Introduction
The initial goal of Rhizoretention was to engineer rhizobacteria to assist canola in the toleration of drought. This would be through increased production of essential plant hormones and regulatory molecules in the chosen plant. Each following iteration of our project improved and specified the delivery mechanisms, host bacteria, and molecular targets. By using the engineering design cycle, we refined our approach from the very first conception to our current project’s design.
Iteration 1
Our team’s first design involved engineering rhizobacteria to produce five essential plant hormones: ABA, proline, trehalose, auxins, and polyamines. The ABA produced by the bacteria would trigger the plant to close its stomata, reducing water loss through transpiration. The proline and trehalose would accumulate in the canola plant cells, helping them retain water and prevent damage from dehydration. The auxins would stimulate the growth of deeper and more extensive roots, and polyamines would protect the plant’s cells from stress and damage. Genes encoding enzymes for these molecules would be inserted into the bacteria’s DNA. The engineered rhizobacteria would be introduced to the roots of plants using inoculation. Each bacterium would be equipped with a natural kill switch to self-destruct after a specific time or upon detecting a specific stimulus. With this design underway, our team sought the feedback of multiple experts through the Tech Futures Challenge (TFC) competition, where we presented our ideas. While the feedback was mostly positive, the judges expressed concerns about feasibility of producing this many hormones as well as about the biosafety of the proposed inoculation method of our project. Using these insights, our team came up with our second iteration.
Iteration 2
Pivoting from complex multi-pathway engineering in this second iteration, our team explored a simplified approach, focusing primarily on ABA production. For possible bacteriums, we identified E. coli as a candidate capable of expressing parts of the ABA biosynthetic pathway, particularly the zeaxanthin epoxidase enzyme. Other candidate bacteria such as Bacillus spp. and Azospirillum brasilense were considered for their root-colonizing ability. From our feedback, our team also decided to shift to a hydrogel-based water retention seed coating for both germination support and bacterial delivery instead of our first proposed delivery system. Inspired by MIT’s gel-like mucilage-mimicking coating, we selected sodium alginate for the outer layer due to its water retention properties. This encapsulation method involves alginate beads created via endogenous emulsification and nutrients like chitosan were planned to be added to sustain bacterial viability. Our team also began exploring the integration of RNA interference (RNAi), aiming to knock down plant genes that contribute to drought sensitivity. To further improve upon our project, the team reached out and consulted multiple experts, who reviewed what we had accomplished and gave us feedback. These reviewers asked whether canola might have different stomata specialized for gas exchange versus water regulation, and how this could influence plant respiration under drought conditions. They also recommended exploring other pathways regulated by abscisic acid (ABA), as well as understanding any potential side effects when manipulating drought-response genes. Additionally, we were encouraged to investigate whether ABA production could be naturally triggered by drought stress and how this might interact with our RNAi strategy. This feedback guided us to further refine our testing framework and to explore a more comprehensive analysis of stress-response pathways in canola.
Iteration 3
In our most current iteration of RhizoRetention, we plan to engineer the root-colonizing bacterium Arthrobacter globiformis to produce small interfering RNA (siRNA) designed to silence the bHLH61 gene. Previous work in the model plant Arabidopsis thaliana has demonstrated that overexpression of the transcription factor bHLH61 reduces drought tolerance, whereas plants with mutations in this gene exhibit improved drought resistance. Since an 86% homologous gene is present in canola, we hypothesize that targeted knockdown of bHLH61 expression can enhance drought tolerance in this crop.The bacteria will be incorporated into a seed coating, allowing them to establish themselves in the rhizosphere of developing canola plants. Once colonized, the bacteria will deliver double-stranded RNA (dsRNA) to the host, triggering RNAi and suppressing expression of the drought-sensitivity gene. In addition, the bacteria are expected to stimulate host stress response pathways, providing further protection under adverse conditions. To avoid unnecessary energy expenditure and ensure precise regulation, the siRNA expression cassette will be driven by the heat-inducible dnaK promoter. This promoter is activated during elevated temperatures associated with drought and heat stress, ensuring that siRNA is produced only when plants are most vulnerable. This stress-responsive system provides a targeted, sustainable approach to enhancing drought tolerance in canola, supporting food security in a changing climate. Once we had firmly established this design, our team authored a paper through Biotreks, where scientific experts of their respective fields reviewed our writings and provided feedback. Through the Biotreks platform, our team was provided valuable feedback and insights through detailed comments and questions. During the process, reviewers highlighted the need for deeper research into how bHLH61 functions in drought-response pathways while also encouraging exploring additional drought-related genes. The feedback also emphasized considering safe bacterial concentrations in seed coatings to avoid disrupting the soil microbiome and recommended clear testing strategies. Lastly, reviewers noted that methods should also be explored for greater efficiency and stability. With the help of the judge’s feedback and our testing progress (below) , we continue to modify our project to allow it to function in the most effective way possible
As a proof of concept for our current project, our team designed an engineered plasmid (pPSLA) which contains the heat-inducible dnaK promoter, RBS, tsPurple coding sequence and the sense, loop, antisense sequences for siRNA production. To test the functionality of our proposed design, we plan to insert this plasmid into RNase III-deficient E.coli and measure its tsPurple expression under different heat stress conditions in order to verify dnaK control conditions and parameters, and then compare our results to our kinetic models. As of now, our team has performed insertion mutagenesis PCR on the parent pSB1C3-tsPurple plasmid (using NEB Q5 mutagenesis protocol). We used the above primers to introduce two insertions into our parent plasmid. The PCR products were run on 1% agarose and compared to a wildtype plasmid, resulting in inconclusive results prompting further testing.
We also grew Arabidopsis thaliana lines carrying either GA-YFP, ER-YFP or SEC-RFP or AFVY-RFP reporter genes for proof-of-concept models for gene knockdown experiments, where successful silencing would result in a loss of fluorescence. Since these seeds were from older stocks, our first goal was to germinate and grow the plants to confirm their viability before proceeding with RNAi-based assays. The RFP variants grew the best and will be used for future experiments.
Through iterative design, our project evolved from general hormone production to targeted RNAi delivery using a native soil bacterium and hydrogel seed coating. This progression reflects our efforts to balance the biological complexity of our goals with real-world feasibility.