Virus-Based Vector (Plant Virus/Gene Expression/Subgenomic Promoter) J

Home , Brome mosaic virus, Tobacco mosaic virus

Proc. Nati. Acad. Sci. USA Vol. 88, pp. 7204-7208, August 1991 Genetics Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector (plant virus/gene expression/subgenomic promoter) J. DONSON*, C. M. KEARNEY, M. E. HILF, AND W. 0. DAWSON Department of Plant Pathology, University of California, Riverside, CA 92521 Communicated by George A. Zentmyer, May 24, 1991

ABSTRACT Tobacco mosaic virus (TMV) produces large genomic RNA, whereas the 30-kDa cell-to-cell movement quantities of RNA and protein on infection of plant cells. This protein and 17.5-kDa coat protein are translated from two and other features, attributable to its autonomous repfication, 3'-coterminal subgenomic RNAs produced during replication make TMV an attractive candidate for expression of foreign (12, 13). sequences in plants. However, previous attempts to construct Previous attempts to express foreign open reading frames expression vectors based on plant RNA viruses, such as TMV, (ORFs) by gene replacement in a plant RNA virus have either have been unsuccesfl in obtaining systemic and stable move- resulted in failure to infect plants (7) or loss of long distance ment offoreign genes to uninoculated leaves in whole plants. A movement (3, 14-16). Vectors have also been constructed hybrid viral RNA (TB2) was constructed, containing sequences with an additional viral subgenomic promoter to express a from two tobamoviruses (TMV-U1 and odontoglossum ring- foreign gene (17). However, on infection of plants these spot virus). Two bacterial sequences inserted independently vectors had the added sequences deleted and failed to be into TB2 moved systemically in Nicotiana benthamna, al- transported systemically. This was hypothesized to be from though they differed in their stability on serial passage. Sys- recombination between the two repeated subgenomic pro- temic expression of the bacterial protein neomycin phospho- moter sequences within the viral constructs. To circumvent transferase was demonstrated. Hybrid RNAs containing both this problem the expression vector described here was con- TMV-U1 and the inserted bacterial gene sequences were en- structed. capsidated by the odontoglossum ringspot virus coat protein, Because the 126- and 183-kDa proteins are required for facilitating their transmission and amplification on pgin TMV RNA replication (18), we decided to express the foreign to subsequent plants. The vector TB2 provides a rapid means sequences by means of a subgenomic RNA 3' ofthese genes. of expressing genes and gene variants in plants. For brome mosaic virus, subgenomic RNA synthesis has been shown to be internally initiated on minus-sense RNA The expression of foreign genes in plants has proven advan- (19), and sequences that promote this synthesis have been tageous to the study of molecular biology (1, 2). Stable gene described (20, 21). Functionally similar sequences occur transfer to whole plants can be achieved by a combination of upstream of the highly expressed (8) TMV coat protein ORF DNA transformation and tissue culture techniques or by (14, 17) and are within the 30-kDa protein-coding region. The virus transfection. With viral-based vectors the ease of 203 bases upstream of the respective coat protein initiation infection avoids the time-consuming procedures, "position" codons of TMV-U1 (10) and odontoglossum ringspot virus effects, and "somaclonal" variation seen when foreign genes (ORSV), a tobamovirus isolated from Cymbidium spp. (22), are integrated into the plant genome (1). The merits of using exhibit a remarkably low sequence similarity [45% compared have with 65% for the 3' 2.4 kilobases (kb) of the respective a systemically expressing plant RNA virus-based vector genomes; data not shown]. The expression vector pTB2 (Fig. been extensively reviewed (3-7). Several features oftobacco 