Project Description

Helping Canola overcome drought using synthetic biology

Intro

In Alberta, more than 14,000 farmers grow over 6.6 million acres of canola every year. Canola is Canada's most valuable crop, as it contributes over $29.9 billion annually to the economy. However, increasing challenges with climate change threaten the productivity of this sector. Conditions such as persistent drought have become a great concern to canola producers. Drought-resistant canola can be engineered through a collective approach combining plant-growth-promoting rhizobacteria and RNA interference (RNAi) to silence negative regulators of drought tolerance. A 2020 study using the model plant Arabidopsis thaliana showed that overexpression of bHLH61 impaired plant drought tolerance. However, the plants showed increased drought tolerance when this gene was mutated. There is a homologous gene found in canola, and we predict that by knocking down the expression of bHLH61 in canola, we can improve the drought tolerance of the crop. We will engineer Arthrobacter globiformis to express small-interfering RNA (siRNA) targeting the bHLH61 gene and incorporate the bacteria into the seed coating. The engineered bacteria can colonise the canola roots, delivering double-stranded RNA (dsRNA) to silence the bHLH61 gene to improve drought tolerance.

Project Overview

Southern Alberta is distinguished by its extensive farmland, variety of crops, and adoption of advanced farming methods. With over 10 million acres in production—more than 20% of Alberta’s farmland—the sector generates over $2.6 billion annually (Stahl & Gething, 2024). Between 2021 and 2023, Alberta’s agriculture industry expanded by 24%, highlighting the growing importance of protecting this sector (Stahl & Gething, 2024). However, climate change continues to place increasing stress on the region. Persistent drought, intensifying pest pressures, strong winds, and limited water availability have become major threats to productivity (Nickel & Williams, 2024). Historically, the fertile soils of Southern Alberta have supported crop and livestock production as well as agricultural processing, contributing $17.9 billion to Canada’s agri-food exports in 2023 (Calgary Economic Development, n.d.). These strengths, combined with a skilled workforce and cross-sector collaboration, position the region as a cornerstone of Canadian food security and economic resilience. Prolonged drought has become one of the most pressing issues in recent years. Consecutive dry seasons, diminished snowpack, and declining reservoir levels have resulted in Southern Alberta entering Stage 4 (of 5) in the province’s Water Shortage Management Plan (Government of Alberta, 2025). This stage signals extensive water shortages across multiple management areas, long-term deficits in supply, and severe impacts for agricultural producers and households (Government of Alberta, 2023). As a result, the agriculture sector is increasingly constrained by water allocation limits and faces higher risks of soil erosion (Stephenson, 2024). Among the region’s leading crops is canola. Cultivated on more than 6.6 million acres by roughly 14,000 farmers, it contributes $4 billion annually to Alberta’s economy (Canola Statistics - Alberta Canola, n.d.). As a valuable export for both domestic and international markets, canola is especially vulnerable to drought stress. Forecasts suggest a 30–40% decline in national production due to water shortages, with Southern Alberta at particular risk because of its semi-arid conditions and limited natural water bodies (Farmonaut, 2024). Water deficits during early plant development reduce both yield and oil quality, with serious financial consequences. Although research into drought-resistant canola varieties is underway, the crop’s water requirement of at least 400 mm throughout its lifecycle underscores the challenge (Alberta Agriculture & Rural Development, 2011). Current strategies include government water-sharing agreements and the adoption of agricultural technologies. In April 2024, Alberta secured a major agreement among South Saskatchewan River Basin water users to manage drought risk (Alberta Law Review, 2024). While these arrangements provide a temporary framework for distributing scarce water resources, their reliability is limited. Dependence on other jurisdictions and the unpredictability of climate patterns mean that these agreements may quickly lose effectiveness.

Schematic diagram

At the physiological level, drought stress in canola (Brassica napus) disrupts a range of processes such as photosynthesis, stomatal function, transpiration, protein synthesis, and metabolite accumulation (Zhu et al., 2016). These disruptions ultimately lower seed yield and quality. Transcription factors in the bHLH protein family are known to regulate plant stress responses, though their precise mechanisms are not fully understood. In Arabidopsis thaliana, overexpression of bHLH61 reduced drought tolerance, whereas loss of function mutations enhanced tolerance. A homologous gene in canola has been linked to cold tolerance, suggesting a similar role in stress adaptation (Wang et al., 2022). Based on this evidence, targeting bHLH61 expression in canola may provide a viable strategy for enhancing drought tolerance (Wang et al., 2021).

