blot analysis of BNYW infected protoplasts from experiment 4 and isolated from the CP expressing line 1~14 ~ashed) and the non expressing P l l line (solid). 1 x 10 protoplasts were inoculated with 30 tag BNYVV by the PEG method of inoeulation.
Densitometric profiles of the dots are represented in Fig 4. The high signal at 0 time is due to the very high amount of inoculum used in this experiment. Comparison of both profiles show that protection is not overcome by increased concentrations of virus in the inoculum. BNYVV RNA do not replicate efficiently in protoplasts isolated from transformed sugarbeet cells in contrast to those from the non-expressing line. Our results are in agreement with those of Register and Beachy (1988) who have shown that protoplasts isolated from transgenic plants expressing TMV CP were protected from infection by TMV. We have demonstrated that coat protein mediated protection in protoplasts can be extended to sugarbeet and BNYVV, a member of the furovirus group. Whether the high levels of CP mediated protection in protoplasts from sugarbeet suspension cells will also be observed in sugarbeet plants remains to be demonstrated. If so, this could be an effective means of introducing novel traits of resistance to rhizomania in sugarbeet. Acknowledgements. We thank Sue Loesch-Fries for advice on immunofluoreseent staining, Ken Richards for reviewing the text, Monique Alric for antibodies, Fran~oise Rapt for typing the manuscript and Christine Poncetta for skilled technical assistance.
An G (1986) Plant Physiol. 81 : 86-91 Bouzoubaa S, Ziegler V, Beck D, Guilley H, Richards K, Jonard G (1986) J. Gen. Virol. 67:1689-1700 Bouzoubaa S, Guilley H, Jonard G, Jupin I, Quillet L, Richards K, Scheidecker D, Ziegler-Graff V (1989) In : Viruses with fungal vectors. Cooper JI and Asher MJC (eds), Wellesbourne, UK pp 99-110 Dale PJ, Marks MS, Brown MM, Woolston CJ, Gunn HV, Mullineaux PM, Lewis DM, Kemp JM, Chen DF, Gilmour DM, Flavell RB (1989) Plant Science 63:237245 Ditta G, Stanfield S, Corbin D, Helinski DR (1980) PNAS 77:7347-7351 Guerehe P, Bellini C, Le Moullec JM, Caboche M (1987) Biochimie 69:621-628 Hoekema M, Hirsch PR, Hoykaas PJJ, Schillperoort RA (1983) Nature 303:179-180 Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) J. Bact. 168:1291-1301 Jefferson RA, Kavanagh TA, Bevan HW (1987) EMBO J. 6:3901-3907 Maule AJ, Boulton MI, Wood KR (1980) Phytopath. Z. 97:118-126 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Perez P, Tiraby G, Kallerhoff J, Perret J (1989) Plant Mol. Biol. 13:365-373 Powell Abel P, Nelson RS, De B, Hoffmann N, Rogers SG, Fraley RT, Beachy RN (1986) Science 232:738-743 Putz C, Kuszala M (1978) Ann. de Phytopath. 10:247-262 Register III JC, Beachy RN (1988) Virology 166:524-532 Samac DA, Nelson SE, Loesch-Fries LS (1983) Virology 131:455-462 Saunders JW, Doley WP (1986) J. Plant Physiol. 124: 473-479 Stark DM, Beachy RN (1989) Biotechnology 7:1257-1262 Van Den Elzen PJM, Huisman MJ, Willink DPL, Jongedijk E, Hoekema A, Cornelissen BJC (1989) Plant Mol. Biol. 13:337-346 Watts JW, King JM, Stacey NJ (1987) Virology 157:40-46 Widholm JM (1972) Stain Tech. 47:189-194 Ziegler V, Richards K, Guflley H, Jonard G, Putz C (1985) J. Gen. Virol. 66:2079-2087
Plant Cell Reports (1990) 9:224-228
9 Springer-Verlag1990
Beet necrotic yellow vein virus coat protein-mediated protection in sugarbeet (Beta vulgaris L.) protoplasts Jean Kallerhoff 1, Pascual Perez 1, Salah Bouzoubaa 2, Sofia Ben Tahar 1, and Joi~l Perret 1 1 BIOSEM Laboratory, Groupe Limagrain, 24, Avenue des Landais, F-63170 Aubiere, France 2 I.B.M.P., 12 rue du G6n6ral Zimmer, F-67000 Strasbourg, France Received March 29, 1990/Revised version received May 24, 1990 - Communicated by I. Potrykus
Abstract. Transformed Beta vulgaris L. suspension cultures were obtained after cocultivation of sugarbeet cells with Agrobacterium tumefaciens harbouring a binary vector containing the coat protein gene of beet necrotic yellow vein virus inserted between the kanamycin resistance gene and a l~-glucuronidase reporter gene. Protoplasts were isolated both from untransformed cells, and from transformed cells expressing the viral coat protein, and both were then infected with beet necrotic yellow vein virus. Comparison of the levels of infectivity shows that the expression of the coat protein gene in sugarbeet protoplasts mediates high levels of protection against infection by beet necrotic yellow vein virus.
