Two different types of Erwinia amylovora, wild and transformed with our composite aiiA BioBrick (BBa_K2471000), were inoculated on apple leaves. The disease (Fire Blight) began to be visible three days after inoculation and developed in a gradual and similar manner until the eighth day when the disease considerably increased its expansion through the leaf. However, differences in disease development (both the severity and onset time) were seen between the wild-type E. amylovora and the one transformed with the aiiA BioBrick. The assay consisted of cutting leaves and inoculating them with the previously mentioned types of E. amylovora -plus a negative control, which was inoculated with medium without inoculum-, in order to simulate close to real conditions and see how each of them evolved.

Erwinia amylovora’s virulence factors principally depend on its cellular density, N-Acyl homoserine lactones (AHLs) are responsible for and involved in the synthesis of many secondary metabolites, among them, exopolysaccharides like the amylovoran and levan. AHLs in E. amylovora have been proven to contribute to the expression of several virulence factors and disease development; similar to the role they play in other pathogenic bacteria. When incorporating the aiiA BioBrick (BBa_K2471000) into E. amylovora, leaf necrosis was inhibited, as shown in figure 3 when compared with the wild-type E. amylovora in figure 2. The production of the characteristic bacterial exudate of the disease was inhibited, which is composed of the amylovoran and levan EPS, and the infection that is presented by the type III secretion system dismissed.

The gene expressed is an N-Acyl homoserine lactonase, which is involved in the hydrolysis of AHLs. The degradation of these molecules stops the AHL-EamR complex from binding, which in turn, stops the activation of an operon responsible for the EamI transcription of intracellular AHLs and other genes that contribute to several virulence factors.

Phenotypic dismissal of Fire Blight was observed when the disease was caused by E. amylovora electroporated with our aiiA BioBrick compared to the wild-type E. amylovora. As seen in figure 3, these results show a noticeable decrease in disease development in the bacteria transformed with our BioBrick. Thus, we can conclude that our gene does reduce the decay caused by the disease in close-to-reality scenarios.