1) was designed to avoid deletion of foreign inserts due to mosaic virus (TMV) (8, 9) suggest that it might be usefully flanking repeated sequences by using these two heterologous adapted for such a purpose: (i) tobamoviruses have a wide promoters from TMV-U1 and ORSV to synthesize subge- host range; (it) they can move cell-to-cell mediated by a nomic RNAs for the foreign gene and ORSV coat protein virus-encoded peptide; (iii) they exhibit rapid systemic gene, respectively. To determine whether foreign sequences spread in plants; (iv) TMV infections are maintained for the inserted into such a TMV-based expression vector could be lifetime ofthe plant; (v) TMV RNA is replicated to high levels replicated stably and whether they could be propagated as autonomous sequences; (vi) this replication results in rapid systemically, two bacterial sequences were tested in the and productive cytoplasmic gene expression; (vii) tempera- vector. The DHFR sequence from plasmid R67 [ref. 23; ture-sensitive mutations of RNA synthesis are available to chosen for its small size of 238 base pairs (bp)] and the larger modulate expression of foreign genes; (viii) TMV also lacks (832-bp) NPTII ORF from transposon TnS (24) were cloned the packaging constraints found with nonhelical viruses, into the Xho I site ofpTB2, downstream ofthe TMV-U1 coat including existing DNA plant virus vectors; and (ix) the TMV protein subgenomic promoter, to produce pTBD4 and genome can now be manipulated as a DNA copy and then pTBN62, respectively. transcribed in vitro to produce infectious RNA molecules. The TMV genome consists of one 6395-nucleotide (nt) molecule of messenger-sense, single-stranded RNA, encod- MATERIALS AND METHODS ing at least four proteins (10; for review see ref. 9; Fig. 1). It Construction of Bacterial Plasmids. Numbers in parenthe- has similarities of sequence and replication strategy with a ses refer to the TMV-U1 sequence (10). DNA manipulations series of plant and animal RNA viruses (11). The 126- and 183-kDa replicase proteins are translated directly from the Abbreviations: TMV, tobacco mosaic virus; ORSV, odontoglossum ringspot virus; NPTII, neomycin phosphotransferase II; DHFR, The publication costs of this article were defrayed in part by page charge dihydrofolate reductase; ORF, open readingframe; nt, nucleotide(s); payment. This article must therefore be hereby marked "advertisement" MS medium, Murashige and Skoog medium. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 7204 Downloaded by guest on September 25, 2021 Genetics: Donson et al. Proc. Natl. Acad. Sci. USA 88 (1991) 7205

-126K , 183K 130K cp pTKU1 II N;I~ K( pTKU 1

XI K pTMVS3-28 XI K, 30K xXI-U 1-cp pTB2

pTBD4

NPTII }LIs=Z7J-_pTBN62

FIG. 1. Construction of expression vector pTB2 and derivatives containing dihydrofolate reductase (DHFR; pTBD4) and neomycin phosphotransferase II (NPTII; pTBN62) genes. Thin lines and open boxes (ORFs) are sequences transcribed in vitro by E. coli RNA polymerase from its promoter (hatched box in top diagram). ORFs for the 126-, 183-, 30-kDa and coat proteins (126K, 183K, 30K, and cp, respectively) are TMV-U1 sequences, except where noted as being from ORSV (o). Thick lines represent bacterial cloning vector sequences. Narrow open box (o) delineates 203 ORSV bases upstream of coat protein initiation codon. Vertical lines in pTBD4 and pTBN62 indicate extent of foreign sequences. Selected restriction sites are shown (B, BamHI; K, Kpn I; N, Nsi I; Nc, Nco I; P, Pst I; S, Spi I; and X, Xho I). were done essentially as described in ref. 25. All plasmids from pTB281 into BamHI/Spl I-digested pTKU1 (replacing were propagated in Escherichia coli strain JM109 (26), except the corresponding wild-type fragment 3332-6245). for pTBN62 [DH5a; GIBCO/BRL (27); and HB101 (28)]. pNC4X (33). This plasmid consisted ofthe R67 DHFR gene pTKUJ (Fig. 1). The 7.3-kb pTMV204 (29) Pst I fragment cloned into pUC8X. The plasmid