Restoring (E)-ß-Caryophyllene Production in a Non-producing Maize Line Compromises its Resistance against the Fungus Colletotrichum graminicola.

The sesquiterpene (E)-ß-caryophyllene is emitted from maize (Zea mays) leaves and roots in response to herbivore attack. This compound serves as a signal for the attraction of herbivore enemies and is present in most European maize varieties. However, most North American maize lines have lost the ability to produce (E)-ß-caryophyllene. Previously, we showed that restoring the ability to synthesize (E)-ß-caryophyllene in a non-producing maize line improved its resistance against the root herbivore Diabrotica virgifera virgifera. However, it is largely unknown whether this modification affects the resistance to other pests. In this study, we investigated the response of constitutively (E)-ß-caryophyllene-producing transgenic lines to infection by a hemibiotrophic fungus Colletotrichum graminicola. Our results showed that restoring (E)-ß-caryophyllene synthesis in a Hi-II genetic background enhanced the susceptibility of the plant to C. graminicola infection rather than increasing its resistance. This modification did not alter the baseline levels of plant defense hormones and metabolites. Nor did (E)-ß-caryophyllene production modify the expression of anti-fungal defense genes. Instead, the addition of (E)-ß-caryophyllene seemed to directly stimulate fungal growth. In an in vitro antifungal assay, we found that (E)-ß-caryophyllene stimulated hyphal growth of C. graminicola and Fusarium graminearum. Thus, although restoring (E)-ß-caryophyllene production in a non-producing maize line may improve the resistance of the plant against herbivores, it may compromise its resistance to major fungal pathogens. This might explain the loss of (E)-ß-caryophyllene during maize breeding in environments where C. graminicola and Fusarium graminearum are prevalent.

Can insect egg deposition 'warn' a plant of future feeding damage by herbivorous larvae?

Plant anti-herbivore defence is inducible by both insect feeding and egg deposition. However, little is known about the ability of insect eggs to induce defences directed not against the eggs themselves, but against larvae that subsequently hatch from the eggs. We studied how oviposition (OP) by the sawfly Diprion pini on Pinus sylvestris foliage affects the plant's defensive potential against sawfly larvae. Larvae that initiated their development on P. sylvestris twigs on which they hatched from eggs gained less weight and suffered higher mortality than those fed on egg-free twigs. The poor performance of these larvae also affected the next herbivore generation since fecundity of resulting females was lower than that of females which spent their larval development on egg-free pine. Transcript levels of P. sylvestris sesquiterpene synthases (PsTPS1, PsTPS2) were increased by D. pini OP, reached their highest levels just before larval hatching, and decreased when larvae started to feed. However, concentrations of terpenoid and phenolic metabolites presumed to act as feeding deterrents or toxins for herbivores did not change significantly after OP and feeding. Nevertheless, our performance data suggest that insect egg deposition may act to 'warn' a plant of upcoming feeding damage by larvae.

Species-specific responses of pine sesquiterpene synthases to sawfly oviposition.

Pinus sylvestris (Scots pine) is known to respond to eggs laid by the sawfly Diprion pini on its needles by releasing a blend of terpenoids, including the sesquiterpene (E)-beta-farnesene. These compounds attract a wasp, Closterocerus ruforum, which parasitizes sawfly eggs. D. pini oviposition also enhances the transcription of two sesquiterpene synthases, an (E)-beta-caryophyllene/alpha-humulene synthase (PsTPS1) and a 1(10),5-germacradiene-4-ol synthase (PsTPS2). To gain a better understanding of the function of these sesquiterpenes in promoting insect egg parasitism, we compared the outcome of D. pini oviposition on P. sylvestris with interactions between other pine and sawfly species: Neodiprion sertifer eggs on P. sylvestris, Gilpinia pallida eggs on P. sylvestris, D. pini eggs on Pinus nigra. The first of these attracts the parasitoid C. ruforum, while the latter two do not. As determined by quantitative real-time PCR, both PsTPS1 and PsTPS2 transcripts increased significantly only for those species combinations where the odor of egg-laden pine needles was attractive to C. ruforum. Moreover, enhanced transcription of these genes was found only at those time periods when odor was attractive, i.e. 3days after oviposition. Thus, the PsTPS1 and PsTPS2 genes are good markers for parasitoid attraction. We also characterized a sesquiterpene synthase from P. sylvestris (PsTPS5) which produces (E)-beta-farnesene, the compound previously determined to be responsible for C. ruforum attraction. However, transcript levels of PsTPS5 were not enhanced by oviposition of sawfly species that cause C. ruforum attraction. More research on this experimental system is required to determine the role of oviposition-induced sesquiterpenes in attracting egg parasitoids and the role of sesquiterpene synthases in regulating sesquiterpene formation.

Does egg deposition by herbivorous pine sawflies affect transcription of sesquiterpene synthases in pine?

Scots pine (Pinus sylvestris; Pinaceae, Pinales) is known to defend against egg deposition by herbivorous sawflies by changing its terpenoid volatile blend. The oviposition-induced pine odor attracts egg parasitoids that kill the sawfly eggs. Here, we investigated whether sawfly egg deposition activates genes encoding pine terpene synthases by extracting mRNA from oviposition-induced P. sylvestris. Three new sesquiterpene synthases, PsTPS 1, PsTPS 2, and PsTPS 3, were isolated that were shown on heterologous expression in Escherichia coli to produce (E)-beta-caryophyllene and alpha-humulene (PsTPS 1), 1(10),5-germacradiene-4-ol (PsTPS 2), and longifolene and alpha-longipinene (PsTPS 3) as their principal products. Quantitative RT-PCR analyses revealed that transcript levels of PsTPS 1 and PsTPS 2 were significantly higher in oviposition-induced twigs that were attractive to the parasitoids than in non-attractive, artificially damaged twigs. Thus, our results demonstrate a specific transcription response to egg deposition, distinct from that caused by artificial wounding. Transcripts of PsTPS 3 did not change in response to egg deposition. The transcript levels of PsTPS 1, PsTPS 2, and PsTPS 3 were also determined in relation to time after egg deposition, since pine odor is attractive to the parasitoid only 72 h after egg deposition. Transcription rates of PsTPS 1 and PsTPS 2 were significantly enhanced only 72 h after egg deposition, thus matching the timing of odor attractiveness, while for PsTPS 3, enhanced transcription was not detected at any time period studied after egg deposition. The ecological significance of the oviposition-induced increase of sesquiterpene synthase transcripts is discussed.