Priya, S.; Rossbach, S.; Eng, T.; Andeer, P.; Lin, H.-H.; Mortimer, J.; Northen, T.; Mukhopadhyay, A.

Horizontal gene transfer (HGT) is a major process by which genes are transferred within microbes in the rhizosphere. However, examining HGT remains challenging due to the complexity of mimicking conditions within the rhizosphere. Fabricated ecosystems (EcoFABs) have been used to investigate several complex processes in plant associated environments. Here we show that EcoFABs are efficient tools to examine and measure HGT frequency in the rhizosphere. We provided the first demonstration of gene transfer via triparental conjugation system in the Brachypodium distachyon rhizosphere in the EcoFABs using Pseudomonas putida KT2440 as both donor and recipient bacterial strain with the donor having the mobilizable and non-self-transmissible plasmid. We also observed that the frequency of conjugal plasmid transfer in the rhizosphere is potentially dependent on the plant developmental stage, and composition and amount of root exudates. The frequency of conjugation also increased with higher numbers of donor cells. We have also shown the transfer of plasmid from P. putida to another B. distachyon root colonizer, Burkholderia sp. showing the possibility of HGT within a rhizosphere microbial community. Environmental stresses also influence the rate of HGT in the rhizosphere between species and genera. Additionally, we observed transfer of a non-self transmissible donor plasmid without the helper strain on agar plates when supplemented with environmental stressors, indicating reduced dependency on the helper plasmid under certain conditions. This study provides a robust workflow to evaluate conjugal transfer of engineered plasmids in the rhizosphere when such plasmids are introduced in a field or plant associated environment.