Team:Groningen/Engineering
Choosing our host
The main idea of RootPatch is to engineer a bacterium that can efficiently express and secrete neuropeptide-like proteins (NLPs) that will repel the parasitic nematodes. Several factors may get in the way of this plan.
First of all, survivability of the host bacterium is key. If the bacterium is unable to survive in the harsh conditions in the soil, even a high NLP production will not be sufficient to act on all the nematodes. In order to give RootPatch a higher chance of success, we need our host bacterium to have the following characteristics:
▪ The bacterium is a natural soil isolate from the potato rhizosphere. This will give it a competitive advantage over non-native bacteria to the potato rhizosphere . The soil is a tough environment for bacteria and hence bacterial competition may be the biggest challenge for RootPatch to succeed. Our dry lab model and several of our stakeholders have indicated this as well.
▪ The bacterium needs to be able to proliferate at both high humidity and low humidity. According to our dry lab model, the lower the minimum water activity at which the bacteria in RootPatch can grow, the higher the chance of survival throughout the potato season. This minimal water activity is a characteristic that is typical for bacterial species but it has not been measured for our potential host bacterium.
▪ The bacterium is capable of sporulation. This will give it a higher survival probability under harsh conditions but will also guarantee a better shelf life of the eventual product. More about this issue can be found in the potato “Choosing the method of application”.
▪ The bacterium is capable of forming a biofilm layer at the roots. This will give RootPatch structural strength against severe environmental conditions such as heavy rainfall. However, this property is also questionable and should be further researched, likely bacteria in a biofilm alter their metabolic activity and that could significantly impact neuropeptide production (Pisithkul et al., 2019; Vlamakis et al., 2013).
Secondly, we need a bacterium that is good at producing and secreting peptides at high quantities to create the desired NLP environment around the roots. A bacterium that can be easily genetically modified is therefore of importance. Most wild-type bacteria from the soil are difficult to genetically manipulate.
Many different bacteria could be chosen as a host. In the end, only lab and field tests will tell which bacterium checks all the boxes: amenable to genetic engineering, suitable for high production of the neuropeptides, and high survival rate in the soil after having been genetically modified. We suggest four bacteria that could potentially be used for RootPatch. Bacillus mycoides is our first choice since, on paper, it shows the best characteristics. While reading through the other parts of this RootMap, you will notice that some of the parts are designed for Bacillus mycoides, as it is our primary target host bacterium.
Bacillus mycoides M2E15
Bacillus mycoides M2E15 is an endophytic soil bacterium that has been isolated in high abundance from the potato rhizosphere (Yi et al., 2018). Because it appears to be dominant in the potato rhizosphere, we believe it has a competitive advantage over other bacteria spp. and could, therefore, give RootPatch a higher probability of survival throughout the season. In addition, it can be genetically engineered, is able to sporulate and is capable of forming biofilm-like layers (Yi et al., 2018). The minimal water activity has never been investigated for Bacillus mycoides and remains to be tested.
We plan to introduce the gene coding for the NLP in the chromosome of Bacillus mycoides using the CRISPR-Cas9 technique. In order to achieve this, we will use the pYCR vector and the one-step CRISPR-based integration approach that has been described by Yi and colleagues in 2018. This technology has proven to be successful in this wildtype Bacillus species. The gene will be expressed under the strong constitutive promoter Ppta (Frenzel et al., 2018). Click on this link for more detailed information about this cloning strategy.
Bacillus subtilis HS3
Bacillus subtilis is a commonly used model of Gram-positive bacteria from the soil. Genetic engineering in Bacillus subtilis is easy and the organism is used as an efficient protein production chassis (Dong & Zhang, 2014). Genome integration can be achieved via a CRISPR-Cas9 method that relies on the vector pJOE8999 described by Altenbuchner in 2016.
Bacillus subtilis is known to form biofilms on plant roots (Posado et al., 2018), is very susceptible to genetic modification, and could be a very good host for RootPatch. Notwithstanding this, the question is whether it will survive in the soil since most strains are only used in the laboratory. There are natural soil isolates of Bacillus subtilis (such as strain HS3) that have been successfully manipulated using the pJOE8999 vector (Yi et al., 2018). Bacillus subtilis HS3 would, therefore, be a good potential host but we put it as a second option because it has not been isolated from the potato rhizosphere.
