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.