Aug 4, 2005

Research Projects

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Development of a new biosafety system for the production of proteins in plants with modified viruses

(2001 – 2004) Federal Biological Research Centre for Agriculture and Forestry (BBA); Institute for Plant Virology, Microbiology and Biosafety, Braunschweig

Topic

Plants can be made to produce specific proteins with the help of modified viruses. Such procedures can be used to obtain a high protein yield of one per cent and over. This will be of particular economic interest if high-grade proteins such as pharmaceutical components are synthesised in plants in the future.

However, the use of modified viruses raises new biosafety issues. There are concerns that the modified viruses could spread and infect other plants.

The aim of this research project was to develop and test combinations of modified viruses and customised transgenic plants and so prevent the undesirable spread of the viruses.

The procedure should facilitate the synthesis of proteins in plants whilst alleviating concerns over biosafety.

Summary

Regulated expression of the viral full-length clone in transgenic plants was obtained with the first system (TOP10/TetVP16, see below). A risk remains because the virus particles produced in these plants are capable of multiplying in wild type plants. The regulation of the gene expression does not work perfectly.

The second system works as planned. The transport-defective virus can systemically infect only those transgenic plants which express the transport protein. So the undesirable spread of the virus is reliably prevented and safe synthesis of foreign proteins using viral full-length clones is made possible.

Experiment description

A full-length clone of potato virus X (PVX) was used to develop the safety system. The full-length clone carries a reporter gene (GUS gene) as an example of the type of foreign protein to be produced. GUS is an enzyme that converts a colourless dye to a blue dye. So a simple colour test is all that is needed to check whether the GUS protein is being produced in the plant.

Approach 1: A full-length clone is modified so that it cannot produce an infection in a normal plant. A promoter (TOP10) is added before the viral DNA sequence. This promoter is only “switched on” if a specific protein (TetVP16) is present. Since this protein is not normally present in plants, the virus cannot develop.

However, the “switch protein” which is required to activate the modified full-length clone is present in transgenic tobacco plants. They are produced solely for this purpose.

Only when a full-length clone under the control of the TOP10 promoter is inserted into this transgenic tobacco plant by means of agroinfection does the TetVP16 protein produced in the plant activate the TOP10 promoter. As a result, the viral sequence is expressed and viral protein, including the desired foreign protein, is produced in the plant.

This approach also works when the full-length clone under the control of the TOP10 promoter is inserted into non-transgenic tobacco plants in parallel with the gene for the TetVP16 protein and results in a transient expression (temporary, unstable) of the TetVP16 protein.

The disadvantage of this approach is that the virus can spread once it has been produced in a TetVP16-expressing plant.

Approach 2: In the second approach, then, the idea was to develop a system with a higher degree of safety, where there is no risk of the virus spreading outside the transgenic plant.

This is achieved by removing the viral transport protein gene, which is responsible for the systemic spread of the virus in the plant. The modified virus is then no longer capable of spreading in normal (wild type) plants. If, on the other hand, this transport-defective virus is inserted into transgenic plants which express the viral transport protein, this “defect” can be remedied, enabling these plants to be systemically infected by the virus.

Procedures and safety systems are to be transferred to other viral/host systems and used to produce foreign proteins. In addition the biological safety of the two approaches will be investigated.

Results

Approach 1: Regulated expression of the viral full-length clone was obtained in TetVP16-expressing plants using the TOP10/TetVP16 system. A risk remains because the virus particles produced in these plants are capable of multiplying in wild type plants.

However, over an extended period of observation it became apparent that the regulation of the gene expression via the TetVP16 “switch protein” does not work perfectly. The TOP10 promoter, which should allow expression only in the presence of the switch protein, does not constitute a sufficient obstacle. When the viral full-length clone is integrated into the plant genome during agrobacterial infection, it seems that the plant’s own promoters also trigger expression. The project is currently attempting to increase the functionality of the TOP10 promoter with additional “stop” sequences.

Approach 2: Full-length clones were produced, in which the transport protein required for systemic infection was destroyed (PVX GUS Δ25). In addition, transgenic tobacco plants (Nicotiana benthamiana) were produced which carry this viral transport protein gene.

It was demonstrated that the system functions as planned.

1. Infection of non-transgenic tobacco plants with agrobacteria that carry the viral full-length clone, including the gene for the transport protein (PVX GUS), results in systemic infection i.e. one that affects the entire plant.
2. If the non-transgenic tobacco plant is infected with the Agrobacterium that carries the full-length clone without the transport protein gene (PVX GUS Δ25), systemic infection does not occur.
3. However, if transgenic tobacco plants that produce the transport protein themselves are infected with Agrobacteria that carry the full-length clone without the transport protein gene (PVX GUS Δ25), then this again results in a systemic infection.

Photographs showing symptoms on tobacco plants 21 days after infection (upper) Leaf from the upper region of each plant after GUS test (lower).

Since the transport-defective virus can systemically infect only transgenic plants that express the transport protein, an undesirable spread of the virus is reliably prevented and safe synthesis of foreign proteins using viral full-length clones is made possible.