Bacillus megaterium DSM319
Bacillus megaterium, a well-known host for extracellular protein production in industry, is another option (Stammen et al., 2010). The wild-type strain DSM319 has been isolated from soil, is commercially available and is amenable to genetic manipulation. Although no CRISPR-integration systems were described for this strain yet, we suggest using plasmid pJOE8999 for this purpose as the authors describe it as a universal CRISPR-Cas system for firmicutes (Altenbuchner, 2016). Bacillus megaterium seems to be a suitable choice but its protein secretion capacity has only been described under laboratory conditions. As extracellular protease knock-out strains have been used, we are skeptical about its ability to survive the microbial competition in the soil while secreting a high amount of NLP. Therefore, Bacillus megaterium was placed as a third option for RootPatch.
Bacillus amyloliquefaciens 205
Bacillus amyloliquefaciens is a species that is used by well-known agricultural companies such as Bayer Crop Science as an inoculant to protect plants. Apparently, it survives well in the plant rhizosphere and already provides protection against other potato plant pathogens (Lin et al., 2018). In addition, it is widely used for the production of industrial enzymes, indicating that it might be well suited for the production of our NLP. Importantly, the CRISPR-Cas9 system seems to work in Bacillus amyloliquefaciens strain 205 (Zhao et al., 2020). However, as Bacillus amyloliquefaciens has never been used as a recombinant protein production host, and as having to genetically repair any deficiencies in the secretion system would be a long-term endeavor, we placed this bacterium as a fourth option.
Required Protocols
References
Altenbuchner, J. (2016). Editing of the Bacillus subtilis genome by the CRISPR-Cas9 system. Applied and Environmental Microbiology, 82(17), 5421–5427. https://doi.org/10.1128/AEM.01453-16
Dong, H., & Zhang, D. (2014). Current development in genetic engineering strategies of Bacillus species. Microbial Cell Factories, 13(1), 1–11. https://doi.org/10.1186/1475-2859-13-63
Lin, C., Tsai, C. H., Chen, P. Y., Wu, C. Y., Chang, Y. L., Yang, Y. L., & Chen, Y. L. (2018). Biological control of potato common scab by Bacillus amyloliquefaciens Ba01. PLoS ONE, 13(4), 1–17. https://doi.org/10.1371/journal.pone.0196520
Pisithkul, T., Schroeder, J. W., Trujillo, E. A., Yeesin, P., Stevenson, D. M., Chaiamarit, T., … Amador-Noguez, D. (2019). Metabolic Remodeling during Biofilm Development of Bacillus subtilis. MBio, 10(3), e00623-19. http://mbio.asm.org/content/10/3/e00623-19.abstract
Posada, L. F., Álvarez, J. C., Romero-Tabarez, M., De-Bashan, L., & Villegas-Escobar, V. (2018). Enhanced molecular visualization of root colonization and growth promotion by Bacillus subtilis EA-CB0575 in different growth systems. Microbiological Research, 217, 69–80. https://doi.org/10.1016/j.micres.2018.08.017
Yi, Y., Frenzel, E., Spoelder, J., Elzenga, J. T. M., van Elsas, J. D., & Kuipers, O. P. (2018). Optimized fluorescent proteins for the rhizosphere-associated bacterium Bacillus mycoides with endophytic and biocontrol agent potential. Environmental Microbiology Reports, 10(1), 57–74. https://doi.org/10.1111/1758-2229.12607
Yi, Y., Li, Z., Song, C., & Kuipers, O. P. (2018). Exploring plant-microbe interactions of the rhizobacteria Bacillus subtilis and Bacillus mycoides by use of the CRISPR-Cas9 system. Environmental Microbiology, 20(12), 4245–4260. https://doi.org/10.1111/1462-2920.14305
Vlamakis, H., Chai, Y., Beauregard, P., Losick, R., & Kolter, R. (2013). Sticking together: Building a biofilm the Bacillus subtilis way. Nature Reviews Microbiology, 11(3), 157–168. https://doi.org/10.1038/nrmicro2960
Zhao, X., Zheng, H., Zhen, J., Shu, W., Yang, S., Xu, J., … Ma, Y. (2020). Multiplex genetic engineering improves the endogenous expression of the mesophilic α-amylase gene in a wild strain Bacillus amyloliquefaciens 205. International Journal of Biological Macromolecules, 165, 609–618. https://doi.org/10.1016/j.ijbiomac.2020.09.210
Choosing the secretion method
After ensuring the strong expression of the neuropeptide, we need to confirm that it will be secreted into the environment. Proteins destined to be secreted by bacteria are usually equipped with an N-terminal secretion signal. Selecting the proper secretion-signal is a crucial step in optimizing protein secretion yield while other characteristics of the protein may also play a role. Our main strategy is to employ a secretion tag known to drive efficient protein secretion in a variety of Gram-positive hosts. As an alternative, we describe a library of five secretory signals that appear to be promising for NLP secretion.
