Team:Valencia UPV

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Project overview (Draft)

Pests are one of the major problems in agriculture because their effect cause huge losses in this sector. Since pest control techniques relying on the use of pesticides are becoming less and less popular, because of the environmental damages they can cause, treatment with pheromones are gaining ground among pest management. Nowadays insect pheromones are chemically synthesised, which happens to be an expensive and complex process because of manufacturing costs and purification steps. In pursuit this line of thinking, our main efforts are aimed at avoiding the damage produced by pests in crops, specifically moths, which cause significant damage in crops all around the world. Moths lay eggs on the crops after mating, then the larvae eat the crops causing considerable damage. Our objective is to find an alternative method of pest control using pheromones.

Male moths find the females by tracing the pheromones they release into the air. For the attraction to be effective, female of a certain species must release the exact proportion of pheromone components. However, the strategy to be used is not based in attraction but in confusion. When the major pheromone component concentration in the environment reaches high levels, males are not able to find the females. This strategy is called mate disruption.

Our final objective is to create a synthetic plant which can produce a determined pheromone specifically in their trichomes, allowing its diffusion and secretion to the environment, causing mating disruption among moths (Lepidoptera). These plants would allow pest control simply sowing some of them around a crop field and inducing the pheromone production by applying a copper-rich nutrient solution before the insects mating season. In addition, due to their induced sterility, these plants would be completely safe to cultivate in the field and easily recognizable due to their anthocyanin production which would turn them purple.

PHEROMONE BIOSYNTHESIS

The first step in the creation of this synthetic plant is to define and construct the metabolic pathway of pheromone production.

"Most moth species utilize Type I pheromones that consist of straight-chain compounds 10-18 carbons in length with a functional group of a primary alcohol, aldehyde, or acetate ester, and usually with several double bonds, de novo synthesized in the pheromone gland through modifications of fatty acid biosynthetic pathways" [1].

Following the principle of modularity in synthetic biology, Hagström et al [2] produced the insect pheromone Z-11:160H in yeast from palmitic acid by introducing a delta-11 fatty-acyl desaturase and a fatty-acyl reductase from Agrotis segetum. Recently, Ding et al [3] had already done the same work taking into account that palmitate is synthesised in the chloroplast, leading to successful results.

In Ding's work they use a delta 11 desaturase mRNA from Amyelois transitella (accession number JX964774); HarFAR_KKYR, an improved version of HarFAR-3 fatty acid reductase from Helicoverpa armigera (accession number JF709978) with an almost five folds conversion rate, and 1,2-diacyl-sn-glycerol:acetyl-CoA acetyltransferase from Euonymus alatus (accession number GU594061). With these three enzymes we would obtain two pheromones: Z-11:16OH and Z-11:16OAc.

Moreover, inspired by Hagström's discussion we also thought about using an alcohol oxidase in order to try to obtain pheromone Z11-16:Ald from Z11-16:OH. According to the SolGenomics database, there is already a fatty-acid alcohol oxidase in Nicotiana benthamiana but there was no certainty that the enzyme would work in in vivo conditions. On the other hand, we learned that Candida tropicalis had a fatty alcohol oxidase that catalysed the conversion from Z11-16:Ald to Z11-16:O [4,5,6,7]. (At the moment it is not clear from which organism will the fatty acid alcohol oxidase be obtained).

At the same time we were planning which pheromones could we produce, we also took into account on which insects will they have effect. We did a thorough search in the Pherobase database (http://www.pherobase.com) and we checked which insect met the conditions of having one of those as a major pheromone component and being a plague.

TRICHOMES

Once the metabolic pathway needed to synthetize the desired pheromone has been properly described and characterized, a method that allows pheromone diffusion from the plant to the exterior environment must be implemented. With this objective in mind, we decided to express the enzymes required to produce the pheromones specifically in glandular trichomes, which are secretory structures naturally present in the plant. For doing so, the enzyme coding sequence would be under the control of CSP2 promoter from Nicotiana tabacum [8], inducing their specific expression in trichomes and allowing the pheromones secretion.

SWITCH

The pheromone production would suppose an additional energy cost to the plant, therefore the objective is to activate this pathway and the subsequent pheromone production only when necessary, upon Lepidoptera mating season. The regulation mechanism chosen with that purpose has been the one naturally controlling the CUP1 metallothionein gene in S. cervisiae [9]. CUP1 function is to chelate copper ions to avoid its toxicity to the yeast; therefore it is only expressed under high copper concentration. This regulation mechanism relies on the expression of a transcription factor (ACE1 or CUP2) that changes its conformation under high copper concentrations allowing its interaction with a regulating region (Metal Responsive Element, MRE) in the promoter of the CUP1 gene.

Taking as a reference this mechanism, Mett et al adapted it so it could be used in whole plants. Takanori Saijo and Akitsu Nagasawa [10] posteriorly improved its efficiency. We decided to take their work as an example and use a system in which the transcription factor CUP2 or ACE1 is expressed under the constitutive promoter P35S, and the enzymes responsible for the pheromone production are expressed under the regulation of a inducible promoter, composed of the MRE domain fused with the P35S minimal promoter. Therefore, CUP2 transcription factor would only interact with MRE and the pheromone will be produced under high copper concentrations. A copper-rich solution will be applied to the plant by foliar spraying or soil drenching during Lepidoptera mating season allowing pheromone production on demand.

BIOSAFETY

One of the concerns about having transgenic plants in the field involves its uncontrolled spread. It is being developed a biosecurity module containing two parts: a male sterility submodule and an identity preservation submodule.

The male sterility submodule is based on a barnase, an RNase gene from Bacillus amyloliquefaciens, specifically expressed in anthers under the regulation of the TA29 tapetum-specific promoter. As result, pollen from these plants will not be capable of fertilize plants. The identity preservation submodule will express Rosea1, a transcription factor that activates anthocyanin synthesis pathways, leading to their accumulation, so that plants containing the biosafety module will be easily differentiated from non-transgenic plants.

FINAL PLANT

Finally, our purpose is to find an ideal plant which gathers the special characteristics to lay out the implementation of the plant as a real solution of the problem. This ideal plant is still to be determined, and will probably vary depending on the geographical situation of the crop.

References