Replication of an incomplete alfalfa mosaic virus genome in plants transformed with viral replicase genes

VIROLOGY

181,

445-450

(1991)

Replication of an incomplete Alfalfa Mosaic Virus Genome in Plants Transformed with Viral Replicase Genes PETER E. M. TASCHNER, ANTOINETTE Gorlaeus

Laboratories,

Leiden

Received

C. VAN DER KUYL, LYDA NEELEMAN,

University,

October

P.O.

3, 1990;

Box 9502, accepted

2300

RA Leiden,

December

AND

JOHN F. BOL’

The Netherlands

4, 1990

RNAs 1 and 2 of alfalfa mosaic virus (AIMV) encode proteins Pl and P2, respectively, both of which have a putative role in viral RNA replication. Tobacco plants were transformed with DNA copies of RNA1 (Pl -plants), RNA2 (P2-plants) or a combination of these two cDNAs (Pl P-plants). All transgenic plants were susceptible to infection with the complete AIMV genome (RNAs 1,2, and 3). Inoculation with incomplete mixtures of AIMV RNAs showed that the Pl-plants were able to replicate RNAs 2 and 3, that the PS-plants were able to replicate RNAs 1 and 3, and that the P12-plants were able to replicate RNA3. Initiation of infection of nontransgenic plants, Pl-plants, or P2-plants requires the presence of AIMV coat protein in the inoculum, but no coat protein was required to initiate infection of P12-plants with RNA3. Results obtained with P12-protoplasts supported the conclusion that coat protein plays an essential role in the replication cycle of AIMV RNAs 1 and 2. o 1991 Academic PESS. I~C.

INTRODUCTION

other viruses (see Beachy, 1990). No resistance to AIMV infection was observed with plants transformed with the genes encoding Pl (Pl-plants) or P2 (P2plants) (Van Dun et a/., 1988), or with plants transformed with the P3 gene (Dore et al., 1991). However, only for the Pl-plants was evidence obtained that an active viral gene was expressed. In the present study we proved that our P2-plants also expressed an active viral gene product. To obtain plants expressing both AIMV replicase genes, the transgenic P2-plants were transformed with the Pl gene. When the resulting plants, termed P12-plants, were inoculated with RNA3, this genome segment replicated at normal levels indicating that active Pl and P2 are produced in these transgenic plants. The observation that CP is not required to initiate RNA3 replication in P12-plants sheds new light on the early function of AIMV CP in the viral replication cycle.

The tripartite RNA genome of alfalfa mosaic virus (AIMV) is believed to encode four proteins. RNAs 1 and 2 encode proteins Pl and P2, respectively. RNA3 is dicistronic and encodes protein P3 and the viral coat protein (CP). The observation that RNAs 1 and 2 are able to replicate in protoplasts in the absence of RNA3 indicates that Pl and P2 are involved in RNA replication (Nassuth and Bol, 1983). The genome structure of AIMV is similar to that of bromo- and cucumoviruses; for these viruses Pl and/or P2 have been identified as subunits of the viral replicase (Quadt et a/., 1988; Horikoshi et al., 1988; Hayes and Buck, 1990). P3 of AIMV is believed to have a function in cell-to-cell transport of the virus (Stussi-Garaud et al., 1987) but CP may also be required for this process (Dore et al., 1991). CP is translated from a subgenomic messenger, RNA4. In addition to its structural role, CP has an early function in the replication cycle of AIMV: a mixture of the three genomic RNAs is not infectious to plants (Bol et a/., 1971) or protoplasts (Nassuth eta/., 1981) unless a few molecules of CP or CP-mRNA are added per RNA molecule. To further study their role in the viral replication cycle, we have transformed tobacco with DNA copies of all four AIMV genes fused to the CaMV 35 S promoter. Plants transformed with the CP gene were resistant to infection with AIMV particles but susceptible to infection with AIMV RNA (Van Dun et a/., 1987). A similar CP-mediated resistance has been reported for several ’ To whom

requests

for reprints

should

MATERIALS

AND

METHODS

Plant material Mcotiana tabacum cv. Samsun NN plants transformed with AIMV cDNA1 (Pl-plants) and cDNA2 (P2plants) were lines S10.1 and S20.2, respectively (Van Dun et a/., 1988). Plants transformed with both AIMV cDNA1 and cDNA2 (P12-plants) were obtained as follows. BamHl linkers were added to a HindIll fragment containing cDNA to the Pl gene (Van Dun eta/., 1988). This fragment was inserted in the sense orientation in the BamHl site downstream of the CaMV 35 S promoter in the transformation vector pMBV5EH (kindly

be addressed. 445

0042.6822191

$3.00

Copyright Q 1991 by Academic Press. Inc. All rights of reproduction I” any form resewed.

