Control of T-DNA Transfer from Agrobacterium tumefaciens to Plants Based on an Inducible Bacterial Toxin-Antitoxin System

Abstract

High-value pharmaceutical products are already successfully produced in contained facilities using Agrobacterium-mediated transient transformation of plants. However, transfection methods suitable for open field applications are still desirable as a cheaper alternative. Biosafety concerns related to the use of recombinant agrobacteria in an industrial transfection process include possible transformation or transfection of unintended hosts or spread of the genetically modified agrobacteria in the environment. In this paper, we explored a novel biocontrol approach resulting in greater biosafety of the transient expression process in plants. Our proposed solution involves inducible expression of Agrobacterium tumefaciens toxin PemK and antitoxin PemI that provides for strictly regulated T-DNA transfer from agrobacteria to plants. We also identified several other toxins from putative Agrobacterium toxin-antitoxin modules and demonstrate their potential usefulness in the control of Agrobacterium tumefaciens as a DNA vector.

Biology and evolution of bacterial toxin–antitoxin systems

“…toxin-antitoxin systems can be adopted as kill-switch modules, providing an attractive solution to contain various genetically modified organisms 217,226 . Gene-containment systems usually comprise toxin and antitoxin genes under the control of different promoters 217,227 . For example, the GeneGuard system encodes the toxin on the plasmid and the antitoxin on the chromosome, therefore coupling the plasmid to a specific host 228 .…”

Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

“…MazF-at (Atu0940) was identified as pemIK TA system in the previous study [15]. They found six candidates of TA modules in A. tumefaciens C58, and five of those six candidates showed growth inhibition in A. tumefaciens gv3101 strains [15]. In this study, we further evaluated whether the two genes, mazE-at and mazF-at, function as a TA system.…”

“…The two proteins, MazE-at and MazF-at, consist of 89 and 119 amino acid residues, respectively, and the pI values are 5.35 and 8.31, respectively (Figure 1a). MazF-at (Atu0940) was identified as pemIK TA system in the previous study [15]. They found six candidates of TA modules in A. tumefaciens C58, and five of those six candidates showed growth inhibition in A. tumefaciens gv3101 strains [15].…”

Microbial Biocontainment Systems for Clinical, Agricultural, and Industrial Applications

“…This gene is kept silent until desired and is stably controlled by an inducible promoter. To enhance the steadiness of the system, an antitoxin gene is also integrated under the control of a constitutive promoter (Contreras et al, 1991;Denkovskienė et al, 2020;Hayes and Kędzierska, 2014;Molin et al, 1987;Morita et al, 2001;Pasotti et al, 2011;Peng et al, 2019;Stirling et al, 2017;Zhou et al, 2019). The antitoxin prevents any leaked toxin from triggering cell death, and its suppression is only overcome upon the expression of the toxin (Figure 2B).…”

“…Hence, the delivery of this bacterium in crop fields is strictly controlled by daylight, which interferes with the development of new spores for propagation (Park et al, 2017). In parallel, other organisms like Agrobacterium tumefaciens has been engineered to integrate a toxin-antitoxin system based on the inducible expression of PemK and PemI; while this system has been employed so far for the in situ engineering of plants, it could be used for the delivery of synthesized hormones and stimulants (Denkovskienė et al, 2020).…”