: TMV : Tobacco Mosaic Virus, CP : Coat Protein, BNYVV : Beet Necrotic Yellow Vein Virus, l~-Glu : l~-glucuronidase, MS: Murashige and Skoog (1962), PEG : Polyethylene glycol, npt : neomycin phosphotransferase, nos: nopaline synthase, FITC : fluoresceine isothiocyanate, IAA : indole acetic acid, BAP : benzyl amino purine, MES : 2-[N-Morpholinolethane sulfonic acid, IgG : Immunoglobulin G, nt : nucleotide.
Abbrevations
Introduction
Powell Abel et al. (1986) first demonstrated that transgenic tobacco plants expressing Offprint requests to: J. Kallerhoff
TMV CP gene were protected against infection by TMV. This approach has since been extended to other viruses in tobacco, tomato and potato (Stark and Beachy 1989 ; Van Den Elzen et al. 1989). Sugarbeet is an agricultural crop of considerable economic importance and is severely damaged by rhizomania. This disease is characterized by massive lateral proliferation of rootlets on the main root resulting in severe stunting and consequent reduction in the sugar content. The causal agent is BNYVV, a well characterized multieomponent RNA virus (Bouzoubaa et al. 1989). There is to date no known source of natural resistance in the germplasm of breeding varieties of sugarbeet. As a first step towards obtaining sugarbeet resistant to BNYVV by means of nonconventional approaches, we have investigated whether engineered CP protection could be extended to sugarbeet. To test this hypothesis, we have expressed BNYVV CP gene in sugarbeet suspension cells and compared the ability of BNYW to infect protoplasts isolated from high CP expressing cells to the ability to infect protoplasts isolated from the non-expressing cells. M a t e r i a l s a n d methods Virus. BNYVV isolates (F13, $2) were gifts from Dr K. Riehards (IBMP, Strasbourg). Virus was purified as described by Putz and Kuszala (1978). Cell cultures. Sugarbeet suspension cultures were established from friable callus induced as described by Saunders and Doley (1986). This callus was cultured in MS liquid medium containing 1 rag/1 BAP and IAA for
225 one month and thereafter subcultured weeldy by 10 fold dilution with the same medium. Cultures were shaken at 200 rpm under continuous light at 25~ Protoplast isolation. Five days after subculture, cells were digested in 20 ml Caylase enzyme solution (0.25 % cellulase 345S ; 0.25 % hemi-cellulase T and 0.08 % pectolyase M2L) obtained from Soci6t6 CAYLA, 20 avenue de Larrieu, Cent~e Commercial de Gros, 31094 Toulouse Cedex, France. Enzymes were dissolved in 0.7 M mannitol containing 0.08 mM NaH2PO4, 0.03 mM MES, 0.68 mM CaC12 and adjusted to 760 mosm/kg at pH 5.8. After 18 h digestion at 24~ in the dark, protoplasts were filtered through 100 and 50 Izm sieves and sedimented at 50 g for 5 rain with an equal volume of iso-osmotic KC1 solution. Protoplasm were purified from debris by centrifuging twice in iso-osmotie sucrose. The protoplast band was removed and washed with mannitol. Viability was assessed by fluoresceine diacetate staining (Widholm 1972). Bacterial strains and plamids. E. Coll HBI01 was used for in vitro DNA transformation. The binary vector pGA 492 (An 1985) and derivatives were mobilized into A. tumefaciens LBA 4404 (Hoekema et al. 1983), EHA 101 (Hood et al. 1986) and C58'3 (Dale et al. 1989) by triparental mating (Ditta et al. 1980). Transformation of suspension cells. 50 pl of an overnight culture of each Agrobacterium swain harbouring binary vectors was shaken for 3 days at 24~ with 2 ml of a 7 day old cell culture diluted 2-fold. Following cocultivation, cells were washed twice in culture medium containing 600 and 300 rag/1 cefotaxim respectively and were plated on two 7 cm Whatman filter paper discs on solid MS medium containing 300 mg/1 cefotaxim and 200 mg/1 kanamycin. Transformed colonies appearing on solid selective medium were cultured in kanamyein containing liquid medium. They were tested for l~-glu activity according to Jefferson et al. (1987) and for the expression of fgglu and BNYVV CP by standard western blotting techniques using purified rabbit polyclonal antibodies and alkaline phosphatase conjugated antirabbit IgC made in goats. Inoculation of protoplasts. Protoplasts were inoculated by the PEG method essentially as described by Samac et al. (1983) except that 5 p g virus was used as inoeulum for a pellet of 1 x 1 0 v protoplasts. Inoculation of protoplasm by electroporation was done according to Watts et al. (1987) using a capacitor discharge apparatus and chamber already described (Guerche et al. 1987). Two pulses were delivered at 15 s interval from 100 nF to 200 pF capacitors charged at different voltages. Assay of infected protoplasts. The percentage of infected protoplasts was quantified by using an indirect fluorescent antibody staining technique (Maule et al. 1980). BNYVV polyclonal antibodies produced in rabbits were used as primary antibodies and FITC conjugated