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provided by MOGEN International, Leiden), which contains the hygromycin resistance gene for the selection of transformed plant cells. The resulting plasmid pPV58, was mobilized into Agrobacterium tumefaciens LBA4404 containing pAL4404 by triparental mating as described by Van Dun et al. (1987). Transconjugants were selected by resistance to rifampicin and kanamytin. S20.2 plants were retransformed with A. tumefaciens containing pPV58 by the leaf disk transformation procedure (Horsch et al., 1985). Retransformed plant cells were selected on media containing hygromycin (20 pg/ml). Several hygromytin-resistant plants were obtained and checked by Southern and Northern blot analysis for the presence and expression of the chimeric genes. Of the plants tested, transformant SH2058-2 gave the highest expression of Pl and P2 transcripts, and plants of this line were designated P12-plants. Untransformed N. tabacum cv. Samsun NN plants were used as controls. Seeds of Pl- and P2-plants were germinated on MS medium containing kanamycin (100 pg/ml). In addition, the medium for P12-plants contained hygromycin (20 dml).

ET Al

Inoculation

of protoplasts

Protoplasts were isolated and inoculated as described by Van Dun et al. (1988). Per sample, 1 O5 protoplasts were inoculated with purified RNAs or transcripts of cDNA3 in the presence or absence of AIMV CP (2 pg/sample). As indicated in the text, the inocula contained various combinations of RNA1 (1.5 pg), RNA2 (600 ng), RNA3 (300 ng), or RNA3 transcripts (1.5 pg). lnoculum RNAs were purified by agarose gel electrophoresis and isolated from the gel by the freeze-squeeze method (Tautz and Renz, 1983). The RNA3 transcripts were made in vitro by T7 RNA polymerase run-off transcription from a full-length cDNA clone of RNA3. The construction of this infectious clone will be published elsewhere. Protoplasts were incubated for 42 hr at 25” and total RNA was isolated from each sample according to Sarachu et al. (1985). One-third of the RNA isolated from 1 O5protoplasts was analyzed by Northern blot hybridization using random primed cDNA as a probe (Feinberg and Vogelstein, 1984). Scanning of autoradiograms was done with a LKB 2222-020 Ultrascan XL Laser Densitometer. RESULTS

inoculation

of plants

Seedlings from the original transformants were grown to the six-leaf stage and mechanically inoculated using Carborundum as an abrasive, after shading the plants for 2 days (Van Dun et a/., 1988). Unless mentioned otherwise, inoculation was done with strain 425 which causes mild chlorotic symptoms on Samsun NN tobacco. In a few experiments AIMV strain YSMV, which causes yellow necrotic lesions on this host, was used. Inoculation with virus particles was done on eight half-leaves per sample with 20 PI/half-leaf of a solution containing 2.5 pg/ml of viral nucleoprotein. This viral nucleoprotein was purified as described by Bol et al. (197 1). RNAs extracted from virus particles were separated by agarose gel electrophoresis and isolated from the gel by the freeze-squeeze method (Tautz and Renz, 1983). Inoculation with viral RNAs was done on three half-leaves per sample with 20 PI/half-leaf of a solution containing the indicated combination of RNA1 (30 pg/ml), RNA2 (10 pg/ml), and RNA3 (5 pg/ml). Four days after inoculation virus particles were isolated from inoculated leaves (Bol et a/., 1971) and RNA extracted from 20 ng virus was analyzed by Northern blot hybridization using random primed cDNA as probe (Feinberg and Vogelstein, 1984). Protein from 0.15 mg leaf material was analyzed by the Western blot technique (Towbin et a/., 1979).

