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Expression of the 16K cistron of tobacco rattle virus in protoplasts
VIROLOGY
169,305-311
(1989)
Expression
of the 16K Cistron of Tobacco
Rattle Virus in Protoplasts
GERCO C. ANGENENT, HANS B. M. VERBEEK, AND JOHN F. BOL’ Department
of Biochemistry,
Leiden University,
P.O. Box 9505, 2300 RA Leiden, The Netherlands
Received July 18, 1988; accepted November
8, 1988
An antiserum was raised against a synthetic peptide corresponding to the 18 C-terminal amino acids of a putative 16K protein encoded by the 3’-terminal open reading frame of tobacco rattle virus (TRV) RNA-l. This antiserum was used to demonstrate expression of the 16K cistron in vivo. TRV-infected tobacco protoplasts accumulated similar amounts of 16K protein and viral coat protein but in tobacco plants only the coat protein was detectable. Time course experiments revealed that in protoplasts the accumulation of 16K protein lagged somewhat behind that of coat protein. The 16K protein was incorporated in a high-molecular-weight cellular component that was resistant to treatment with 0 1989 Academic Press, Inc. nOniOniC detergents.
INTRODUCTION
we have determined the nucleotide sequence of RNA2 of TRV strain PLB (Angenent et al., to be published). Because the 3’-terminal sequence of PLB RNA-2 is identical to the 3’-end of PLB RNA-l over a length of 820 nucleotides, this strain is also diploid for the 16K gene. None of the nonstructural TRV proteins has been detected in infected cells so far. Homologies with corresponding gene products of tobacco mosaic virus (TMV) indicate that the 134K and 194K proteins are involved in viral replication and suggest a role for the 29K protein in cell-to-cell transport of infectious material (Hamilton et a/., 1987). The combination of these functions in TRV RNA-l could explain the ability of this genome segment to replicate and spread in plants in the absence of RNA-2 (Harrison and Robinson, 1978). Although the 16K cistron is present in all TRV strains sequenced to date, no function of this gene is known. The amino acid sequence homology between the 16K proteins of strains PSG, PLB, TCM, and SYM is approximately 90%. To permit an analysis of the expression of the 16K gene in infected cells, we raised an antiserum to a synthetic peptide corresponding to the conserved C-terminus of the 16K protein. Here we repot-t that the 16K gene is expressed to relatively high levels in tobacco protoplasts infected with TRV strain PLB. In addition, a preliminary characterization of the cellular structure in which the 16K protein is incorporated is described.
Tobacco rattle virus (TRV) is the type member of the Tobraviruses. The genome of these viruses consists of two plus-stranded RNA molecules, which are separately encapsidated into rod-shaped particles (Harrison and Robinson, 1986). RNA-l of the SYM-strain (6791 nucleotides) has been completely sequenced (Hamilton et al., 1987) and the 3’-terminal sequences of RNA1 of strains PSG and TCM have been reported (Cornelissen et a/., 1986; Angenent et al., 1986). The sequence data indicate that TRV RNA-l encodes four proteins. The 5’-proximal open reading frame encodes a protein with a molecular weight of 134,000 (134K) and read-through of a “leaky” termination codon results in an extension of this protein to a 194K polypeptide. These two proteins are directed by RNA-l in an in vitro translation system (Fritsch et al., 1977). The third and fourth reading frames in RNA-l encode 29K and 16K proteins, respectively. Subgenomic RNAs that may function as messenger for these proteins have been identified (Cornelissen et a/., 1986; Bocarra et a/., 1986). RNA-2 of tobraviruses varies in length between 1800 and 4000 nucleotides, depending on the strain (Harrison and Robinson, 1978). Part of this variability is due to differences in length of the 3’-terminal sequence of RNA-2 that is identical to that of the corresponding RNA-l (Angenent et a/., 1986). The sequence that is unique to RNA-2 encodes the coat protein (CP). In addition, RNA-2 of strain TCM encodes a 29.1 K protein (Angenent et al,, 1986). Because the sequence of the 3’terminal 1099 nucleotides of TCM RNAs 1 and 2 are identical, both RNAs encode the 16K protein. Recently,
MATERIALS Purification
AND METHODS
of viral nucleoprotein
and RNA
TRV strain PLB (Angenent eta/., to be published) was purified from Samsun NN tobacco according to Hut-
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ANGENENT,
306
VERBEEK, AND BOL
tinga (1972) and sedimented in sucrose gradients. RNA was extracted with phenol/chloroform (1: 1) at 65” from purified virus suspensions that had been incubated in 1o/oSDS for 15 min at 37”. Ethanol precipitated RNA was dissolved in H,O. Peptide synthesis, immunization
antigen preparation,
and
Preparation
The peptide was synthesized essentially according to the solid-phase method of Barany and Merrifield (1980). The sequence of the synthetic peptide was NH,-KRFLRDDVPLGIDQLFAF-COOH. This sequence is identical to that of the C-terminal 18 residues of the 16K protein of strain PLB (Angenent et a/., to be published) and shows only one mismatch with the amino acid sequences of the 16K proteins of strains PSG and TCM (Cornelissen et a/., 1986; Angenent et al., 1986). The synthetic peptide was coupled to ovalbumin by glutaraldehyde as described by Pfaff et a/. (1982). Purified virus particles were used for the production of antiserum against the viral CP. New Zealand White rabbits were immunized with 500 pg peptide-ovalbumin conjugate or 500 pg virus particles emulsified in complete Freund’s adjuvant. A sample of serum was collected prior to immunization. The subcutaneous injections were repeated in incomplete Freund’s adjuvant 2,4, and 6 weeks after the first immunization and antiserum was collected 7 days after the last injection. In vitro transcription
and translation
A PLB cDNA clone corresponding to the 1070 3’-terminal nucleotides of PLB-2 was used for in vitro expression of the 16K cistron. By exonuclease III and Sl nuclease treatment (Henikoff, 1984) the 5’-terminal366 nucleotides were removed, resulting in a clone with the 16K cistron preceded by an upstream sequence of 24 nucleotides including the putative leader sequence of 21 nucleotides of the subgenomic RNA-lb (formerly called RNA-5) (Cornelissen et a/., 1986; Angenent et a/., 1986). This cDNA was fused to the T7 promotor and in vitro run-off transcription with T7 polymerase was carried out as described by Tabor et al. (I 985). Transcription products and RNA-2a, the putative subgenomic mRNA for the CP (formerly called RNA-4) (Cornelissen et a/., 1986; Angenent et a/., 1986) were translated in rabbit reticulocyte lysates as described by Van Tol and Van Vloten-Doting (1979). RNA-2a and RNA-2 were extracted from virions that had been purified by zonal gradient centrifugation. Isolation,
inoculation,
Van Dun eT a/. (1988). The cells (1 05) were inoculated by means of the PEG method with 5 pg of total PLB RNA and incubated for 40 hr at 25” under constant illumination. Proteins were labeled during the incubation with [35S]methionine (5 &i/sample) and the protoplasts were collected by centrifugation.
