Insects are capable of causing a tremendous amount of damage to plants, both in crop production and in ornamental uses. The losses caused by insects led to the development of insecticides, starting with DDT as the first widely used synthetic insecticide which came on to the market in the 1950s. Before we consider how biotechnology is being used to improve the insect resistance of plants, I will first provide a very brief introduction to the damage caused by insects, how they can be controlled, and the problems with current control methods.
Insects can damage crops at many different stages of development including:
Insects can attack different parts of the plant:
It is usual to think of the damage that insects can cause during growth of the plant. However, there are many circumstances where the most serious damage can occur after harvest, for example when the grain is stored. Insects lay their eggs in the stored seed. When the eggs hatch, larvae feed on the nutrients available in the seed and this can lead to very large losses. Controlling this type of insect damage is difficult, especially in regions where it is either difficult to obtain pesticides or they are prohibitively expensive. Adequate storage facilities can also help reduce crop losses after harvest, not only by reducing insect infestations but also by reducing losses caused by rodents (mice and rats).
What methods are currently used to control damage caused by insects?
Insecticides are very widely used and can be extremely effective. Approximately 75% of all the insecticides used in the US are applied to cotton and corn. However, there are a number of concerns and limitations regarding the use of insecticides as outlined below.
What improvements can be made to the current systems that are used to control insect pest populations in crops? There is certainly room to improve the current control practices, including greater use of IPM, incorporation of more resistance genes into crops, and development of insecticides that have less impact on the environment and are safer. In addition, gene transfer affords some new opportunities to control insect pests.
I will discuss two strategies that are being used to develop transgenic plants with improved resistance to insects:
Legume seeds and many other stored grains are attacked by a variety of insects. In many cases the attack takes the form of female insects laying their eggs in the seed. As the insect larvae develop, they feed on the seed and destroy it. The ability of the larvae to feed on the seed is dependent on their ability to digest the carbohydrates and proteins in the seed. An insect digests these dietary components in a manner that is similar to how you digest your food: it secretes into its gut a variety of enzymes that break down carbohydrates and proteins. These enzymes include proteases, enzymes that can break down the proteins into individual amino acids, and alpha amylases, enzymes that metabolize starches.
Many organisms that might be attacked by an organism that produces these digestive enzymes have developed a mechanism to resist this attack. They defend themselves by make other proteins that can inhibit the action of these enzymes. In the case of proteases, these proteins are known as protease inhibitors. For protection against alpha amylase, they produce alpha-amylase inhibitors. If a food source contains a supply of digestible protein or carbohydrate, but also an inhibitor that prevents the insect larva from digesting the food and obtaining any nutrition, the larva will not grow and develop through its normal cycle.
The seeds of many plants contain both protease inhibitors and alpha-amylase inhibitors, probably to provide just this type of protection. The common bean seed contains an alpha-amylase inhibitor. When this protein was included in an artificial diet for insects in feeding trials, the alpha-amylase inhibitor protein reducedthe growth of larvae of beetles that infest other legumes such as peas and cowpeas. Similar results have been obtained when insects are fed diets that contain protease inhibitor proteins.
From these studies the idea was developed that engineering plants to express these proteins that inhibit digestive enzymes would be a potential strategy to make plants resistant to some insects. A group at Purdue, in collaboration with scientists from other institutions, followed the plan outlined below:
When seeds from these transgenic pea plants were infested with beetle larvae, there was a negative correlation between growth of the larvae and the amount of alpha-amylase inhibitor that was present in the seed. In other words, the more alpha-amylase inhibitor present in the seed, the slower the growth of the larvae.
Similar approaches have been used to express genes encoding protease inhibitor proteins in transgenic plants. These transgenes have been expressed not only in seeds but also in vegetative parts of the plant. In general, these studies have been demonstrated to give some protection to plants from insect predation in lab assays, against a small number of insects. However, I am not aware of any plans to devlop this method into a commercial application at the moment. It appears that insect's digestive systems are somewhat flexible and can adapt to the presence of these types of inhibitors to allow them to continue feeding and digesting plant material that contains these engineered inhibitors.
In general, the use of protein inhibitors of insect digestive enzymes to control insects in transgenic plants is designed not to kill the insects that feed, but to retard their development. In many cases, control of insect pests is designed to linit the growth of insect populations so they do not develop to a level where they cause economic losses. If the rate at which insects develop is slowed down, and the time it takes for the insects to complete their life cycle is increased, then it will take a longer time for the insect populations to reach this critical level. Ideally, this will not occur before the crop is harvested. This strategy is based on reducing insect growth rather than elimination of insect populations.
