Team:Valencia UPV/Project/results/biosafety

Project > Results > Biosafety

Our goal was to develop a sterile and easily identifiable plant. In order to do this, we created a module in collaboration with NRP-UEA-Norwich which incorporated an RNAse (barnase) under the regulation of a tapetum specific promoter (TA29) and a chromoprotein

Male Sterility strategy was not possible to test in a transient approach, but both components are well documented (see Biosafety Module).

The NRP-UEA-Norwich provided us with blue and yellow chromoproteins transcriptional units (TU) and we agroinfiltrated both TUs in N. benthamiana along with a GFP control. Chromoprotein detection was impossible by the naked eye (Figure 1) even though GFP control was expressed (Figure 2). Plants agroinfiltration was correct since GFP was expressed in the leaf.

Nevertheless, identity preservation is an important part of the biosafety module, and it must be happen efficiently. NRP-UEA-Norwich team suggested leaf degreening in order to observe the chromoproteins, but we don’t consider this strategy convenient for our purpose since identity should be easily recognisable in plants without any kind of treatment. As an alternative to chromoproteins, we propose the use of two transcriptional factors to enhance anthocyanins production.

Our identity preservation construct consists of two transcriptional units carrying the tomato (Solanum lycopersicum) transcriptional factors (SlANT1, SlJAF13) which are regulated by the 35S constitutive promoter. Both transcriptional factors are involved in flavonoids biosynthetic pathway regulation; it is that they enhance anthocyanin accumulation.

SlANT1 and SlJAF13 are S. lycopersicum orthologous of the Antirrhinum majus Rosea1 and Delila genes. In previous researchs, ectopic over-expression of these transcription factors under the control of the E8 fruit-specific promoter increases the transcript levels of most of the anthocyanin biosynthetic genes in tomato fruit leading to high levels of anthocyanins [1,2]. In N. benthamiana, transient expression of SIANT1 and SIJAF3 activates the expression of several flavonoid biosynthetic genes leading to a change of the colour on the leaf due to accumulation of anthocyanins.

We transiently transformed this new identity preservation module (containing SIANT1 and SIJAF13 transcriptional units) by agroinfiltration into N. benthamiana. As result, the anthocyanin accumulation drives our plant to a violet colour change that can be observed by the naked eye (Figure 3).

As explained in the methodology section (see Methodology: Electroantennography) we performed an electroantennography (EAG) to test the moth response to pheromones. Insects can detect pheromones through their antennae, then an electrical impulse is transmitted from them to the brain in order to trigger moth response to the pheromones. The EAG allows us to detect these electrical impulses by connecting one insect antenna to two electrodes that will amplify this impulse in order to be detected.

We connected one antenna from a male moth, Sesamia nonagrioides , with the two electrodes. Then , an air current with a leaf extract containing our pheromones was applied (Figure 2. Signal 1). As it can be appreciated, as the extract was applied the antenna transmitted an electrical impulse. This was the moth response to our insect pheromones produced in plant.

Figure 1. Electroantennography.

As a control, we also applied an air current with no pheromones in suspension. (Figure 2. Signal 2) The antena did not transmit any electrical signal.

With these results, we can positively say that moths respond to our pheromones produced in genetically engineered Nicotiana benthamiana plants.