and incubation
of protoplasts
The isolation of tobacco protoplasts (Mcotiana tabacum cv. Samsun NN) was performed as described by
of subcellular
fractions
The pelleted tobacco protoplasts (1 05) were ground in a Potter mini-homogenizer at 4” in 0.5 ml of 100 mM Tris-HCI, pH 8.0, 10 mll/l KCI, 5 mM MgCl*, 10% glycerol, 10 mlVI P-mercaptoethanol and centrifuged at 1000 g for 15 min. The supernatant was subsequently centrifuged at 30,000 g for 30 min at 4”. The different fractions and culture medium were immunoprecipitated with antisera against the CP and 16K protein. Detergent treatment of the 1000 g supernatant was done by adding Triton X-l 00 and sodium deoxycholate to final concentrations of 0.5% each. After incubation of 30 min at 37” the supernatant was centrifuged at 30,000 g as mentioned above. Sucrose gradient extracts
centrifugation
of protoplast
The 1000 g supernatant (0.5 ml) of homogenized protoplasts was loaded onto a 10 to 40% sucrose gradient (4.5 ml). The gradient was centrifuged at 25,000 rpm (Beckman SW50.1 rotor) for 2 hr at 4” and 10 fractions of 0.5 ml were taken. The fractions were dialyzed against 10 mM NaH,PO,, 10 mll/l Na,HPO,, 0.99/o (WI v) NaCI, pH 7.6 (PBS), before immunoprecipitation. lmmunoprecipitation Samples were incubated with preimmune serum (diluted 1:25) in 200 ~1 of 10 mll/l NaH,PO,, 10 mNI Na,HPO,, 0.9% (w/v) NaCI, 1% (v/v) Triton X-l 00, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.6 (PBSTDS) for 1 hr at 4”. Protein A-Sepharose (1 mg) was added and the incubation was continued for one hour at room temperature. The Sepharose-bound material was removed by centrifugation and the supernatant was incubated with the antisera against CP and 16K protein (dilution 1:25) overnight at 4’. Unlabeled healthy protoplast extract was added (100/o) to the antisera in order to reduce the precipitation of labeled plant proteins. Immunoglobulins were bound to the protein A-Sepharose as described above and washed three times with 5x PBSTDS and once with 1X PBSTDS. Laemmli sample buffer was added to the collected Protein A-Sepharose and boiled for 3 min, and the supernatant was electrophoresed in a 12.5% polyacrylamide gel under denaturing conditions (Laemmli, 1970). The proteins
307
TRV 16K PROTEIN
were transferred to a PVDF membrane (Millipore) and detected by autoradiography. Immunological
Ml
2
detection
Immunological detection on Western blots and ELISA experiments were carried out as described by Van Pelt-Heerschap et al. (1987). The samples were taken from protoplasts or from leaves, which were squeezed with a glass rod in the presence or absence of 0.5% SDS. The leaves were also treated with cellulase and pectinase, analogous to the protoplast isolation, to obtain cell wall bound proteins. Labeling of proteins in infected leaf tissue Small disks of leaf tissue were taken from PLB-infected plants 24 hr after inoculation. The small disks were vacuum infiltrated with [35S]methionine as described by Bujarski et a/. (1982). Incubation of the leaf tissue was continued for 2 days at 25” under constant illumination in 1 ml H,O containing 10 &i [35S]methionine. The disks were ground with 200 ~1 PBSTDS in an Eppendorf tube and centrifuged and the supernatant was used for immunoprecipitation. RNA isolation,
Northern
blotting,
and hybridization
RNA isolation from infected tobacco leaves was carried out essentially as described by Hooft van Huijsduinen et a/. (1985). Total RNA isolated from tobacco protoplasts was transferred to a Gene Screen membrane and hybridized with 32P-labeled viral cDNA (Sarachu et al., 1985). RESULTS Reactivity
and specificity
FIG. 1. lmmunoprecipitation of in vitro synthesized 16K protein. cDNA containing the 16K gene was transcribed in vitro with T7 polymerase and the transcript was translated in a reticulocyte lysate. %Labeled proteins were precipitated with preimmune serum (lane 1) or with an antiserum to the C-terminal peptide of the 16K protein (lane 2). Lane M contains ‘Wabeled molecular weight markers. Proteins were analyzed by polyacrylamide gel electrophoresis.
of the antiserum
A peptide of 18 amino acids was synthesized corresponding to the sequence of the C-terminus of the 16K protein. This sequence was chosen because Van PeltHeerschap et a/. (1987) showed that C-termini of several viral proteins were effective antigens. Moreover, the C-terminus of the 16K protein is hydrophilic, suggesting that this part is located on the outside of the protein. The antiserum against the 16K protein was tested in ELISA and immunoprecipitation experiments. In ELISA an antiserum dilution of 1: 10,000 was able to detect as little as 1 ng of uncoupled peptide (data not shown). Figure 1 shows the immunoprecipitation of in vitro synthesized 16K protein. The mRNA was an in vitro transcript of a cDNA clone containing the 16K cistron. The clone was truncated in such a way that the leader of the transcript did not contain internal AUG condons. Preimmune serum was not able to precipitate the 16K
protein (Fig. 1, lane l), while incubation of the labeled product with the 16K antiserum yielded a unique protein band of the predicted size (lane 2). Detection of the 16K protein in infected tobacco protoplasts Tobacco protoplasts were inoculated with PLB RNA and tested for the production of CP and 16K protein. The protoplasts were incubated for 40 hr at 25” and homogenized in immunoprecipitation buffer (PBSTDS). Lanes A and B of Fig. 2 show the controls with immunoprecipitates of in vitro synthesized 16K protein and CP, respectively. Genomic RNA-2 did not show any messenger activity in the reticulocyte lysate (result not shown) but subgenomic RNA-2a was efficiently translated into CP. This RNA lacks the 5’-terminal474 nucleotides of the PLB RNA-2 leader sequence, including all eight AUG codons occurring in this leader sequence (Angenent eta/., to be published). A mixture of the antisera to the 16K protein and CP did not precipitate any labeled protein from mock-inoculated protoplasts (Fig. 2, lane l), nor did preimmune serum react with proteins from TRV-infected protoplasts (lane 2). However, with the mixture of the two antisera, CP and 16K protein were clearly detectable in infected protoplasts (Fig. 2, lane 3). Preincubation of the mixed antiserum with an excess of synthetic peptide corresponding to the C-terminus of the 16K protein resulted in a specific loss of
308
ANGENENT,
VERBEEK, AND BOL
Detection of the 16K protein in subcellular of tobacco protoplasts
AB1234
16kFIG. 2. Detection of TRV coat protein (CP) and 16K protein in protoplasts. Tobacco protoplasts were mock-inoculated (lane 1) or inoculated with TRV RNA (lanes 2. 3, and 4). %-Labeled proteins extracted from the protoplasts were immunoprecipitated with preimmune serum (lane 2) or a mixture of antisera to CP and 16K protein (lanes 1, 3, and 4). In lane 4, the antiserum was preincubated with 100 pg of synthetic peptide corresponding to the C-terminus of the 16K protein. Lanes A and B show the controls of in vitro synthesized 16K protein and CP, respectively, immunoprecipitated with the mixture of antisera. Proteins were analyzed by polyacrylamide gel electrophoresis.