contained a Xho I site 8 (TMV-U1 genome and A phage promoter from pPM1; ref. 30) bases upstream ofthe initiation codon for the DHFR gene. In was subcloned into pUC19 (26) to give pTP5. pTMV204 Apa addition, the stop codon and 5 bases of carboxyl-terminal I fragment (5455-6389) was ligated to oligonucleotides pd- DHFR sequence were deleted and replaced by a Sal I site. (CAGGTACCC) and d(GGGTACCTGGGCC), digested with pNUJ 16. A 315-bp pNEO Sau3A (Kienow polymerase Kpn I (underlined within oligonucleotide sequence) and Nco in-filled) fragment (amino terminus of TnS NPTII gene) was I (5459), and ligated into Nco I/Kpn I-digested pTP5 to ligated to Sal I [pd(GGTCGACC)] linkers, digested with Sal produce pTPK10. pTKU1 was constructed by subcloning the I/Pst I, and inserted into Pst I/Sal I-digested pUC128 (34) to 7.3-kb Pst I-Kpn I fragment from pTPK10 into Pst I/Kpn give pNU10. pNEO was digested with Asu II, in-filled with I-digested pUC118 (31). pTKU1 contained a DNA copy of Klenow polymerase, and ligated to Xho I linkers [pd(CCTC- the entire TMV-U1 genome downstream of the A phage GAGG)] to generate pNX1. pNU116 was constructed by promoter from pPM1. Kpn I digestion and in vitro transcrip- digesting pNX1 with Xho I, in-filling with Klenow polymer- tion of pTKU1 gave infectious TMV RNA (data not shown). ase, digesting with Pst I, and ligating the resulting 632-bp pTKU1 was constructed because Pst I sites in the ORSV coat fragment (carboxyl terminus of the Tn5 NPTII gene) into Pst protein, DHFR, and NPTII ORFs prohibited the use of this I/Sma I-digested pNU10. This manipulation of the NPTII restriction enzyme (employed to linearize pTMV204; ref. 29) gene removed an additional ATG codon 16 bases upstream of to digest plasmid DNA of the hybrid constructs and produce the initiation codon, the presence of which decreased NPTII activity in transformed plant cells (35). infectious in vitro transcripts. pTBD4 and pTBN62 (Fig. 1). Xho I-Sal I fragments from pTB2 (Fig. 1). pTMVS3-28 (32) was a derivative of pNC4X (DHFR sequence) and pNU116 (NPTII sequence), pTMV204 in which the coat protein initiation codon was respectively, were ligated into the Xho I site of pTB2 in the mutated to ACG and a Xho I site replaced the entire coat same sense as the TMV coding sequences. protein-coding sequence. The 1.9-kb Nco I-Sal I fragment (nt In Vitro Transcription and Inoculation of Plants. Plants 5459-Sal I site in pBR322) from pTMVS3-28 was ligated into grown as in ref. 32 were inoculated with in vitro transcripts Nco I/Sal I-digested pNEO (24) to give pNS283. pBabsl was TB2 (6602 nt), TBD4 (6840 nt), and TBN62 (7434 nt) from a 2.4-kb EcoRI cDNA clone from ORSV virion RNA with Kpn I-digested pTB2, pTBD4, and pTBN62, respectively. nucleotide, ORF, and amino acid sequence similarities to The in vitro transcription method was as described (29). TMV-U1 nt 4254-6370 (data not shown). A 680-bp pBabsl Analysis of Progeny Virion RNA. Virus purification was HincII-Ear I (Klenow polymerase in-filled) fragment (con- essentially as described by Gooding and Hebert (36) with one taining the ORSV coat protein ORF and 203 bases upstream precipitation with polyethylene glycol (8% polyethylene gly- of its AUG) was ligated into the Nsi I site (6202; blunt-ended col/0.1 M NaCl; 0°C for 1 hr) and one ultracentrifugation with T4 DNA polymerase) of pNS283 to produce pB31. The (151,000-235,000 x g; 90 min). Virion RNA was extracted by Nco I-Sal I fragment from pB31 was then ligated into Nco dige- ting 1 mg of virus with 0.2 ,ug of proteinase K in 10 mM I/Sal -digested pTMV204 (replacing the corresponding wild- Tris-HCl, pH 7.5/1 mM EDTA/0.1% SDS at 37°C for 1 hr, type fragment nt 5459-Sal I site in pBR322) to give pTB281. followed by phenol/chloroform extractions. RNA samples pTB2 was constructed by ligating the BamHI-Spl I fragment were dimethyl sulfoxide-denatured, glyoxylated, electropho- Downloaded by guest on September 25, 2021 7206 Genetics: Donson et al. Proc. Natl. Acad. Sci. USA 88 (1991)

