Hexima’s insect resistance technology or ‘PI technology’ was discovered during research on the female sexual tissues of an ornamental tobacco (Nicotiana alata) as a model. A gene that was very highly expressed in the female stigma of the plant was identified. This gene was shown to code for a protein which included six proteinase inhibitor peptides. As the flower matures, the six individual peptides are released from the protein.
Figure 4.1 NaPI is a circular protein with six domains. It is processed in the plant to release four trypsin inhibitors and two chymotrypsin inhibitors.
These proteinase inhibitor peptides isolated from Nicotiana alata (NaPIs) are capable of inhibiting enzymes (proteinases) that are critical to digestion of proteins. Two of the proteinase inhibitors inhibit the digestive enzyme, chymotrypsin, and the remaining four inhibit another digestive enzyme, trypsin.
If an insect were to attack and feed on the female stigma of the plant, the insect’s digestive proteinases would be inhibited by the proteinase inhibitors. The insect would then be unable to digest protein normally and its growth and development would be adversely affected. The presence of high concentrations of proteinase inhibitors in floral parts of the plant is one of the mechanisms which have evolved to protect the reproductive capacity of the plant.
Hexima’s strategy was to take the gene encoding the suite of six proteinase inhibitors and transfer it to cotton, which is vulnerable to attack by insect pests. One preliminary experiment to test the PI technology was to feed the products of the genes (proteinase inhibitors) isolated from stigmas of Nicotiana alata, to insect larvae, in an artificial diet. The growth of larvae of Helicoverpa species, the major pest affecting cotton, was significantly inhibited. The gene was then transferred to cotton plants which were grown in the glasshouse. The proteinase inhibitor peptides accumulated in the leaves, where the gene was expressed. The leaves were fed to the larvae of Helicoverpa species, and their growth and development were inhibited. These and other experiments gave confidence to invest in testing the technology in field grown plants.
Three field trials of the PI technology in cotton have now been conducted in Queensland at different sites during 2005/06, 2006/07 and 2007/08 growing seasons. Insect pressure during the three season differed, as did the scale of the trial. In each trial, the plants containing the PI technology out-performed the plants without the technology. Overall, plants containing the technology produced, without any insecticide spray treatment, a higher number of bolls per plant and matured earlier than plants not carrying the gene.
We have also demonstrated that the proteinase inhibitor genes act together with other insect resistance genes. The results indicate that proteinase inhibitors can be combined with other molecules to give more effective control of insect growth. Such combinations may also be useful in reducing the chance of insects developing resistance to available technologies. These experiments were conducted in collaboration with three major agribusiness companies. We are now focused on developing the next generation of our own technology, based on the use of our proteinase inhibitor technology with other inhibitory molecules.