Usp45 signal peptide
Usp45 is the major secreted protein of the lactic acid bacterium Lactococcus lactis. It is equipped with a signal peptide that has been extensively used for secreting various heterologous peptides not only in Lactococcus lactis but also in other Gram-positive bacteria (Borrero et al., 2011; Marciniak et al., 2012). Because we suggest using alternative hosts for NLP synthesis, we decided to employ this signal sequence as our main strategy. However, although it has been described as a universal signal peptide, we do not have experimental data on its efficiency in Bacilllus mycoides. We also have to keep in mind that the NLP14a peptide is significantly smaller than the proteins that have been secreted with the help of the Usp45 secretion signal.
Alternative secretion signals
In case the Usp45 signal peptide is not efficient for secreting neuropeptides, we suggest to use several alternative signal peptides that might induce strong and consistent secretion of the neuropeptides.
Phr are small peptides secreted by Bacillus subtilis that regulate cell density. Although the amino acid sequences of the peptides are quite different, their lengths are comparable and similar to that of the NLPs (between 5 and 30 amino acid residues) (Tjalsma et al, 2000). Based on this reasoning we suggest using different signal sequences of the Phr class of peptides as they may turn out to be suitable options. As a last alternative, we propose using a signal peptide prediction algorithm on secreted proteins of all the suggested host bacteria.
For a high-throughput method of screening the feasibility of the signal sequences in secreting the NLP, we suggest using a dot blot assay with an antibody that targets the His-tag which is attached to the C-terminus of the NLP sequence for the purpose of this experiment. This is a fast immunological technique that will hopefully allow identifying the most suitable signal peptide from the ones suggested.
Table of the possible secretion signals
| Name | Host organism | Amino acid residue sequence | Reference |
|---|---|---|---|
| Usp45 | Lactococcus lactis | MKKKIISAILMSTVILSAAAPLSGVYA | van Asseldonk et al., 1990 |
| PhrH | Bacillus subtilis | MPIKKKVMMCLAVTLVFGSMSFPTLTNSG | Bolotin & Borchert, 1997 |
| PhoD Tat type signal | Bacillus subtilis | MPKQAVPVKWEVAKDEHFRKIVRKGTEMAKP SLAHSVHVEADGLEPNKVYYYRFKT |
Eder et al., 1996 |
| AmyE | Bacillus megaterium | MKGKKWTALALTLPLAASLSTGVHA | Jong et al., 2016 |
| AmyE | Bacillus mycoides | MFAKRFKTSLLPLFAGFLLLFHLVLAG | van Asseldonk et al., 1990 |
Required Protocols
References
Borrero, J., Jiménez, J. J., Gútiez, L., Herranz, C., Cintas, L. M., & Hernández, P. E. (2011). Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis. Applied Microbiology and Biotechnology, 89(1), 131–143. https://doi.org/10.1007/s00253-010-2849-z
Marciniak, B. C., Trip, H., van-der Veek, P. J., & Kuipers, O. P. (2012). Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins, or membrane proteins. Microbial Cell Factories, 11, 1–13. https://doi.org/10.1186/1475-2859-11-66
Tjalsma, H., Bolhuis, A., Jongbloed, JDH., Bron, S., & van Dijl, JM. (2000). Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiology and Molecular Biology Reviews, 64(3), 515-547. https://doi.org/(...)BR.64.3.515-547.2000
Choosing the integration method
The engineering strategy we have chosen is to integrate the gene for NLP synthesis in the genome of our host bacteria using CRISPR-Cas9 technology. We will describe this strategy for Bacillus mycoides, our preferred host by characterizing the main parts of the pYCR system. As an alternative, we suggest using a different chromosomal integration site.