Replication of incomplete transgenic plants

viral genomes

in

Previously, we have transformed tobacco with cDNA to AIMV RNAs 1 and 2, giving Pl- and P2-plants, respectively. In these cDNAs the 5’-terminal 36 nucleotides of RNA1 and the 3’-terminal 10 nucleotides of RNA2 were lacking (Van Dun et al., 1988). To see whether the viral genes expressed in the Pl- and P2plants were functional, the plants were inoculated with incomplete mixtures of AIMV genomic RNAs supplemented with CP. Production of viral RNA and CP in these plants was monitored by the Northern and Western blot techniques, respectively (Figs. 1A and 1 B). Plplants inoculated with a mixture of RNAs 2 and 3 and CP were able to support replication of the inoculum RNAs and produced the subgenomic CP messenger (RNA4), and CP (Fig. 1, lanes 1). A similar observation was made for the P2-plants inoculated with a mixture of RNAs 1 and 3 and CP (Fig. 1, lanes 3). Apparently, a functional replicase activity is assembled in these transgenic plants when one of the viral replicase subunits is encoded by the plant genome and the other is encoded by an inoculum RNA. This polymerase activity does not replicate the transcripts from the chimeric nuclear genes, possibly because the cDNAs that were used for transformation were incomplete. To check the purity of the inoculum RNAs nontransgenie plants were inoculated with a mixture containing

REPLICASE-TRANSFORMED

Pl

P2

P12

c

B 123456

769

FIG. 1. RNA and protein synthesis in transgenic plants inoculated with complete and incomplete mixtures of AIMV genomic RNAs. Pl -plants (lanes 1, 2) P2-plants (lanes 3, 4) P12-plants (lanes 5. 6) and nontransformed control plants (lanes 7-9) were inoculated with the complete AIMV genome (lanes 2, 4, 6, 9) with mixtures of RNAs 2 and 3 (lanes 1, 7) with RNAs 1 and 3 (lanes 3, 8) or with RNA3 (lane 5). All inocula were supplied with AIMV CP. Four days after inoculation, protein and virus particles were isolated from the plants. RNA extracted from these virus particles was analyzed by Northern blot hybridization (A) and protein was analyzed by the Western blot technique (B). The Northern blot was hybridized to random primed AIMV cDNA; the Western blot was incubated with antiserum against AIMV CP. The positions of AIMV RNAs and CP are indicated in the margin.

RNAs 2 and 3 and CP (Fig. 1, lanes 7) or RNAs 1 and 3 and CP (Fig. 1, lanes 8). The former inoculum did not induce the production of detectable amounts of virus material but a longer exposure of the blot of Fig. 1A revealed that the inoculum containing RNAs 1 and 3 induced the synthesis of trace amounts of all four AIMV RNAs. Moreover, some CP is detectable in lane 8 of Fig. 1 B, illustrating that the inoculum RNAs were not absolutely pure. When virus particles were isolated from Pl- and P2-plants which had been inoculated with incomplete mixtures of AIMV genome segments, the yield of viral nucleoprotein ranged between 30 and 70% of that routinely isolated from nontransgenic plants infected with the complete AIMV genome. Analysis by electron microscopy showed that these particles had the bacilliform morphology characteristic of AIMV virions (result not shown). The progeny of Plplants which had been inoculated with RNAs 2 and 3 (Fig. 1, lane 1) was not infectious to nontransgenic control plants at concentrations ranging from 2.5 to 400 pg/ml, but became so when RNA1 was added to these progeny virus particles. Similarly, the progeny of P2-plants which had been inoculated with RNAs 1 and 3 (Fig. 1, lane 3) was infectious to control plants only

