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.

农杆菌（Agrobacterium）不仅是一类引发重大经济损失的植物病原菌，同时也是植物转化领域不可或缺的核心工具。尽管农杆菌介导转化（Agrobacterium-mediated transformation, AMT）已得到广泛研究，但其多基因特性与复杂的转录调控机制，使得通过传统遗传学与生物信息学方法解析其转化效率的遗传基础仍颇具挑战。
本研究采用自下而上的合成生物学策略，对编码绝大多数毒力系统的诱瘤质粒（tumor-inducing plasmid, pTi）开展系统性工程化改造。我们借助一套经过验证的工具套件在植物体内调控农杆菌的基因表达，对AMT过程进行定量解析，以探究不同表达水平下关键毒力基因（vir-genes）的功能贡献。
我们构建了可实现瞬时植物转化与稳定真菌转化的合成型pTi，并对复杂多基因合成型pTi的设计瓶颈与优化方案进行了系统表征。本研究的还原论研究方法证实，自下而上的工程化策略可用于解析并阐明复杂生物性状的遗传基础，为未来实现对AMT这一关键过程的全合成调控奠定了重要基础。