Quantitative Dissection of Agrobacterium Virulence to Generate a Synthetic Ti Plasmid

<i>Agrobacterium</i> is not only a costly plant pathogen but is also an essential tool for plant transformation. Though <i>Agrobacterium</i>-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 <i>Agrobacterium</i> gene expression <i>in planta</i>, we perform a quantitative dissection of AMT to investigate the contributions of critical <i>vir</i>-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.