The α-amylase gene as integration site
The NLP14a site in the pYCR plasmid which contains the repair fragment and Cas9 protein.
Yi and colleagues designed a CRISPR-Cas9 system for Bacillus mycoides starting with plasmid pJOE originally established for Bacillus subtilis (Yi et al., 2018). The vector they constructed, pYCR, allows performing a one-step integration as the plasmid contains both the cas9 gene and the required DNA repair fragment. The method has a transformation efficiency of 77% (Yi et al., 2018). We designed the spacer sequences required for the system using Benchling and opted for the α-amylase gene as the site for integration. The neuropeptide gene is used as a ‘’repair fragment’’ and the expression of this gene will be controlled by the strong constitutive promoter pPta. For detection purposes, a 6 residue His-tag was introduced at the C-terminus of the NLP sequence. In parallel, we also created a plasmid that encodes the neuropeptide without the His-tag to test whether the stretch of 6 histidine residues influences the biological activity of the peptide. For more detailed information regarding the cloning strategy, click here.
Alternative integration site
Even though replacing the α-amylase gene with other sequences has never been reported to inhibit the growth of the bacteria in the laboratory, we do not have experimental evidence that this will also be the case in nature. It has also been shown that different integration sites may yield different levels of expression (Jeong et al., 2018). Common sites for chromosomal integration in Bacillus subtilis are nonessential genes such as lacA, gltA, pyrD, sacA and thrC. All of these are parts of amino acid or sugar metabolism. Nevertheless, we suggest using gltA (glutamate synthase gene). The potato rhizosphere is rich in glutamate so we expect that by deleting this gene, the survivability of Bacillus mycoides will not be affected. The removal of the glutamate synthase gene may also improve the containment of the bacteria in RootPatch as is described in the potato “Safety mechanism”.
Required Protocols
References
Jeong, D. E., So, Y., Park, S. Y., Park, S. H., & Choi, S. K. (2018). Random knock-in expression system for high yield production of heterologous protein in Bacillus subtilis. Journal of biotechnology, 266, 50–58. https://doi.org/10.1016/j.jbiotec.2017.12.007
Yi, Y., Li, Z., Song, C., & Kuipers, O. P. (2018). Exploring plant-microbe interactions of the rhizobacteria Bacillus subtilis and Bacillus mycoides by use of the CRISPR-Cas9 system. Environmental Microbiology, 20(12), 4245–4260. https://doi.org/10.1111/1462-2920.14305
Choosing the neuropeptide
The core of RootPatch is the utilization of neuropeptides to influence the behavior of parasitic nematodes. This approach is based on the pioneering study by Warnock and colleagues in 2017. These authors showed that by presenting the proper neuropeptides in the environment of nematodes, one can alter their chemoattraction towards root exudate. Several potential candidates from the class of NLPs were examined. The study shows that NLPs can be used to reverse the chemoattraction of the parasitic nematode of interest (Globodera pallida) towards root exudate; whereas in a normal situation, the nematode is attracted by the exudate, it now avoids it.
From a metabolic point of view, NLP appears to be a great choice for RootPatch because its production does not require energy-consuming and complicated post-translational modifications. Thus, the metabolic burden of NLP synthesis and secretion by the bacteria that we are planning to employ, should not greatly affect the survivability of RootPatch. However, the NLPs are rather unexplored and there are still a lot of questions around this very interesting class of peptides. Most likely this type of neuropeptide will bind to G-protein-coupled receptors of the anterior neurons of the nematodes.
To learn more about this pioneering study from Warnock and colleagues, what neuropeptides are and how they affect the nervous system, visit the project description page.
We will explore several NLPs by testing their efficiency in a simple chemosensory assay. Thus, we will be able to find out how well the different NLPs affect nematode behavior and also which peptide concentrations are required for such an effect. It is important to understand in which concentration range the NLPs, produced by the bacteria in RootPatch, will be effective. Our main neuropeptide candidate is NLP14a as it showed the highest potential in creating the root exudate avoidance behavior (see Figure 1). In case this NLP does not produce the desired effect in the nematodes, we propose two other peptide candidates: NLP21e and NLP15c.
