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Expression of alfalfa mosaic virus and tobacco rattle virus coat protein genes in transgenic tobacco plants
VIROLOGY
159, 299-305 (1987)
Expression of Alfalfa Mosaic Virus and Tobacco Rattle Virus Coat Protein Genes in Transgenic Tobacco Plants CEES M. P. VAN DUN, JOHN F. BOL,’ Department
of Biochemistry,
State University Received February
AND
LOUS VAN VLOTEN-DOTING’
of Leiden, P.O. Box 9505, 2300 RA Leiden, The Netherlands 10, 1987; accepted April 7, 1987
Using the Agrobacrerium rumefaciens binary vector system, a chimeric gene consisting of the cauliflower mosaic virus 35 S promoter, alfalfa mosaic virus (AIMV) coat protein (CP) cistron, and the nopaline synthase polyadenylation signal was integrated into the genome of Nicoriana rabacum cv. Samsun NN. In 7070 of the transgenic tobacco plants the chimeric mRNA and its translation product could be detected. CP accumulated to levels up to 0.05% of the soluble leaf protein. The accumulation was independent of leaf age. The same approach was undertaken for the CP of tobacco rattle virus (TRV). The chimeric gene was integrated in the genome of Nicoriana rabacum cv. Xanthi nc. The results with respect to the accumulation of the chimeric mRNA and TRV CP in leaves of transgenic tobacco plants were comparable to those with AIMV transformed plants. Plants accumulating AIMV CP were highly resistant to infection with AIMV nucleoproteins but could be infected with a mixture of AIMV RNAs l-4. Moreover, a mixture of AIMV RNAs 1, 2, and 3 was infectious to these plants but not to nontransformed control plants. o 1987 Academic PWSS, hc.
INTRODUCTION
been demonstrated (Bevan et al., 1985). Abel et al. (1986) showed that expression of the TMV CP gene in transgenic tobacco plants caused a delay in symptom development when these plants were inoculated with TMV. To permit a further investigation of the mechanism of cross-protection and to construct plants that are useful in a study of the viral replication cycle, we have transformed tobacco plants with cDNA to various parts of the genome of several RNA plant viruses. Here we report the expression of the CP genes of alfalfa mosaic virus (AIMV) and tobacco rattle virus (TRV) in Nicotiana tabacum Samsun NN and Xanthi nc plants, respectively. Plants accumulating AIMV CP were tested for their susceptibility to infection with AIMV nucleoproteins and RNAs. The genome of AIMV consists of three positivestranded RNAs. RNAs 1 and 2 encode 126- and 90kDa proteins, respectively, that are involved in viral RNA replication (Nassuth and Bol, 1983; Cornelissen and Bol, 1984). RNA 3 is dicistronic and encodes a 32-kDa protein with a putative role in cell-to-cell transport and the viral CP (Cornelissen and Bol, 1984). The CP is translated from a subgenomic messenger, RNA 4, that is homologous to the 3’-terminal 881 nucleotides of RNA 3. In addition to its structural role, the CP has an early function in the replication cycle: a mixture of the AIMV genomic RNAs is not infectious unless a few CP subunits are added per RNA molecule (Bol et a/., 197 1). Transgenic plants expressing the CP may be helpful in elucidating this early function. The genome of TRV consists of two RNAs of plus polarity. The CP gene is located on RNA 2. Here we
Genetic engineering of plants has advanced to a level permitting a study of mechanisms that could control the spread of viruses. Recently, promising results have been obtained with plants transformed with viral cDNAs by the route of “agroinfection” (Grimsley et al., 1986). Cucumber mosaic virus (CMV) was shown to acquire its satellite, CARNA 5, from transgenic tobacco plants transcribing satellite RNA from a chimeric nuclear gene (Baulcombe er a/., 1986). As most CARNA 5 strains interfere with the CMV symptom expression, this approach could be of economical importance. However, this approach is limited to the very few viruses that are accompanied by interfering satellites. A more general phenomenon that may be employed to control virus infections is cross-protection. The term cross-protection is used for the observation that plants infected with a mild strain of a given virus do not develop additional symptoms upon inoculation with a severe strain of the same virus (Fulton, 1982). The use of crossprotection in the field has been limited by certain risks such as mutation of the mild strain to a severe phenotype, synergism of the mild strain with other viruses, and spread of the mild strains to other crops. Possibly, these disadvantages could be circumvented by using transgenic plants that express an incomplete noninfectious viral genome. Transformation with the coat protein (CP) gene of tobacco mosaic virus (TMV) has ’ To whom reprint requests should be addressed. ’ Present address: Research Institute Ital, P.O. Box 48, 6700 A4 Wageningen, The Netherlands.
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used RNA 2 of strain TCM (3389 nucleotides) which contains reading frames for the CP, a 29.1 -kDa protein and a 16-kDa protein, the CP gene being 5’-proximal (Angenent et al., 1986). A subgenomic messenger lacking the 5’-terminal 437 nucleotides of the leader sequence of 546 nucleotides has been identified, thus avoiding a number of AUG codons in this noncoding region. A unique property of TRV is the ability of RNA 1 to replicate and spread in plants independently of RNA 2 (Harrison and Robinson, 1978). It will be interesting to see the effect of CP, produced endogenously in transgenic plants, on RNA 1 replication. MATERIALS AND METHODS Construction of chimeric genes Plasmid pDH51 (Pietrzak et al., 1986) was obtained from the Friedrich Miescher Institute (Basel) and plasmid pMONl77 was obtained from Monsanto (St. Louis). A BarnHI fragment of pMON 177, containing the nopaline synthase (nos) polyadenylation signal, was inserted into the BarnHI site just downstream from the cauliflower mosaic virus (CaMV) 35 S promoter in pDH51, yielding plasmid pDH52 (Fig. 1). An EcoRl fragment of pDH52, containing the 35 S promoter and nos polyadenylation signal, was inserted into the binary transformation vector pAGSHB. This vector was derived
FIG. 1. Construction of the chimeric genes consisting of the CaMV 35 S promoter, CP gene of AIMV or TRV and nos-polyadenylation signal. P35 S, CaMV 35 S promoter; Pnos, nopaline synthase promoter; Tnos, nopaline synthase poly(A) signal; Tots, octopine synthase poly(A) signal; NPT, neomycin phosphotransferase; cos, CDS site; TcR,tetracycline resistance gene; LB, left border; RB, right border; E, EcoRI; B, BarnHI; H, Hindlll.