resed in 1% agarose gels, and transferred to nitrocellulose A (pore size, 0.45 um; Schleicher & Schull; ref. 25). The 1 2 3 4 5 6 7 8 9 10 transfers were probed with [a-35S]dATP (New England Nu- clear)-labeled (37) restriction fragments. RNase protection assays were as described in ref. 25. TBD4-3B and pTBN62-3B contained BamHI-Kpn I fragments (nt 3332-

63%) from pTBD4 and pTBN62, respectively, cloned into NW -1_N- -4 BamHI/Kpn I-digested pBluescript SK- (Stratagene). I. I Immunological Detection ofNPTII. Sample preparation and -4 immunoblot analysis were as described (32). Leaf samples were ground in liquid N2 and extraction buffer [1o (vol/vol) glycerol/62.5 mM Tris HCl, pH 7/5% 2-mercaptoethanol/5 mM phenylmethylsulfonyl fluoride]. Equivalent protein concentrations were determined, and absolute concentrations were estimated by Bradford assay (Stratagene; ref. 38), with bovine serum albumin as standard. Immunoblot transfers were probed with antiserum to NPTII (1:500; 5 Prime -+ 3 Prime, Inc.) and then with alkaline phosphatase-conjugated goat anti-rabbit IgG (1:1000). B NPIIM Activity Assays. NPTII activity was detected by its a b c d e f 9 h phosphorylation ofneomycin sulfate. Enzyme assays were as described in ref. 39, except the extraction buffer was as described above and dilution series of purified NPTII (5 Prime -+ 3 Prime, Inc.) in healthy tissue were included. [U.- Leaf Disc Assays to Screen for Resistance to Kanamycin Sulfate. NPTII confers resistance to the aminoglycoside kanamycin (24). Young systemic leaves 12 days after inoculation were surface-sterilized and washed in =0.01% Tween 20 (5 min)/0.25% sodium hypochlorite (2 min)/70%o ethanol (30 sec)/distilled water (4 x 10 sec). Leafdiscs were cut from FIG. 2. Analysis of progeny viral RNA. (A) Northern transfer of a leafin pairs; one was placed on Murashige and Skoog (MS) 1% agarose gel containing dimethyl sulfoxide-denatured virion RNA medium (40) alone and the other on kanamycin sulfate- (0.25 ,ug per lane) from TB2 (lanes 2, 4, 7, and 9), TBD4 (lanes 3 and supplemented MS medium. Plates were incubated at 32°C 5), and TBN62 (lanes 8 and 10) systemically infected N. benthamiana with a photoperiod of 16 hr. Leaf discs were transferred to tissue 11 days after inoculation with in vitro transcripts. Lanes: 1 and freshly prepared medium every 7 days. 6, dimethyl sulfoxide-denatured pTMV204 restriction fragments [0.58, 0.98, 2.2, 3.1, 4.2, 5.1, 6.5, 7.4, 8.6, 9.4 kb indicated (-) in order of decreasing mobility]. Transfers were hybridized with 35S- RESULTS AND DISCUSSION labeled 7.3-kb pTMV204 Pst I fragment (TMV-U1 genome plus pPM1 promoter; lanes 1-3 and 6-8); 238-bp pNC4X Xho I-Sal I Mechanical inoculation of N. benthamiana plants with in fragment (DHFR sequence; lanes 4 and 5); 832-bp pNU116 Sal vitro transcripts derived from the DNA constructs pTB2, I-XhoI fragment (NPTII ORF; lanes 9 and 10). (B) RNase protection pTBD4, and pTBN62, respectively, resulted in symptomatic assays of progeny virus RNA samples from part A electrophoresed infection with virus of typical TMV shape (data not shown) in 1.2% agarose gels. For lanes a-c the probe was a 32P-labeled and yield (1.5-5.8 mg of virus per g of tissue). Symptoms transcript (3.5 kb) complementary to the 3' half of TBD4 viral were less severe compared with TMV-U1-infected plants and genome, synthesized from BamHI-digested pTBD4-3B. Lanes: a, and probe without RNase treatment (D) and marker transcripts of 3.1, consisted of plant stunting with mild chlorosis distortion 2.1, and 0.9 kb; b, probe protected by virion RNA from TBD4- of systemic leaves (data not shown). The sizes ofvirion RNA infected plant; c, probe alone with RNase treatment; d, marker from systemically infected tissue of plants inoculated with transcripts of 3.1, 2.1, and 0.9 kb; e-h, probe was a 32P-labeled TB2, TBD4, and TBN62, respectively, were