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The role of isomerisation of green leaf volatiles in fine-tuning tritrophic interactions

Final Report Summary - ISOSPIT (The role of isomerisation of green leaf volatiles in fine-tuning tritrophic interactions)

At first glance, plants are easy food for hungry herbivores. While carnivores often have to chase their prey and put up a struggle before they can eat, herbivores seem to have it much easier because plants don’t run away. However, herbivores don’t get their meals for free: plants do fight back. Plants can amongst others produce toxic compounds which are ingested by the herbivore during feeding, but they also have much more subtle defensive strategies: plants emit volatiles in response to herbivore damage thereby attracting predators of the herbivores as part of an indirect defense. One main group of plant volatiles is called green leaf volatiles (GLVs). These volatiles are known for their characteristic smell of freshly cut grass. GLVs consist of C6-aldehydes, alcohols and esters and are released in two different isomeric forms – cis (Z) and trans (E).
We showed that mechanically damaged leaves of the wild tobacco Nicotiana attenuata release high amounts of (Z)-3-GLVs and low amounts of (E)-2-GLVs. However, when the plant is attacked by its specialist herbivore, the tobacco hornworm (Manduca sexta), the chemical composition of the GLV bouquet dramatically changes characterized by a distinct shift from (Z)-3- to (E)-2-GLVs. This herbivore-induced change in the (Z)/(E)-ratio attracts the generalist predator Geocoris spp., and the approaching predator decreases the herbivore load on the plant by feeding on caterpillar eggs and early instar larvae. In this specific tritrophic interaction (N. attenuata – M. sexta – Geocoris), it is not the plant that is responsible for the conversion of the leaf aldehyde (Z)-3-hexenal, but an enzyme present in the oral secretions of Manduca caterpillars.
These results are quite surprising since it shows that caterpillars are betraying themselves by making the volatile bouquet more attractive to their own enemy. This raises an important question: why does Manduca produce an enzyme that generates volatiles that attract its own enemy and why did evolution not select against it?

We aimed (1) to identify and characterize the M. sexta GLV-isomerase enzyme responsible for the conversion of (Z)-3-hexenal to (E)-2-hexenal, and (2) to determine the physiological role of this enzyme during M. sexta’s life cycle and its ecological role in natural tritrophic interactions.
Our consecutive experimental objectives were: (1) to purify the isomerase from the oral secretions of M.sexta and to clone the corresponding cDNA and gene; (2) to characterize the GLV-isomerase enzyme and to search for homologs of the isomerase gene in other Lepidoptera and in plants; (3) to express the isomerase in transgenic plants; (4) to knock-down expression of the GLV-isomerase in M. sexta caterpillars and (5) to assess the effect of this on the caterpillars performance.

During the ISOSPIT project we have partially purified (3Z):(2E)-enal isomerase from 100mL oral secretions of Manduca sexta caterpillars by classical biochemical fractionation techniques. The purified fractions were analyzed by LC-MS/MS and resulted in the identification of 5 candidates. We amplified the full-length cDNAs of 4 candidates and cloned them in several destination vectors for expression in E.coli and plants. The recombinant protein of one of the candidates (MsISO1) showed isomerase activity, and this was confirmed by stably expressing the full-length cDNA of this candidate in two ecotypes of Arabidopsis (Col-0 and Ler) and transiently in Nicotiana benthamiana. The identification of the isomerase was a very important requirement for the whole project and we are pleased that we succeeded to do so, especially since the abundance of the enzyme in the oral secretion of the caterpillar appeared to be pretty low. We have furthermore determined MsISO1 expression in M. sexta during larval development and in different tissues of the caterpillar i.e. the midgut, malphigian tubuli and salivary glands. We are currently busy with the generation of caterpillars that either lack isomerase activity or show severely reduced activity.
Not only insects but also some plants are able to convert Z-3-hexenal to E-2-hexenal. Since blasting of MsISO1 against publicly available databases did not lead to any close orthologs we initiated a collaboration with a postdoctoral co-worker, dr. Eleni Spyropoulou, who, in an NWO-funded project,partially purified (3Z):(2E)-enal isomerase activity from cucumber fruits and identified several candidates. During the ISOSPIT project we expressed the most promising candidate cDNA from cucumber in E. coli and demonstrated that the recombinant protein has isomerase activity. Interestingly, insect and plant isomerase belong to two completely different gene families and show hardly any sequence homology.

The identification of an insect- and plant-derived (3Z):(2E)-enal isomerase is a major step forward for the research on GLV biosynthesis and the results of this analysis will facilitate our knowledge on the complexity and the mechanisms shaping plant-insect interactions in nature.
Although crop protection by various chemicals has been and is very successful, society demands less use of these chemicals. Using a plant’s own naturally evolved solutions to cope with biotic stress is one of the areas that can be explored for this purpose. By understanding the natural defense systems better, one can start to use this knowledge in breeding programs to address these issues. (Z)-3-hexenal is a good candidate compound for creating “green booby traps” due to its possibly multifunctional role in direct and indirect defense responses after conversion to (E)-2-hexenal. Since we have identified a MsISO1 homologous counterpart in plants (cucumber fruit), this can be the lead to novel insect defense strategies for a variety of crop plants. Especially tomato and potato (close relatives of Nicotiana), crops that suffer severe damage from insect larvae could benefit from these novel approaches.