Our project proposes to knock down the expression of bHLH61 in canola using bacteria-mediated RNAi. Our engineered bacteria will be incorporated into the canola seed coating, then incorporated into the plant’s rhizosphere, where they will produce double-stranded hairpin RNA that targets the bHLH61 gene in canola, thus increasing drought tolerance.

Project Design

Seed Coating

Seed coating involves applying small amounts of external materials—such as insecticides, fungicides, nutrients, and biostimulants—onto seeds. Including plant-beneficial microbes in these coatings can enhance germination rates, increase crop yields, and reduce the need for additional fertilization. In this project, engineered bacteria would be incorporated into the canola seed coating, providing a cost-effective method for microbial delivery. For improving water retention in plants, biopolymer-based hydrogel coatings are particularly effective, especially those designed to mimic natural seed mucilage. These programmable coatings can activate rhizobacteria after sowing, leading to the formation of symbiotic, water-stress-tolerant root nodules—a feature especially valuable in semi-arid conditions (Zvinavashe et al., 2021). Hydrogels made from sodium alginate and pectin have demonstrated strong water-holding capacity while maintaining favorable microbial activity for over a month. When enriched with NPK and micronutrients, these coatings substantially promote root and shoot development (Skrzypczak et al., 2021). Starch-based hydrogels can support initial growth under moderate water stress, but their benefits diminish after root establishment. Overall, hydrogel-based seed coatings can be customized with beneficial microbes to improve plant growth and optimize water use efficiency.


Arthrobacter EM

Microorganisms naturally colonize plant root environments and can establish symbiotic relationships with their host plants. Certain microbes assist with nutrient uptake, suppress pathogenic colonization, and help plants cope with environmental stresses (Lay et al., 2018). A 2018 study of canola root microbiomes identified numerous rhizobacteria species, including Amycolatopsis, Serratia proteamaculans, Pedobacter, Arthrobacter, Stenotrophomonas, Fusarium merismoides, and Fusicolla (Lay et al., 2018). For this project, we plan to engineer Arthrobacter globiformis to express siRNA targeting canola stress response genes, and these bacteria will be incorporated into the seed coating.

Arthrobacter is a genus of Gram-positive soil bacteria commonly found in the indigenous microbial communities of soil and rhizospheres. Being a native component of the canola root microbiome, it supports efficient root colonization and maintains long-term stability in the soil. Standard microbiology and molecular biology methods can be used to culture and transform Arthrobacter species with plasmids carrying genes of interest. It is classified as a biosafety level one organism, posing no risk to human health or the environment. To enable siRNA production, however, the RNase III gene must be deleted from the Arthrobacter genome.

Canola Knockdown RNAi

RNAi is a naturally occurring immune response against viral infections and can occur through multiple pathways (Baulcombe, 2004). Our team plans to utilize the siRNA pathway to target a gene within canola that is related to the expression of stress response proteins, specifically bHLH61. The siRNA is generated through transcription from a plasmid as a double-stranded hairpin precursor. It is then processed into the active siRNA sequence by Dicer. The siRNA is then integrated into the RNA-induced silencing complex (RISC), which can then scan mRNA to find the correct corresponding target sequence. Upon base pairing, the messenger RNA (mRNA) is degraded, leading to a loss of protein expression in the target plant (Svoboda, 2020).


arabidopsis knockdown

The basic helix-loop-helix (bHLH) gene family codes for the largest group of transcription factors in plants, meaning they help turn genes on and off in plants, but their functions are poorly characterized. In canola (Brassica napus), more than 600 potential bHLHs have been identified and categorized into 35 subfamilies (Ke et al., 2020). The bHLH gene family is highly conserved across plant species and is involved in growth and development, responses to light, drought, and pests, among others.
A 2020 study using the model plant Arabidopsis thaliana showed that overexpression of bHLH61 impaired plant drought tolerance, but when this gene was mutated, the plants showed increased drought tolerance (Wang et al., 2021). There is a homologous gene found in canola that codes for a hypothetical transcription factor (NIH, n.d.) and has previously been shown to be involved in cold tolerance in the related cultivar Brassica campestris (Wang et al., 2022).
Since transcription factors, like bHLH61, often have multiple roles, and knocking them down could lead to unintended consequences, transcriptomic profiling will be used to assess global gene expression changes and compared to the control samples. Additionally, phenotypic analyses under different stress conditions will be completed to hopefully catch any context-specific effects like cold sensitivity.

