Team:Rice/Description

Inspiration

The Intergovernmental Panel on Climate Change predicts a global surface temperature increase of 1.8°C to 4.0°C by the end of the century, accompanied by increased drought and heat waves.¹ This has had devastating effects on food production for the past 30 years, decreasing agricultural yields by 1-5% per decade, which is expected to continue in the coming years.² With the world population on the rise,³ this decline in food resources is expected to leave millions of additional people at risk of hunger by 2080. Our project aims to tackle this projected food insecurity from the bottom up by conferring heat and drought resistance to plants via the associated engineered rhizobacteria.

Why rhizobacteria?

Since agriculture is particularly affected by climate change, we want to design a product that makes plants more resistant to extreme temperatures. Genetically modified plants are an avenue that many researchers and farmers have already turned to in order to increase crop resilience, but engineering rhizobacteria can be a more modular avenue to conserve the wide array of crop species and cultivars that are needed to maintain biodiversity and culinary variety. Given the extensive characterization of plant-microbial interactions, engineered bacteria provide a promising route to achieving crop climate resilience. Altering the rhizobacteria, rather than the plant itself, will hopefully avoid some of the negative connotations associated with genetically modified organisms and increase the accessibility of our product.

Why use synthetic biology?

There have always been manual attempts to mitigate the effects of drought and other stressors on plants, such as irrigation and raised beds. However, they tend to be costly and not readily adaptable to changes in weather patterns. Another approach many researchers have used is transplanting bacteria evolved for extreme climates into the rhizosphere of another crop.⁴ While this method has seen positive results on the growth of the plant, a synthetic biology-focused approach provides a cheaper, more convenient, and optimizable solution.⁵

What is an RNA thermometer?

RNA thermometers (RNAT) are a form of translational regulation, in which a stem-loop structure forms within the mRNA and renders the ribosome binding site (RBS) inaccessible to the ribosome. The melting point or melting temperature is often used to describe the temperature at which the RNAT changes conformation, allowing the ribosome to bind to the RBS and initiate translation.

Why make a new thermometer?

Our project aims to perfect the thermosensor capabilities of RNA thermometers within bacteria. Many RNA thermometers that have been well characterized are optimized for 37°C ⁶ or higher. However, this temperature is well above the typical growth conditions plants experience. For example, rice grows best between 24° and 30°C, and wheat grows best between 19°C and 25°C, indicating a greater need for thermosensors that can alter gene expression at lower, specific temperatures.⁷

Project Proposal

We aim to design a synthetic circuit in Pseudomonas putida that will confer greater stress resistance to Arabidopsis thaliana. We chose P. putida because it is naturally found in many rhizospheres and has a symbiotic relationship with plants, including A. thaliana;⁸ this means metabolite exchange is native between A. thaliana roots and P. putida. The enzymes used to mediate climate resilience only need to be expressed once the plant begins to feel heat-induced stress. To achieve this, our circuit will be temperature-induced by an RNA thermometer. Since A. thaliana grows best between 18 and 23 °C, we decided to optimize our thermometer for 30°C, which is considerably lower than what most RNA thermometers are currently optimized for. At around 30°C, our thermometer will begin to melt, which will allow for araC to be transcribed and induce expression of the downstream genes. Complete circuits can be found on the project design page.

The genes we are expressing have all been previously shown to help plants cope with environmental stresses. IAA, commonly known as auxin, works to expand lateral root growth and the plant's nutrient access. ACC deaminase reduces the plant's cytosolic concentration of ethylene, a compound shown to hinder plant growth by triggering the plant's stressed response, by sequestering a precursor to ethylene⁹. Trehalose synthase (otsB) helps mitigate the effects of drought in plants by accumulating in the root nodules and promoting an increased flow of water from the soil to the plant.¹⁰An image depicting how the three enzymes work together is shown below. More explanations regarding the enzymes and their respective pathways can be found in project design.

References

[1] Schmidhuber, J., Tubiello, F. Global food security under climate change 2007. PNAS 2007, 104(50), 19703-19708. https://www.pnas.org/content/104/50/19703#ref-5
[2] Newberry, F., Qi, A., Fitt, B. Modelling impacts of climate change on arable crop diseases: progress, challenges and applications. Current Opinion in Plant Biology 2016, 32, 101-109. https://doi.org/10.1016/j.pbi.2016.07.002
[3] United Nations, Department of Economic and Social Affairs, Population Division. World Population Prospects: The 2017 Revision, Key Findings and Advance Tables 2017, 1-3. https://population.un.org/wpp/Publications/Files/WPP2017_KeyFindings.pdf
[4] Vurukonda, S. S. K. P.; Vardharajula, S.; Shrivastava, M.; SkZ, A. Enhancement of Drought Stress Tolerance in Crops by Plant Growth Promoting Rhizobacteria. Microbiological Research 2016, 184, 13–24. https://doi.org/10.1016/j.micres.2015.12.003.
[5] Suarez, R., Wong, A., Ramirez, M., Barraza A., Orozco, M. d. C., Cevallos, M. A., Lara, M., Hernandez, G., Iturriaga, G. (2008). Improvement of Drought Tolerance and Grain Yield in Common Bean by Overexpressing Trehalose-6-Phosphate Synthase in Rhizobia. Molecular Plant-Microbe Interactions, 20(7), 958-966, DOI: 10.1094/ MPMI -21-7-0958
[6] Neupert, J., Karcher, D., Bock, R. (2008). Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli. Nucleic Acids Research, 36(19), pp e124, DOI: 10.1093/nar/gkn545
[7] Nagai, T., Makino, A. (2009). Differences Between Rice and Wheat in Temperature Responses of Photosynthesis and Plant Growth. Plant and Cell Physiology, 50(4), pp 744-755, DOI: 10.1093/pcp/pcp029
[8] Ramos-González, M. I., Campos M. J., Ramos, J. L. (2005). Analysis of Pseudomonas putida KT2440 Gene Expression in the Maize Rhizosphere: In Vitro Expression Technology Capture and Identification of Root-Activated Promoter. Journal of Bacteriology, 187(12) pp 4033-4041, DOI: 10.1128/JB.187.12.4033-4041.2005
[9] Glick B. R. (2012). Plant growth-promoting bacteria: mechanisms and applications. Scientifica, 2012, 963401. doi:10.6064/2012/963401
[10] Sugawara, M., Cytryn, E. J., Sadowsky M. J. (2010). Functional Role of Bradyrhizobium japonicum Trehalose Biosynthesis and Metabolism Genes during Physiological Stress and Nodulation. Applied and Environmental Microbiology, 76(4), pp 1071-1081; DOI: 10.1128/AEM.02483-09