There are a large number of viruses that infect plants and these can cause serious losses in production, both in terms of yield and quality. First I will describe the structure of most typical plant viruses, then the methods that are currently used to control viruses, and finally how biotechnology may provide new approaches to developing resistance.
The vast majority of viruses that infect plants are RNA viruses. This means that the nucleic acid carried in the virus particle, the viral genome, is comprised of RNA. There are a small number of plant DNA viruses, including Cauliflower Mosaic Virus (CaMV) from which the 35S promoter was obtained. Plant viruses are grouped into families based on their physical structure and the organization of their genomes.
Plant viruses are fairly simple creatures. The virus particle is made up of a protein shell or coat which surrounds the RNA or DNA genome. The shell is made up of one or two proteins, and these proteins assemble to form the coat. These proteins are known as coat protein.
What is the life cycle of a typical plant virus?
The genomes of plant viruses contain only a small number of genes. These encode for the coat protein, a replicase (a protein to aid in replication of the RNA genome) and maybe a small number of other proteins. Many viruses produce a protein that helps the virus move through the plant from the original site of infection. This movement protein enlarges the small passageways between cells (the plasmodesmata), so that virus particles can get through, allowing them to spread systemically from cell to cell.
What strategies are currently used to control plant viruses? This is not going to be an exhaustive survey of viral pathology and control, just a few pertinent and important examples.
This formed the basis for experiments to examine this phenomenon and led to the development of a general method to make plants resistant to viruses. The hypothesis was put forward that some component of the virus was responsible for triggering this resistance phenomenon.
How could this hypothesis be tested? As I said earlier, plant viruses are simple and have few components. Perhaps one of these major components was responsible for cross protection. If the different genes that comprise the virus genome were expressed in transgenic plants, it might be possible to show that a single part of the virus was able to induce this resistance in plants. How was this done?
Plant viruses contain only a few genes: a replicase, coat protein, movement protein, perhaps a few others. The genome of the virus is comprised of RNA. In order to construct chimeric genes that direct the expression of individual viral proteins in transgenic plants, the RNA first had to be converted into a DNA copy. An RNA sequence can be copied into a DNA molecule, maintaining the correct sequence of bases, by reverse transcriptase. This is the first step towards making the chimeric genes to express viral proteins, as outlined below.
This was first tested with transgenic tobacco plants engineered to express the coat protein of tobacco mosaic virus (TMV). When transgenic tobacco plants that express the coat protein of TMV were exposed to the virus, they were found to be resistant to infection by the virus. The inoculated transgenic plants did not develop symptoms, while the control non-transformed plants did become infected. However, the transgenic plants expressing the TMV coat protein were not completely immune; they would become infected if inoculated with a high dose of the virus, but it took longer for symptoms to develop and the symptoms were less severe.
This protection works against the virus from which the coat protein was isolated, as well as against closely related viruses. Having been shown to work providing protection against TMV in tobacco, the same strategy (expression of the virus coat protein in transgenic plants) was then applied to other plant viruses. This approach has now been shown to work providing resistance against a large number of different plant viruses. This is called coat protein-mediated resistance.
How does coat protein-mediated resistance work? It is not exactly clear, but it appears to block some early stage in the infection process, maybe the uncoating of the virus when it first enters the plant cell. If you infect transgenic plants expressing coat protein with normal viruses, the plants are protected, but if the plants are infected with the RNA of the virus, the plants will develop symptoms. So it appears that expressing the coat protein in the plant prevents the virus from uncoating, expressing the viral genome and causing disease symptoms.
This method has been used successfully in greenhouse and field tests to obtain resistance to many plant viruses. While the first, and the majority, of experiments and trials have been conducted with model systems such as tobacco, the strategy appears to be equally successful with crop plants. It appears to be a technique which can be applied very widely to obtain resistance.
The first product developed utilizing this approach was a summer squash with resistance to 2 viruses. Seed for this squash was available in 1996 but I believe it has been withdrawn. It appears that the squash was still susceptible to another virus, and the company responsible (Asgrow Vegetables) is developing an improved version with resistance to 3 viruses.
Another vegetable crop that has been developed with resistance to a virus using coat protein mediated resistance is potato. These are being developed by Monsanto with resistance to potato leafroll virus and potato virus Y.
Perhaps the best example to date of this strategy is in trying to develop papaya trees with resistance to papaya ringspot virus (PRSV). This is essentially a lethal disease for papaya and prevents commercial production of these tropical fruits. There was a thriving papaya industry in Hawaii which had to move from one region of the island to another because of the spread of PRSV. Over the last ten years researchers at a number of institutions have been developing transgenic papaya that express the coat protein of PRSV. The strategy is the same as I have outlined previously. Clone a cDNA for the PRSV coat protein gene. Construct a chimeric gene where the open reading frame of this coat protein is placed under the control of a high level promoter. Transfer this chimeric gene into papaya by transformation. Regenerate transgenic plants and evaluate for resistance.
This has led to the release of a transgewic papaya with resistance to PRSV which is now being planted in Hawaii. A detailed description of this research program and the development of this variety can be viewed here. This product has been approved by USDA, EPA and FDA. However, there is no long term research to indicate how stable this resistance will be, and whether or not viruses will adapt and develop strategies to thwart this mechanism of resistance.
While coat protein-mediated resistance has proven to be quite effective, other methods have also been tested to develop plants with resistance to viruses. When the replicase of the virus is expressed in transgenic plants, this also provides resistance to the virus from which the replicase was cloned.
Expression of coat protein and replicase are the best examples of what is called pathogen-derived resistance, where part of the virus is used to make plants resistant to that pathogen.
Other strategies that have been used include:
All of these have been shown to work to some degree, but none have been developed into a commercial application at this time.
The final method I will discuss for making plants resistant to viruses targets the movement of viruses from one plant cell to another. This intercellular movement of viruses requires that the plasmodesmata, the pores between cells, are enlarged. Many viruses produce a movement protein that is responsible for this modification of plasmodesmata. From detailed studies on viruses like TMV, it was shown that some strains of virus were unable to move from cell to cell and produced very limited disease symptoms. The reason these viruses could not move was because they have a defective movement protein, one that prevented the plasmodesmata from enlarging. When this defective movement protein was expressed in transgenic plants, it was found to provide resistance against viruses, not by preventing the infection of plant cells but by preventing the spread of virus from cell to cell.
In summary, it is clear that there are a number of strategies that can be used to produce transgenic plants with resistance to viruses. However, there are few examples of the commercial or practical use of any of these methods. I am not sure if this is because of difficulties in moving this research from the lab to the field, or if this problem (virus diseases) is of more concern in the horticulture sector, where it is more difficult to get a return on the research investment because of the fragmentation of the market between different crops and varieties.