from pAGS127 (Van den Elzen et al., 1985) by deleting the HindIll and BarnHI sites. The unique BarnHI site between the 35 S promoter and polyadenylation signal was converted into a HindIll site using a linker. This HindIll site was subsequently used to insert the cDNAs of AIMV and TRV encoding the viral coat proteins (Fig. 1). The AIMV cDNA is a full-length copy of RNA 4 of strain 425; first- and second-strand cDNA syntheses were primed with oligonucleotides containing the restriction sites Smal (3’) and Pstl (53, respectively (Langereis et al., 1986). These sites were converted into HindIll sites. The cDNA encoding the CP of TRV strain TCM is a fragment of cDNA 2 cloned by Angenent et a/. (1986). The 5’-end of the fragment is located at position 441 which is four nucleotides downstream from the start site of the putative subgenomic messenger for the CP. The 3’-end of the fragment extends into the 29.1 K cistron to position 1440. The transformation vector containing the AIMV CP chimeric gene was designated pCA40, whereas that containing the TRV CP chimeric gene was designated pCTR20. Due to the cloning procedure the expected chimeric mRNA in transgenic plants is extended with approximately 20 nucleotides at the 5’-end and 150 nucleotides at the 3’-end excluding the poly(A) tail. The plasmids pCA40 and pCTR20 were mobilized into Agrobacterium tumefaciens strain LBA 4404 containing PAL 4404 (virulence region) by a triparental mating. Briefly, 1 ml of a logarithmically growing culture of Escherichia colistrain 490, containing either pCA40 or pCTR20, was mixed with 1 ml of logarithmically growing E. co/i strain HBl 01 containing pRK2013 (Ditta et a/., 1980) and 1 ml of logarithmically growing A. tumefaciens strain LBA4404 (Hoekema et al., 1983). After incubation to allow plasmid transfer, transconjugants resistant to rifampicin (to select A. tumefaciens) and tetracycline (to select for the presence of plasmids pCA40 or pCTR20) were isolated. Plant transformation Leaf disks of N, tabacum cvs. Samsun NN and Xanthi nc were infected with A. tumefaciens LBA 4404 containing pCA40 or pCTR20 (Horsch et al., 1985). Transformed cells were selected on shooting medium containing kanamycin (100 pglml). Kanamycin-resistant shoots were transferred to rooting medium containing kanamycin (100 pglml). After development of the root system (2-3 weeks) plantlets were cultured in the greenhouse. After about 6 weeks, when the plants were in the four to six leaf stage, the assay for the presence of virus-specific RNA or protein was performed. Plants containing AIMV cDNA are designated S40, whereas plants containing TRV cDNA are designated XTRV.
301
VIRUS TRANSFORMED TOBACCO
12
RNA analysis of transgenic plants RNA was isolated from transgenic plants essentially as described by Hoof? van Huijsduijnen et a/. (1985). Polyadenylated RNA was isolated by poly(U)-Sepharose chromatography. The RNA was denatured in glyoxal/DMSO, run on a 1% agarose gel, and transferred to a nitrocellulose filter (Sarachu et a/., 1985). The Northern blot was probed with purified 32P-nicktranslated cDNA fragments corresponding to the insert in the transformation vector (Hooft van Huijsduijnen et a/., 1985). Protein analysis of transgenic plants Protein containing sap was squeezed from young leaves of the transgenic plants. The sap was clarified by centrifugation. The protein concentration of the clarified supernatant was determined by the Bradford method (Bradford, 1976). Western blots were made essentially as described by Towbin eta/. (1979). Equal amounts of protein (50 pg) from different transgenic plants were electrophoresed on gels containing 11% acrylamide and 0.1% SDS and subsequently transferred to nitrocellulose. The nitrocellulose filters were incubated with antiserum raised against AIMV or TRV. The filters were then incubated with peroxidase-conjugated goat anti-rabbit antibodies. Staining was performed with 3,3’-diaminobenzidine. Expression levels were estimated by comparing the intensity of the obtained bands with the band intensities of known quantities of CP added to juice of control plants. Infectivity assay Tobacco leaf material (3 cm2) was homogenized in 2 ml 0.01 M phosphate buffer, pH 7.0, and inoculated in several dilutions on the leaves of Samsun NN tobacco or bean plants (Phaseohs vulgaris L. var. “Berna”).
123456 -CP
FIG. 2. Accumulation of AIMV CP in transgenic tobacco plants. Lanes 1 to 3: protein extracts from untransformed plants to which 0, 10, and 100 ng of AIMV CP had been added, respectively. Lane 4: protein extract from a vector transformed plant. Lanes 5 and 6: protein extracts from plants S40-7 and S40-8, respectively. The Western blot was analyzed with an antiserum to AIMV CP.
3L5678
-CP
FIG. 3. Accumulation of AIMV CP in different leaves of a tobacco plant transformed with the AIMV CP gene. Lanes 1 to 5: protein extracts from lower to upper leaves, respectively. Lanes 6 to 8: protein extracts from untransformed plants to which 0, 5, and 50 ng of AIMV CP were added, respectively. The Western blot was analyzed with an antiserum to AIMV CP.
RESULTS Expression of coat protein A total of 15 kanamycin-resistant plants, transformed with the chimeric AIMV CP gene, were tested for the presence of CP. Eleven plants were found to accumulate detectable amounts of CP. The accumulated level ranged between 0.01 and 0.05% of the extracted protein. Two plants, S40-7 and S40-8, were chosen for further analysis. Figure 2 shows a Western blot containing protein from S40-7 and S40-8, from a control plant transformed with the vector only, and from an untransformed plant. The antiserum to AIMV CP clearly binds to a protein in the extract from plants transformed with viral cDNA (lanes 5 and 6) but not to the proteins in the controls (lanes 1 and 4). The CP molecules produced by the transgenic plants comigrate with native AIMV CP. Levels of accumulation were estimated by comparing the signals to those obtained in lanes run with 10 and 100 ng of native AIMV CP added to extracts from untransformed plants (Fig. 2, lanes 2 and 3). These control signals were similar to those of the bands obtained by loading samples of 10 and 100 ng of pure protein to the gel (result not shown). Analysis of AIMV CP accumulation in different leaves of a transformed tobacco plant showed that the accumulation was independent of leaf age (Fig. 3). Besides AIMV CP, a band of lower mobility was detected in the older leaves. As this band was not reproducibly seen in other experiments, its nature was not further analyzed. Six kanamycin-resistant plants transformed with the chimeric TRV CP gene were assayed for the presence of CP. Four of them showed the accumulation of detectable levels of TRV CP, ranging between 0.01 and 0.05% of the extracted protein. The protein analysis of two of these plants XTRVl and XTRVB is depicted in Fig. 4. This Western blot shows the presence of TRV
VAN DUN, BOL, AND VAN VLOTEN-DOTING
302
1
2
3
4
5
6
1234
-CP
FIG. 4. Accumulation of TRV CP in transgenic tobacco plants. Lanes 1 to 3: protein extracts from untransformed plants to which 0, 10, and 100 ng of TRV CP had been added, respectively. Lane 4: protein extract from a vector transformed plant. Lanes 5 and 6: protein extracts from XTRVl and XTRV3, respectively. The Western blot was analyzed with an antiserum to TRV CP.