anti-rabbit IgG made in goats, were used as secondary antibodies to reveal BNYVV infected protoplasts. Replication of viral RNA was followed by isolating total RNA from protoplasts lysed in 0.2 M borate buffer pH 9, containing 1 % (w/v) SDS followed by phenol extraction and selective precipitation by 2 M LIE1. RNA was spotted on nitrocellulose and hybridised with 32p-labelled cDNA reversed transcribed from total viral RNA using standard techniques.
Results and discussion
Binary vectors containing BNYVV CP pGA.3.1.b
30 kd) was treated with f~-glu antibodies and the lower part with BNYW antibodies. Numbers 1 to 17 correspond to different transformed cell lines. 2 and 20 ng of purified BNYVV were loaded as controls. WT is a non transformed line.
Optimisation of the method was performed on protoplasts isolated from non transformed cells.
0
Approximately 2 % of the co-cuttivamd cells were transformed and could grow on 200 mg/l kanamycin containing media. Transformed colonies showed different levels of g-Glu activity ranging from 10 to 50 fold induction when compared to levels obtained with non transformed colonies. The latter did not grow on selective media. Western blotting analysis of transformed cells demonstrated the expression of g-Glu as well as the BNYVV CP gene (Fig. 2). In addition to the 22 kd CP band, transformed cells also express a 29 kd protein which immuno-reacts with specific BNYVV antibodies. The BNYVV coat protein cistron ends with an amber termination codon (UAG) at nt. 709 which sometimes undergoes translational suppression to produce a longer protein of 85 kd apparent molecular weight having the coat protein aminoacid sequence at its N-terminus (Ziegler et a'l. 1985, Bouzoubaa et al. 1986). The BNYVV coat protein sequence in plasmid pGA g.3.1.b, also contains the first 163 residues of the readthrough domain following this amber termination codon (Fig. 2). Suppression of the UAG codon in transcripts of this region would produce a polypeptide with a calculated molecular weight of 29 kd containing the coat protein plus this truncated readthrough domain and ending at a termination codon in the nopaline synthase termination signal. We suggest that the immunoreactive 29 kd polypeptide observed in the transformed cell lines arises from such
25 50 (7~ l i m e in culture
Fig. 3. Time course of infection of sugarbeet protoplasts with F13 ( H and V ) and $2 ( O ) isolates of BNYVV by the PEG method. 9 represents results obtained with inactivated F13 isolate.
Fluorescent antibody staining was used to monitor the frequency of infection. BNYVV infected protoplasts display a characteristic bright green fluorescence dispersed through the cytoplasm ; non infected protoplasts are dull brown in appearance. Fig 3 shows results obtained in 4 experiments using the PEG method of inoculation. More than 50 % of the protoplasts were infected 24 h post-inoculation. Inactivated virus obtained after repeated freezing and thawing does not replicate in protoplasts. Weakly fluorescent protoplasts detected at early stages of infection may be due t o the inoculum retained by the protoplasts. This varied from one experiment to another and based on data from more than 50 successful experiments, we found no correlation between the amount of retained inoculum and the level of infectivity. On the average, 40 to 50 % of the protoptasts were infected in these experiments. Higher levels of infectivity occured rarely, probably in cases where protoplast preparations were exceptionally competent for infection and/or when the virus in the inoculum had very high specific infectivity. Heterologous antibodies and non-immune serum did not stain BNYVV infected protoplasts.