consistent with transcript (4.1 kb), complementary to the 3' half of TBN62 viral the predicted lengths of RNA transcribed in vitro from the genome, synthesized from BamHI-digested pTBN62-3B. Probe pro- respective plasmids (Fig. 2A). These RNA species contained tected by virion RNA from TBN62- (lane e) and TMV-204- (ref. 29; TMV sequences (Fig. 2, lanes 2, 3, 7, and 8) plus their lane f) infected plants is shown. Probe alone with (lane g) and without respective bacterial gene inserts (Fig. 2, lanes 5 and 10). (lane h) RNase treatment is shown. Probes complementary to the manipulated portion of the respective genomes were protected in RNase protection The sequence stability of TBD4 and TBN62 virion RNA assays by progeny TBD4 and TBN62 viral RNAs (Fig. 2B). was examined in serial passages through N. benthamiana. This result indicated that the precise and rapid deletion of Plants were inoculated with two and four independent in vitro inserted sequences that was a problem with previous con- transcription reactions from pTBD4 and pTBN62, respec- structs (17) did not occur with TBD4 or TBN62. It was tively, and systemically infected leaf tissue was serially hypothesized that with the previously reported constructs passaged every 11-12 days. After 48 days of systemic infec- foreign inserts were deleted due to recombination between tion, full-length virion RNA of TBD4, including the DHFR repeated subgenomic promoter sequences (17). With TBD4 sequences, was still detected by Northern (RNA) transfer and TBN62, such repeated sequences were reduced by using hybridization and still protected probes complementary to heterologous subgenomic mRNA promoters. Additional the manipulated portion of the genome in RNase protection bands smaller than the probe in Fig. 2B, lane e, and smaller assays (data not shown). Five clonal populations of virion than the full-length viral RNA in Fig. 2A, lanes 8 and 10, RNA were derived from TBD4-infected plants propagated for might represent alterations within a portion of the TBN62 170 days (one series involving 10 passages) by isolation of population, although in this case the relative proportion of local lesions on Nicotiana tabacum Xanthi-nc. The consen- full-length and additional smaller bands was unchanged after sus DHFR sequence for three of the populations corre- a subsequent passage (data not shown). sponded with the published DHFR sequence (23, 33), except Downloaded by guest on September 25, 2021 Genetics: Donson et al. Proc. Natl. Acad. Sci. USA 88 (1991) 7207

for a translationally silent third base change (U -> C) at nt 72 TIB2 TBN62 of the coding sequence. Sequence data were not obtained ,...-.:.. from the other two RNA populations. The reason for this, whether technical or a consequence of the sequence compo- i of sition ofthese two populations, has not been determined. The MS nucleotide change at position 72 of the DHFR-coding se- to % quence was not evident in progeny RNA from TBD4-infected wt- plants propagated for 48 days. Virion RNA from plants *A-~t~o serially infected with TBN62 was less stable with different \-c1 portions of the NPTII sequence being deleted in each of the /~~~~~~~~~~~~~~~~~~~~~~~t independent series of passages. The time of loss of these MS sequences varied between after the first passage (12-24 days) KAN and the third passage (36-47 days; data not shown). The reason for deletions occurring in the NPTII sequence of / ,- , sl. .. TBN62 is not known. However, on the basis of the stability I ofthe DHFR sequences in TBD4, such instability ofinserted foreign sequences would not seem an intrinsic feature of FIG. 4. N. benthamiana leaf disc assay to screen for resistance expression vector TB2. In contrast, such deletions might be to kanamycin sulfate conferred by NPTII. Petri dishes contained dictated by the nucleotide composition ofthe inserted foreign systemically infected leaf tissue from plants inoculated with in vitro transcripts