the detection of the 16K protein (Fig. 2, lane 4). Taking into account that the antisera to the 16K protein and CP have similar titers, that they precipitate in vitro made proteins with similar efficiency, and that the two proteins contain two and four methionine residues, respectively, the results of Fig. 2 suggest that in infected protoplasts the 16K protein is produced in excess over CP at the time point analyzed. Time course of CP and 16K production protoplasts
Tobacco protoplasts were inoculated with TRV RNA and harvested after 40 hr of incubation. To investigate the location of the 16K protein and CP, the infected protoplasts were homogenized and the homogenate was fractionated into a 1000 g pellet, 30,000 g pellet, and a 30,000 g supernatant. In such a fractionation the 1000 g pellet contains nuclei and chloroplasts, whereas membranes and virus particles are present in the 30,000 g pellet. These fractions were immunoprecipitated with antisera against the 16K protein and CP. Neither of the two proteins could be detected in the protoplast culture medium (Fig. 4, lane l), indicating that there is no transport of these proteins over the cellular membrane. Figure 4, lane 2, shows the immunoprecipitated proteins present in the 1000 g pellet. Small amounts of CP are present in this fraction but no 16K protein was detectable. Virus particles attached to cell organelles as found by Harrison et a/. (1977) may explain the occurrence of CP in the 1000 g fraction. The majority of the CP is present in the 30,000 g pellet (Fig. 4, lane 3) and a small amount occurs in the cytoplasmic fraction possibly as free CP (lane 4). For unknown reasons the CP appeared as a double band in this experiment. Almost all of the 16K protein was found in the 30,000 g pellet. To see if the appearance of the 16K protein in the 30,000 g pellet fraction is due to an interaction with membranes, we treated the 1000 g supernatant with
in tobacco
Tobacco protoplasts were infected with PLB RNA and incubated in medium containing [35S]methionine. The protoplasts were collected at various times after inoculation and proteins were immunoprecipitated with antisera against the CP and the 16K protein (Fig. 3). The CP is already detectable 8 hr postinoculation and has reached its maximum level after 20 hr. This is in agreement with the results of Harrison et a/. (1976), who detected infectious virus in tobacco protoplasts 9 hr after TRV infection. The CP migrated as a 29-kDa protein in a 12.5% SDS-polyacrylamide gel, while the calculated molecular weight deduced from the amino acid sequence is 22,856 (Angenent et a/,, to be published). The 16K protein was detectable after 20 hr and continued to accumulate in the next 28 hr. The double band in lane A is probably due to an artifact or degradation of the 16K protein.
fractions
MA
B
0
4
8
20
48
30k
14kFIG. 3. Time course of the synthesis of TRV coat protein and 16K protein in infected tobacco protoplasts. 35S-Labeled proteins were extracted at 0, 4, 8, 20. and 48 hr after inoculation of the protoplasts. The proteins were immunoprecipitated with a mixture of antisera against coat protein and 16K protein and analyzed by polyacrylamide gel electrophoresis. Lanes A and B were run with in vitro synthesized 16K protein and coat protein, respectively. Lane M shows ‘%-labeled marker proteins.
TRV 16K PROTEIN
309
Assay of infected plants for the 16K protein
CP-
16K-
FIG. 4. Detection of TRV coat protein (CP) and 16K protein in subcellular fractions of infected tobacco protoplasts. %Z-Labeled proteins from the protoplast medium (lane 1) or from the 1000 g pellet (lane 2) 30,000 g pellet (lane 3) and 30,000 g supernatant fraction of homogenated protoplasts were immunoprecipitated with a mixture of antisera against CP and 16K protein, The proteins were analyzed by polyacrylamide gel electrophoresis. Lanes A and B were run with in v&o synthesized 16K protein and CP, respectively.
detergents (0.5% Triton X-100 and 0.5% sodium deoxycholate) before centrifugation at 30,000 g (Vu et a/., 1973). This treatment did not affect the distribution of the 16K protein over the respective fractions (results not shown). Therefore, the 16K protein is probably not bound to membrane structures. To further investigate the localization of the 16K protein, the 1000 g supernatant fraction from infected protoplasts was sedimented in a sucrose gradient. Ten fractions were taken and immunoprecipitated with the mixture of the 16K and CP antisera. Figure 5 shows the distribution of the CP and 16K protein over the gradient. The CP is detectable in fractions 3 to 10, corresponding to the known sedimentation of the virions containing the genomic and subgenomic RNAs (Robinson and Mayo, 1978). The major amount of the 16K protein is present in fractions 4 and 5, indicating that in infected tobacco protoplasts the 16K protein is incorporated in a high-molecular-weight complex. The relatively low production of CP in this experiment may be due to a high proportion of protoplasts infected with RNA-l only (NM-infection) (Harrison and Robinson, 1978). The proteins marked by an asterisk are also precipitated from homogenates of mock-inoculated protoplasts and probably represent the small and large subunits of ribulose bisphosphate carboxylase. When the total proteins present in the gradient fractions of Fig. 5 were analyzed on a SDS-polyacrylamide gel, no labeled host proteins were detectable in fractions 4 and 5 (result not shown). This indicates that the high-molecular-weight structure in these fractions consists of a multimer of 16K subunits, or that the 16K protein associates with preexisting nonlabeled host proteins.
Leaf disks from PLB-infected Samsun NN tobacco plants were vacuum-infiltrated with a [35S]methionine solution and after a labeling period of 2 days proteins were extracted. As a control, the production of viral RNA was monitored by Northern blot hybridization using a cDNA probe complementary to the homologous 3’-terminus of TRV RNA capable of detecting all genomic and subgenomic TRV RNAs. Figure 6A shows the production of PLB RNAs in tobacco protoplasts (lane 1) and plants (lane 2). The subgenomic mRNAs for the 29K protein (RNA-l a) and coat protein (RNA-2a) of PLB have approximately the same length (Angenent et a/., to be published) and are not separated in Fig. 6A. The subgenomic mRNA for the 16K protein is termed RNA1b according to the nomenclature proposed by Robinson et al. (1987). However, because PLB is diploid for the 16K gene, it is impossible to say whether this RNA is derived from RNA-l or RNA-2 or both. Although all RNA species are detectable both in protoplasts and plants their relative proportions in the two preparations are clearly different. Figure 6B shows an analysis of proteins synthesized in vivo. As controls, lanes 1 and 2 show the in vitro synthesized 16K protein and CP while lanes 3 and 4 show the proteins from PLB-infected protoplasts precipitated with preimmune serum and the mixture of the 16K protein and CP antisera, respectively. Lanes 5 and
FIG. 5. Sucrose gradient centrifugation of subcellular structures containing TRV coat protein (CP) or 16K protein. A homogenate of infected protoplasts was sedimented in a sucrose gradient and fraction 1 (bottom) to fraction 10 (top) were collected. Y-Labeled proteins in these fractions were immunoprecipitated with a mixture of antisera to CP and 16K protein and analyzed by polyacrylamide gel electrophoresis. Lanes A and B were run with in vitro synthesized 16K protein and CP, respectively.
310
ANGENENT,
VERBEEK, AND BOL
B
A 1
2
123456
FIG. 6. Detection of TRV RNAs, coat protein (CP) and 16K protein in infected tobacco protoplasts and leaves. Panel (A) shows a Northern blot with RNAfrom TRV-infected tobacco protoplasts (lane 1) and leaves (lane 2). Nick-translated cDNA corresponding to the 3’.terminai 1070 nucleotides of PLB RNA-2 was used as probe. The position of TRV genomic and subgenomic RNAs is indicated in the margin. Panel (B) shows 35S-labeled proteins that were extracted from infected tobacco protoplasts (lanes 3 and 4) or leaves (lanes 5 and 6) and were precipitated with preimmune serum (lanes 3 and 5) or a mixture of antisera against CP and 16K protein (lanes 4 and 6). The proteins were analyzed by polyacrylamide gel electrophoresis. Lanes 1 and 2 were run with in vitro synthesized 16K protein and CP. respectively.