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after addition of RNA2 (results not shown). Apparently, the inoculation of Pl- and P2-plants with incomplete mixtures of AIMV genome segments results in the production of biologically active RNA molecules that are normally encapsidated. It has been reported by Van Dun et a/. (1988) that after inoculation with virus particles containing the complete genome of AIMV strain YSMV, similar numbers of yellow necrotic lesions developed on the inoculated leaves of Pl-, P2-, and control plants. In agreement with this, Fig. 1 shows that inoculation with the complete genome of AIMV strain 425 of Pl -plants (lanes 2) P2-plants (lanes 4) and control plants (lanes 9) induces the synthesis of similar levels of all four AIMV RNAs and CP. From these data we conclude that the chimeric nuclear genes in the Pland P2-plants are expressed into functional products which can substitute for inoculum RNAs 1 and 2, respectively, but which do not interfere with virus multiplication. Because of a lack of suitable antisera, the Pl and P2 proteins could not be detected by serology in these plants (Van Dun et a/., 1988). To combine the two putative replicase functions of AIMV in one transgenic plant, the P2-plants were transformed with the Pl gene. The presence and expression of the Pl and P2 genes in the resulting P12plants was confirmed by Southern and Northern blot hybridization (result not shown). After inoculation with a mixture of RNA3 and CP, the Plil-plants accumulated RNA3, RNA4, and CP (Fig. 1, lanes 5) and virus particles containing RNAs 3 and 4 could be isolated from these plants. These progeny virus particles were not infectious to control plants but induced the synthesis of RNAs 3 and 4 and CP when inoculated to P12plants (result not shown). Lane 6 of Fig. 1A shows that after inoculation of P12-plants with a mixture of RNAs 1, 2, and 3 and CP of AIMV (strain 425) all four viral RNAs are produced although the relative amounts of RNAs 1 and 2 were reduced compared to that in control plants (lane 9). This reduction was consistently observed in several experiments. After inoculation of P12-plants and control plants with virus particles of AIMV strain YSMV at a concentration of 2.5 pglml, an average of 600 lesions per half-leaf was induced on the inoculated leaves of P12-plants whereas only 20 lesions per half-leaf developed on nontransformed control plants. Both type of plants developed the systemic yellowing characteristic for this strain. When the virus concentration in the inoculum was reduced from 2.5 /*g/ml to 0.1 pg/ml, the P12-plants still showed symptoms of the YSMV-type whereas symptoms were no longer detectable on the controls. We have shown earlier that symptom formation by strain YSMV on tobacco is determined by RNA3 (Dingjan-Versteegh et a/., 1972). Inoculation of P12-plants

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protoplasts

plants

12

34

CP 34-

FIG. 2. CP dependency of AIMV RNA3 replication in P12-protoplasts and P12-plants. P12-protoplasts (lanes 1, 2) and P12-plants (lanes 3, 4) were inoculated with RNA3 plus CP (lanes 1, 3) or minus CP (lanes 2, 4). The RNA3 was transcribed in vitro with T7 RNA polymerase from a full-length DNA copy of RNAB. RNA was isolated from protoplasts 42 hr after inoculation and virus particles were isolated from plants 4 days after inoculation. RNA extracted from these virus particles and from protoplasts was analyzed by Northern blot hybridization using random primed AIMV cDNA as probe. The positions of RNAs 3 and 4 are indicated In the margin.

with purified RNA3 of strain YSMV resulted in the formation of symptoms indistinguishable from those induced by the complete genome of this strain (Neeleman et a/., unpublished observation). Thus, symptom formation induced by strain YSMV on P12-plants probably reflects the replication of RNA3 only. Requirement plants

of CP for the infection

of transgenic

Because CP is required for infection of nontransgenie plants with AIMV, it was routinely added to the inocula used to initiate the infections shown in Fig. 1. To test whether CP is also required for the initiation of infection of replicase-transformed plants, the P12plants and protoplasts from these plants were inoculated with RNA3 in the presence or absence of CP. To rule out the possibility that RNA3 in the inoculum was contaminated with RNA4 (which can replace CP in the inoculum), the RNA used in this experiment was transcribed in vitro with T7 RNA polymerase from a fulllength cDNA clone of RNA3. Transcripts thus obtained have authentic 5’- and 3’-termini and resemble native RNA3 in infectivity (Neeleman et al., unpublished observation). P12-plants produced similar levels of RNAs 3 and 4 whether or not the RNA3 in the inoculum was supplemented with CP (Fig. 2, lanes 3 and 4). In several experiments the RNA synthesis induced in protoplasts by RNA3 was found to be reduced 2-to 3-fold when CP was omitted from the inoculum (Fig. 2, lanes 1 and 2). However, the RNA synthesis induced by a mixture of RNAs 1, 2, and 3 in nontransgenic protoplasts, if any, is at least 250-fold lower than that induced by an inocu-

