Abstract
In 2020, Canada produced over 679,000 tonnes of greenhouse produce, with a farmgate value of 1.8 billion CAD (Agriculture and Agri-food Canada, 2021). In Ontario, Leamington alone is home to more greenhouse acreage than the entire United States (Municipality of Leamington, 2020). However, many factors influence yield of these crops, and growers can experience massive yield losses due to insect damage. To meet the demand for innovative strategies to control insect pests, we engineered Bacillus subtilis to inducibly express insect Cytotoxic (Cyt) proteins through an IPTG responsive promoter, Pgrac. When “off”, the modified B. subtilis behaves normally and may have a positive effect on plant growth. When “on,” the bacterial biofilm produces Cyt proteins, creating a pesticide barrier to any Dipteran insect larvae that may be present in the soil. This powerful bioinsecticide designed by iGEM Guelph will place a new tool in the hands of growers to combat harmful greenhouse insect pathogens.
The greenhouse environment
Originally made popular in England by a desire to display exotic plants, greenhouses have become a valuable tool in agriculture (Petruzello, 2019). Greenhouse agriculture offers a way to get higher crop yields from smaller spaces, all while reducing environmental impact. It also creates opportunities for agriculture in non-traditional spaces, like polar regions or cities! Greenhouse growers are constantly searching for ways to improve their output, and as such have become an environment of constant innovation. Every greenhouse is unique, but they are almost always high tech! Some growers use soil as a substrate for their plants, but many grow hydroponically, with plants rooted in rockwool or free-floating in water. Many greenhouses are also equipped with lighting and heating systems so that they can be productive for a larger part of the year. Newer robotic technology is also being implemented in some greenhouses, and has potential for early detection of insect and microbial diseases. Greenhouse growers are open to working with new tools and cropping systems within their greenhouses, and this is reflected in their willingness to speak with a team like iGEM Guelph!
However, while greenhouse agriculture presents a promising tool for facing the global challenge of food supply and sustainability, it also has its own set of challenges that need to be overcome. If a greenhouse creates the perfect environment for plants to thrive, it also tends to create the perfect environment for insect pests and plant diseases to run wild! Current pest management in greenhouses involves two main categories. These are the use of pesticides (chemical or biological) and biocontrol such as predatory mites or wasps. One often limits the other, since if growers use predatory insects, they cannot use pesticides that might also kill these beneficial biocontrol insects. Because of how quickly harmful insects can develop resistance to pesticides, many greenhouse growers are implementing biocontrols, which is why modern pesticides need to be adaptable and precise for growers to use them effectively (Smith, 2015).
A possible scenario involves application of a pesticide initially to kill off harmful insects in the greenhouse, with biocontrol predatory insects added later when the pesticide is no longer present. This means that any biopesticide cannot be constitutively expressing a toxin. Our solution to this problem is therefore to genetically engineer a non-pathogenic bacterial strain (Bacillus subtilis) to produce specific insecticidal proteins that target insect pests in greenhouses at the desired time using an inducible promoter.
Why Cytotoxic proteins
Since its discovery in 1901, the entomopathogenic bacteria Bacillus thuringiensis (Bt) has been a powerful tool to combat insect pests related crop loss in Agriculture. Bt naturally carries a mega plasmid that encodes genes which produces delta endotoxins of the crystal (Cry) and cytotoxic (Cyt) family that directly target insects (Valtierra-de-luis et al., 2020). These protein toxins are expressed as protoxins that are proteolytically cleaved and activated by midgut specific proteases (Valtierra-de-luis et al., 2020). The active protein then binds to or forms aggregates in the membrane that result in pore formation, cell death, and the death of the insect (Soberón et al., 2013).
Both families of proteins are non-toxic to normal human cells but compared to Cyt protein, Cry proteins have a more broad insect range that includes major and minor pollinators such as bees and butterflies (respectively) (Valtierra-de-luis et al., 2020). Cyt proteins however can only target Dipteran species such as Drosophila melanogaster and Bradysia spp (Soberón et al., 2013). The latter insect species is a very common greenhouse pest that directly feed on plants. They can also spread fungal diseases. For these reasons, Cyt1Aa and Cyt2Ba were chosen as the insecticides for this project.
