Introduction
Plant virus diseases are responsible for significant crop losses throughout the world. For example, it has been estimated that viruses in potatoes cause annual losses of between $US60 and 120 million in the USA alone. Annual losses in field crops are generally placed at between 5 and 10% in the USA with significantly higher losses in other parts of the world. Crop-yield losses are especially significant in tropical regions, where insect vectors survive year-round and where alternate hosts provide year-round reservoirs for both virus and insect vectors.
In the USA and Europe, research in plant breeding has led to the development of crop varieties with high levels of disease resistance against specific viruses. Many resistance genes are not durable, but others are highly durable and have been deployed in agricultural regions for 15-20 years. For example, the Tm-2 gene for resistance against tomato mosaic virus (ToMV) has been used in both greenhouse and field situations for more than 15 years, following its introduction by seed companies in Holland. Tm-2 has found its way into tomato varieties around the world and has been a highly durable gene. Recently, however, strains of ToMV that overcome the Tm-2 gene have been isolated.
There are, on the other hand, numerous examples in which breeding for virus disease resistance has been notably unsuccessful. For example, the disease caused by the papaya ringspot virus (PRV) has largely eliminated commercial production of papaya in many parts of the world, due to the lack of genes for resistance. Perhaps more importantly, the geminiviruses have emerged as significant pathogens in many parts of Asia and Latin America and recently in the southern USA.
These agents comprise a genome of single-stranded deoxyribonucleic acid (DNA), and they are borne by whiteflies. Whiteflies have been difficult to control by chemical means, and little or no genetic resistance has been found to control the disease. These characteristics have contributed to the increasing importance of geminiviruses in agriculture.
In the mid-1980s, several research laboratories began experiments to prevent the replication and disease-causing cycle of plant viruses through the use of transgenic plants. Several different approaches were taken to interrupt the disease cycle, as shown in Fig. 14.1. This illustrates virus entry into the cell through a wound caused by either mechanical damage or by insects. After the virus is placed within the cell, an as-yet uncharacterized reaction causes the release of viral capsid (coat) proteins (CPs) from the viral genome, releasing the genome for translation and replication.
During replication, complementary strands of the viral genome are synthesized by a replica encoded either wholly or in part by the viral genome. As this enzyme replicates the genome, it produces new mRNAs, messenger ribonucleic acids which use the host translation machinery to produce the viral proteins. Some of these proteins may be important for the insect vector’s acquisition and spread of the virus.
Other gene products may be proteases, which cleave a virus preprotein to release active viral protein(s). Some proteins are responsible for modifying or creating the channels that enable the virus to move from cell to cell. Virus mRNAs produce additional CP which is responsible for encapsidating the replicated virus and the accumulation of new virus particles.
Since 1986, novel transgenes have caused the interruption of the virus replication and life cycle in several ways, as indicated in Fig. 14.1. These will be briefly reviewed here, with emphasis on studies with the virus CP.
Plant (Virus-resistant Transgenic Plants)
How to Develop Virus Disease Resistance in Transgenic Plants:
The first example of interference with virus infection and disease was reported by Powell et al. (1986), who described transgenic plants that express the CP of tobacco mosaic virus (TMV). Plants that express the CP gene are resistant to infection by the strain of TMV from which the CP gene was isolated and to closely related strains.
This resistance was subsequently described as CP-mediated resistance (CP-MR). The effects of the expression of the gene were to reduce the number of sites where the infection occurred and to reduce the rate of spread of the virus from the inoculated leaf to the upper leaves. This resulted in decreased severity of infection in plants that accumulate CP.
It was subsequently shown that this approach can be effective for the control of a large number of different classes of viruses. In each of these cases, the CP gene provides resistance against the virus from which the CP sequence was isolated and against closely related strains, but not too different groups of viruses. In some cases, there was significant resistance against distantly related serotypes (reviewed by Fitchen and Beachy, 1993).
To date, CP-MR has been reported to be effective against viruses in at least 11 different classification groups, and in a variety of dicotyledon and monocotyledon crop species, including potato, tomato, papaya, squash, cucumber, melon, alfalfa, sugar beet, rice, and maize.
