PhD Dissertation Defense: Vivian Zhong

Title: Building Synthetic Biology Tools Towards Precision Engineering in Plants

Abstract: Agrobacterium-mediated transformation (AMT) has become the principal method for delivering DNA payloads into plant cells over the last few decades, underpinning both fundamental research and crop improvement across a wide range of species. In my first project, I developed a synthetic genetic tool to measure the impact of different transformation variables on the outcome of AMT as a step towards efficient site-specific genome engineering. In my second project, I leveraged AMT to generate transgenic plants capable of controlling the activity of associated soil microbes. 

Despite its versatility and utility, AMT has presented persistent problems for precision plant engineering. The integration locus of the transferred DNA (T-DNA) within the plant genome is effectively random, leading to variation in transgene expression strength, as well as potential knockout of native genes or even rearrangement of entire chromosomes. Thus, inhibiting non-specific T-DNA integration would greatly facilitate plant genome engineering. Site-specific approaches such as CRISPR editing cannot prevent this alone. We therefore developed T-SWITCH (T-DNA Sensing With Integrase Chassis) in the model organism Arabidopsis thaliana. Using the T-SWITCH system, we measured the effect of plant DNA repair factors, Agrobacterium virulence proteins, promoter choice, and binary vector architecture on T-DNA delivery efficiency and integration rate as distinct contributors to transformation efficiency. We found that most factors alter T-DNA delivery, but not integration. Finally, we leveraged T-SWITCH to achieve integration-free CRISPR editing in a single generation, introducing heritable mutations but not Cas9 T-DNA into the plant genome.

One application of genetically engineered plants that could enable a new generation of precision agricultural biotechnology is in controlling the activity of associated engineered microbes. Using O-methyl-L-tyrosine (OMY) as a model compound, we established non-canonical amino acids (ncAAs) as a tool for programming plant-microbe partnerships. We engineered plants to biosynthesize OMY for uptake by soil bacteria engineered via genetic code expansion for OMY-dependent protein synthesis. We showed that plant-derived OMY can stimulate gene expression in both model and wild soil bacteria while also demonstrating that inducible and tissue-specific expression of a single biosynthetic enzyme by the plant enables on-demand control over microbial activity. 

Please contact Sofia Rakicevic-More for the Zoom link.