6 of Fig. 6B show the proteins from PLB-infected plants that are precipitated with preimmune serum and the mixture of the 16K protein and CP antisera, respectively. Although approximately similar amounts of the CP are detected in the extracts from protoplasts and plants, the 16K protein was found exclusively in the extract from protoplasts. To preclude the possibility that the 16K protein is trapped in cell wall structures, proteins were extracted from unlabeled infected plant material using buffers containing cellulase (2%) and pectinase (0.1 o/b)or various extraction buffers with or without SDS (0.5%). However, an analysis of these extracts on Western blots failed to detect the 16K protein whereas the CP was readily observed under all conditions (results not shown). DISCUSSION The observation that the 16K protein accumulates to high levels in TRV-infected protoplasts corroborates the notion that this conserved TRV gene has a function in the viral replication cycle. Because the 16K protein continues to accumulate when CP synthesis has already leveled off, it may not be involved in the production of viral RNA or virion assembly but may have a function for example in virus spread. It has been suggested that the 16K protein plays a role in transmission of the virus by nematodes (Boccara et a/., 1986). Highly specific relationships have been found to exist between TRV isolates and the species of trichodorid nematodes involved in their transmission (Brown et al., 1988). Because in a number of TRV isolates the 16K
protein is encoded by both RNA-l and RNA-2, it would be interesting to investigate the nematode transmissibility of pseudo-recombinants with 16K genes from different parents. In this respect it is noteworthy that a pseudo-recombinant containing RNA-l of strain TCM and RNA-2 of strain PLB was found to be unchanged in sequence after numerous passages in tobacco (Angenent eta/., to be published). Apparently, RNA recombination restoring the perfect homology at the 3’-termini of the genome segments is not a frequent event under greenhouse conditions. The finding that RNA-l b, the putative messenger for the 16K protein, is produced in infected tobacco protoplasts as well as in leaves indicates that the 16K gene is expressed under both conditions. Our inability to detect the 16K protein in leaf material may have several explanations. It is possible that in intact plants the turnover of the 16K protein is much higher than in protoplasts. Alternatively, a modification of the protein or its inclusion in a structure that is inaccessible to our extraction procedures may interfere with the detection of the 16K protein in plants. Theoretically, regulation at the translation level could be involved, resulting in a tissue-specific expression. At present, the possibility of detecting the 16K protein in plant tissues by immunoelectron microscopy is being investigated. ACKNOWLEDGMENTS We thank Dr. Huub Linthorst for the antiserum against the coat protein and for critical reading of the manuscript. This work was sponsored in part by the Netherlands Organization for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Research (NWO).
REFERENCES ANGENENT. G. C., LINTHORST. H. J. M., VAN BELKUM, A. F., CORNELISSEN. B. J. C., and BOL, J. F. (1986). RNA-2 of tobacco rattle virus strain TCM encodes an unexpected gene. Nucleic Acids Res. 14, 4673-4682. BARANY,G., and MERRIFIELD,R. B. (1980). Solid phase peptide synthesis. ln “The Peptides” (E. Gross and J. Meienhofer, Eds.), Vol. 2, pp. l-284. Academic Press, New York/London. BOCCARA, M., HAMILTON, W. D. O., and BAULCOMBE,D. C. (1986). The organisation and interviral homologies of genes at the 3’ end of tobacco rattle virus RNA-l. En/lBO/. 5, 223-229. BROWN, D. J. F., PLOEG,A. T., and ROBINSON,D. J. (1988). A review of reported associations between Trichodorus and Paratrichodorus species and tobraviruses with a description of laboratory methods for examining virus transmission by trichodorids. Rev. Nematol. In press. BUJARSKI.J. I., HARDY, S. F., MILLER, W. A., and HALL, T. C. (1982). Use of dodecyl-P-o-maltoside in the purification and stabilization of RNA polymerase from brome mosaic virus-infected barley. Viology 119,465-473. CORNELISSEN,B. J. C., LINTHORST,H. J. M., BREDERODE,F. T., and BOL, J. F. (1986). Analyses of the genome structure of tobacco rattle virus strain PSG. Nucleic Acids Res. 14. 2157-2169.
TRV 16K PROTEIN FRITSCH, C., MAYO, M. A., and HIRTH, L. (1977). Further studies on the translation products of tobacco rattle virus in vitro. Virology 77, 722-732. HAMILTON, W. D. O., BOCARRA,M., ROBINSON,D. J., and BAULCOMBE, D. C. (1987). The complete nucleotide sequence of tobacco rattle virus RNA-l. /. Gen. Viral. 68, 2563-2575. HARRISON,B. D., HUTCHESON,A. M., and BARKER,H. (1977). Association between the particles of rasberry ringspot and tobacco rattle viruses in doubly infected Nicofiana benthamiana cells and protoplasts. /. Gen. Viral. 36, 535-539. HARRISON, B. D., Kuso, S., ROBINSON, D. J., and HUTCHESON, A. M. (1976). The multiplication cycle of tobacco rattle virus in tobacco mesophyll protoplasts. /. Gen. l&o/. 33, 237-248. HARRISON,B. D., and ROBINSON, D. 1. (1978). The tobraviruses. Adv. Virus Res. 23,25-77. HARRISON, B. D., and ROBINSON, D. J. (1986). Tobraviruses. In “The Plant Viruses” (M. H. V. van Regenmortel and H. Fraenkel-Conrat, Eds.), Vol. 2, pp. 339-369. Plenum, New York. HENIKOFF,S. (1984). Bidirectional digestion with exonuclease Ill creates breakpoints for DNA sequencing. Gene 28, 351-359. HOOFT VAN HUIJSDUINEN,R. A. M., CORNELISSEN,B. J. C., VAN LOON, L. C.. VAN BOOM, J. H., TROMP, M., and BOL, J. F. (1985). Virusinduced synthesis of messenger RNAs for precursors of pathogenesis-related proteins in tobacco. EM50 J. 4, 2 167-217 1, HU~INGA, H. (1972). “Interaction between Long and Short Particles of Tobacco Rattle Virus.” PhD. Thesis, University of Wageningen. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685.
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PFAFF, E., MUSSGAY, M. H. O., BOHM, G. E., and SCHALLER,H. (1982). Antibodies against preselected peptides recognize and neutralize foot and mouth disease. EMBOJ. 1,875-881. ROBINSON, D. I., HAMILTON, W. D. O., HARRISON, B. D., and BAULCOMBE, D. C. (1987). Two anomalous tobravirus isolates: Evidence for RNA recombination in nature. /. Gem Viral. 68,2551-2561. ROBINSON, D. J., and MAYO, M. A. (1978). “Separation of Long and Short Particles of Tobacco Rattle Virus (TRV).” Annual Report, Scottish Horticultural Research Institute, 1977, pp. 90-9 1. SARACHU, A. N.. HUISMAN, M. J., VAN VLOTEN-DOTING, L., and BOL, J. F. (1985). Alfalfa mosaic virus temperature-sensitive mutants. L%ology 141, 14-22. TABOR, S. (1985). A bacteriophage T7 RNA polymerase/promotor system for controlled exclusive expression of specific genes. Proc. Nat/. Acad. Sci. USA 82, 1074-l 078. VAN DUN, C. M. P., VAN VLOTEN-DOTING, L., and BOL, J. F. (1988). Expression of alfalfa mosaic virus cDNA1 and 2 in transgenic tobacco plants. Virology 163, 572-578. VAN PELT-HEERSCHAP,H., VERBEEK.H., HUISMAN, M. J., LOESCH-FRIES. L. S., and VAN VLOTEN-DOTING, L. (1987). Non-structural proteins and RNAs of alfalfa mosaic virus synthesized in tobacco and cowpea protoplasts. Virology 161, 190-l 97. VAN TOL, R. G. L., and VAN VLOTEN-DOTING, L. (1979). Translation of alfalfa mosaic virus RNA1 in the mRNA-dependent translation system from rabbit reticulocyte lysates. Eur. /. Biochem. 93, 461468. Yu, J.. FISCHMAN. D. A., and STECK, T. L. (1973). Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. J. Supramol. Srruct. 1, 233-249.