CP in these transgenic plants comigrating with native TRV CP. The accumulation of TRV CP was not affected by the age of the leaves (result not shown). Expression of RNA The expression of viral genes in transgenic plants was analyzed at the RNA level by hybridizing Northern blots, loaded with purified poly(A)-containing RNA, to probes corresponding to the respective viral cDNAs. Figure 5, lanes 3 and 4, shows RNA extracted from plants S40-7 and S40-8 which produce AIMV CP. After hybridization to labeled AIMV cDNA 4, a distinct band is seen in each lane. The controls, containing polyadenylated RNA from untransformed and vector transformed plants, did not give any signal (Fig. 5, lanes 1 and 2). The AIMV-specific transcripts have an approximate length of 1300 bases. If transcription is initiated at the CaMV 35 S promoter and is terminated behind the nos-polyadenylation site of the transformation vector, the transcript is expected to contain a 5’-nonviral sequence of approximately 20 nucleotides, an RNA 4specific sequence of 881 nucleotides, a 3’-nonviral sequence of 150 nucleotides, and a poly(A) tail. This would suggest that the average length of the poly(A) tail in the in viva produced transcripts is 250 residues.
1
2
3
4
FIG. 5. Autoradiograph of a Northern blot containing polyadenylated RNA from transgenic plants accumulating AIMV CP. Lane 1: RNA from an untransformed plant. Lane 2: RNAfrom a vectortransformed plant. Lanes 3 and 4: RNA from plants S40-7 and S40-8, respectively. The positions of AIMV RNAs 3 and 4 are indicated. The blot was probed with 32P-labeled cDNA to AIMV RNA 4.
FIG. 6. Autoradiograph of a Northern blot containing polyadenylated RNA from transgenic plants accumulating TRV CP. Lane 1: RNA from an untransformed plant. Lane 2: RNA from a vector transformed plant. Lanes 3 and 4: RNA from plants XTRVl and XTRV3, respectively. The position of AIMV RNAs 3 and 4 are indicated. The blot was probed with 3zP-labeled cDNA to TRV-RNA 2 (strain TCM).
Figure 6 shows the analysis of poly(A)-containing RNA from transgenic plants XTRVI and XTRV3. Hybridization of the Northern blot with a cDNA probe corresponding to the TRV CP cistron showed clear bands with the preparations from virus-transformed plants (Fig. 6, lanes 3 and 4) which are absent in the controls (Fig. 6, lanes 1 and 2). The estimated length of the TRV-specific transcript is 1400 nucleotides. This compares well with the size of the RNA that was expected to be transcribed from the chimeric TRV CP gene (999 bases of viral RNA, flanked by 5’- and 3’-nonviral sequences of approximately 20 and 150 bases, respectively, and a 3’-poly(A) tail). Susceptibility of transgenic plants to virus infection Seed collected from plants S40-7 and S40-8 was germinated on medium containing kanamycin. Over 95% of the seedlings from S40-7 were found to be resistant to the antibiotic. This non-Mendelian segregation of the NPT gene indicates the occurrence of multiple integrations during the transformation procedure. On the other hand, none of the S40-8 seedlings showed resistance to kanamycin. Differences in the integration events in plants S40-7 and S40-8 are presently being analyzed. Plants obtained from S40-7 seedlings accumulated AIMV CP to the same level as the original transformant and were tested for their susceptibility to infection with AIMV nucleoproteins or RNAs. As controls, plants SC1 and SV-1 were used, which were regenerated from Samsun NN leaf disks dipped into a suspension of A. tumefeciens containing no transformation vector or a transformation vector without viral cDNA, respectively. Six plants of each group (SC-l, SV-1, and S40-7) were inoculated with AIMV strain YSMV which induces necrotic yellow lesions on Samsun NN tobacco. Plants SC-l and SV-1 developed lesions after a few days, whereas all S40-7 plants remained free of any symptoms of infection during a 4-week observation period. On the control plants, inocula containing 0.25 or 2.5
VIRUS TRANSFORMED
pglml virus induced an average lesion number per half leaf of 40 and 430, respectively. Table 1 lists the lesion numbers obtained with the highest virus concentration. Two weeks after inoculation with YSMV (2.5 pg/ml), leaf material was taken from four SC-l, SV-1, and S407 plants and a homogenate of this material was inoculated to nontransformed tobacco plants. Table 2 shows that the homogenates from SC-l and SV-1 were highly infectious, whereas the homogenate from S407 did not induce any lesions. This demonstrates that after infection with YSMV the symptomless plants do not accumulate detectable amounts of infectious nucleoprotein. The above results indicate that the CP of AIMV strain 425, that accumulates in S40-7 plants, interferes with one or more steps in the replication cycle of the closely related strain YSMV. When SC-l, SV-1, and S40-7 were inoculated with TMV, the plants developed similar numbers of local lesions (result not shown), demonstrating that the protection is specific for the homologous virus. To obtain insight in the replication event that is inhibited by the endogenously produced CP and to analyze its biological activity, plants of SC-l, SV-1, and S40-7 were inoculated with a mixture of AIMV genomic RNAs of strain 425 plus or minus subgenomic RNA 4. On Samsun NN tobacco strain 425 induces mild chlorotic symptoms which are difficult to quantitate. Therefore, leaf material was collected 1 week after inoculation of the plants, and homogenates of this material were inoculated in several dilutions to bean plants, which is a local lesion host for strain 425. Table 3 lists the lesions induced by appropriate dilutions. The mixture of all four AIMV RNAs is equally infectious to the control plants (SC-l and SV-1) and to the transgenic S40-7 plants. This shows that the endogenously produced CP does not protect against infection with viral
303
TOBACCO
TABLE2 PRODUCTIONOF INFECTIOUSVIRUSIN TRANSFDRMEDAND NONTRANSFORMEDTOBACCO PLANTSINOCULATEDWITHAIMV STRAINYSMV” Lesions induced by homogenates
of
Plant No.