from pTB2 (TB2) and pTBN62 (TBN62), respectively. sequences themselves. Similar instabilities among DNA Petri dishes (H) contained comparable tissue from uninoculated plant virus vectors have been reviewed (6). plants. The top row of dishes (MS) contained MS medium alone, A commercial source ofantiserum and sensitive enzymatic whereas the bottom row (MS + KAN) contained kanamycin sulfate assays for the extensively used selectable marker NPTII (39) at 100 ,ug/ml. The photograph was taken 25 days after start ofthe leaf allowed further analysis of tissue infected with TBN62. disc assay. Immunoblot analysis (Fig. 3A), enzyme activity (Fig. 3B), and leaf disc assays (Fig. 4) demonstrated the presence of proteins in plant cells. Alternatively, this result might be from functional NPTII enzyme and its phenotypic expression in one or more aspects of the vector or foreign gene sequences plant tissue systemically infected with TBN62 but not in affecting synthesis of the subgenomic mRNA or posttran- TB2-infected or healthy plants. NPTII protein and enzyme scriptional expression ofthe reporter gene (9). The relatively activity were even detected in some TBN62-infected plants high yield of virus from plants infected with the vector propagated for 36 days. constructs would seem to preclude a dramatic reduction in It was evident from Fig. 3 that levels of extractable NPTII the efficiency of virus replication. However, one possibility protein were considerably lower than levels of coat protein, for the low expression might be the position of the reporter the most highly expressed TMV protein (8). This result could gene relative to the 3' terminus of the genome. The amount reflect the relative stabilities or partitioning of the respective of 30-kDa protein produced by different mutants ofTMV has been shown to be inversely proportional to the distance the A 1 2 3 4 5 6 7 ORF for the 30-kDa protein was from the 3' terminus of the genome (J. N. Culver, K. Lehto, and W.O.D., unpublished data). This result was consistent with the observations of French and Ahlquist (21) that the level of subgenomic RNA -43 0 from brome mosaic virus RNA 3 was progressively greater the closer promoter insertion was to the 3' terminus. -29 0 Plant virus RNA polymerases have been proposed to have a low fidelity from the absence of proofreading and, as such, would result in foreign sequences in the viral genome only -184 being maintained under constant selection pressure (6, 41). -14 3 However, the systemic spread of the foreign sequences in TBD4 and TBN62 RNA without selection suggests that sequence fidelity was possible despite replication of the B a b c d e f g h foreign gene by RNA-dependent RNA polymerase. * 0 Animal RNA virus-based vectors (42, 43) that require helper viruses to make infectious particles have been produced, but only tested in tissue culture. The data presented FIG. 3. Detection of NPTII in TBN62-infected N. benthamiana. above demonstrated that a RNA virus-based vector can (A) NPTII antiserum-probed immunoblot transfer of 15% denaturing systemically express a foreign gene in intact plants. We polyacrylamide gel containing 100 gg of plant protein per lane. foresee a wide use for such a vector in the systemic expres- Markers in kDa are at right. (B) NPTII dot blot assay [each dot sion of foreign sequences and as a means to investigate the contained extract from -4 mg oftissue (fresh weight)]. Samples were molecular biology of TMV. extracted from inoculated (lanes 2 and 3; dots c and e) and systemically infected (lanes 6 and 7; dots d and f) leaves ofN. benthamiana plants 14 days after inoculation with in vitro transcripts from pTB2 We thank Thomas Hohn for pNC4X, Dennis Lewandowski and (lanes 2 and 6; dots c and d) and pTBN62 (lanes 3 and 7; dots e and Gael Kurath for discussions, and John Kitasako for photography. f). Lanes 1 and 5 and dots a and b contain comparable samples from This work was supported by the National Science Foundation healthy plants. Lane 4 contains 30 ng of purified NPTII protein (

