Rubén Mateos-Fernández, Elena Moreno-Giménez, Silvia Gianoglio, Alfredo Quijano-Rubio, Jose Gavaldá-García, Lucía Estellés, Alba Rubert, José Luis Rambla, Marta Vazquez-Vilar, Estefanía Huet, Asunción Fernández-del-Carmen, Ana Espinosa-Ruiz, Mojca Juteršek, Sandra Vacas, Ismael Navarro, Vicente Navarro-Llopis, Jaime Primo,⁵ and Diego Orzáez

BioDesign Research; Published 12 Oct 2021; Article ID 9891082 | https://doi.org/10.34133/2021/9891082

Plant-based bioproduction of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here, we describe the engineering of transgenic plants producing (Z)-11-hexadecenol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them into Nicotiana benthamiana plants. The constructs contained the Amyelois transitella AtrΔ11 desaturase gene, the Helicoverpa armigera fatty acyl reductase HarFAR gene, and the Euonymus alatus diacylglycerol acetyltransferase EaDAct gene in different configurations. All the pheromone-producing plants showed dwarf phenotypes, the severity of which correlated with pheromone levels. All but one of the recovered lines produced high levels of Z11-16OH, but very low levels of Z11-16OAc, probably as a result of recurrent truncations at the level of the EaDAct gene. Only one plant line (SxPv1.2) was recovered that harboured an intact pheromone pathway and which produced moderate levels of Z11-16OAc (11.8 μg g^-1 FW) and high levels of Z11-16OH (111.4 μg g^-1). Z11-16OAc production was accompanied in SxPv1.2 by a partial recovery of the dwarf phenotype. SxPv1.2 was used to estimate the rates of volatile pheromone release, which resulted in 8.48 ng g^-1 FW per day for Z11-16OH and 9.44 ng g^-1 FW per day for Z11-16OAc. Our results suggest that pheromone release acts as a limiting factor in pheromone biodispenser strategies and establish a roadmap for biotechnological improvements.

See: https://spj.sciencemag.org/journals/bdr/2021/9891082/

Figure 1

Stable and transient expression in Nicotiana benthamiana of the synthetic moth pheromone pathway. (a) Schematic view of the T-DNA construct used for transient expression, carrying the three transgenes AtrΔ11, HarFAR, and EaDAct, each under the control of the constitutive CaMV35s promoter and terminator, and the biosynthetic route of the moth pheromones. (b) GC/MS analysis of the volatile profile of N. benthamiana transiently expressing the transgenes (blue line) and a mock infiltrated plant with only P19 (red line). Peaks corresponding to the target insect pheromones are indicated with a label. Highlighted in red is the region of the (Z)-11-hexadecenal peak. (c) Schematic view of the T-DNA construct for the SxPv1.0 encoding the three transgenes and two selection markers. The two selection markers DsRed and NptII are highlighted in red and purple, respectively. (d) Pheromone content in SxPv1.0 T₀ plants (numbered from 1 to 11). The diameter of each dot corresponds to the (Z)-11-hexadecenal level of each sample. Plants marked with a red cross died before seeds could be collected. (e) Overlapped chromatograms showing the volatile profile of a representative SxPv1.0 T₀ plant (blue line) and a WT plant (red line).