ET At

lum containing CP or CP-mRNA (Nassuth et a/., 1981). Thus, the relatively small effect of CP seen in P12-protoplasts is probably not related to the early function of CP in the initiation of infection. The finding that CP is not required for the initiation of RNA3 replication in P12-plants was rather unexpected and made us reinvestigate the CP dependency of the infection of Pl -and P2-plants. The Pl -plants were inoculated with a mixture of RNAs 2 and 3 plus or minus CP, whereas the CP dependency of P2-plants was tested with an inoculum containing RNAs 1 and 3. Figure 3 shows that an inoculum without CP did not induce a detectable level of RNA synthesis in the P2plants (lane 4) whereas the control with CP was infectious (lane 3). In the Pl -plants inoculated with RNAs 2 and 3 without CP a very low level of RNA synthesis was detectable (Fig. 3, lane 2) compared to the control (lane 1). This may be due to a minor contamination of the inoculum with RNA4. However, the results of Fig. 3 clearly indicate that CP is required for the infection of Pl- and P2-plants in the same way as it is required for infection of nontransgenic plants. Replication

of RNAs 1 and 2 in P12-protoplasts

Because RNAs 1 and 2 do not express the cell-tocell transport function(s) encoded by RNA3, it was expected that these genome segments would not be able to induce a detectable level of replication in P12plants. In agreement with this assumption, no viral RNA synthesis could be detected in P12-plants inoculated with a mixture of RNA1 and CP (results not shown). Therefore, the requirement for CP to initiate replication of RNAs 1 and 2 was studied in P12-proto-

Pl cp:r

P2 +

FIG. 3. CP dependency of the infection of Pl- and P2-plants. Plplants were inoculated with RNAs 2 and 3 (lanes 1, 2) and P2-plants were inoculated with RNAs 1 and 3 (lanes 3. 4). The inocula were made plus CP (lanes 1, 3) or minus CP (lanes 2, 4). Four days after inoculation virus particles were isolated from the plants and RNA extracted from these partrcles was analyzed by Northern blot hybridrzation using random primed AIMV cDNA as probe. The positions of AIMV RNAs I-4 are indicated in the margin.

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FIG. 4. Replication of AIMV RNAs 1 and 2 in P12-protoplasts. P12protoplasts were inoculated with RNA1 (lanes 1, 4) RNA2 (lanes 2, 5) or a mixture of RNAs 1 and 2 (lanes 3, 6) plus CP (lanes l-3) or minus CP (lanes 4-6). RNA extracted from the protoplasts was analyzed by Northern blot hybridization using random primed AIMV cDNA as probe. The position of AIMV RNAs l-4 is indicated in the margin.

plasts. Figure 4 shows the viral RNA synthesis observed in P12-protoplasts inoculated with RNAl, RNA2, or a mixture of these genome segments. When the inoculum contained CP, RNA1 was efficiently replicated by the replicase activity in the P12-protoplasts (Fig. 4, lane 1) but in the absence of CP, RNA1 replication was reduced by more than 1 OO-fold (Fig. 4, lane 4). In contrast, RNA2 replicated poorly if at all, whether or not CP was present in the inoculum (Fig. 4, lanes 2 and 5). The contamination of the RNA2 preparation with RNA3 was less than 0.1% but this contaminant was efficiently replicated by the P12-protoplasts. When these protoplasts were inoculated with a mixture of RNAs 1 and 2 and CP, RNA2 was able to coreplicate with RNAl, confirming that it was biologically active (Fig. 4, lane 3). The replication of RNAs 1 and 2 seen under these conditions is probably independent of the chimeric nuclear viral genes and has also been observed in nontransgenic protoplasts (Nassuth and Bol, 1983). The low background of RNA synthesis seen in the absence of added CP (Fig. 4, lane 6) is believed to be due to CP expressed from the RNA3 contamination. The large differences between the signals seen in lanes 1 and 4 or lanes 3 and 6 of Fig. 4 clearly indicate that the replication of RNA 1 in P12-protoplasts is dependent on the presence of CP in the inoculum. DISCUSSION The AIMV Pl and P2 proteins show extensive sequence similarity to the tobacco mosaic virus (TMV)encoded 126- and 183-kDa proteins, respectively (Goldbach and Wellink, 1988). Recently, it was reported that transformation of tobacco with the TMV 54-kDa gene (i.e., the open reading frame in the readthrough portion of the 183-kDa gene) resulted in a

