Quantitative Dissection of Agrobacterium Virulence to Generate a Synthetic Ti Plasmid

Agrobacterium is not only a costly plant
pathogen
but is also an essential tool for plant transformation. Though Agrobacterium-mediated transformation (AMT) has been heavily
studied, its polygenic nature and complex transcriptional regulation
make identification of the genetic basis of transformational efficiency
difficult through traditional genetic and bioinformatic approaches.
Here, we use a bottom-up synthetic approach to systematically engineer
the tumor-inducing plasmid (pTi), wherein the majority of virulence
machinery is encoded. Using a validated toolkit to control Agrobacterium gene expression in planta, we perform a quantitative dissection of AMT to investigate the
contributions of critical vir-genes at different
expression levels. We construct a synthetic pTi capable of transient
plant and stable fungal transformation and characterize bottlenecks
and solutions for complex polygenic synthetic pTi designs. Our reductionist
approach demonstrates how bottom-up engineering can be used to dissect
and elucidate the genetic underpinnings of complex biological traits,
laying the foundation for future engineering to establish full synthetic
control over the critical process of AMT.