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

Abstract

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...

Tobacco mosaic virus and the virescence of biotechnology

“…The principle that chimeric virus particles had potential as protective immunogens was thus established with TMV, as well as with the more commonly known hepatitis B surface antigen particles (Valenzuela et al 1985). After it became possible to synthesize infectious RNA from cloned cDNA copies of plant viral genomes, chimeric virus particles derived from TMV and other viruses were shown to present foreign peptides on their surface without signi¢cantly inhibiting the ability of the viruses to replicate and to infect whole plants systemically (Porta et al 1994;Turpen et al 1995;Fitchen et al 1995).…”

“…Using these designs, it is now possible to synthesize multiple proteins from a single mRNA. Together with the improved ability to design and use RNA subgenomic promoters (Donson et al 1991) for the expression of added sequences, a family of powerful gene vectors has evolved. These concepts have spread rapidly to in£uence the development of gene vectors from other plant and animal RNA viruses (Bredenbeck & Rice 1992;Scholthof et al 1996).…”

Transient Virus Expression Systems for Recombinant Protein Expression in Dicot- and Monocotyledonous Plants

“…These vectors benefit from the strength of the viral subgenomic promoter's activity to reprogram the translational activities of infected plant cells such that virus-encoded proteins are synthesized at high levels, often similar to the TMV CP [40]. A foreign gene encoding the protein for overexpression is added in place of the virus CP so it will be expressed from the endogenous virus CP promoter [38,40]. A second CP promoter from a different tobamoviruses strain, of sequence divergent to the first CP promotor, is placed downstream of the heterologous coding region and a virus CP gene is then added 3' terminal to the heterologous subgenomic promoter.…”

“…With greater understanding of virus function, plant RNA virus vectors were constructed to express a foreign gene product in addition to all required viral proteins [36,38]. These vectors were the first independent-virus system that expressed recombinant products while moving systemically in a host plant.…”

“…These vectors were the first independent-virus system that expressed recombinant products while moving systemically in a host plant. To construct independent-virus systems for (+) strand RNA viruses, vectors exploit subgenomic mRNA production to express foreign genes by using an additional subgenomic promoter inserted into the virus [38][39][40]. For viruses that used polyprotein processing, the foreign gene was inserted in translational frame with the existing virus open reading frame (ORF) and peptide sequences that facilitate the proteolytic processing of the fusion protein were present to insure release of the recombinant protein.…”