227 Eleetroporation also proved to be an efficient means of introducing BNYVV particles into sugarbeet protoplasts (data not shown). Highest levels of infectivity (55 ~ were obtained when using two 100 ms pulses delivered by a 63 ~F capacitor charged to 220 V to protoplasts suspended in mannitol containing 5 mM CaC12. Omission of CaCI~ led to a three fold decrease in infectivit~ levels. Under all other conditions tested, electroporation did not prove to be superior to the PEG inoculation method to achieve higher levels of infectivity. In order to determine whether BNYVV CP expressed in sugarbeet protoplasts could confer protection against infection by the virus, we inoculated protoplasts isolated from transformed and non transformed cells strictly in parallel and cultured them on nonselective media. Indeed, on medium without kanamycin, the plating efficiencies of protoplasts from transformed and nontransformed cells were not significantly different to those of protoplasts from transformed cells on medium containing 200 rag/1 kanamycin. Protoplasts from untransformed cells did not survive under selective conditions. Inoculation of protoplasts was performed using PEG and eleetroporation, and infection was monitored by fluorescent antibody staining 30 h postinoculation. No background immunofluorescence was detected in protoplasts isolated from the CP expressing lines prior to inoculation. Table 1 summarizes typical results obtained with the two highest CP expressing lines (f~7 and 1~14), with the non transformed line (Pll) and with a transformed line (f~D) not expressing BNYVV CP. The I~D line was obtained after transformation of sugarbeet cells with A. tumefaciens harbouring a binary vector containing the kanamycin and phleomycin resistance genes (Perez et al. 1989). In all cases and irrespective of virus isolate, or of the inoculation method, the percentage of protoplasts issued from transformed cells expressing CP and which were infected by BNYVV, was significantly lower than that from the non-transformed line. Experiments 7 and 8 reveal that transformed protoplasts not expressing CP also become infected by BNYVV. Thus, it can
be concluded that the observed protection is indeed due to the expression of viral coat protein and that the expression of foreign genes other than the BNYVV CP gene is not responsible for the protective effects.
Infected protoplasts
Protection
(%)
Protoplast source
nontransformed CPPl i
/~7
(%)
transformed
CP + 814
CPI~D
Exp 1
30
1
97
2
68
10
85
3
70
4
94
4
27
5
35
5
85
6
25
2
92
7
37
10
8
1
12
96
36
73
48
75
Table 1. Infection of wansformed (1~7, 1~14, I~D) and nontransformed ( P l l ) sugarbeet protoplasm by BNYVV. CP + and CP- are transformed lines expressing and not expressing BNYVV CP respectively. 1 x 105 protoplasts were inoculated by PEG (exp. 1, 2, 4, 6, 7) or electroporation (exp. 3, 5, 8) with 5 pg BNYW (F13 or $2) except for experiment 4 where inoeulum consisted of 30 lag BNYVV. Numbers represent the % of fluorescent protoplasm detected 30 h post-inoeulatlon.
The effect of using high amolants of inoculum (30 lag instead of 5 ~tg/10" protoplasts) is shown m expertment 4. Total RNA was extracted from infected protoplasts and approximately 500 ng of total RNA, determined spectrophotometrically, was spotted on nitrocellulose, followed by hybridization with a radioactive cDNA probe complementary to viral RNA.
228 References
Relative 3 30 ..50
optical density 'p
J
Fig 4. Densitometric profiles of RNA dot-blot analysis of BNYW infected protoplasts from experiment 4 and isolated from the CP expressing line 1~14 ~ashed) and the non expressing P l l line (solid). 1 x 10 protoplasts were inoculated with 30 tag BNYVV by the PEG method of inoeulation.
Densitometric profiles of the dots are represented in Fig 4. The high signal at 0 time is due to the very high amount of inoculum used in this experiment. Comparison of both profiles show that protection is not overcome by increased concentrations of virus in the inoculum. BNYVV RNA do not replicate efficiently in protoplasts isolated from transformed sugarbeet cells in contrast to those from the non-expressing line. Our results are in agreement with those of Register and Beachy (1988) who have shown that protoplasts isolated from transgenic plants expressing TMV CP were protected from infection by TMV. We have demonstrated that coat protein mediated protection in protoplasts can be extended to sugarbeet and BNYVV, a member of the furovirus group. Whether the high levels of CP mediated protection in protoplasts from sugarbeet suspension cells will also be observed in sugarbeet plants remains to be demonstrated. If so, this could be an effective means of introducing novel traits of resistance to rhizomania in sugarbeet. Acknowledgements. We thank Sue Loesch-Fries for advice on immunofluoreseent staining, Ken Richards for reviewing the text, Monique Alric for antibodies, Fran~oise Rapt for typing the manuscript and Christine Poncetta for skilled technical assistance.
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