A new generation of insect resistant GM crops: transgenic wheat synthesising the aphid alarm signal

When aphids (greenfly) are attacked by predators they release a warning signal to alert other neighbouring aphids. This is achieved by a chemical called an alarm pheromone that other aphids can smell. This alarm pheromone is completely non-toxic, even at levels far higher than would be used in this project. As well as being produced by aphids, it is produced naturally by some non-crop plants. One such plant is peppermint and we have isolated the gene responsible for the production of pure aphid alarm pheromone in peppermint plants. By inserting this gene into other plants we can make them produce the pheromone and we have recently performed this transformation with a simple plant called thale cress which is widely used by biologists as a model. When we did this aphid pests were no longer attracted to the transformed cress. Furthermore, natural insect control was improved because a key natural enemy of aphids, called a parasitoid, searched for aphids for a longer period of time on the transformed thale cress. Following this success we now need to carry out a similar transformation with wheat, a crop plant of worldwide importance, so as eventually to exploit this system for aphid pest control and breed a new generation of environmentally friendly GM crops. Other insect pests also have pheromones and the approach developed here should lead to possibilities for replacing toxic insecticides with non-toxic insect behaviour signal chemicals in other crops too. Studies will initially be conducted under contained laboratory conditions and then extended to carefully regulated field trials. We propose to transform wheat plants with the aphid alarm pheromone gene, check that the transformed wheat plants are emitting the pheromone and then examine the effects on aphid pests and their natural enemies. Groups of aphids on leaves will be exposed to the odour of the transformed wheat and the proportion of aphids scuttling away will be recorded. Special devices called olfactometers will then be used to give aphids and their parasitoids a choice between air passing from the transformed wheat or a blank airflow. We expect that aphids will be attracted to normal wheat odour but repelled by odours of the transformed plants and that the aphid parasitoids will be more attracted to the transformed wheat. Parasitoid behaviour on the transformed plants will also be compared with behaviour on normal plants. Field simulator containers will be used to measure aphid settlement on normal and transformed wheat. Once we have identified the most promising wheat varieties under lab conditions we will test them outdoors in field plot trials. Aphid numbers will be counted in samples of 100 plants per plot every week during the growing season. We expect that aphid settlement will be reduced on the transformed wheat plants and that any aphids that do settle will be more exposed to attack by natural enemies. The combined effect of an aphid repellent odour and attraction of natural enemies will mean lower aphid infestations on the transformed wheat plants. This will provide a new option for reducing insecticide use on crops in the future.

The alarm pheromone for many aphids is a volatile sesquiterpene (E)-beta-farnesene (EBf). Here we propose to develop EBf emitting wheat, Triticum aestivum, by transforming plants with Mentha x piperita EBf synthase fused to a strong constitutive promoter (maize Ubi1). Molecular and biochemical characterisation of transformed phenotypes will be used to check the presence of the EBf synthase gene, its expression levels and EBf emission from the plants. We will also enhance EBf emission via targeting EBf synthase to plastids and also via the co-expression of a farnesyl diphosphate synthase (both strategies have been shown to enhance the transgenic accumulation of terpenoids). Specialised insect behavioural bioassays developed by the Chemical Ecology Group will then be used to determine responses of cereal aphids Sitobion avenae and Rhopalosiphum padi and their parasitoids Aphidus ervi and Aphidius rhopalosiphi to the wheat plants. These insects are known to respond to EBf. Bioassays will comprise: 1) an aphid alarm response bioassay in which colonies of aphids are exposed to volatiles and disturbance is quantified; 2) an olfactometer repellency and attraction bioassay in which walking insects are introduced into an arena where they have a choice between test volatiles or control areas (without the volatiles); 3) a no-choice field simulator test for aphid settlement on plants in special containers with a gentle air-flow in which large numbers of aphids are released, and 4) a parasitoid foraging bioassay where parasitoid behaviour is quantified. These bioassays will allow us to compare responses to wild-type and transformed EBf emitting wheat plants to determine whether transgenic plants subvert the aphid alarm signal to deter aphids and attract their enemies. Furthermore, the project includes two field trials in which aphid infestation levels of two GM lines under typical field conditions will be determined and compared with wild type control plots.