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complete resistance of the plants to TMV infection (Golemboski et al., 1990). In contrast, expression of AIMV Pl and P2 genes in transgenic plants did not result in a detectable resistance (Van Dun et al., 1988). Here we showed that these Pl and P2 genes are expressed into functional products which permit the replication of incomplete mixtures of genomic RNAs. There is no proof yet that the 54-kDa protein has a role in TMV replication and its interference with virus multiplication could be due to a possible defective nature of this protein. The ability of the P12-plants to replicate AIMV RNA3 may explain the observation that relatively low concentrations of virus particles induced more severe symptoms on these transgenic plants than on nontransformed control plants. On the P12-plants every infection site occupied by RNA3 has the potential to develop into a lesion, whereas on control plants RNAs 1 and 2 have to be present also at such a site. The reduction in the accumulation of RNAs 1 and 2 in P12plants seen in lane 6 of Fig. 1 (4 days after inoculation) was even more pronounced when the plants were analyzed 1 or 2 weeks after inoculation. We have not investigated whether this reduction is accompanied by a gradual loss of infectious virus in these plants. The finding that CP is not required to initiate replication of RNA3 in P12-plants sheds new light on the early function of this structural protein. It has been proposed that binding of CP to the 3’-termini of AIMV RNAs is a prerequisite for recognition of the RNAs by the viral replicase (Houwing and Jaspars, 1978). Our data indicate that recognition of RNA3 by the endogenous replicase activity in P12-plants can occur in the absence of CP. The observation that in the absence of CP, inoculation of nontransgenic plants with a mixture of AIMV RNAs l-3 does not result in infection whereas transgenie plants expressing Pl and P2 are efficiently infected with RNA3, indicates that in the former situation the Pl and P2 genes in the inoculum RNAs are not expressed. Possibly, CP in the inoculum is required for expression of these genes from the inoculum RNAs but not for expression of Pl and P2 from the chimeric nuclear genes mediated by host RNA polymerase II. It has been proposed that, in addition to its early function in the viral replication cycle, AIMV CP is involved in the regulation of the balance between the synthesis of plus- and minus-strand viral RNAs (Nassuth and Bol, 1983). In P12-protoplasts inoculated with RNA3 this regulatory function could be performed by CP that is translated from the subgenomic RNA4. In P12-protoplasts inoculated with RNA1 there will be no synthesis of CP and addition of CP to the inoculum is required to permit RNA1 synthesis (Fig. 4). The synthesis of trace amounts of RNA induced by inocula with-

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out CP may reflect a low level of CP-independent replication or may be due to contaminants in the inocula. Discrimination between these two possibilities must await the construction of infectious clones of RNAs 1 and 2. The inability of RNA2 to replicate in P12-protoplasts in the absence of RNA1 was reproducibly observed in several experiments and is not yet understood. We are currently investigating the replication of mutated AIMV genome segments in P12-plants and P12-protoplasts to obtain further insight into the role of cis- and trans-acting sequences in the viral replication cycle. ACKNOWLEDGMENTS The plants. tlon for dation Sctentiflc

authors thank Aad Wessellng for cultivating the transgenic This work was supported in part by the Netherlands FoundaChemical Research (SON) and the Dutch Technology Foun(STW) with financial aid from the Netherlands Organization for Research (NWO).