SC-1
sv-1
s40-7
1 2 3 4
269 186 140 200
180 240 80 185
0 0 0 0
BTwo weeks after inoculation, leaf material of plants 1 to 4 of Table 1 was homogenized and the homogenates were inoculated to nontransformed Samsun NN tobacco plants (two leaves per homogenate). Lesions were counted 1 week after inoculation; average lesion number per half leaf is given.
RNA. The mixture of AIMV RNAs 1, 2, and 3 is not infectious to the control plants, demonstrating that the mixture is not contaminated with detectable amounts of RNA 4. However, the infectivity of this inoculum toward S40-7 plants is comparable to that of the inoculum containing all four AIMV RNAs. This proves that the endogenously produced CP is biologically active and is able to replace the RNA 4 or CP that is required in the inoculum for infection of nontransformed plants (601 eta/., 1971). DISCUSSION Viral CP was produced in about 70% of the kanamycin-resistant plants transformed with the AIMV and TRV CP genes. This indicates that, generally, during transformation the construct between the border sequences in the vector remains intact. The observation that both AIMV- and TRV-specific transcripts copurify with the poly(A)-containing cellular RNAs on poly(U)-
TABLE 1 INDUCTIONOF LOCAL LESIONSBYAIMV STRAINYSMV ON TRANSFORMED AND NONTRANSFORMED TOBACCO PLANTS’
TABLE 3 PRODUCTIONOF INFECTIOUSVIRUSIN TRANSFORMEDAND NONTRANSFORMEDTOBACCO PLANTSINOCULATEDWITHAIMV RNA9
Lesions induced on Plant No.
SC-1
sv- 1
s40-7
1 2 3 4 5 6
635 510 350 690 625 505
745 420 260 355 110 370
0 0 0 0 0 0
@Tobacco plants accumulating AIMV CP (S40-7) and control plants (SC-l and SV-1) were inoculated with 2.5 pg/ml YSMV (two leaves per plant). Lesions were counted 2 weeks after inoculation; average lesion number per half leaf is given.
Lesions induced by homogenates
of
lnoculum
SC-I
sv-1
s40-7
RNAs 1+2+3+4 RNAs 1+2+3
171 0
124 0
251 207
8 Tobacco plants SC-l, SV-1, and S40-7 were inoculated with 40 ~1 per half leaf containing 100 ng RNAs 1+2+3, plus or minus 100 ng RNA 4. After 10 days leaf material of each plant was homogenized and the homogenates were inoculated in several dilutions on bean leaves. Lesions were counted after 3 days; the number per half leaf is given.
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VAN DUN, BOL, AND VAN VLOTEN-DOTING
Sepharose columns indicates that they are transcribed from a nuclear gene. Preliminary data indicate that a plus-stranded RNA transcribed in plants transformed with a DNA copy of AIMV RNA 3 is being processed to a smaller molecule (Van Dun et al., in preparation). Apparently, this is not the case with the AIMV RNA 4 and partial TRV RNA 2 transcripts that were transcribed from nuclear genes, as their estimated sizes were in agreement with those expected from the sequences of the respective transformation vectors. At present we are determining the copy number of the NPT and CP genes in the respective AIMV and TRV transformed plants. This may give a clue to the observation that the progeny of S40-8 plants had lost their kanamycin resistance. The progeny of TRV transformed plants is not yet available for further analysis. In vitro, AIMV RNA 4 is efficiently translated into viral CP (Van Vloten-Doting and Jaspars, 1977). ln viva, it can replace the CP in the “activation” of the genome (Bol et al., 1971), suggesting that AIMV RNA 4 is also an active messenger in virus-infected cells. The functional messenger RNA for TRV CP is not known. In several strains a subgenomic RNA has been identified that lacks the eight AUG codons that are present in the 5’-leader sequence preceding the CP cistron in TRVRNA 2 (Cornelissen et al., 1984; Angenent et a/., 1986). These AUG codons are also absent in the TRV-RNAs transcribed in plants XTRVl and XTRV3. Apparently, the 20 nonviral nucleotides that are present at the 5’termini of the AIMV and TRV specific transcripts in transgenic plants do not grossly affect the messenger activity of the transcripts. In both AIMV and TRV CP gene transformed plants, the accumulation of CP varied between 0.01 and 0.05% of the soluble leaf protein. This indicates that the two types of genes are expressed with similar efficiencies. The inverse relationship between the synthesis of AIMV mRNA and coat protein that is suggested by a comparison of the bands in Figs. 2 and 5 was not reproducibly obtained in other experiments. In general, a parallel was observed between the level of mRNA and CP synthesis. Although the accumulation of AIMV CP in S40-7 plants is about 1O-fold lower than it is in AIMV-infected plants, it is apparently sufficient to block infection by viral nucleoprotein. The finding that these plants are susceptible to infection with viral RNA indicates that the endogenous CP interferes with an early step of the replication cycle, e.g., absorption to specific receptor sites in the cell or uncoating. To initiate infection, each AIMV genomic RNA must have bound a few CP subunits (Smit et a/., 1981). The detection of a major CP binding site at the 3’-end of the viral RNAs has led to the suggestion that this binding is required for recognition of the RNAs by the viral replicase (Houwing and
Jaspars, 1978). At the final stage of the replication cycle this binding site may act as a nucleation site for encapsidation of the viral RNAs. Because a mixture of the AIMV genomic RNAs is infectious to S40-7 plants, it may be concluded that the CP accumulating in these plants is biologically active and is able to associate with the incoming viral RNAs. Apparently, the level of endogenous CP is such that the parental RNAs are not inactivated by encapsidation and that no other possible regulatory function is blocked in which the CP might be involved. It has been suggested that CP is the RNA 3 encoded protein that regulates the balance between AIMV plus- and minus-strand RNA synthesis (Nassuth and Bol, 1983). We have not yet analyzed the offspring of XTRVl and XTRV3 for possible resistance to TRV infection. Evidence has been presented that infection of plants with TRV RNA 1 alone results in the induction of cross-protection (Cadman and Harrison, 1959). Thus, it will be interesting to study the phenotype of transgenic plants expressing nonstructural viral genes. ACKNOWLEDGMENTS The authors thank Dr. Peter van den Elzen for the gift of pAGS 127, Dr. Maciej Pietrzak for the gift of the CaMV 35 S promoter, Corrie Houwing for providing the YSMV inoculum, and Bert Overduin for technical assistance. This research was supported in part by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO).