169,305-311
(1989)
Expression
of the 16K Cistron of Tobacco
Rattle Virus in Protoplasts
GERCO C. ANGENENT, HANS B. M. VERBEEK, AND JOHN F. BOL’ Department
of Biochemistry,
Leiden University,
P.O. Box 9505, 2300 RA Leiden, The Netherlands
Received July 18, 1988; accepted November
8, 1988
An antiserum was raised against a synthetic peptide corresponding to the 18 C-terminal amino acids of a putative 16K protein encoded by the 3’-terminal open reading frame of tobacco rattle virus (TRV) RNA-l. This antiserum was used to demonstrate expression of the 16K cistron in vivo. TRV-infected tobacco protoplasts accumulated similar amounts of 16K protein and viral coat protein but in tobacco plants only the coat protein was detectable. Time course experiments revealed that in protoplasts the accumulation of 16K protein lagged somewhat behind that of coat protein. The 16K protein was incorporated in a high-molecular-weight cellular component that was resistant to treatment with 0 1989 Academic Press, Inc. nOniOniC detergents.
INTRODUCTION
we have determined the nucleotide sequence of RNA2 of TRV strain PLB (Angenent et al., to be published). Because the 3’-terminal sequence of PLB RNA-2 is identical to the 3’-end of PLB RNA-l over a length of 820 nucleotides, this strain is also diploid for the 16K gene. None of the nonstructural TRV proteins has been detected in infected cells so far. Homologies with corresponding gene products of tobacco mosaic virus (TMV) indicate that the 134K and 194K proteins are involved in viral replication and suggest a role for the 29K protein in cell-to-cell transport of infectious material (Hamilton et a/., 1987). The combination of these functions in TRV RNA-l could explain the ability of this genome segment to replicate and spread in plants in the absence of RNA-2 (Harrison and Robinson, 1978). Although the 16K cistron is present in all TRV strains sequenced to date, no function of this gene is known. The amino acid sequence homology between the 16K proteins of strains PSG, PLB, TCM, and SYM is approximately 90%. To permit an analysis of the expression of the 16K gene in infected cells, we raised an antiserum to a synthetic peptide corresponding to the conserved C-terminus of the 16K protein. Here we repot-t that the 16K gene is expressed to relatively high levels in tobacco protoplasts infected with TRV strain PLB. In addition, a preliminary characterization of the cellular structure in which the 16K protein is incorporated is described.
Tobacco rattle virus (TRV) is the type member of the Tobraviruses. The genome of these viruses consists of two plus-stranded RNA molecules, which are separately encapsidated into rod-shaped particles (Harrison and Robinson, 1986). RNA-l of the SYM-strain (6791 nucleotides) has been completely sequenced (Hamilton et al., 1987) and the 3’-terminal sequences of RNA1 of strains PSG and TCM have been reported (Cornelissen et a/., 1986; Angenent et al., 1986). The sequence data indicate that TRV RNA-l encodes four proteins. The 5’-proximal open reading frame encodes a protein with a molecular weight of 134,000 (134K) and read-through of a “leaky” termination codon results in an extension of this protein to a 194K polypeptide. These two proteins are directed by RNA-l in an in vitro translation system (Fritsch et al., 1977). The third and fourth reading frames in RNA-l encode 29K and 16K proteins, respectively. Subgenomic RNAs that may function as messenger for these proteins have been identified (Cornelissen et a/., 1986; Bocarra et a/., 1986). RNA-2 of tobraviruses varies in length between 1800 and 4000 nucleotides, depending on the strain (Harrison and Robinson, 1978). Part of this variability is due to differences in length of the 3’-terminal sequence of RNA-2 that is identical to that of the corresponding RNA-l (Angenent et a/., 1986). The sequence that is unique to RNA-2 encodes the coat protein (CP). In addition, RNA-2 of strain TCM encodes a 29.1 K protein (Angenent et al,, 1986). Because the sequence of the 3’terminal 1099 nucleotides of TCM RNAs 1 and 2 are identical, both RNAs encode the 16K protein. Recently,
MATERIALS Purification
AND METHODS
of viral nucleoprotein
and RNA
TRV strain PLB (Angenent eta/., to be published) was purified from Samsun NN tobacco according to Hut-
’ To whom requests for reprints should be addressed. 305
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tinga (1972) and sedimented in sucrose gradients. RNA was extracted with phenol/chloroform (1: 1) at 65” from purified virus suspensions that had been incubated in 1o/oSDS for 15 min at 37”. Ethanol precipitated RNA was dissolved in H,O. Peptide synthesis, immunization
antigen preparation,
and
Preparation
The peptide was synthesized essentially according to the solid-phase method of Barany and Merrifield (1980). The sequence of the synthetic peptide was NH,-KRFLRDDVPLGIDQLFAF-COOH. This sequence is identical to that of the C-terminal 18 residues of the 16K protein of strain PLB (Angenent et a/., to be published) and shows only one mismatch with the amino acid sequences of the 16K proteins of strains PSG and TCM (Cornelissen et a/., 1986; Angenent et al., 1986). The synthetic peptide was coupled to ovalbumin by glutaraldehyde as described by Pfaff et a/. (1982). Purified virus particles were used for the production of antiserum against the viral CP. New Zealand White rabbits were immunized with 500 pg peptide-ovalbumin conjugate or 500 pg virus particles emulsified in complete Freund’s adjuvant. A sample of serum was collected prior to immunization. The subcutaneous injections were repeated in incomplete Freund’s adjuvant 2,4, and 6 weeks after the first immunization and antiserum was collected 7 days after the last injection. In vitro transcription
and translation
A PLB cDNA clone corresponding to the 1070 3’-terminal nucleotides of PLB-2 was used for in vitro expression of the 16K cistron. By exonuclease III and Sl nuclease treatment (Henikoff, 1984) the 5’-terminal366 nucleotides were removed, resulting in a clone with the 16K cistron preceded by an upstream sequence of 24 nucleotides including the putative leader sequence of 21 nucleotides of the subgenomic RNA-lb (formerly called RNA-5) (Cornelissen et a/., 1986; Angenent et a/., 1986). This cDNA was fused to the T7 promotor and in vitro run-off transcription with T7 polymerase was carried out as described by Tabor et al. (I 985). Transcription products and RNA-2a, the putative subgenomic mRNA for the CP (formerly called RNA-4) (Cornelissen et a/., 1986; Angenent et a/., 1986) were translated in rabbit reticulocyte lysates as described by Van Tol and Van Vloten-Doting (1979). RNA-2a and RNA-2 were extracted from virions that had been purified by zonal gradient centrifugation. Isolation,
inoculation,
Van Dun eT a/. (1988). The cells (1 05) were inoculated by means of the PEG method with 5 pg of total PLB RNA and incubated for 40 hr at 25” under constant illumination. Proteins were labeled during the incubation with [35S]methionine (5 &i/sample) and the protoplasts were collected by centrifugation.
