Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.
Our project aims to increase drought tolerance in Brassica napus (canola) by deploying engineered Arthrobacter globiformis as a delivery vehicle for siRNAs. A heat-inducible promoter will regulate siRNA production, ensuring that gene silencing is activated only under environmental stress conditions such as elevated soil temperatures during drought. The target gene, bHLH61, negatively regulates drought tolerance, and its knockdown is predicted to improve crop resilience.
To avoid antibiotic markers in the engineered bacteria, plasmid maintenance will rely on a non-antibiotic selection system based on thymidine auxotrophy. The engineered bacteria will be applied via seed coatings, allowing for colonization of the rhizosphere and localized siRNA delivery to plant roots, while minimizing environmental dispersal.
A pUB110 plasmid (or similar) compatible with Gram-positive soil bacteria will encode our genetic circuit: a heat-inducible promoter (dnaK) upstream of the siRNA hairpin targeting bHLH61, and a functional thyA expression cassette that complements a chromosomal thyA knockout in the host. Only cells that retain the plasmid express thyA and can synthesize dTMP, creating plasmid dependence without antibiotics. The heat-inducible promoter restricts siRNA transcription to drought-associated temperature conditions.
Seed coatings, such as alginate or pectin hydrogel, will be optimized to preserve bacterial viability and control initial exposure to the soil environment.
While we would like to build on our design of bacteria-mediated RNAi in canola, it has not been carried out yet as it involves multiple complex steps beyond the scope of our team. Our biggest hurdles are constructing a thyA auxotrophic Arthrobacter globiformis strain and knocking out RNase III. These steps require more time and skills than currently available. Therefore, we decided to pursue an alternative route to test some of our project ideas.
Our proof-of-concept will test whether bacterial systems can produce functional siRNAs that induce gene silencing in plants. RNase III-deficient Escherichia coli HT115 will be engineered to produce siRNAs against both RFP (in transgenic Arabidopsis) and bHLH61 (endogenous target). The bacterial production of siRNAs will be monitored via tsPurple expression. siRNAs purified from bacterial cultures will be applied to Arabidopsis thaliana seedlings to evaluate uptake and knockdown efficiency.
Our plasmid construct places a hairpin RNA sequence targeting RFP or bHLH61 downstream of the tsPurple reporter gene. Successful transcription produces both the chromoprotein and siRNA hairpin, linking reporter presence to siRNA output. siRNAs can be purified from E. coli cultures and applied to Arabidopsis by foliar spraying or root soaking. Knockdown will be measured by reduced RFP fluorescence in transgenic lines and phenotypic stress-response assays in wild-type plants targeting bHLH61. We will also test dnaK promoter activation by measuring tsPurple expression under different heat conditions.
Our experimental protocols can be found here. The first goal was to insert the siRNA sequences into the tsPurple plasmid using PCR; however, we were unable to verify successful insertion and must troubleshoot.
Concurrently, four Arabidopsis variants (GA-YFP, ER-YFP, SEC-RFP, AFVY-RFP) were grown to test viability (kind donation from Dr. Elizabeth Schultz, University of Lethbridge). Plants were placed in a growth chamber with a 22/8 hour light/dark cycle at 22°C. The RFP variants grew best.
We will continue plasmid engineering to insert the siRNA sequences and exchange promoters. Then we will test dnaK heat activation by quantifying tsPurple expression under different heat conditions. Additionally, we aim to produce large amounts of siRNA in E. coli, purify it, and apply it to Arabidopsis seedlings. These findings will support downstream engineering of field-deployable bacteria.