Gluzman, Y. & Hughes, S. H. (Cold Spring Harbor Lab., Cold eds. Harrison, B. D. & Murant, A. F. (CAB/Assoc. Appl. Spring Harbor, NY), pp. 183-189. Biol., Kew, U.K.), no. 155. 4. Siegel, A. (1985) Plant Mol. Biol. 4, 327-329. 23. Brisson, N. & Hohn, T. (1984) Gene 28, 271-275. 5. Ahlquist, P. & Pacha, R. F. (1990) Physiol. Plant. 79, 163-167. 24. Beck, E., Ludwig, G., Auerswald, E. A., Reis, B. & Schaller, 6. Hull, R. (1990) in Recognition and Response in Plant-Virus H. (1982) Gene 19, 327-336. Interactions, NATO Adv. Study Inst. Ser., ed. Fraser, R. S. S. 25. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., (Springer, Berlin), Vol. H41, pp. 443-457. Seidman, J. G., Smith, J. A. & Struhl, K., eds. (1988) Current 7. Joshi, R. L., Joshi, V. & Ow, D. W. (1990) EMBO J. 9, Protocols in Molecular Biology (Wiley, New York). 2663-2669. 26. Yanisch-Perron, C., Vieira, J. & Messing, J. (1985) Gene 33, 8. Matthews, R. E. F. (1981) Plant Virology (Academic, New 103-119. York). 27. Hanahan, D. (1983) J. Mol. Biol. 166, 557-580. 9. Dawson, W. 0. & Lehto, K. (1990) Adv. Virus Res. 38, 28. Boyer, H. W. & Roulland-Dussoix, D. (1969) J. Mol. Biol. 41, 307-342. 459-470. 10. Goelet, P., Lomonossoff, G. P., Butler, P. J. G., Akam, 29. Dawson, W. O., Beck, D. L., Knorr, D. A. & Grantham, G. L. M. E., Gait, M. J. & Karn, J. (1982) Proc. Natl. Acad. Sci. (1986) Proc. Natd. Acad. Sci. USA 83, 1832-1836. 30. Ahlquist, P. & Janda, M. (1984) Mol. Cell. Biol. 4, 2876-2882. USA 79, 5818-5822. 31. Vieira, J. & Messing, J. (1987) Methods Enzymol. 153, 3-11. 11. Haseloff, J., Goelet, P., Zimmern, D., Ahlquist, P., Dasgupta, 32. Dawson, W. O., Bubrick, P. & Grantham, G. L. (1988) Phy- R. & Kaesberg, P. (1984) Proc. Natl. Acad. Sci. USA 81, topathology 78, 783-789. 4358-4362. 33. Brisson, N., Paszkowski, J., Penswick, J. R., Gronenborn, B., 12. Hunter, T. R., Hunt, T., Knowland, J. & Zimmern, D. (1976) Potrykus, I. & Hohn, T. (1984) Nature (London) 310, 511-514. Nature (London) 260, 759-764. 34. Keen, N. T., Tamaki, S., Kobayashi, D. & Trollinger, D. (1988) 13. Beachy, R. N. & Zaitlin, M. (1977) Virology 81, 160-169. Gene 70, 191-197. 14. Takamatsu, N., Ishikawa, M., Meshi, T. & Okada, Y. (1987) 35. Rogers, S. G., Fraley, R. T., Horsch, R. B., Levine, A. D., EMBO J. 6, 307-311. Flick, J. S., Brand, L. A., Fink, C. L., Mozer, T., O'Connell, 15. French, R., Janda, M. & Ahlquist, P. (1986) Science 231, K. & Sanders, P. R. (1985) Plant Mol. Biol. Rep. 3, 111-116. 1294-1297. 36. Gooding, G. V., Jr., & Hebert, T. T. (1967) Phytopathology 57, 16. Takamatsu, N., Watanabe, Y., Yanagi, H., Meshi, T., Shiba, 1285. T. & Okada, Y. (1990) FEBS Lett. 269, 73-76. 37. Feinberg, A. P. & Vogelstein, B. (1984) Anal. Biochem. 137, 17. Dawson, W. O., Lewandowski, D. J., Hilf, M. E., Bubrick, P., 266-267. Raffo, A. J., Shaw, J. J., Grantham, G. L. & Desjardins, P. R. 38. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. (1989) Virology 172, 285-292. 39. McDonnell, R. E., Clark, R. D., Smith, W. A. & Hinchee, 18. Ishikawa, M., Meshi, T., Motoyoshi, F., Takamatsu, N. & M. A. (1987) Plant Mol. Biol. Rep. 5, 380-386. Okada, Y. (1986) Nucleic Acids Res. 14, 8291-8305. 40. Murashige, T. & Skoog, F. (1962) Physiol. Plant. 1S, 473-497. 19. Miller, W., Dreher, T. & Hall, T. (1985) Nature (London) 313, 41. Van Vloten-Doting, L., Bol, J. & Cornelissen, B. (1985) Plant 68-70. Mol. Biol. 4, 323-326. 20. Marsh, L. E., Dreher, T. W. & Hall, T. C. (1988) Nucleic 42. Xiong, C., Levis, R., Shen, P., Schlesinger, S., Rice, C. M. & Acids Res. 16, 981-995. Huang, H. V. (1989) Science 243, 1188-1191. 21. French, R. & Ahlquist, P. (1988) J. Virol. 62, 2411-2420. 43. Luytjes, W., Krystal, M., Enami, M., Pavin, J. D. & Palese, P. 22. Paul, H. L. (1975) CMI/AAB Descriptions of Plant Viruses, (1989) Cell 59, 1107-1113. Downloaded by guest on September 25, 2021