Testing

arabidopsis knockdown growth chamber

For our proof of concept, we plan to create our siRNA by utilizing RNase III-deficient E. coli HT115. This strain does not break down the siRNA once transcribed. The bacteria will then be used to treat Arabidopsis under normal and drought conditions. This will allow us to monitor siRNA uptake, siRNA levels in the soil, and observe gene knockdown in a controlled environment before field deployment.



For more information on our Proof-of-Concept Testing, please visit our Engineering page.

Sustainability

UN Sustainability Goals

RhizoRetention supports UN Sustainability Goal 2, to promote sustainable agriculture. Our project also addresses Goal 12, by reducing the need for intensive irrigation and fertilization and UN Sustainability Goal 13, Climate Action by strengthening agricultural resistance to drought and reducing crop losses. Our project avoids genetic modification of the crop while also encouraging sustainable and environmentally friendly practices. Arthrobacter globiformis is also non toxic and poses no threat to human individuals or surrounding environments (Environment and Climate Change Canada, 2018).

References

  1. Baulcombe, D. (2004, November 16). RNA silencing in plants. Nature, 431, 356-363.
  2. Calgary Economic Development. (n.d.). Agribusiness: Calgary is an agriculture-based city. https://www.calgaryeconomicdevelopment.com/sectors/agribusiness/
  3. Callaghan, M., Wright, D., Swaminathan, J., Young, S., & Wessman, P. (2012). Microbial inoculation of seed - issues and opportunities. Agronomy New Zealand, 42.
  4. Canola Statistics - Alberta Canola. (n.d.). Alberta Canola Producers Commission. Retrieved April 25, 2025, from https://albertacanola.com/about/canola-statistics/
  5. Darlington, M., Jurat-Fuentes, J. L., Kogel, K.-H., Rathore, K. S., Smagghe, G., & Whyard, S. (2024). RNA Interference in Agriculture: Methods, Applications, and Governance. CAST. https://doi.org/10.62300/IRNE9191
  6. Environment and Climate Change Canada. (2018). Final screening assessment for Arthrobacter globiformis. https://www.canada.ca/en/environment-climate-change/services/evaluating-existing-substances/screening-assessment-arthrobacter-globiformis.html
  7. Gong, M., He, J., Kong, M., Huo, Q., Jiang, Y., Song, J., Han, W., & Lv, G. (2023). A microencapsulation approach to design microbial seed coatings to boost wheat seed germination and seedling growth under salt stress. Frontiers Plant Science, 14.
  8. Goodfellow, S., Zhang, D., Wang, M.-B., & Zhang, R. (2019). Bacterium-Mediated RNA Interference: Potential Application in Plant Protection. Plants, 8(12). https://www.mdpi.com/2223-7747/8/12/572
  9. Government of Alberta. (2023, August). Water Shortage Management Stages. PDF
  10. Government of Alberta. (2025). Drought - Current Conditions. https://www.alberta.ca/drought-current-conditions
  11. Halvorsen, T. M., Ricci, D. P., Park, D. M., Jiao, Y., & Yung, M. C. (2022). Comparison of kill switch toxins in plant-beneficial Pseudomonas fluorescens reveals drivers of lethality, stability, and escape. ACS Synthetic Biology, 11(11), 3785–3796. https://doi.org/10.1021/acssynbio.2c00386
  12. Ke, Y.-Z., Wu, Y.-W., Zhou, H.-J., Chen, P., Wang, M.-M., Liu, M.-M., Li, P.-F., Yang, J., Li, J.-N., & Du, H. (2020). Genome-wide survey of the bHLH super gene family in Brassica napus. BMC Plant Biology, 20. https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-020-2315-8#:~:text=Conclusion,in%20future%20gene%20function%20research
  13. Kleter, G. A. (2020). Food safety assessment of crops engineered with RNA interference and other methods to modulate the expression of endogenous and plant pest genes. Pest Management Science, 10.1002/ps.5957
  14. Lay, C.-Y., Bell, T. H., Hamel, C., Harker, K. N., Mohr, R., Greer, C. W., Yergeau, E., & St-Arnaud, M. (2018, June). Canola Root–Associated Microbiomes in the Canadian Prairies. Frontiers Microbiology, 9. https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.01188/full#B73