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159, 299-305 (1987)
Expression of Alfalfa Mosaic Virus and Tobacco Rattle Virus Coat Protein Genes in Transgenic Tobacco Plants CEES M. P. VAN DUN, JOHN F. BOL,’ Department
of Biochemistry,
State University Received February
AND
LOUS VAN VLOTEN-DOTING’
of Leiden, P.O. Box 9505, 2300 RA Leiden, The Netherlands 10, 1987; accepted April 7, 1987
Using the Agrobacrerium rumefaciens binary vector system, a chimeric gene consisting of the cauliflower mosaic virus 35 S promoter, alfalfa mosaic virus (AIMV) coat protein (CP) cistron, and the nopaline synthase polyadenylation signal was integrated into the genome of Nicoriana rabacum cv. Samsun NN. In 7070 of the transgenic tobacco plants the chimeric mRNA and its translation product could be detected. CP accumulated to levels up to 0.05% of the soluble leaf protein. The accumulation was independent of leaf age. The same approach was undertaken for the CP of tobacco rattle virus (TRV). The chimeric gene was integrated in the genome of Nicoriana rabacum cv. Xanthi nc. The results with respect to the accumulation of the chimeric mRNA and TRV CP in leaves of transgenic tobacco plants were comparable to those with AIMV transformed plants. Plants accumulating AIMV CP were highly resistant to infection with AIMV nucleoproteins but could be infected with a mixture of AIMV RNAs l-4. Moreover, a mixture of AIMV RNAs 1, 2, and 3 was infectious to these plants but not to nontransformed control plants. o 1987 Academic PWSS, hc.
INTRODUCTION
been demonstrated (Bevan et al., 1985). Abel et al. (1986) showed that expression of the TMV CP gene in transgenic tobacco plants caused a delay in symptom development when these plants were inoculated with TMV. To permit a further investigation of the mechanism of cross-protection and to construct plants that are useful in a study of the viral replication cycle, we have transformed tobacco plants with cDNA to various parts of the genome of several RNA plant viruses. Here we report the expression of the CP genes of alfalfa mosaic virus (AIMV) and tobacco rattle virus (TRV) in Nicotiana tabacum Samsun NN and Xanthi nc plants, respectively. Plants accumulating AIMV CP were tested for their susceptibility to infection with AIMV nucleoproteins and RNAs. The genome of AIMV consists of three positivestranded RNAs. RNAs 1 and 2 encode 126- and 90kDa proteins, respectively, that are involved in viral RNA replication (Nassuth and Bol, 1983; Cornelissen and Bol, 1984). RNA 3 is dicistronic and encodes a 32-kDa protein with a putative role in cell-to-cell transport and the viral CP (Cornelissen and Bol, 1984). The CP is translated from a subgenomic messenger, RNA 4, that is homologous to the 3’-terminal 881 nucleotides of RNA 3. In addition to its structural role, the CP has an early function in the replication cycle: a mixture of the AIMV genomic RNAs is not infectious unless a few CP subunits are added per RNA molecule (Bol et a/., 197 1). Transgenic plants expressing the CP may be helpful in elucidating this early function. The genome of TRV consists of two RNAs of plus polarity. The CP gene is located on RNA 2. Here we
Genetic engineering of plants has advanced to a level permitting a study of mechanisms that could control the spread of viruses. Recently, promising results have been obtained with plants transformed with viral cDNAs by the route of “agroinfection” (Grimsley et al., 1986). Cucumber mosaic virus (CMV) was shown to acquire its satellite, CARNA 5, from transgenic tobacco plants transcribing satellite RNA from a chimeric nuclear gene (Baulcombe er a/., 1986). As most CARNA 5 strains interfere with the CMV symptom expression, this approach could be of economical importance. However, this approach is limited to the very few viruses that are accompanied by interfering satellites. A more general phenomenon that may be employed to control virus infections is cross-protection. The term cross-protection is used for the observation that plants infected with a mild strain of a given virus do not develop additional symptoms upon inoculation with a severe strain of the same virus (Fulton, 1982). The use of crossprotection in the field has been limited by certain risks such as mutation of the mild strain to a severe phenotype, synergism of the mild strain with other viruses, and spread of the mild strains to other crops. Possibly, these disadvantages could be circumvented by using transgenic plants that express an incomplete noninfectious viral genome. Transformation with the coat protein (CP) gene of tobacco mosaic virus (TMV) has ’ To whom reprint requests should be addressed. ’ Present address: Research Institute Ital, P.O. Box 48, 6700 A4 Wageningen, The Netherlands.
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used RNA 2 of strain TCM (3389 nucleotides) which contains reading frames for the CP, a 29.1 -kDa protein and a 16-kDa protein, the CP gene being 5’-proximal (Angenent et al., 1986). A subgenomic messenger lacking the 5’-terminal 437 nucleotides of the leader sequence of 546 nucleotides has been identified, thus avoiding a number of AUG codons in this noncoding region. A unique property of TRV is the ability of RNA 1 to replicate and spread in plants independently of RNA 2 (Harrison and Robinson, 1978). It will be interesting to see the effect of CP, produced endogenously in transgenic plants, on RNA 1 replication. MATERIALS AND METHODS Construction of chimeric genes Plasmid pDH51 (Pietrzak et al., 1986) was obtained from the Friedrich Miescher Institute (Basel) and plasmid pMONl77 was obtained from Monsanto (St. Louis). A BarnHI fragment of pMON 177, containing the nopaline synthase (nos) polyadenylation signal, was inserted into the BarnHI site just downstream from the cauliflower mosaic virus (CaMV) 35 S promoter in pDH51, yielding plasmid pDH52 (Fig. 1). An EcoRl fragment of pDH52, containing the 35 S promoter and nos polyadenylation signal, was inserted into the binary transformation vector pAGSHB. This vector was derived
FIG. 1. Construction of the chimeric genes consisting of the CaMV 35 S promoter, CP gene of AIMV or TRV and nos-polyadenylation signal. P35 S, CaMV 35 S promoter; Pnos, nopaline synthase promoter; Tnos, nopaline synthase poly(A) signal; Tots, octopine synthase poly(A) signal; NPT, neomycin phosphotransferase; cos, CDS site; TcR,tetracycline resistance gene; LB, left border; RB, right border; E, EcoRI; B, BarnHI; H, Hindlll.