Aims for Project Ceres
iGEM Guelph 2022 was motivated to create a project that targets greenhouse pests that are common and can introduce diseases in the greenhouse environment. During our search for potential candidates, we reached out to individuals in the pest management sector. We were informed on how common fungus gnats infections are and how quickly they can transmit and spread fungal disease. Although we reached out to local members of pest management, fungus gnat infections can occur worldwide. We also learned from government staff and greenhouse growers that whiteflies and thrips cause significant damage to plants. For these reasons, we wanted to target fungus gnats starting in tomato greenhouses. Since Ontario is the number one global producer of greenhouse tomatoes (Government of Canada, Statistics Canada, 2022).iGEM Guelph 2022 plans to create two recombinant Bacillus subtilis (B.subtilis) cells. B.subtilis is a motile, biofilm, and spore forming soil dwelling microbe that has the ability to act as a barrier in plant root systems. The use of a biofilm forming soil microbe was inspired by team Groningen 2020’s work on plant protection, although the project from Groningen targets parasitic nematodes (worms) instead of insects.
We will create one recombinant B.subtilis containing genes for a 20 kDa helpher protein (P20), Cyt1Aa, and red fluorescent protein mScarlet (Cyt1Aa cassette) and a second recombinant B. subtilis containing genes for a Cyt2Ba protein, and a blue fluorescent protein (BFP) (Cyt2Ba cassette). The P20 helper protein is necessary for proper expression and stability of Cyt1Aa.
We have created plasmid constructs to express the two Cyt gene cassettes mentioned above through the use of the strong IPTG-inducible promoter, grac (Pgrac). This will be achieved through the digestion with BsaI of our cassettes and the E.coli-Bacillus subtilis shuttle plasmid, pCG004 (Gilbert et al., 2017), that contains a gene for repressor LacI and green fluorescent protein gene (GFP mut3b). The GFP gene was replaced with our gene cassettes using BsaI. BsaI is a type IIS restriction enzyme and in this case two different single stranded overhangs on each end of the digested cassettes and plasmid will be produced after restriction digestion. Following the digestion of our Cyt cassettes and pCG004, the complementary ends can therefore be ligated together in a directional manner to produce the two plasmids Cyt1Aa-pCG004 and Cyt2Ba-pCG004. These gene cassettes are located directly downstream of the Pgrac promoter within the plasmid. The ligated plasmids were first transformed into E. coli DH5alpha. Positive transformants were confirmed by plasmid miniprep and analytical digest with BsaI showing the expected sizes of the pCG004 plasmid and the gene cassettes. The positive plasmids Cyt1Aa-pCG004 and Cyt2Ba-pCG004 were then transformed separately into B subtilis. Recombinant B. subtilis expressing either toxin through IPTG induction were found to be toxic to wingless Drosophila melanogaster. Specifically, toxicity assays show that we have produced a biopesticide (B. subtilis containing Cyt1Aa-pCG004 and Cyt2Ba-pCG004) that is significantly more toxic than controls (B. subtilis containing pCG004 only), leading to a 65-70% mortality rate of D. melanogaster within 20 minutes of exposure (more details of toxicity experiments are indicated under the modelling section of the wiki). We have therefore successfully genetically engineered a valuable tool for greenhouse farmers to target harmful Dipteran insects. Although we focused on tomato greenhouses, our project can be applicable to other Dipteran insects in cucumber and lettuce greenhouses; it can also be refined to target whiteflies and aphids.
In summary, iGEM Guelph has successfully built two constructs and transformed these into two separate populations of B. subtilis. Initial toxicity assays show that we have produced a biopesticide which is significantly more toxic than controls. Because we cannot test our project in a greenhouse setting due to safety restrictions and the timeframe of the iGEM competition, our Modeling team ran experiments as proof of concept. They determined that key characteristics of B. subtilis which make it uniquely suitable for a soil-borne biopesticide were not lost over the course of our modifications. They also did further tests to better characterize the toxicity of our biopesticide, the results of which confirm our engineering success.
Works Cited
- Crops and Horticulture Division, Agriculture and Agri-Food Canada. (2021). Statistical overview of the canadian greenhouse vegetable industry, 2020. Government of Canada.
- Gilbert, C., Howarth, M., Harwood, C.R. and Ellis, T. (2017) Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis. ACS Synth Biol 6:957-967.
- Government of Canada, Statistics Canada. (2022, April 26). Production and value of greenhouse fruits and vegetables. Retrieved October 11, 2022.
- Municipality of Leamington. (2020). Community profile.
- Petruzzello, M. (2019). Greenhouse. Encyclopedia Britannica.
- Soberón, M., López-Díaz, J. A., & Bravo, A. (2013). Cyt toxins produced by Bacillus thuringiensis: A protein fold conserved in several pathogenic microorganisms. Peptides, 41, 87–93.
- Smith, T. (2015). Biological control: Greenhouse pests and their natural enemies. University of Massachusetts Amherst, Center for Agriculture, Food, and the Environment.
- Valtierra-de-Luis, D., Villanueva, M., Berry, C., & Caballero, P. (2020). Potential for Bacillus thuringiensis and other bacterial toxins as biological control agents to combat dipteran pests of medical and agronomic importance. Toxins, 12(12), 773.