and incubation
of protoplasts
The isolation of tobacco protoplasts (Mcotiana tabacum cv. Samsun NN) was performed as described by
of subcellular
fractions
The pelleted tobacco protoplasts (1 05) were ground in a Potter mini-homogenizer at 4” in 0.5 ml of 100 mM Tris-HCI, pH 8.0, 10 mll/l KCI, 5 mM MgCl*, 10% glycerol, 10 mlVI P-mercaptoethanol and centrifuged at 1000 g for 15 min. The supernatant was subsequently centrifuged at 30,000 g for 30 min at 4”. The different fractions and culture medium were immunoprecipitated with antisera against the CP and 16K protein. Detergent treatment of the 1000 g supernatant was done by adding Triton X-l 00 and sodium deoxycholate to final concentrations of 0.5% each. After incubation of 30 min at 37” the supernatant was centrifuged at 30,000 g as mentioned above. Sucrose gradient extracts
centrifugation
of protoplast
The 1000 g supernatant (0.5 ml) of homogenized protoplasts was loaded onto a 10 to 40% sucrose gradient (4.5 ml). The gradient was centrifuged at 25,000 rpm (Beckman SW50.1 rotor) for 2 hr at 4” and 10 fractions of 0.5 ml were taken. The fractions were dialyzed against 10 mM NaH,PO,, 10 mll/l Na,HPO,, 0.99/o (WI v) NaCI, pH 7.6 (PBS), before immunoprecipitation. lmmunoprecipitation Samples were incubated with preimmune serum (diluted 1:25) in 200 ~1 of 10 mll/l NaH,PO,, 10 mNI Na,HPO,, 0.9% (w/v) NaCI, 1% (v/v) Triton X-l 00, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.6 (PBSTDS) for 1 hr at 4”. Protein A-Sepharose (1 mg) was added and the incubation was continued for one hour at room temperature. The Sepharose-bound material was removed by centrifugation and the supernatant was incubated with the antisera against CP and 16K protein (dilution 1:25) overnight at 4’. Unlabeled healthy protoplast extract was added (100/o) to the antisera in order to reduce the precipitation of labeled plant proteins. Immunoglobulins were bound to the protein A-Sepharose as described above and washed three times with 5x PBSTDS and once with 1X PBSTDS. Laemmli sample buffer was added to the collected Protein A-Sepharose and boiled for 3 min, and the supernatant was electrophoresed in a 12.5% polyacrylamide gel under denaturing conditions (Laemmli, 1970). The proteins
307
TRV 16K PROTEIN
were transferred to a PVDF membrane (Millipore) and detected by autoradiography. Immunological
Ml
2
detection
Immunological detection on Western blots and ELISA experiments were carried out as described by Van Pelt-Heerschap et al. (1987). The samples were taken from protoplasts or from leaves, which were squeezed with a glass rod in the presence or absence of 0.5% SDS. The leaves were also treated with cellulase and pectinase, analogous to the protoplast isolation, to obtain cell wall bound proteins. Labeling of proteins in infected leaf tissue Small disks of leaf tissue were taken from PLB-infected plants 24 hr after inoculation. The small disks were vacuum infiltrated with [35S]methionine as described by Bujarski et a/. (1982). Incubation of the leaf tissue was continued for 2 days at 25” under constant illumination in 1 ml H,O containing 10 &i [35S]methionine. The disks were ground with 200 ~1 PBSTDS in an Eppendorf tube and centrifuged and the supernatant was used for immunoprecipitation. RNA isolation,
Northern
blotting,
and hybridization
RNA isolation from infected tobacco leaves was carried out essentially as described by Hooft van Huijsduinen et a/. (1985). Total RNA isolated from tobacco protoplasts was transferred to a Gene Screen membrane and hybridized with 32P-labeled viral cDNA (Sarachu et al., 1985). RESULTS Reactivity
and specificity
FIG. 1. lmmunoprecipitation of in vitro synthesized 16K protein. cDNA containing the 16K gene was transcribed in vitro with T7 polymerase and the transcript was translated in a reticulocyte lysate. %Labeled proteins were precipitated with preimmune serum (lane 1) or with an antiserum to the C-terminal peptide of the 16K protein (lane 2). Lane M contains ‘Wabeled molecular weight markers. Proteins were analyzed by polyacrylamide gel electrophoresis.
of the antiserum
A peptide of 18 amino acids was synthesized corresponding to the sequence of the C-terminus of the 16K protein. This sequence was chosen because Van PeltHeerschap et a/. (1987) showed that C-termini of several viral proteins were effective antigens. Moreover, the C-terminus of the 16K protein is hydrophilic, suggesting that this part is located on the outside of the protein. The antiserum against the 16K protein was tested in ELISA and immunoprecipitation experiments. In ELISA an antiserum dilution of 1: 10,000 was able to detect as little as 1 ng of uncoupled peptide (data not shown). Figure 1 shows the immunoprecipitation of in vitro synthesized 16K protein. The mRNA was an in vitro transcript of a cDNA clone containing the 16K cistron. The clone was truncated in such a way that the leader of the transcript did not contain internal AUG condons. Preimmune serum was not able to precipitate the 16K
protein (Fig. 1, lane l), while incubation of the labeled product with the 16K antiserum yielded a unique protein band of the predicted size (lane 2). Detection of the 16K protein in infected tobacco protoplasts Tobacco protoplasts were inoculated with PLB RNA and tested for the production of CP and 16K protein. The protoplasts were incubated for 40 hr at 25” and homogenized in immunoprecipitation buffer (PBSTDS). Lanes A and B of Fig. 2 show the controls with immunoprecipitates of in vitro synthesized 16K protein and CP, respectively. Genomic RNA-2 did not show any messenger activity in the reticulocyte lysate (result not shown) but subgenomic RNA-2a was efficiently translated into CP. This RNA lacks the 5’-terminal474 nucleotides of the PLB RNA-2 leader sequence, including all eight AUG codons occurring in this leader sequence (Angenent eta/., to be published). A mixture of the antisera to the 16K protein and CP did not precipitate any labeled protein from mock-inoculated protoplasts (Fig. 2, lane l), nor did preimmune serum react with proteins from TRV-infected protoplasts (lane 2). However, with the mixture of the two antisera, CP and 16K protein were clearly detectable in infected protoplasts (Fig. 2, lane 3). Preincubation of the mixed antiserum with an excess of synthetic peptide corresponding to the C-terminus of the 16K protein resulted in a specific loss of
308
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Detection of the 16K protein in subcellular of tobacco protoplasts
AB1234
16kFIG. 2. Detection of TRV coat protein (CP) and 16K protein in protoplasts. Tobacco protoplasts were mock-inoculated (lane 1) or inoculated with TRV RNA (lanes 2. 3, and 4). %-Labeled proteins extracted from the protoplasts were immunoprecipitated with preimmune serum (lane 2) or a mixture of antisera to CP and 16K protein (lanes 1, 3, and 4). In lane 4, the antiserum was preincubated with 100 pg of synthetic peptide corresponding to the C-terminus of the 16K protein. Lanes A and B show the controls of in vitro synthesized 16K protein and CP, respectively, immunoprecipitated with the mixture of antisera. Proteins were analyzed by polyacrylamide gel electrophoresis.