from pAGS127 (Van den Elzen et al., 1985) by deleting the HindIll and BarnHI sites. The unique BarnHI site between the 35 S promoter and polyadenylation signal was converted into a HindIll site using a linker. This HindIll site was subsequently used to insert the cDNAs of AIMV and TRV encoding the viral coat proteins (Fig. 1). The AIMV cDNA is a full-length copy of RNA 4 of strain 425; first- and second-strand cDNA syntheses were primed with oligonucleotides containing the restriction sites Smal (3’) and Pstl (53, respectively (Langereis et al., 1986). These sites were converted into HindIll sites. The cDNA encoding the CP of TRV strain TCM is a fragment of cDNA 2 cloned by Angenent et a/. (1986). The 5’-end of the fragment is located at position 441 which is four nucleotides downstream from the start site of the putative subgenomic messenger for the CP. The 3’-end of the fragment extends into the 29.1 K cistron to position 1440. The transformation vector containing the AIMV CP chimeric gene was designated pCA40, whereas that containing the TRV CP chimeric gene was designated pCTR20. Due to the cloning procedure the expected chimeric mRNA in transgenic plants is extended with approximately 20 nucleotides at the 5’-end and 150 nucleotides at the 3’-end excluding the poly(A) tail. The plasmids pCA40 and pCTR20 were mobilized into Agrobacterium tumefaciens strain LBA 4404 containing PAL 4404 (virulence region) by a triparental mating. Briefly, 1 ml of a logarithmically growing culture of Escherichia colistrain 490, containing either pCA40 or pCTR20, was mixed with 1 ml of logarithmically growing E. co/i strain HBl 01 containing pRK2013 (Ditta et a/., 1980) and 1 ml of logarithmically growing A. tumefaciens strain LBA4404 (Hoekema et al., 1983). After incubation to allow plasmid transfer, transconjugants resistant to rifampicin (to select A. tumefaciens) and tetracycline (to select for the presence of plasmids pCA40 or pCTR20) were isolated. Plant transformation Leaf disks of N, tabacum cvs. Samsun NN and Xanthi nc were infected with A. tumefaciens LBA 4404 containing pCA40 or pCTR20 (Horsch et al., 1985). Transformed cells were selected on shooting medium containing kanamycin (100 pglml). Kanamycin-resistant shoots were transferred to rooting medium containing kanamycin (100 pglml). After development of the root system (2-3 weeks) plantlets were cultured in the greenhouse. After about 6 weeks, when the plants were in the four to six leaf stage, the assay for the presence of virus-specific RNA or protein was performed. Plants containing AIMV cDNA are designated S40, whereas plants containing TRV cDNA are designated XTRV.
301
VIRUS TRANSFORMED TOBACCO
12
RNA analysis of transgenic plants RNA was isolated from transgenic plants essentially as described by Hoof? van Huijsduijnen et a/. (1985). Polyadenylated RNA was isolated by poly(U)-Sepharose chromatography. The RNA was denatured in glyoxal/DMSO, run on a 1% agarose gel, and transferred to a nitrocellulose filter (Sarachu et a/., 1985). The Northern blot was probed with purified 32P-nicktranslated cDNA fragments corresponding to the insert in the transformation vector (Hooft van Huijsduijnen et a/., 1985). Protein analysis of transgenic plants Protein containing sap was squeezed from young leaves of the transgenic plants. The sap was clarified by centrifugation. The protein concentration of the clarified supernatant was determined by the Bradford method (Bradford, 1976). Western blots were made essentially as described by Towbin eta/. (1979). Equal amounts of protein (50 pg) from different transgenic plants were electrophoresed on gels containing 11% acrylamide and 0.1% SDS and subsequently transferred to nitrocellulose. The nitrocellulose filters were incubated with antiserum raised against AIMV or TRV. The filters were then incubated with peroxidase-conjugated goat anti-rabbit antibodies. Staining was performed with 3,3’-diaminobenzidine. Expression levels were estimated by comparing the intensity of the obtained bands with the band intensities of known quantities of CP added to juice of control plants. Infectivity assay Tobacco leaf material (3 cm2) was homogenized in 2 ml 0.01 M phosphate buffer, pH 7.0, and inoculated in several dilutions on the leaves of Samsun NN tobacco or bean plants (Phaseohs vulgaris L. var. “Berna”).
123456 -CP
FIG. 2. Accumulation of AIMV CP in transgenic tobacco plants. Lanes 1 to 3: protein extracts from untransformed plants to which 0, 10, and 100 ng of AIMV CP had been added, respectively. Lane 4: protein extract from a vector transformed plant. Lanes 5 and 6: protein extracts from plants S40-7 and S40-8, respectively. The Western blot was analyzed with an antiserum to AIMV CP.
3L5678
-CP
FIG. 3. Accumulation of AIMV CP in different leaves of a tobacco plant transformed with the AIMV CP gene. Lanes 1 to 5: protein extracts from lower to upper leaves, respectively. Lanes 6 to 8: protein extracts from untransformed plants to which 0, 5, and 50 ng of AIMV CP were added, respectively. The Western blot was analyzed with an antiserum to AIMV CP.
RESULTS Expression of coat protein A total of 15 kanamycin-resistant plants, transformed with the chimeric AIMV CP gene, were tested for the presence of CP. Eleven plants were found to accumulate detectable amounts of CP. The accumulated level ranged between 0.01 and 0.05% of the extracted protein. Two plants, S40-7 and S40-8, were chosen for further analysis. Figure 2 shows a Western blot containing protein from S40-7 and S40-8, from a control plant transformed with the vector only, and from an untransformed plant. The antiserum to AIMV CP clearly binds to a protein in the extract from plants transformed with viral cDNA (lanes 5 and 6) but not to the proteins in the controls (lanes 1 and 4). The CP molecules produced by the transgenic plants comigrate with native AIMV CP. Levels of accumulation were estimated by comparing the signals to those obtained in lanes run with 10 and 100 ng of native AIMV CP added to extracts from untransformed plants (Fig. 2, lanes 2 and 3). These control signals were similar to those of the bands obtained by loading samples of 10 and 100 ng of pure protein to the gel (result not shown). Analysis of AIMV CP accumulation in different leaves of a transformed tobacco plant showed that the accumulation was independent of leaf age (Fig. 3). Besides AIMV CP, a band of lower mobility was detected in the older leaves. As this band was not reproducibly seen in other experiments, its nature was not further analyzed. Six kanamycin-resistant plants transformed with the chimeric TRV CP gene were assayed for the presence of CP. Four of them showed the accumulation of detectable levels of TRV CP, ranging between 0.01 and 0.05% of the extracted protein. The protein analysis of two of these plants XTRVl and XTRVB is depicted in Fig. 4. This Western blot shows the presence of TRV
VAN DUN, BOL, AND VAN VLOTEN-DOTING
302
1
2
3
4
5
6
1234
-CP
FIG. 4. Accumulation of TRV CP in transgenic tobacco plants. Lanes 1 to 3: protein extracts from untransformed plants to which 0, 10, and 100 ng of TRV CP had been added, respectively. Lane 4: protein extract from a vector transformed plant. Lanes 5 and 6: protein extracts from XTRVl and XTRV3, respectively. The Western blot was analyzed with an antiserum to TRV CP.