Results from Warnock and colleagues in 2017 showing the disruption of normal chemotaxis behavior of Globodera pallida second-stage juveniles by exogenous neuropeptide-like proteins (NLPs). 100 juveniles were incubated with the selected NLPs, and subsequently released to an agar plate divided into three parts; agar mixed with root exudate, water control, and a normal stroke of agar in the middle. This was repeated 10 times for each neuropeptide. After 3 hours their location was used to determine the chemotaxis score.
NLP14a
One of the predicted structures for NLP14a with its amino acid residue sequence below. Note that due to the short length, the peptide probably does not have a fixed structure. Structure is predicted using the PEP-FOLD server (Shen et al., 2014).
NLP14a is a peptide of 13 amino acid residues, which is predicted from the genome of the Globodera pallida (see Figure 2). The work of Warnock et al. shows that this NLP has the highest potential in creating the avoidance behavior towards the root exudate in Globodera pallida in nature (see Figure 1). For our proof-of-concept, it is important to replicate these findings to make sure that they are valid, but also to investigate whether this NLP may have additional side-effects on other nematodes in the soil. Therefore, we plan not only to perform this experiment on Globodera pallida, but also on other non-parasitic nematodes such as Caenorhabditis elegans.
NLP21e or NLP15c
NLP21e and NLP15c are two NLPs consisting of, respectively, 21 and 14 amino acid residues (see Figure 3). Both NLPs also showed potential avoidance effects. Should NLP14a fail (e.g. in recombinant extracellular production by our host bacteria or in in vivo efficiency) these two neuropeptides may be possible alternatives that can be explored.
Predicted structures for NLP21e & NLP15c with their amino acid residue sequence below. Note that due to the short length, the peptides probably do not have a fixed structure. Structure is predicted using the PEP-FOLD server (Shen et al., 2014).
Required Protocols
References
Warnock, N. D., Wilson, L., Patten, C., Fleming, C. C., Maule, A. G., & Dalzell, J. J. (2017). Nematode neuropeptides as transgenic nematicides. PLoS Pathogens, 13(2). https://doi.org/10.1371/journal.ppat.1006237
Shen Y, Maupetit J, Derreumaux P, Tufféry P. (2014). Improved PEP-FOLD approach for peptide and miniprotein structure prediction. J. Chem. Theor. Comput. 20;10(10):4745-4758. http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD
Choosing the promoter
The NLPs in RootPatch will be secreted into the soil by a bacterium that is described in the “Creating our chassis” pathway. While this production may work very well in the laboratory, there are no guarantees that it is sufficient for application in nature. Many environmental factors such as rain and temperature, but also possible proteases in the soil may influence the fate of the NLPs. For RootPatch to be effective, it needs to produce a stable concentration of NLPs around the root system of the potato plant. This means that the breakdown of the neuropeptide should not be too fast and the NLPs should not diffuse too far away from the roots. Because for an iGEM project, it is almost impossible to get to the stage of a contained field trial, we propose two alternative experiments that could give indications as to how the NLPs will behave in a soil environment:
▪ Soil thin layer chromatography is a technique that can give a relative indication of diffusion of peptides, protein, and other compounds in soils. By comparing the diffusion with two root exudate compounds (glucose, which diffuses far away from the roots, and glutamic acid, which does not diffuse far) with the diffusion of the NLPs in this experiment, we can get an indication of its diffusion in nature. Check out the protocol given below for more information.
▪ Using His-tag purification of soil samples from the potato rhizosphere, we can investigate the rate of breakdown of NLPs by proteases in the soil. By taking random time point samples from the soil that is inoculated with the NLPs, purifying them using a nickel column, and performing SDS polyacrylamide gel electrophoresis, we can determine the breakdown of the His-tagged NLPs over time. Since the quantity of the neuropeptides in the sample may be very low, western blot hybridization may be necessary with anti-his tag antibodies to detect a proper signal.
When the breakdown of NLPs is too fast and/or they diffuse too far, we need to adjust the setup of RootPatch. These problems could be solved by changing the promoter driving NLP gene expression. Higher production would lead to a higher NLP concentration in the soil, which could counteract the fast breakdown and high diffusion rate.