Another source of genes that might provide resistance to insects is a bacterium called Bacillus thuringiensis. One of the characteristics of this bacterium is that it produces spores when conditions are not suitable for normal growth. As part of the normal program of sporulation, the bacterium also makes a crystal structure. This crystal is comprised of a small number of proteins (two or three, usually) that have insecticidal activity. These proteins are referred to as Bt toxins and have been the basis for developing transgenic crop plants with resistance to some insects. First I will describe how these toxins function before considering how they have been used to make insect-resistant plants.
Bt toxin proteins are therefore able to stop insect growth. The toxin proteins also function as feeding deterents for insects in artificial diet feeding trials. How an insect can detect the toxin in the first place is not clear. I don't know if these toxins function as deterents when expressed in whole plants.
How are Bt toxins used to protect plants from insect damage? They have been in use now for about 20 years. The bacteria are grown in fermentation systems and the spores harvested. The spores are then used for insect control by spraying or dusting the spores onto plants (e.g. Dipel). Bt spores are normally effective only for a few days, they are washed off plants by rain, can be inactivated in other ways and in general do not remain on the plant very long. A single strain of Bacillus thuringiensis is only active against a small number of insects, these are not broad spectrum insecticides. However, there are many different strains of this bacterial species, and they are characterized by accumulating a variety of different toxin proteins that have activity against different insects. So different strains of Bacillus thuringiensisare used to control specific insects. There is a great deal of interest in collecting strains of Bacillus thuringiensis to identify new strains with novel toxins that have activity against other insects. It has been reported recently that some strains even have activity against some nematodes, soil-borne worms that can cause serious crop losses.
The general approach to expressing Bt toxin proteins in plants is quite straightforward, one with which you should be very familiar by this point in the class.
Sounds very straightforward, but it didn't work to provide plants with resistance to insects. In most transgenic plants no Bt toxin could be detected, and in those that did express the toxin, the level of expression was so low that it was insufficient to provide ny protection against insects.
The low level of expression was shown to be caused by a subtle difference between the ORF of the bacterial toxin gene and most plant genes. The Bt toxin gene tended to use codons (sequence of 3 bases) for amino acids that, while still correct in the genetic code, were used very rarely in plant genes. This difference in "codon usage" means that translation of the bacterial ORF into protein was extremely inefficient in the plant resulting in very low levels of expression of the Bt toxin.
As a hypothetical example, there are two codons for the amino acid tyrosine in the genetic code, UAU and UAC. For illustration only (I don't know the frequency with which each codon is used in bacteria or plants), let's say that in plants 95% of the codons for tyrosine are encoded by UAU, and only 5% are UAC. However, in Bacillus thuringiensisthe frequency is reversed, 5% are UAU and 95% UAC. When the Bt toxin RNA is expressed in plants, and the plant ribosome tries to add a tyrosine to the protein during translation, the plant has very few transfer RNAs for this codon because the plant normally uses the UAU codon for tyrosine. So the plant can only translate the protein from the unmodified Bt toxin ORF very inefficiently. This accounted for the low levels of expression in the first attempts to make Bt toxins in plants.
This was overcome by redesigning the ORF of the toxin gene so that it resembled more closely a typical plant gene. In the example given above, the UAC codons for tyrosine in the bacterial Bt toxin gene would have been changed to UAU codons. The protein encoded by this modified open reading frame was the same as that expressed in bacteria, but it used codons for amino acids that were more typical of those found in plants. Such modification to the gene can be made in a number of ways, including synthesizing the gene, the sequence of bases in DNA, on a machine and then incrporating these sequences into a plasmid.
Chimeric genes were then produced consisting of a promoter, a modified ORF for the toxin, and a terminator. Instead of using the ORF for the protoxin, only the mature toxin part is produced in plants. I don't know the advantage of this modification. Transgenic plants have been produced where the Bt toxin protein is successfully expressed. The toxin protein may account for 0.1% or more of the total protein in any tissue. This level of expression is then sufficient to give these plants protection against some insects.
Currently there are 3 crops on the market that use Bt toxin expression for insect control:
These are the first crops that have been genetically engineered to control a pest. They may offer some new approaches to controlling insect damage and reducing insecticide usage. Next time we will discuss the potential advantages and disadvantages afforded by this new technology.