CP in these transgenic plants comigrating with native TRV CP. The accumulation of TRV CP was not affected by the age of the leaves (result not shown). Expression of RNA The expression of viral genes in transgenic plants was analyzed at the RNA level by hybridizing Northern blots, loaded with purified poly(A)-containing RNA, to probes corresponding to the respective viral cDNAs. Figure 5, lanes 3 and 4, shows RNA extracted from plants S40-7 and S40-8 which produce AIMV CP. After hybridization to labeled AIMV cDNA 4, a distinct band is seen in each lane. The controls, containing polyadenylated RNA from untransformed and vector transformed plants, did not give any signal (Fig. 5, lanes 1 and 2). The AIMV-specific transcripts have an approximate length of 1300 bases. If transcription is initiated at the CaMV 35 S promoter and is terminated behind the nos-polyadenylation site of the transformation vector, the transcript is expected to contain a 5’-nonviral sequence of approximately 20 nucleotides, an RNA 4specific sequence of 881 nucleotides, a 3’-nonviral sequence of 150 nucleotides, and a poly(A) tail. This would suggest that the average length of the poly(A) tail in the in viva produced transcripts is 250 residues.
1
2
3
4
FIG. 5. Autoradiograph of a Northern blot containing polyadenylated RNA from transgenic plants accumulating AIMV CP. Lane 1: RNA from an untransformed plant. Lane 2: RNAfrom a vectortransformed plant. Lanes 3 and 4: RNA from plants S40-7 and S40-8, respectively. The positions of AIMV RNAs 3 and 4 are indicated. The blot was probed with 32P-labeled cDNA to AIMV RNA 4.
FIG. 6. Autoradiograph of a Northern blot containing polyadenylated RNA from transgenic plants accumulating TRV CP. Lane 1: RNA from an untransformed plant. Lane 2: RNA from a vector transformed plant. Lanes 3 and 4: RNA from plants XTRVl and XTRV3, respectively. The position of AIMV RNAs 3 and 4 are indicated. The blot was probed with 3zP-labeled cDNA to TRV-RNA 2 (strain TCM).
Figure 6 shows the analysis of poly(A)-containing RNA from transgenic plants XTRVI and XTRV3. Hybridization of the Northern blot with a cDNA probe corresponding to the TRV CP cistron showed clear bands with the preparations from virus-transformed plants (Fig. 6, lanes 3 and 4) which are absent in the controls (Fig. 6, lanes 1 and 2). The estimated length of the TRV-specific transcript is 1400 nucleotides. This compares well with the size of the RNA that was expected to be transcribed from the chimeric TRV CP gene (999 bases of viral RNA, flanked by 5’- and 3’-nonviral sequences of approximately 20 and 150 bases, respectively, and a 3’-poly(A) tail). Susceptibility of transgenic plants to virus infection Seed collected from plants S40-7 and S40-8 was germinated on medium containing kanamycin. Over 95% of the seedlings from S40-7 were found to be resistant to the antibiotic. This non-Mendelian segregation of the NPT gene indicates the occurrence of multiple integrations during the transformation procedure. On the other hand, none of the S40-8 seedlings showed resistance to kanamycin. Differences in the integration events in plants S40-7 and S40-8 are presently being analyzed. Plants obtained from S40-7 seedlings accumulated AIMV CP to the same level as the original transformant and were tested for their susceptibility to infection with AIMV nucleoproteins or RNAs. As controls, plants SC1 and SV-1 were used, which were regenerated from Samsun NN leaf disks dipped into a suspension of A. tumefeciens containing no transformation vector or a transformation vector without viral cDNA, respectively. Six plants of each group (SC-l, SV-1, and S40-7) were inoculated with AIMV strain YSMV which induces necrotic yellow lesions on Samsun NN tobacco. Plants SC-l and SV-1 developed lesions after a few days, whereas all S40-7 plants remained free of any symptoms of infection during a 4-week observation period. On the control plants, inocula containing 0.25 or 2.5
VIRUS TRANSFORMED
pglml virus induced an average lesion number per half leaf of 40 and 430, respectively. Table 1 lists the lesion numbers obtained with the highest virus concentration. Two weeks after inoculation with YSMV (2.5 pg/ml), leaf material was taken from four SC-l, SV-1, and S407 plants and a homogenate of this material was inoculated to nontransformed tobacco plants. Table 2 shows that the homogenates from SC-l and SV-1 were highly infectious, whereas the homogenate from S407 did not induce any lesions. This demonstrates that after infection with YSMV the symptomless plants do not accumulate detectable amounts of infectious nucleoprotein. The above results indicate that the CP of AIMV strain 425, that accumulates in S40-7 plants, interferes with one or more steps in the replication cycle of the closely related strain YSMV. When SC-l, SV-1, and S40-7 were inoculated with TMV, the plants developed similar numbers of local lesions (result not shown), demonstrating that the protection is specific for the homologous virus. To obtain insight in the replication event that is inhibited by the endogenously produced CP and to analyze its biological activity, plants of SC-l, SV-1, and S40-7 were inoculated with a mixture of AIMV genomic RNAs of strain 425 plus or minus subgenomic RNA 4. On Samsun NN tobacco strain 425 induces mild chlorotic symptoms which are difficult to quantitate. Therefore, leaf material was collected 1 week after inoculation of the plants, and homogenates of this material were inoculated in several dilutions to bean plants, which is a local lesion host for strain 425. Table 3 lists the lesions induced by appropriate dilutions. The mixture of all four AIMV RNAs is equally infectious to the control plants (SC-l and SV-1) and to the transgenic S40-7 plants. This shows that the endogenously produced CP does not protect against infection with viral
303
TOBACCO
TABLE2 PRODUCTIONOF INFECTIOUSVIRUSIN TRANSFDRMEDAND NONTRANSFORMEDTOBACCO PLANTSINOCULATEDWITHAIMV STRAINYSMV” Lesions induced by homogenates
of
Plant No.
