A plant factory for moth pheromone production

Abstract

Moths depend on pheromone communication for mate finding and synthetic pheromones are used for monitoring or disruption of pheromone communication in pest insects. Here we produce moth sex pheromone, using Nicotiana benthamiana as a plant factory, by transient expression of up to four genes coding for consecutive biosynthetic steps. We specifically produce multicomponent sex pheromones for two species. The fatty alcohol fractions from the genetically modified plants are acetylated to mimic the respective sex pheromones of the small ermine moths Yponomeuta evonymella and Y. padella. These mixtures are very efficient and specific for trapping of male moths, matching the activity of conventionally produced pheromones. Our long-term vision is to design tailor-made production of any moth pheromone component in genetically modified plants. Such semisynthetic preparation of sex pheromones is a novel and cost-effective way of producing moderate to large quantities of pheromones with high purity and a minimum of hazardous waste.

Figure 1. Production of moth pheromones in N. benthamiana leaf cells by transient expression of introduced genes.

Introduced enzymes and pathways are depicted in magenta. 16C pheromones are produced from preexisting cytosolic pool of 16:CoA whereas 14C pheromones are produced from a cytosolic 14:CoA pool created by introduction of a Cuphea palustris thioesterase intersecting chloroplast de novo acyl synthesis at 14C level. The genes for the enzymes involved in the pheromone production in this study were cloned from various moth and plant species: CpFATB2, Cuphea palustris 14:ACP thioesterase; AveΔ11, Argyrotaenia velutinana desaturase; CroΔ11, Choristoneura rosaceana desaturase; CpaE11, Choristoneura parallela desaturase; OnuΔ11, Ostrinia nubilalis desaturase; AtrΔ11, Amyelois transitella desaturase, HarFAR, Helicoverpa armigera acyl reductase; YroFAR, Yponomeuta rorellus acyl reductase, OnuFAR_E/Z, Ostrinia nubilalis acyl reductase, EaDAcT, Euonymus alatus acetyltransferase. Acyl intermediates in the pathway (also throughout the article) are given as short forms, for instance, E/Z11-14:CoA refers to the fatty-acyl coenzyme A with a chain length of 14-carbon atoms and a double bond at Δ11 position in ‘E’ or ‘Z’ configuration. ACP, acyl carrier protein; FFA, free fatty acid; OH, fatty alcohol; OAc, acetate.

Figure 2. Optimized pathways for moth pheromone production.

Expression of four (CpFATB2, AveΔ11, HarFAR, EaDAcT) or three (AtrΔ11, HarFAR, EaDAcT) genes in two different tobacco plants, led to the production of 14-carbon (14C) or 16-carbon (16C) pheromone components. Amounts (numbers in magenta) are μg of products recovered from one gram of fresh leaf tissue in the respective treatments.

Figure 3. Test of attractiveness of plant-derived moth pheromones.

Trap catches of males of two small ermine moth species obtained with synthetic pheromone (positive control), plant-derived pheromone and blank traps (negative control) (N=5). Plant-derived pheromones were prepared by acetylation of corresponding alcohols produced from the 14C and 16C treatment, respectively. The treatment producing 14C compounds was used alone for trapping Yponomeuta evonymella, and a mixture of the two treatments producing 14C and 16C compounds was used for trapping Y. padella. The synthetic baits for Y. evonymella contained 14:OAc/E11-14:OAc/Z11-14:OAc in a blend ratio of 187:45:100, and the synthetic baits for Y. padella contained 14:OAc/E11-14:OAc/Z11-14:OAc/Z11-16:OAc in a ratio of 187:45:100:400. The baits of plant-derived compounds contained above-mentioned acetates and minor by-products. Moth catches were species specific with both synthetic and plant-derived pheromone. In the case of Y. evonymella, the plant-derived pheromone was not as effective as the synthetic pheromones (t-test, P=0.012), whereas for trapping of Y. padella, the plant-derived pheromones showed no difference from the synthetic pheromones (t-test, P=0.896). The blank control traps did not catch any moths. Error bars represent s.e.m. (standard error of mean). NS, not significant; *, significant (P<0.05).