Expanding the Utility and Host Range of Ensifer-Mediated Transformation (EMT): A Novel Platform for Engineering Plant Genomes

To identify new plant associated bacteria strains that could be used as a substitute for Agrobacterium tumefaciens in existing transforming protocols, a functional screen using the open source vector pCAMBIA5105 was performed. “OV14”, a strain of the Gram negative, non‑pathogenic species Ensifer adhaerens was successfully isolated and has been confirmed to have the ability to stably integrate T‑DNA into dicotyledonous and monocotyledonous genomes^{1, 2}. Comparative sequencing of the 7.7Mb genome of OV14 (versus A. tumefaciens C58) revealed that the OV14 genome contains two plasmids and two chromosomes in which several homologs to chromosomally based Agrobacterium genes that support T-DNA transfer were present³. Antibiotic profiling of OV14 showed its susceptibility/resistance to 14 antibiotics, with a strong resistance to kanamycin identified⁴. This is currently being addressed by editing (via CRISPR/Cas9) two homologs (AHK42288 and AHK42618) whose transcription was significantly elevated within 2h exposure to kanamycin⁴. To date the confirmed host range of Ensifer-Mediated Transformation (EMT) includes potato, tobacco, canola, rice, cassava and safflower (verified via southern/sequencing) with transient assays indicating potential with wheat, barley, maize and soybean. Present research is focussed on (i) expanding the host range of EMT (ii) confirming EMT as a suitable platform for crop genome editing tools and (iii) examining the presence/absence of genotype dependency in select crops.

 1: Wendt et al., Transgenic research 21, no. 3 (2012): 567-578

2: Zuniga-Soto et al., SpringerPlus 4.1 (2015): 1-10

3: Rudder et al. BMC Genomics, (2014): 15, 268, 1-17.

4: Rathore et al., FEMS Microbiology Letters 362.17 (2015): fnv126.