SC-1
sv-1
s40-7
1 2 3 4
269 186 140 200
180 240 80 185
0 0 0 0
BTwo weeks after inoculation, leaf material of plants 1 to 4 of Table 1 was homogenized and the homogenates were inoculated to nontransformed Samsun NN tobacco plants (two leaves per homogenate). Lesions were counted 1 week after inoculation; average lesion number per half leaf is given.
RNA. The mixture of AIMV RNAs 1, 2, and 3 is not infectious to the control plants, demonstrating that the mixture is not contaminated with detectable amounts of RNA 4. However, the infectivity of this inoculum toward S40-7 plants is comparable to that of the inoculum containing all four AIMV RNAs. This proves that the endogenously produced CP is biologically active and is able to replace the RNA 4 or CP that is required in the inoculum for infection of nontransformed plants (601 eta/., 1971). DISCUSSION Viral CP was produced in about 70% of the kanamycin-resistant plants transformed with the AIMV and TRV CP genes. This indicates that, generally, during transformation the construct between the border sequences in the vector remains intact. The observation that both AIMV- and TRV-specific transcripts copurify with the poly(A)-containing cellular RNAs on poly(U)-
TABLE 1 INDUCTIONOF LOCAL LESIONSBYAIMV STRAINYSMV ON TRANSFORMED AND NONTRANSFORMED TOBACCO PLANTS’
TABLE 3 PRODUCTIONOF INFECTIOUSVIRUSIN TRANSFORMEDAND NONTRANSFORMEDTOBACCO PLANTSINOCULATEDWITHAIMV RNA9
Lesions induced on Plant No.
SC-1
sv- 1
s40-7
1 2 3 4 5 6
635 510 350 690 625 505
745 420 260 355 110 370
0 0 0 0 0 0
@Tobacco plants accumulating AIMV CP (S40-7) and control plants (SC-l and SV-1) were inoculated with 2.5 pg/ml YSMV (two leaves per plant). Lesions were counted 2 weeks after inoculation; average lesion number per half leaf is given.
Lesions induced by homogenates
of
lnoculum
SC-I
sv-1
s40-7
RNAs 1+2+3+4 RNAs 1+2+3
171 0
124 0
251 207
8 Tobacco plants SC-l, SV-1, and S40-7 were inoculated with 40 ~1 per half leaf containing 100 ng RNAs 1+2+3, plus or minus 100 ng RNA 4. After 10 days leaf material of each plant was homogenized and the homogenates were inoculated in several dilutions on bean leaves. Lesions were counted after 3 days; the number per half leaf is given.
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Sepharose columns indicates that they are transcribed from a nuclear gene. Preliminary data indicate that a plus-stranded RNA transcribed in plants transformed with a DNA copy of AIMV RNA 3 is being processed to a smaller molecule (Van Dun et al., in preparation). Apparently, this is not the case with the AIMV RNA 4 and partial TRV RNA 2 transcripts that were transcribed from nuclear genes, as their estimated sizes were in agreement with those expected from the sequences of the respective transformation vectors. At present we are determining the copy number of the NPT and CP genes in the respective AIMV and TRV transformed plants. This may give a clue to the observation that the progeny of S40-8 plants had lost their kanamycin resistance. The progeny of TRV transformed plants is not yet available for further analysis. In vitro, AIMV RNA 4 is efficiently translated into viral CP (Van Vloten-Doting and Jaspars, 1977). ln viva, it can replace the CP in the “activation” of the genome (Bol et al., 1971), suggesting that AIMV RNA 4 is also an active messenger in virus-infected cells. The functional messenger RNA for TRV CP is not known. In several strains a subgenomic RNA has been identified that lacks the eight AUG codons that are present in the 5’-leader sequence preceding the CP cistron in TRVRNA 2 (Cornelissen et al., 1984; Angenent et a/., 1986). These AUG codons are also absent in the TRV-RNAs transcribed in plants XTRVl and XTRV3. Apparently, the 20 nonviral nucleotides that are present at the 5’termini of the AIMV and TRV specific transcripts in transgenic plants do not grossly affect the messenger activity of the transcripts. In both AIMV and TRV CP gene transformed plants, the accumulation of CP varied between 0.01 and 0.05% of the soluble leaf protein. This indicates that the two types of genes are expressed with similar efficiencies. The inverse relationship between the synthesis of AIMV mRNA and coat protein that is suggested by a comparison of the bands in Figs. 2 and 5 was not reproducibly obtained in other experiments. In general, a parallel was observed between the level of mRNA and CP synthesis. Although the accumulation of AIMV CP in S40-7 plants is about 1O-fold lower than it is in AIMV-infected plants, it is apparently sufficient to block infection by viral nucleoprotein. The finding that these plants are susceptible to infection with viral RNA indicates that the endogenous CP interferes with an early step of the replication cycle, e.g., absorption to specific receptor sites in the cell or uncoating. To initiate infection, each AIMV genomic RNA must have bound a few CP subunits (Smit et a/., 1981). The detection of a major CP binding site at the 3’-end of the viral RNAs has led to the suggestion that this binding is required for recognition of the RNAs by the viral replicase (Houwing and
Jaspars, 1978). At the final stage of the replication cycle this binding site may act as a nucleation site for encapsidation of the viral RNAs. Because a mixture of the AIMV genomic RNAs is infectious to S40-7 plants, it may be concluded that the CP accumulating in these plants is biologically active and is able to associate with the incoming viral RNAs. Apparently, the level of endogenous CP is such that the parental RNAs are not inactivated by encapsidation and that no other possible regulatory function is blocked in which the CP might be involved. It has been suggested that CP is the RNA 3 encoded protein that regulates the balance between AIMV plus- and minus-strand RNA synthesis (Nassuth and Bol, 1983). We have not yet analyzed the offspring of XTRVl and XTRV3 for possible resistance to TRV infection. Evidence has been presented that infection of plants with TRV RNA 1 alone results in the induction of cross-protection (Cadman and Harrison, 1959). Thus, it will be interesting to study the phenotype of transgenic plants expressing nonstructural viral genes. ACKNOWLEDGMENTS The authors thank Dr. Peter van den Elzen for the gift of pAGS 127, Dr. Maciej Pietrzak for the gift of the CaMV 35 S promoter, Corrie Houwing for providing the YSMV inoculum, and Bert Overduin for technical assistance. This research was supported in part by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO).
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