the detection of the 16K protein (Fig. 2, lane 4). Taking into account that the antisera to the 16K protein and CP have similar titers, that they precipitate in vitro made proteins with similar efficiency, and that the two proteins contain two and four methionine residues, respectively, the results of Fig. 2 suggest that in infected protoplasts the 16K protein is produced in excess over CP at the time point analyzed. Time course of CP and 16K production protoplasts
Tobacco protoplasts were inoculated with TRV RNA and harvested after 40 hr of incubation. To investigate the location of the 16K protein and CP, the infected protoplasts were homogenized and the homogenate was fractionated into a 1000 g pellet, 30,000 g pellet, and a 30,000 g supernatant. In such a fractionation the 1000 g pellet contains nuclei and chloroplasts, whereas membranes and virus particles are present in the 30,000 g pellet. These fractions were immunoprecipitated with antisera against the 16K protein and CP. Neither of the two proteins could be detected in the protoplast culture medium (Fig. 4, lane l), indicating that there is no transport of these proteins over the cellular membrane. Figure 4, lane 2, shows the immunoprecipitated proteins present in the 1000 g pellet. Small amounts of CP are present in this fraction but no 16K protein was detectable. Virus particles attached to cell organelles as found by Harrison et a/. (1977) may explain the occurrence of CP in the 1000 g fraction. The majority of the CP is present in the 30,000 g pellet (Fig. 4, lane 3) and a small amount occurs in the cytoplasmic fraction possibly as free CP (lane 4). For unknown reasons the CP appeared as a double band in this experiment. Almost all of the 16K protein was found in the 30,000 g pellet. To see if the appearance of the 16K protein in the 30,000 g pellet fraction is due to an interaction with membranes, we treated the 1000 g supernatant with
in tobacco
Tobacco protoplasts were infected with PLB RNA and incubated in medium containing [35S]methionine. The protoplasts were collected at various times after inoculation and proteins were immunoprecipitated with antisera against the CP and the 16K protein (Fig. 3). The CP is already detectable 8 hr postinoculation and has reached its maximum level after 20 hr. This is in agreement with the results of Harrison et a/. (1976), who detected infectious virus in tobacco protoplasts 9 hr after TRV infection. The CP migrated as a 29-kDa protein in a 12.5% SDS-polyacrylamide gel, while the calculated molecular weight deduced from the amino acid sequence is 22,856 (Angenent et a/,, to be published). The 16K protein was detectable after 20 hr and continued to accumulate in the next 28 hr. The double band in lane A is probably due to an artifact or degradation of the 16K protein.
fractions
MA
B
0
4
8
20
48
30k
14kFIG. 3. Time course of the synthesis of TRV coat protein and 16K protein in infected tobacco protoplasts. 35S-Labeled proteins were extracted at 0, 4, 8, 20. and 48 hr after inoculation of the protoplasts. The proteins were immunoprecipitated with a mixture of antisera against coat protein and 16K protein and analyzed by polyacrylamide gel electrophoresis. Lanes A and B were run with in vitro synthesized 16K protein and coat protein, respectively. Lane M shows ‘%-labeled marker proteins.
TRV 16K PROTEIN
309
Assay of infected plants for the 16K protein
CP-
16K-
FIG. 4. Detection of TRV coat protein (CP) and 16K protein in subcellular fractions of infected tobacco protoplasts. %Z-Labeled proteins from the protoplast medium (lane 1) or from the 1000 g pellet (lane 2) 30,000 g pellet (lane 3) and 30,000 g supernatant fraction of homogenated protoplasts were immunoprecipitated with a mixture of antisera against CP and 16K protein, The proteins were analyzed by polyacrylamide gel electrophoresis. Lanes A and B were run with in v&o synthesized 16K protein and CP, respectively.
detergents (0.5% Triton X-100 and 0.5% sodium deoxycholate) before centrifugation at 30,000 g (Vu et a/., 1973). This treatment did not affect the distribution of the 16K protein over the respective fractions (results not shown). Therefore, the 16K protein is probably not bound to membrane structures. To further investigate the localization of the 16K protein, the 1000 g supernatant fraction from infected protoplasts was sedimented in a sucrose gradient. Ten fractions were taken and immunoprecipitated with the mixture of the 16K and CP antisera. Figure 5 shows the distribution of the CP and 16K protein over the gradient. The CP is detectable in fractions 3 to 10, corresponding to the known sedimentation of the virions containing the genomic and subgenomic RNAs (Robinson and Mayo, 1978). The major amount of the 16K protein is present in fractions 4 and 5, indicating that in infected tobacco protoplasts the 16K protein is incorporated in a high-molecular-weight complex. The relatively low production of CP in this experiment may be due to a high proportion of protoplasts infected with RNA-l only (NM-infection) (Harrison and Robinson, 1978). The proteins marked by an asterisk are also precipitated from homogenates of mock-inoculated protoplasts and probably represent the small and large subunits of ribulose bisphosphate carboxylase. When the total proteins present in the gradient fractions of Fig. 5 were analyzed on a SDS-polyacrylamide gel, no labeled host proteins were detectable in fractions 4 and 5 (result not shown). This indicates that the high-molecular-weight structure in these fractions consists of a multimer of 16K subunits, or that the 16K protein associates with preexisting nonlabeled host proteins.
Leaf disks from PLB-infected Samsun NN tobacco plants were vacuum-infiltrated with a [35S]methionine solution and after a labeling period of 2 days proteins were extracted. As a control, the production of viral RNA was monitored by Northern blot hybridization using a cDNA probe complementary to the homologous 3’-terminus of TRV RNA capable of detecting all genomic and subgenomic TRV RNAs. Figure 6A shows the production of PLB RNAs in tobacco protoplasts (lane 1) and plants (lane 2). The subgenomic mRNAs for the 29K protein (RNA-l a) and coat protein (RNA-2a) of PLB have approximately the same length (Angenent et a/., to be published) and are not separated in Fig. 6A. The subgenomic mRNA for the 16K protein is termed RNA1b according to the nomenclature proposed by Robinson et al. (1987). However, because PLB is diploid for the 16K gene, it is impossible to say whether this RNA is derived from RNA-l or RNA-2 or both. Although all RNA species are detectable both in protoplasts and plants their relative proportions in the two preparations are clearly different. Figure 6B shows an analysis of proteins synthesized in vivo. As controls, lanes 1 and 2 show the in vitro synthesized 16K protein and CP while lanes 3 and 4 show the proteins from PLB-infected protoplasts precipitated with preimmune serum and the mixture of the 16K protein and CP antisera, respectively. Lanes 5 and
FIG. 5. Sucrose gradient centrifugation of subcellular structures containing TRV coat protein (CP) or 16K protein. A homogenate of infected protoplasts was sedimented in a sucrose gradient and fraction 1 (bottom) to fraction 10 (top) were collected. Y-Labeled proteins in these fractions were immunoprecipitated with a mixture of antisera to CP and 16K protein and analyzed by polyacrylamide gel electrophoresis. Lanes A and B were run with in vitro synthesized 16K protein and CP, respectively.
310
ANGENENT,
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B
A 1
2
123456
FIG. 6. Detection of TRV RNAs, coat protein (CP) and 16K protein in infected tobacco protoplasts and leaves. Panel (A) shows a Northern blot with RNAfrom TRV-infected tobacco protoplasts (lane 1) and leaves (lane 2). Nick-translated cDNA corresponding to the 3’.terminai 1070 nucleotides of PLB RNA-2 was used as probe. The position of TRV genomic and subgenomic RNAs is indicated in the margin. Panel (B) shows 35S-labeled proteins that were extracted from infected tobacco protoplasts (lanes 3 and 4) or leaves (lanes 5 and 6) and were precipitated with preimmune serum (lanes 3 and 5) or a mixture of antisera against CP and 16K protein (lanes 4 and 6). The proteins were analyzed by polyacrylamide gel electrophoresis. Lanes 1 and 2 were run with in vitro synthesized 16K protein and CP. respectively.
