Agrobacterium tumefaciens: A Bacterium Primed for Synthetic Biology

Abstract

Agrobacterium tumefaciens is an important tool in plant biotechnology due to its natural ability to transfer DNA into the genomes of host plants. Genetic manipulations of A. tumefaciens have yielded considerable advances in increasing transformational efficiency in a number of plant species and cultivars. Moreover, there is overwhelming evidence that modulating the expression of various mediators of A. tumefaciens virulence can lead to more successful plant transformation; thus, the application of synthetic biology to enable targeted engineering of the bacterium may enable new opportunities for advancing plant biotechnology. In this review, we highlight engineering targets in both A. tumefaciens and plant hosts that could be exploited more effectively through precision genetic control to generate high-quality transformation events in a wider range of host plants. We then further discuss the current state of A. tumefaciens and plant engineering with regard to plant transformation and describe how future work may incorporate a rigorous synthetic biology approach to tailor strains of A. tumefaciens used in plant transformation.

Figure 1

Future steps to improve AMT via synthetic biology: (a) development of a synthetic biology toolkit for A. tumefaciens will require the characterization of both constitutive and inducible promoters. Inducible promoter systems will need to have the dose response of inducers tested, in addition to precise titration of TF expression. Toolkits will also require distinct compatible origins of replication that should vary in copy number. (b) Once in place, vir genes can be precisely expressed to achieve desired titers of key virulence genes. Depending on the desired transformation outcome and host plant, the virulence complement can be engineered to achieve specific goals. (c) Complimentary synthetic biology tools encoded by transferred T-DNA will further modulate host gene expression and plant regeneration. Catalytically inactive or mRNA-targeted Cas9 variants can be used to silence multiple host genes without generating heritable DNA editing. In parallel, expression of Bbm and WUS increases plant regeneration and can be combined in tandem with other host regulatory proteins to manipulate pathogenesis. Expression of Cre recombinase under the control of a seed-specific or chemically inducible promoter excises T-DNA regions flanked by loxP sites to generate marker-free plants in the T1 generation.