  15. Nickel, R., & Williams, N. (2024, March 18). Farmers, oil drillers in parched Alberta brace for water shortage. Reuters. https://www.reuters.com/business/environment/farmers-oil-drillers-parched-alberta-brace-water-shortage-2024-03-18/
  16. NIH. (n.d.). Nucleotide BLAST. https://www.ncbi.nlm.nih.gov/nuccore/XM_013876193.3?report=fasta
  17. NIH. (n.d.). https://www.ncbi.nlm.nih.gov/gene/830922
  18. O'Kennedy, S. (2022). Soil pH and its impact on nutrient availability and crop growth. International Journal of Geography, Geology and Environment, 4(2), 236-238.
  19. Rajput, M., Choudhary, K., Kumar, M., Vivekanand, V., Chawade, A., Ortiz, R., & Pareek, N. (2021). RNA Interference and CRISPR/Cas Gene Editing for Crop Improvement: Paradigm Shift towards Sustainable Agriculture. Plants, 10(9). https://www.mdpi.com/2223-7747/10/9/1914
  20. Roberts, A., Devos, Y., Lemgo, G. N.Y., & Zhou, X. (2015). Biosafety research for non-target organism risk assessment of RNAi-based GE plants. Frontiers Plant Science, 6. https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2015.00958/full
  21. Rueda-Silva, J. C., Gonzalez-Campos, L. I., Duran-Armenta, L. F., Karam-Coppola, A., Antonio-Perez, A., Ordonez-Rodriguez, J., Saucedo-Tavitas, N. E., Rico-Torres, V., Cruz-Cruz, C., Garcia-Huante, Y. G., Amador-Molina, J. C., Tapia-Ramirez, J. I., & Torres-Huerta, A. L. (2023). Novel bacterial plasmid produces small interfering RNAs (siRNAs) that induce effective gene silencing in the Asian citrus psyllid Diaphorina citri. Electronic Journal of Biotechnology, 64, 59-68. https://doi.org/10.1016/j.ejbt.2023.03.006
  22. Scheberl, L., Scharenbroch, B. C., Werner, L. P., Prater, J. R., & Fite, K. L. (2019). Evaluation of soil pH and soil moisture with different field sensors: Case study urban soil. Urban Forestry & Urban Greening, 38, 267-279. https://doi.org/10.1016/j.ufug.2019.01.001
  23. Skrzypczak, D., Jarzembowski, L., Izydorczyk, G., Mikula, K., Hoppe, V., Mielko, K. A., Pudelko-Malik, N., Mlynarz, P., Chojnacka, K., & Witek-Krowiak, A. (2021). Hydrogel Alginate Seed Coating as an Innovative Method for Delivering Nutrients at the Early Stages of Plant Growth. Polymers, 13. https://doi.org/10.3390/polym13234233
  24. Stahl, H., & Gething, K. (2024). Economic Impact of Agriculture on Southeast Alberta (2024). C4A Centre for Innovation, 1(1). PDF
  25. Stephenson, A. (2024, January 22). Farmers in Alberta face a growing risk of soil erosion events as drought persists. The Canadian Press. https://globalnews.ca/news/10241453/alberta-farmers-soil-erosion-risk/
  26. Svoboda, P. (2020). Key mechanistic principles and considerations concerning RNA interference. Frontiers in Plant Science, 11, 1237. https://doi.org/10.3389/fpls.2020.01237
  27. Vélez Arango, A. M., Darlington, M., Jurat-Fuentes, J. L., Kogel, K.-H., Rathore, K. S., Smagghe, G., & Whyard, S. (2024). RNA Interference in Agriculture: Methods, Applications, and Governance. Council for Agricultural Science and Technology. https://doi.org/10.62300/IRNE9191
  28. Wang, C., Zhang, M., Zhou, J., Gao, X., Zhu, S., Yuan, L., Hou, X., Liu, T., Chen, G., Tang, X., Shan, G., & Hou, J. (2022). Transcriptome analysis and differential gene expression profiling of wucai (Brassica campestris L.) in response to cold stress. BMC Genomics, 23. https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-022-08311-3
  29. Wang, Y., Fang, Z., Yang, L., & Chan, Z. (2021). Transcriptional variation analysis of Arabidopsis ecotypes in response to drought and salt stresses dissects commonly regulated networks. Physiologia Plantarum, 172(1), 77-90. https://onlinelibrary.wiley.com/doi/10.1111/ppl.13295
  30. Zvinavashe, A. T., Laurent, J., Mhada, M., Sun, H., Fouda, H. M. E., Kim, D., Mouhib, S., Kouisni, L., & Marelli, B. (2021, July). Programmable design of seed coating function induces water-stress tolerance in semi-arid regions. Nature Food, 2, 485-493. https://doi.org/10.1038/s43016-021-00315-8