In the last lecture I talked about how plants could be protected from viruses by expressing the coat protein for that virus in transgenic plants. In addition to the coat protein, expression of other components of the virus (replicase protein, mutant movement protein) can also provide protection against some viruses. However, viruses are not the only pathogens that cause diseases in plants.
Bacteria and fungi are the other two classes of microbial pathogens. Together, these microbial pathogens are estimated to cause a reduction in crop production of approximately 12% overall. This translates into an estimated annual loss of approximately $42 billion dollars worldwide, quite a major impact. Under some conditions pathogens can cause epidemics that result in the total loss of a crop. In addition to reduced crop yields, pathogens can also affect the quality of the crop. For example, some fungi produce toxins that contaminate grain. An example is aflatoxin which is harmful both to humans and livestock.
The increasing use of monoculture systems in agriculture is also reducing genetic diversity and can contribute to the problem of susceptibility to diseases. An excellent example of this occurred in the US in the 1970s. A disease called Southern Corn Leaf Blight was causing dramatic losses in the US corn crop. The reason for this lay in the use of a particular genetic background that was very widely used to produce hybrid corn seed. A strain of the pathogen appeared that was particularly damaging to corn hybrids that were developed in this background, which was used on a large fraction of the corn acreage. Once the seed companies moved to a different method to produce hybrids that didn't rely on this disease susceptible genetic background, the problem went away.
What are the current methods to control bacterial and fungal pathogens?
Cultural practices, e.g. in a horticulture greenhouse operation, soil mixes are usually sterilized by high temperatures to eliminate as many pathogens as possible, which can be a problem especially for germinating seeds.
Planting varieties with genetic resistance to specific pathogens. As I have mentioned before, these genetic determinants may consist of a single gene or be controlled by many genes.
Using pesticides, especially fungicides. Pesticides are used much more widely on grain crops in Europe than in North America. In the US most small grains are produced under fairly low input management systems (also low cost), whereas in Europe these crops are much more intensively managed.
What makes a microbe a pathogen? All plants are exposed to, and are probably covered by, hundreds or thousands of different bacterial species. But these organisms are not causing any disease and are not pathogens. What is it that makes a disease causing organism a pathogen?
Pathogens must be able to penetrate the physical barriers of the plant, such as the cuticle and cell wall.
Once the pathogen has entered the plant, it may lyse (burst) plant cells and live off the nutrients that are available from the plant.
What does the plant do to protect itself from these types of pathogens? Plants do not have an immune system in the sense that we think of for ourselves. However, they don't just sit around passively waiting for the next pathogen to attack. Instead, plants are equipped with a variety of mechanisms to fend off such attackers, listed below.
I hope you can see that plants are equipped with a great many methods to combat pathogens. The question then arises as to what the biotechnologist can do to improve the resistance of plants to pathogens.
As these proteins have some antimicrobial properties, it was thought that expressing one of these PR proteins all the time (constitutively) might be able to protect plants from pathogens. Transgenic plants have been produced that express many different PR proteins. In most cases, these individual PR proteins have little or no effect. In some cases it could be shown in the lab or greenhouse that these plants had some modest increase in resistance to one or two pathogens, but this was never enough to be effective in the field.
If one isn't enough, try two or more PR proteins. In some cases, combinations of PR proteins that are expressed constitutively are much more effective than single PR proteins. They have a synergistic effect, i.e. the combination is better than you would expect if you just added together the effects seen with both expressed independently. Examples of the combinations that have been used are chitnase and glucanase, and a ribosome inactivating protein (RIP) with a cell wall hydrolase. The general conclusion from these studies appears to be that the cocktail of PR proteins that the plant normally produces is required to provide effective control of a wide range of pathogens. More on this at the end of the lecture.
Many organisms other than plants produce proteins or small peptides with antimicrobial activity. These compounds either slow down growth of microbes or kill them. Because these are composed of a chain of amino acids, they are encoded by genes. And as we have learned in this class, genes can be transferred from other organisms into plants. So a variety of genes expressing these antimicrobial peptides or proteins have been expressed in different transgenic plants. Among those that have been tested are:
While some of these have a modest effect on plant resistance to some pathogens, the results have not been very promising overall.
The enzyme glucose oxidase (from Aspergillus) produces hydrogen peroxide as part of its normal activity. This gene has been expressed in potato with some clear effect on resistance to bacteria that cause soft rot disease. The hydrogen peroxide is able to restrict the growth of the pathogen and also activates the expression of some PR proteins.
Resistance genes are responsible for resistance to some pathogens. In general they are effective against only a small subset of pathogens rather than providing resistance to all pathogens. Over the last few years, a number of resistance genes have been cloned. In a small number of cases, these have been transferred between species and have some effect on resistance to a pathogen. However, these are not genes that provide a general resistance to all pathogens.
A rather dramatic title, but it does describe the strategy. A chimeric gene has been constructed that consists of the promoter from a PR protein gene and an open reading frame encoding an enzyme that destroys RNA, a RNase. This gene should be expressed in cells that are infected with a pathogen. When activated, this chimeric gene will synthesize an RNase that will destroy the RNA in the cell and lead to the death of the cell. This is supposed to mimic the cell death/sacrifice strategy that plants use as described above. This tricky approach is more complex than I have outlined, but the strategy to accelerate the death of infected cells has shown promise.
These various approaches have been tried in a number of different species and targeted against a variety of pathogens. There have been some successes but no commercial products have yet been forthcoming. In part I believe this reflects the complexity of the interactions between plants and their pathogens. It is perhaps not so surprising that plants use such complex methods to protect themselves. I imagine that it will be a number of years before transgenic plants with enhanced resistance to bacterial and fungal pathogens will be widely available.
Two new strategies are outlined below to show the thinking of at least some researchers who are involved in this type of research.
First, with the knowledge of how plants respond to pathogens, Novartis screened for chemicals that can induce the systemic acquired resistance response in plants. One compound with these properties, abbreviated as BTH, has been developed for control of powdery mildew in wheat and is on the market in Europe. While this is not a biotech product, it was identified because of the fairly recent (in the last 15 years) understanding of how plants respond to pathogens.
Second, a "master gene" has been identified that initiates and regulates many of the disease responses in plants (well at least in Arabidopsis!). In the one report to date on the manipulation of this gene, overexpression of the master gene had a significant impact on resistance to several pathogens.
in conclusion, studies on the interactions between plants and their pathogens have revealed a great deal about mechanisms plants use to protect themselves. The simple approach of overexpressing individual proteins has had very limited success. However, recent results indicate that activation of the control switches for these resistance pathways may be a better approach to improve resistance of plants to pathogens via biotechnology.