May 23, 2012
Empoasca sp. is not a typical pest of wild tobacco (Nicotiana attenuata). When this plant grows in its natural habitats in North America, however, it is attacked by tobacco hornworm larvae (Manduca sexta). This specialist insect is resistant to the toxic nicotine, which the plant produces as a defence against its enemies. When researchers from the Max Planck Institute for Chemical Ecology used particular transgenic plants in field experiments, they noticed that these plants were heavily infested with Empoasca leafhoppers in comparison to wild-type plants. In the particular transgenic plants used in this study, a certain gene, lox3, had been switched off which is essential for the production of jasmonic acid. Due to their inability to produce jasmonates, the plants could not activate their defences against herbivores, because their hormonal signalling cascades were interrupted. The result of this deficiency was visible and had been expected: a heavy infestation by tobacco hornworm larvae. The occurrence of leafhoppers, however, was a surprise, because these insects are not a part of the plant’s normal herbivore community. The scientists speculated that these insects which are common pests of agricultural crops may have been able to evaluate the defensive potential of their host plants before the plants could activate the production of their defences.
To test this hypothesis, the scientists produced different transgenic tobacco lines and used them in field experiments. In six lines, the expression of specific enzymes involved in jasmonate production was blocked or the perception of the jasmonate signal was inhibited, and in three lines the production of jasmonate-elicited toxins was interrupted. The individual genes were switched off by using the inverted-repeat gene-silencing technique. Together with control plants, all lines were grown in their natural habitat, the Great Basin Desert in Utah, USA.
Leafhoppers were attracted by growing alfalfa (Medicago sativa), one of their favourite host plants. When the alfalfa plants were highly infested, they were cut down to motivate the leafhoppers to move into the tobacco field, which was adjacent to the alfalfa field. The following parameters were recorded: The prevalence and intensity of leaf damage on the individual lines and control plants, the corresponding jasmonate levels, the concentration and the occurrence of defence toxins, and finally the release of specific volatiles to indirectly fend off herbivores. Experiments conducted in the glasshouse back in Jena, Germany, were designed to quantify Empoasca leaf damage on transgenic plants, whose inability to produce jasmonate had been compensated for by applying jasmonate on their leaves. “We were able to demonstrate that leafhoppers’ preferred to feed on plants that were incapable of jasmonate signalling. Whether other defence substances, such as the toxin, nicotine, or digestion inhibitors, were present or not, was entirely irrelevant,” says Mario Kallenbach, who carried out these experiments.
These results demonstrated that Empoasca leafhoppers select their food plants after probing the leaves using their mouthparts to find out whether plants are ready for defence. Or more precisely: whether the jasmonate-based hormonal system responsible for signalling herbivory and initiating defences is functional or even present. If this is the case, the insect leaves the plant and causes no further damage (see picture in the middle). If jasmonate-signalling is defective, the plant is selected for feeding (see picture on the right). Interestingly, like prostaglandins, jasmonates belong to the family of oxygenated fatty acid derivatives and the leafhoppers’ behaviour resembles that of blood-sucking mosquitoes which explore their potential hosts’ functional or non-functional prostaglandin-regulated defence signals after they bite but before they start to take a blood meal. It is still unclear, however, what leafhoppers exactly detect when they probe the plants.
Hence, Empoasca feeding damage to individual plants in native plant populations could be an indicator of natural genetic variation in defence responses. Therefore, the scientists studied three different naturally grown Nicotiana attenuata populations − a total of about 700 plants −.over a period of two field seasons. They examined Empoasca damage in every single plant and found six infested plants. These plants were treated with oral secretions of the tobacco specialist Manduca sexta (tobacco hornworm), a treatment which triggers jasmonate-signalling. As a result, these plants showed a significantly lower jasmonate accumulation than uninfested control plants. Seeds of these plants were germinated and the offspring were again tested − with the same result. “Empoasca has identified for us valuable natural mutants for further experiments,” says Ian Baldwin, leader of the study.
Because Nicotiana attenuata uses fires to synchronize its germination from long-lived seed banks to grow in dense populations characterized by intense intraspecific competition and variable herbivore pressures, the scientists hypothesize that growth-defence tradeoffs are likely severe for this plant species, and these tradeoffs likely provide the selective pressure to maintain these JA-signalling mutants occurring in native populations − despite the clear disadvantages of being defence-impaired. “Once we have completed the sequencing of the Nicotiana attenuata genome, we will characterize in greater detail these JA-signalling mutants and are excited to see which genetic variations we will find”, Ian Baldwin continued.
The native tobacco plant Nicotiana attenuata is a model system studied in the Department of Molecular Ecology to scrutinize ecological interactions. By studying how this native plant has adapted to life in its ecological niche, the scientists hope to find means of increasing the ecological sophistication of our agricultural crops and develop lower input and more sustainable agricultural practices. The study presented here shows that the use of transgenic plants in field experiments is crucial for gaining new insights in the complexity of chemical and ecological interactions.