An alternative approach to conferring resistance has been to express portions of the viral genome that encode the viral replication. It has been shown in several laboratories that resistance against at least several classes of viruses can be engendered by expressing a gene encoding either the full viral replication or a portion thereof (Carr et al., 1992). Such resistance can be extremely high, i.e. near immunity.
The expression of these viral sequences interferes with the replication process. Replicase-mediated resistance, while potentially very strong, has been reported to be very narrow (Mueller et al., 1995). Thus a gene encoding replication is effective against the virus strain from which it was isolated, but not against closely related strains.
Attempts to interfere with virus replication through the application of antisense RNAs have achieved mixed results. Genes that encode sequences complementary to viral RNA are effective in providing resistance to viruses whose genome comprises DNA, such as the geminiviruses (Stanley et al., 1990). However, antisense genes have been less effective against viruses whose genome is single-stranded RNA. In the case of the geminiviruses, whose replication cycle occurs in the nucleus, replication is especially sensitive to inhibition by the expression of antisense genes.
In contrast, replication of viruses that replicate in the cytoplasm, such as those whose genomes comprise single-stranded (+)-sense RNA molecules (representing more than 95% of the plant viruses known) is less susceptible to inhibition by antisense RNA (e.g. Powell et al., 1989). There are, however, some notable exceptions, e.g. antisense strategies have been somewhat effective against the luteoviruses.
Other strategies are predicted to yield likely candidate genes for interfering with virus diseases but have yet to be successfully demonstrated. Among these is the possibility of interfering with the proteins that are necessary for the acquisition of the virus by the insect vector. The so-called ‘helper-component proteins’ are essential for virus acquisition. It is likely. that several research teams are attempting to interfere with this process by expressing mutant acquisition proteins that block the involvement of the virus helper component in the acquisition, or by saying mutant CPs that block transmission of the virus.
Fig 14.1. A generalized virus life cycle and gene products may interrupt the cycle.
1 coat protein
2 (-) sense RNA (+ribozyme)
3 (+) sense RNA (+ribozyme)
4 defective replication
5 modified protease
6 defective coat protein
7 defective transmission factors
8 defective movement protein
Other groups are attempting to block the acquisition of viruses by the express. sion of proteins such as antibodies or other competitors to block the sites in the insect to which viruses bind.
A promising type of disease resistance has recently been described by several research groups. This strategy involves attempting to block the spread of the virus from cell to cell in the host plant, thereby limiting the accumulation of the virus throughout the plant. This function is controlled by one or more virus-encoded protein(s) that modify the function and/or structure of the natural cell-to-cell communication channels between plant cells. For example, TMV encodes a 30 kDa movement protein that modifies the function of the plasmodesmata. This modification is essential for the movement of the virus from cell to cell.
Lapidot et al. (1993) demonstrated that the expression in transgenic plants of a gene that encodes a dysfunctional movement protein from tobacco mosaic tobamovirus can interfere with the movement protein produced by infecting tobamoviruses, thereby limiting the spread of the virus from cell to cell. While this dysfunctional movement protein does not confer immunity to the transgenic plant, it does significantly interfere with the spread of the virus and the systemic effects of the disease.
Furthermore, the modified dysfunctional movement protein used in this study is equally effective against all tobamoviruses thus far tested and may be expected to interfere with the spread of other viruses that use similar mechanisms for cell-to-cell spread (Cooper et al, 1995). Thus a dysfunctional movement protein is likely to provide the most wide-ranging type of resistance yet described. Similar approaches are most probably being taken by other research groups to block the movement of the geminiviruses and other important plant viruses (Beck et al., 1994).
Summary
Several experimental approaches have been taken to block the cycle of virus infection and replication in transgenic plants through the use of genes that are derived from the targeted viral pathogen, Approaches that are most likely to be successful will include those that prevent the spread of the virus and its accumulation in infected cells or tissues. An effective resistance could be engendered by decreasing the accumulation of the virus, thereby limiting the likelihood that the virus could be acquired and transmitted to adjacent plants by insect vectors. This would have the effect of reducing the rate of spread of the virus in the field, thereby reducing losses to the farmer.
Source: Biotechnology and Integrated Pest Management. (Part: Virus-resistant Transgenic Plants)
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