BSMV genome mediated expression of a foreign gene in dicot and monocot plant cells.

212

203

Infectious RNA transcripts were generated from full-length cDNA clones of the tobacco etch potyvirus genome containing an insertion of the bacterial I3-glucuronidase (GUS) gene between the polyprotein-coding sequences for the N-terminal 35-kDa proteinase and the helper componentproteinase. The recombinant virus was able to spread systemically in plants and accumulated to a level comparable with wild-type tobacco etch potyvirus. Proteolytic processing mediated by the 35-kDa proteinase and helper componentproteinase resulted in production of an enzymatically active GUS-helper component-proteinase fusion protein. A virus passage line that retained the GUS insert after numerous plant-to-plant transfers, as well as a line that sui a deletion of the GUS sequence, was recovered. Use of an in situ histochemical GUS assay in time-course experiments allowed the visualization of virus activity in single, mechanically inoculated leaf epidermal cells, in neighboring epidermal and mesophyll cells, in phloem-associated cells after long-ditance transport, and in cells surrounding vascular tissues of organs above and below the site of inoculation. This system represents a powerful tool to study plant virus replication, short-and long-distance virus movement, and virus-host interactions.Additionally, we show that potyviruses may serve as highly efficient, autonomously replicating vectors for the expression of foreign genes in plants.Tobacco etch virus (TEV), a well-characterized potyvirus, contains a positive-strand RNA genome of 9.5 kilobases (kb) coding for a single, large polyprotein that is processed by three virus-specific proteinases ( Fig. 1 and refs. 1 and 2). The nuclear inclusion protein "a" proteinase is involved in the maturation of several replication-associated proteins and capsid protein (3). The helper component-proteinase (HCPro) and 35-kDa proteinase both catalyze cleavage only at their respective C termini (4, 5). The proteolytic domain in each of these proteins is located near the C terminus. For HC-Pro, the N-terminal domain is required for virus transmission by aphids (6). The 35-kDa proteinase and HC-Pro derive from the N-terminal region of the TEV polyprotein (Fig. 1). Unlike the events associated with proteolytic processing, relatively little is known regarding the requirements for potyviral RNA replication and virus transport.The production of full-length, infectious RNA transcripts from cloned cDNAs representing plant positive-strand RNA virus genomes has permitted the analysis of many viral functions, including RNA replication and cell-to-cell movement (e.g., refs. 7-10). In addition, infectious transcripts from cDNA have allowed the construction of RNA-based vectors for expression of foreign genes in plants (11)(12)(13). Although a high level of replication is an attractive feature of a putative RNA virus vector, the use of RNA vectors has been limited due to instability of inserted foreign sequences and disruption of systemic virus spread after replacement of virus genes involved in movement....

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Tagging of plant potyvirus replication and movement by insertion of beta-glucuronidase into the viral polyprotein.

Dolja

McBride

Carrington

1992

212

203

“…Reporter genes have been inserted into full-length clones to visualize virus spread and accumulation following infection (9,10). In addition, viruses have been used as vectors for in vivo expression of foreign proteins (11,12).…”

mentioning

confidence: 99%

Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo.

Johansen

1996

Insertion of introns into cloned cDNA of two isolates of the plant potyvirus pea seedborne mosaic virus facilitated plasmid amplification in Escherichia coli. Multiple stop codons in the inserted introns interrupted the open reading frame of the virus cDNA, thereby terminating undesired translation of virus proteins in E. coli. Plasmids containing the full-length virus sequences, placed under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase termination signal, were stable and easy to amplify in E. coli if one or more introns were inserted into the virus sequence. These plasmids were infectious when inoculated mechanically onto Pisum sativum leaves. Examination of the cDNA-derived viruses confirmed that intron splicing of in vivo transcribed pre-mRNA had occurred as predicted, reestablishing the virus genome sequences. Symptom development and virus accumulation of the cDNA derived viruses and parental viruses were identical. It is proposed that intron insertion can be used to facilitate manipulation and amplification of cloned DNA fragments that are unstable in, or toxic to, E. coli. When transcribed in vivo in eukaryotic cells, the introns will be eliminated from the sequence and will not interfere with further analysis of protein expression or virus infection.

