The Merits And Dangers Of Using Viral Coat Proteins In Transgenic Plants
Paul Yeatman 1993
Plants that have had viral coat proteins introduced into their genome are known as transgenic, and display coat protein-mediated resistance. The mechanisms for such resistance are not known, but are most likely those which follow in this discussion. In order to explain each mechanism, it is listed individually along with the virus or viruses such resistance is shown against. It is important to note that in order for any resistance to occur, the transgene needs to be expressed in some form within the transgenic plant cells.
When one wishes to release a transgenic plant into the environment, the possibility of the introduced gene’s escape into wild plants must be considered. Also, the presence of a large amount of coat protein of a viral origin, may coat previously uncoated viruses, which then may be spread by vectors, where before such spread was not possible.
The actual resistance mechanisms of coat protein-mediated resistance are a matter of conjecture. However, it would appear that this form of resistance is due to multiple resistance mechanisms operating and involves the early events in viral infection (Fitchen etal 1993). The major mechanisms are associated with the prevention of viral entry into the plant cell; inhibition of viral recoating; prevention of infection; mRNA accumulation inside the transgenic plant cells and resistance to spread of the virus within the plant.
Prevention Of Viral Uncoating
If a virus is prevented from uncoating, its genetic material will not be exposed to cellular replication machinery and the virus will not be able to reproduce. This mechanism has been shown to function in resistance to tobacco mosaic virus, TMV, (Fitchen etal 1993), (Nelson etal 1987). The prevention of uncoating may be shown to be operable by treating TMV to pH 8.0 for a brief period of time, which removes about sixty coat protein subunits (Fitchen etal 1993). Inoculation of a tobacco plant with the treated virus causes an infection, but if the virus is untreated, no infection results. This lack of infection is due to the viral mRNA not being translated (Register III etal 1988). This suggests that the removal of early coat protein residues from the infecting virus is prevented by the presence of viral coat protein. Two theories exist to explain why this may be so.
Theory One: TMV has a specific uncoating receptor which is blocked by coat protein (Fitchen etal 1993). This would relate to the replication conditions in the cell due to viral activity. High coat protein concentrations would signal an end to replication and tend to induce coating. By blocking the uncoating receptor, which also controls encapsidation in a normal replication cycle, recoating would result. In a transgenic plant the large coat protein concentration blocks the uncoating receptor of the viral RNA, thus preventing infection. Therefore, if a transgenic plant has a high coat protein number within its cells, coat protein binding to the uncoating receptor would effectively block uncoating (Fitchen etal 1993).
Theory Two: TMV uncoats when it enters a plant cell due to local physiological changes (Fitchen etal 1993). This would suggest that uncoating is the result of an equilibrium shift, meaning uncoating may be reversible. Therefore if a transgenic plant has a high coat protein concentration, it will favor viral coating. This would effectively prevent uncoating of the invading virus. This explanation is an attractive one as it allows for no complicated mechanism. Rather, it bases its explanation on simple equilibrium dynamics. From this, a plant with high TMV coat protein would be expected to have high resistance to infection by TMV. Such has been observed in experiments, (Fitchen etal 1993), but a large innoculum of virus does overcome the resistance (Nelson etal 1987), which is likely indicative of this theory.
Inhibition Of Viral Entry
Investigation shows that the actual entry into the transgenic plant cell, of virus may be blocked, (Register III etal 1988), (Nelson et al 1987). If one removes a minimal amount of coat protein subunit, (less than the amount removed by treatment at pH 8.0), the virus can overcome the protein-mediated resistance. This suggests that stabilizing the coat protein may not be the only mechanism if resistance. If recoating was induced, a virus with some coat protein already removed should reencapsidate with transgenic viral protein. Such a process would render the virus ineffective, but this does not seem to happen. As TMV has coat protein surrounding it, entry into a cell containing a high amount of coat protein may prove difficult for the virus particle. This, like the second theory for uncoating is related to equilibrium processes and is analogous to trying to fit more water into a full glass. By this it is meant that the plant cells are already full of coat protein, so the cells will resist any attempt to place more coat protein in them. Also, this seems to suggest that uncoating is prevented and not reversed.
Prevention Of Infection
Prevention of viral entry is related to prevention of infection. For an infection to occur, the virus needs to be able to enter a cell. Due to this, it is necessary fit the correct cell of the transgenic plant to express the coat protein gene. Most importantly, epidermal and xylem cells should express coat protein, (Reinmann-Philipp etal 1993), as these cells are the most likely to be the first cells to be infected with virus. Thus to simplify matters, all the transgenic plant’s cells should express the viral coat protein gene. This would pose a minor drain on cellular energy at the expense of a more healthy plant.
mRNA Accumulation Within Plant Cells
Some resistance to virus has been shown to be due to mRNA accumulation of the viral coat protein genes, (Fitchen etal 1993). Such has been shown in the case of resistance to potato virus X, PVX and potato virus Y, PVY. Viral mRNA that has accumulated in the plants cells may bid to the introduced viral RNA in its native form, (Farinelli etal 1993) once uncoating has occurred in this case. When such binding occurs, the viral RNA is blocked, so the infection cannot proceed. Another suggestion is that the transgenic cell produces substances that block viral multiplication through saturation of viral protease cleavage sites (Farinelli etal 1993). Such substances may act in the same fashion as the coat protein to cause recoating of TMV. Such substances may also act to prevent the virus from re-encapsidating. This would prevent viral spread in the plant by confining the virus to the initially infected cell.