REFERENCES BEACHY, R. N. (1990). Coat protein mediated resistance in transgenic plants. In “Viral Genes and Plant Pathogenesis” (T. P. Pirone and J. G. Shaw, Eds.), pp. 13-20. Springer-Verlag, New York. BOL, J. F., VAN VLOTEN-DOTING, L., and JASPARS. E. M. 1. (1971). A functional equivalence of top component a RNA and coat protein in the initiation of infection by alfalfa mosaic virus. I/iro/ogy 46, 73-85. DINGJAN-VERSTEEGH, A., VAN VLOTEN-DOTING, L., and JASPARS. E. M. J. (1972). Alfalfa mosaic virus hybrids constructed by exchanging nucleoprotein components. Virology 49, 716-722. DORE, J.-M., VAN DUN, C. M. P., PINCK, L.. and BOL, J. F. (1991). Alfalfa mosaic virus RNA3 mutants do not replicate in transgenic plants expressing RNA3 specific genes. /. Gen. Viral., in press. FEINEERG, A. P., and VOGELSTEIN, B. (1984). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137, 266-267. GOLDBACH, R. W.. and WELLINK, J. (1988). Evolution of plus-strand RNA viruses. lntervirology 29, 260-267. GOLEMBOSKI, D. B., LOMONOSSOFF, G. P., and ZAITLIN. M. (1990).

ET AL Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus. Proc. Nat/. Acad. Sci USA 87, 6311-6315. HAYES, R. J., and BUCK, K. W. (1990). Complete replication of a eukaryotlc virus RNA in vitro by a purified RNA-dependent RNA polymerase. Cell 63, 363-368. HORIKOSHI, M., MISE. K., FURUSAWA, I., and SHISHIYAMA, J. (1988) Immunological analysis of brome mosaic virus replicase. I Gen. V;rol. 69, 3081-3087. HORSCH, R. B., FRY, J. E., HOFFMANN, N. L., EICHHOLTZ, D., ROGERS, S. G., and FRALEY, R. T. (1985). A simple and general method for transferring genes into plants. Science 227, 1229--l 231. HOUWING, C. J., and JASPARS, E. M. J. (1978). Coat protein binds to the 3’.terminal part of RNA4 of alfalfa mosaic virus. Biochemistry 17, 2927-2933. NASSUTH, A., ALBLAS, F., and BOL, J. F. (1981). Localization of genetic information Involved In the replication of alfalfa mosaic virus. /. Gen. Viral. 53, 207-2 14. NASSUTH, A., and BOL, J. F. (1983). Altered balance of the synthesis of plus- and minus-strand RNAs induced by RNA 1 and 2 of alfalfa mosaic virus in the absence of RNA3. Virology 124, 75585. QUADT, R., VERBEEK, H. J. M., and JASPARS, E. M. 1. (1988). Involvement of a nonstructural protein In the RNA synthesis of brome mosaic virus. Virology 165, 256.-26 1. SARACHU, A. N., HUISMAN, M. J., VAN VLOTEN-DOTING, L., and BOL, J. F. (1985). Alfalfa mosaic virus temperature-sensitive mutants. I. Mutants defective In viral RNA and protein synthesis. Virology 141, 14-22. STUSSI-GARAUD, C., GARAUD, J. C., BERNA, A., and GODEFROY-COLBURN, T. (1987). In sifu locatlon of an alfalfa mosaic virus nonstructural protein in plant cell walls: Correlation with virus trans port. J. Gen. Viral. 68, 1779-1784. TAUR. D., and RENZ, M. (1983). An optlmlzed freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal. Biochem. 132, 14-l 9. TOWBIN, H., STAEHELIN, T., and GORDON, J. (1979). Electrophoretlc transfer of proteins from polyacrylamide gels to nltrocellulose sheets: Procedure and some applications Proc. Nat/. Acad. SC/. USA 76, 4350-4354. VAN DUN, C. M. P., BOL, 1. F., and VAN VLOTEN-DOTING, L. (1987). Expression of alfalfa mosaic virus and tobacco rattle virus coat protein genes in transgenic tobacco plants. V/ro/ogy 159, 299305 VAN DUN, C. M P., VAN VLOTEN-DOTING, L.. and BOL, J. F. (1988). Expression of alfalfa mosaic virus cDNA1 and 2 in transgenlc tobacco plants. \/irology 163, 572 578