“…With viral-based vectors the ease of infection avoids the time-consuming procedures, "position" effects, and "somaclonal" variation seen when foreign genes are integrated into the plant genome (1). The merits of using a systemically expressing plant RNA virus-based vector have been extensively reviewed (3)(4)(5)(6)(7). Several features of tobacco mosaic virus (TMV) (8,9) suggest that it might be usefully adapted for such a purpose: (i) tobamoviruses have a wide host range; (it) they can move cell-to-cell mediated by a virus-encoded peptide; (iii) they exhibit rapid systemic spread in plants; (iv) TMV infections are maintained for the lifetime ofthe plant; (v) TMV RNA is replicated to high levels as autonomous sequences; (vi) this replication results in rapid and productive cytoplasmic gene expression; (vii) temperature-sensitive mutations of RNA synthesis are available to modulate expression of foreign genes; (viii) TMV also lacks the packaging constraints found with nonhelical viruses, including existing DNA plant virus vectors; and (ix) the TMV genome can now be manipulated as a DNA copy and then transcribed in vitro to produce infectious RNA molecules.…”

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confidence: 99%

Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector.

Donson

Kearney

Hilf

et al. 1991

189

110

Tobacco mosaic virus (TMV) produces large quantities of RNA and protein on infection of plant cells. This and other features, attributable to its autonomous repfication, make TMV an attractive candidate for expression of foreign sequences in plants. However, previous attempts to construct expression vectors based on plant RNA viruses, such as TMV, have been unsuccesfl in obtaining systemic and stable movement of foreign genes to uninoculated leaves in whole plants. A hybrid viral RNA (TB2) was constructed, containing sequences from two tobamoviruses (TMV-U1 and odontoglossum ringspot virus). Two bacterial sequences inserted independently into TB2 moved systemically in Nicotiana benthamna, although they differed in their stability on serial passage. Systemic expression of the bacterial protein neomycin phosphotransferase was demonstrated. Hybrid RNAs containing both TMV-U1 and the inserted bacterial gene sequences were encapsidated by the odontoglossum ringspot virus coat protein, facilitating their transmission and amplification on pgin to subsequent plants. The vector TB2 provides a rapid means of expressing genes and gene variants in plants.The expression of foreign genes in plants has proven advantageous to the study of molecular biology (1,2). Stable gene transfer to whole plants can be achieved by a combination of DNA transformation and tissue culture techniques or by virus transfection. With viral-based vectors the ease of infection avoids the time-consuming procedures, "position" effects, and "somaclonal" variation seen when foreign genes are integrated into the plant genome (1). The merits of using a systemically expressing plant RNA virus-based vector have been extensively reviewed (3-7). Several features of tobacco mosaic virus (TMV) (8,9) suggest that it might be usefully adapted for such a purpose: (i) tobamoviruses have a wide host range; (it) they can move cell-to-cell mediated by a virus-encoded peptide; (iii) they exhibit rapid systemic spread in plants; (iv) TMV infections are maintained for the lifetime ofthe plant; (v) TMV RNA is replicated to high levels as autonomous sequences; (vi) this replication results in rapid and productive cytoplasmic gene expression; (vii) temperature-sensitive mutations of RNA synthesis are available to modulate expression of foreign genes; (viii) TMV also lacks the packaging constraints found with nonhelical viruses, including existing DNA plant virus vectors; and (ix) the TMV genome can now be manipulated as a DNA copy and then transcribed in vitro to produce infectious RNA molecules.The TMV genome consists of one 6395-nucleotide (nt) molecule of messenger-sense, single-stranded RNA, encoding at least four proteins (10; for review see ref. 9; Fig. 1). It has similarities of sequence and replication strategy with a series of plant and animal RNA viruses (11). The 126-and 183-kDa replicase proteins are translated directly from the genomic RNA, whereas the 30-kDa cell-to-cell movement protein and 17.5-kDa coat protein are translated from two 3'-coterminal su...