Resistance To Viral Spread
Unlike resistance to TMV, the amount of resistance shown is not related to the amount of coat protein in the transgenic cells (Kaniewski etal 1990). The mechanism for resistance to PVX and PVY may be due to inhibition of viral transport and replication suggested by low levels of the virus being detected in infected transgenic plants, (Kaniewski etal 1990). Viral replication would most likely be inhibited due to resistance to uncoating or blockage of uncoating receptors. As low levels of coat protein will still produce resistance, a blockage of an uncoating receptor is likely due to insufficient coat protein being present to force the virus to reverse its uncoating. Transport of the virus would be inhibited by resistance to viral entry into plant cells, either via the presence of coat protein preventing entry or as an as yet undiscovered anti viral mechanism. Such a mechanism could be like the production of interferon gamma in mammals when a viral infection has taken place.
A plant expressing the viral coat protein produced due to the presence of the viral transgene may cause other cells to exclude such protein from entering the cells. Perhaps a transport system is created in order to shunt the virus particles from the plant cells before any infection can be initiated. By removing coat protein from the cell, the virus would be prevented from coating so the rate of infection transfer would be reduced. In order to test these theories, measurement would need to be made to determine the concentration of coat protein intra and extracellularly in a transgenic plant.
Resistance to PVY and PVX is not overcome by a high virus inoculation, (Farinelli etal 1993), unlike what is the case for TMV. This may be due to the blockage of infection, blocking of viral cell to cell movement, or blocking of vascular migration, (Farinelli etal 1993). If the infection is blocked in the inoculated cell, the mechanism may involve the prevention of uncoating or binding to uncoating receptors. If cell to cell movement is inhibited, resistance may be due to elevated coat protein levels in the transgenic cells or the shuttle theory. Blockage of vascular migration appears unlikely as the virus should be inhibited long before it reaches the plant’s vascular system, unless placed there by a vector.
Risks Associated With The Release Of Transgenic Plants
The major risk when introducing a transgenic plant into the environment is whether its foreign genes may be passed on to wild plants. For this to occur, gene flow is necessary, either by seed or pollen. The gene transfer via seeds is considered minimal (Elistrand 1992), so gene flow via pollen shall be considered here.
Anther risk is the transmission of previously non-vectored viruses resulting from heterologous encapsidation and template switching (Gonsalves etal 1992).
Gene Flow Via Pollen
As viral coat protein genes are incorporated into transgenic plant genomes, they may be transferred via cross pollination. This is advantageous in a crop situation as viral resistance may be given to other plants of the same species relatively easily through hybridization of the transgenic plant and the original plant species. The gene flow becomes a problem when the recipient plant is a rare species (Elistrand 1992) or a potential weed. In both cases, sexual compatibility between the transgenic and recipient plant is necessary for the gene transfer to occur.
If a species is rare, gene flow may cause the plant to be outgrown by the hybrid. This would result in the loss of the endangered species eventually and a proliferation of the hybrid. Such would result from the hybrid being better able to survive due to its acquired viral resistance.
In the case of weed formation, the worst case scenario would see an explosive growth of the recipient plant, resulting in the local extinction of competing plants and a biodiversity reduction (Elistrand 1992). The reason for such an increase in the new weed would be due to the traits transferred to it by the viral coat protein gene. A fitness advantage would e conferred to the weed (Elistrand 1992). Normally such a species would be held in check by environmental factors such as viral attack. The gaining of resistance to the virus in question would enable the plant to grow unchecked and thus become a problem. Due to this, other control mechanisms would need to be developed to return the ecology back to its normal state if possible.
Viral Spread Via Vectors.
Due to the transgenic plant possessing viral coat proteins, a virus that was previously non encapsulated, may become encapsidated and be transmitted to other plants. Such transmission may occur via vectors, (Gonsalves etal 1992), or via plant to plant contact. Template switching may also cause a previously non vectored virus to be transmissible via vectors. Thus a virus which previously was not a cause for concern, may create localized disease outbreaks.
Factors to Consider
If one were to field release a transgenic plant, one would need to consider the impact on the environment if the gene in question was transferred to native pants, and if the recipient plants near the transgenic crop were capable of cross-pollinating with the crop. Also the possibility of vectors spreading normally non-vectored viruses would require investigation. Such considerations would determine if the development of a new weed was likely or if important plant species would become extinct. Also, whether or not a previously unproblematic virus would present itself as a pest.
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