6 of Fig. 6B show the proteins from PLB-infected plants that are precipitated with preimmune serum and the mixture of the 16K protein and CP antisera, respectively. Although approximately similar amounts of the CP are detected in the extracts from protoplasts and plants, the 16K protein was found exclusively in the extract from protoplasts. To preclude the possibility that the 16K protein is trapped in cell wall structures, proteins were extracted from unlabeled infected plant material using buffers containing cellulase (2%) and pectinase (0.1 o/b)or various extraction buffers with or without SDS (0.5%). However, an analysis of these extracts on Western blots failed to detect the 16K protein whereas the CP was readily observed under all conditions (results not shown). DISCUSSION The observation that the 16K protein accumulates to high levels in TRV-infected protoplasts corroborates the notion that this conserved TRV gene has a function in the viral replication cycle. Because the 16K protein continues to accumulate when CP synthesis has already leveled off, it may not be involved in the production of viral RNA or virion assembly but may have a function for example in virus spread. It has been suggested that the 16K protein plays a role in transmission of the virus by nematodes (Boccara et a/., 1986). Highly specific relationships have been found to exist between TRV isolates and the species of trichodorid nematodes involved in their transmission (Brown et al., 1988). Because in a number of TRV isolates the 16K
protein is encoded by both RNA-l and RNA-2, it would be interesting to investigate the nematode transmissibility of pseudo-recombinants with 16K genes from different parents. In this respect it is noteworthy that a pseudo-recombinant containing RNA-l of strain TCM and RNA-2 of strain PLB was found to be unchanged in sequence after numerous passages in tobacco (Angenent eta/., to be published). Apparently, RNA recombination restoring the perfect homology at the 3’-termini of the genome segments is not a frequent event under greenhouse conditions. The finding that RNA-l b, the putative messenger for the 16K protein, is produced in infected tobacco protoplasts as well as in leaves indicates that the 16K gene is expressed under both conditions. Our inability to detect the 16K protein in leaf material may have several explanations. It is possible that in intact plants the turnover of the 16K protein is much higher than in protoplasts. Alternatively, a modification of the protein or its inclusion in a structure that is inaccessible to our extraction procedures may interfere with the detection of the 16K protein in plants. Theoretically, regulation at the translation level could be involved, resulting in a tissue-specific expression. At present, the possibility of detecting the 16K protein in plant tissues by immunoelectron microscopy is being investigated. ACKNOWLEDGMENTS We thank Dr. Huub Linthorst for the antiserum against the coat protein and for critical reading of the manuscript. This work was sponsored in part by the Netherlands Organization for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Research (NWO).
REFERENCES ANGENENT. G. C., LINTHORST. H. J. M., VAN BELKUM, A. F., CORNELISSEN. B. J. C., and BOL, J. F. (1986). RNA-2 of tobacco rattle virus strain TCM encodes an unexpected gene. Nucleic Acids Res. 14, 4673-4682. BARANY,G., and MERRIFIELD,R. B. (1980). Solid phase peptide synthesis. ln “The Peptides” (E. Gross and J. Meienhofer, Eds.), Vol. 2, pp. l-284. Academic Press, New York/London. BOCCARA, M., HAMILTON, W. D. O., and BAULCOMBE,D. C. (1986). The organisation and interviral homologies of genes at the 3’ end of tobacco rattle virus RNA-l. En/lBO/. 5, 223-229. BROWN, D. J. F., PLOEG,A. T., and ROBINSON,D. J. (1988). A review of reported associations between Trichodorus and Paratrichodorus species and tobraviruses with a description of laboratory methods for examining virus transmission by trichodorids. Rev. Nematol. In press. BUJARSKI.J. I., HARDY, S. F., MILLER, W. A., and HALL, T. C. (1982). Use of dodecyl-P-o-maltoside in the purification and stabilization of RNA polymerase from brome mosaic virus-infected barley. Viology 119,465-473. CORNELISSEN,B. J. C., LINTHORST,H. J. M., BREDERODE,F. T., and BOL, J. F. (1986). Analyses of the genome structure of tobacco rattle virus strain PSG. Nucleic Acids Res. 14. 2157-2169.
TRV 16K PROTEIN FRITSCH, C., MAYO, M. A., and HIRTH, L. (1977). Further studies on the translation products of tobacco rattle virus in vitro. Virology 77, 722-732. HAMILTON, W. D. O., BOCARRA,M., ROBINSON,D. J., and BAULCOMBE, D. C. (1987). The complete nucleotide sequence of tobacco rattle virus RNA-l. /. Gen. Viral. 68, 2563-2575. HARRISON,B. D., HUTCHESON,A. M., and BARKER,H. (1977). Association between the particles of rasberry ringspot and tobacco rattle viruses in doubly infected Nicofiana benthamiana cells and protoplasts. /. Gen. Viral. 36, 535-539. HARRISON, B. D., Kuso, S., ROBINSON, D. J., and HUTCHESON, A. M. (1976). The multiplication cycle of tobacco rattle virus in tobacco mesophyll protoplasts. /. Gen. l&o/. 33, 237-248. HARRISON,B. D., and ROBINSON, D. 1. (1978). The tobraviruses. Adv. Virus Res. 23,25-77. HARRISON, B. D., and ROBINSON, D. J. (1986). Tobraviruses. In “The Plant Viruses” (M. H. V. van Regenmortel and H. Fraenkel-Conrat, Eds.), Vol. 2, pp. 339-369. Plenum, New York. HENIKOFF,S. (1984). Bidirectional digestion with exonuclease Ill creates breakpoints for DNA sequencing. Gene 28, 351-359. HOOFT VAN HUIJSDUINEN,R. A. M., CORNELISSEN,B. J. C., VAN LOON, L. C.. VAN BOOM, J. H., TROMP, M., and BOL, J. F. (1985). Virusinduced synthesis of messenger RNAs for precursors of pathogenesis-related proteins in tobacco. EM50 J. 4, 2 167-217 1, HU~INGA, H. (1972). “Interaction between Long and Short Particles of Tobacco Rattle Virus.” PhD. Thesis, University of Wageningen. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685.
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PFAFF, E., MUSSGAY, M. H. O., BOHM, G. E., and SCHALLER,H. (1982). Antibodies against preselected peptides recognize and neutralize foot and mouth disease. EMBOJ. 1,875-881. ROBINSON, D. I., HAMILTON, W. D. O., HARRISON, B. D., and BAULCOMBE, D. C. (1987). Two anomalous tobravirus isolates: Evidence for RNA recombination in nature. /. Gem Viral. 68,2551-2561. ROBINSON, D. J., and MAYO, M. A. (1978). “Separation of Long and Short Particles of Tobacco Rattle Virus (TRV).” Annual Report, Scottish Horticultural Research Institute, 1977, pp. 90-9 1. SARACHU, A. N.. HUISMAN, M. J., VAN VLOTEN-DOTING, L., and BOL, J. F. (1985). Alfalfa mosaic virus temperature-sensitive mutants. L%ology 141, 14-22. TABOR, S. (1985). A bacteriophage T7 RNA polymerase/promotor system for controlled exclusive expression of specific genes. Proc. Nat/. Acad. Sci. USA 82, 1074-l 078. VAN DUN, C. M. P., VAN VLOTEN-DOTING, L., and BOL, J. F. (1988). Expression of alfalfa mosaic virus cDNA1 and 2 in transgenic tobacco plants. Virology 163, 572-578. VAN PELT-HEERSCHAP,H., VERBEEK.H., HUISMAN, M. J., LOESCH-FRIES. L. S., and VAN VLOTEN-DOTING, L. (1987). Non-structural proteins and RNAs of alfalfa mosaic virus synthesized in tobacco and cowpea protoplasts. Virology 161, 190-l 97. VAN TOL, R. G. L., and VAN VLOTEN-DOTING, L. (1979). Translation of alfalfa mosaic virus RNA1 in the mRNA-dependent translation system from rabbit reticulocyte lysates. Eur. /. Biochem. 93, 461468. Yu, J.. FISCHMAN. D. A., and STECK, T. L. (1973). Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. J. Supramol. Srruct. 1, 233-249.