Thomas H. Turpen

Tobamovirus Transient Expression Vectors: Tools for Plant Biology and High-Level Expression of Foreign Proteins in Plants

The majority of viruses infecting higher plants have single-stranded, positive (or message-) sense RNA genomes. Infectious transcripts can be synthesized in vitro from full-length cDNA clones to study RNA virus biology, develop methods of disease control, and construct plant expression vectors (10,22). Tobamoviruses have a long history of experimentation and represent efficient genetic systems (2,3,4). Vectors based on the tobacco mosaic virus (TMV) genome were among the first to be developed (8) and have particular advantages for novel application(s) in the expression of foreign sequences in plants. These advantages include: 1. Speed: TMV vectors carrying foreign genes move rapidly and systemically in plants (generally 7–10 days post inoculation). 2. High expression levels: TMV produces more virus-encoded protein per infected cell than any other known plant virus [up to 10% of dry weight in TMV-infected tobacco plants; (1)]. Foreign proteins (18,21) and peptides (26) produced systemically in plants by TMV vectors can accumulate to 1–5% of total soluble protein. The levels of foreign proteins expressed from viral vectors are generally much higher than that obtainable from stably transformed cell lines, transgenic organisms, or plasmid DNA-based transient expression systems. 3. Protein targeting: It is possible to direct the foreign protein to various subcellular locations including the endomembrane system, the cytosol, organelles, or even virion particles. Careful study and control of subcellular targeting of foreign proteins or peptides can maximize their specific activity, stability, and greatly facilitate their purification (21,27). 4. Wide range of potential applications: TMV expression vectors have been used as research tools to study plant biosynthetic pathways, screen gene libraries and to express proteins toxic to plant and non-plant systems. Examples include: 1) The carotenoid biosynthetic pathway in plants has been altered by up or down-regulating enzymes responsible for the synthesis of key isoprenoid intermediates (19). The levels of intermediate products were altered up to 50 fold by expressing sequences in the plant cytoplasm in either sense or anti-sense orientation. These dramatic alterations in secondary metabolite accumulation can only be achieved by regulated induction in vegetative tissues, and are predicted to be lethal if constitutively expressed using transgenic technologies. 2) A plant gene library has been constructed in a TMV vector in order to identify expressed sequences that induce cell death in plants (C. Holt, pers. commun.). 3) Peptides or proteins such as antimicrobial peptides, animal hormones, and growth regulators that would be predicted to be toxic or accumulate poorly in microbial or transgenic expression systems have been expressed in plants from TMV vectors (27). 5. Low frequency of sequence drift: Contrary to initial predictions, foreign sequences propagated in TMV-based vectors accumulate very few point mutations during multiple passages in whole plants (15). 6. Broad host range: cDNA clones are available for many tobamoviruses. Functional hybrid vectors are compatible with an array of plant species. 7. Plants as ideal hosts: The greatest advantages in using plant viruses as expression vectors are the characteristics of their hosts. Transfected leaves are one of the most economical sources of biomass for commercial product development and can be inexpensively scaled to meet production requirements. 8. Proven utility in laboratory and field-scale production: Recombinant TMV vectors have been tested in five outdoor field trials to date. Expression characteristics, host range, persistence in the environment, and large-scale plant extraction procedures have all been evaluated (12). Multi-ton extraction of tobacco tissue grown in the field has resulted in the purification of kilogram (kg) quantities of recombinant viruses for development of vaccines and anti-microbial peptides (27). 9. No biological vector: Because TMV is mechanically spread in nature (28), recombinant vectors are contained to inoculated fields (12).

Viral Vector Expression of Foreign Proteins in Plants

It is now fairly routine to engage plant viruses to express foreign proteins in plants. Plant viruses have several features that make them quite useful for vectoring foreign genes into whole plants. The majority of viruses infecting higher plants have single-stranded, positive-sense RNA genomes. Infectious transcripts can be synthesized in vitro from full-length cDNA clones to study RNA virus biology, develop methods of disease control and construct plant expression vectors (Goldbach and Hohn, 1997; Scholthof et al., 1996). Tobamoviruses are among the most studied and well-understood viruses and represent superbly efficient genetic systems (Dawson et al., 1986; Dawson et al.,1990; Dawson, 1992). Vectors based on the tobacco mosaic virus (TMV) were among the first to be developed (Donson et al., 1991) and have several advantages for novel applications in the expression of foreign sequences in plants. These advantages include speed, high expression levels, broad host-range, and controlled containment, as transmission occurs only by mechanical means.