The breakdown by proteases could also be too slow and the NLPs may barely diffuse at all. This could lead to a very high concentration of NLPs and that might have undesired effects in the nematodes (this will be pointed out by the chemosensory assay in the "choosing the neuropeptide" step). In this case we should probably aim for a promoter that leads to a lower peptide production.
Our primary approach is to use the Ppta promoter because it is strong, stable and has already been used in Bacillus mycoides. If this promoter leads to an undesirable protein concentration, we propose a promoter from a list of promoters that could potentially work better.
Ppta promoter
The Ppta promoter of the housekeeping phosphotransacetylase gene pta from Parageobacillus thermoglucosidasius DSM 2542 is a strong constitutive promoter that has been used before in Bacillus mycoides to produce GFP (Yi et al,. 2018; Frenzel et al., 2018). Since it leads to a high rate of transcription in Bacillus mycoides, this will be our first recommendation for a promoter that should drive NLP gene expression.
Testing out different promoters
If the Ppta promoter does not work in a proper way (see above), we suggest that one of a number of other promoters may be suitable for NLP production in RootPatch (see the Table below).
Table of the possible promoters
| Name | Nucleic acid sequence | Reference |
|---|---|---|
| Ppta | TTTGAATTTGATTGGAAGAAGAGTATGCTA | Presecan-siedel et al., 1999 |
| P43 | ATTTTACATTTTTAGAAATGGGCGTGAAAAAAAGCGCGCGATTATGTAAAATATAA | Wang & Doi, 1984 |
| Psigx | AATAGCCAACATTAATAAAATTTAAGGATATGTTAATATAAATTCCCTT | Song et al., 2016 |
| PgroES | ATCTTATCACTTGAAATTGGAAGGGAGATTCTTTATTATAAGAATTGTG | Song et al., 2016 |
| PdltA | TATGTATGGTTTTCACACCGCGAATACCGGTTCATATATTTATAACGATT | Song et al., 2016 |
Required Protocols
References
Frenzel, E., Legebeke, J., Van Stralen, A., Van Kranenburg, R., & Kuipers, O. P. (2018). In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria. Biotechnology for Biofuels, 11(1), 1–19. https://doi.org/10.1186/s13068-017-1008-5
Yi, Y., Li, Z., Song, C., & Kuipers, O. P. (2018). Exploring plant-microbe interactions of the rhizobacteria Bacillus subtilis and Bacillus mycoides by use of the CRISPR-Cas9 system. Environmental Microbiology, 20(12), 4245–4260.https://doi.org/10.1111/1462-2920.14305
Choosing the method of application
The goal for RootPatch is to grow along with the root system of the potato plant, protecting it throughout the entire potato season. As our dry lab model has pointed out, the start of the season is the most sensitive period for the bacteria in RootPatch, which makes the method of inoculation crucial. Choosing the correct composition of the inoculant and providing the necessary nutritional support will stimulate the outgrowth and survival of the bacteria. In addition, since the inoculation should, in theory, be applied on a large scale (the potato field), it should not contain high-cost compounds (such as yeast extract and tryptone) that are routinely used in the laboratory, in order to keep the costs of applying RootPatch for farmers low.
There are two main types of inoculants: solid and liquid formulations. Both have their advantages and disadvantages. We suggest using a solid fine granulate formulation as that would better fit the characteristics of our host bacteria and is well suited for large-scale application. If this formulation should be too costly, we propose to shift to a liquid formulation.
Farming equipment used to apply granular or liquid fertilizer to the potatoes in the field. The green spray system in the middle is used to apply liquids to the soil and potato, whereas the tube on the right, is used to apply a granulate. Both methods are possible for RootPatch but a granulate formulation is preferred.
Granulate formulation
Our first choice would be to create RootPatch as a powder inoculant. All proposed Bacillus RootPatch host species (see the "Choosing a host" potato) can form spores, which make them very suitable for this type of application. Because the bacteria are available as spores, the shelf life will be more than 1 year (Martinez-Alvarez et al., 2016; Cabrefiga et al., 2014). In addition, when talking with farmers about our project, they showed the equipment they use to plant the seed potato tubers. That equipment is already set up to apply granulates to the tubers as they often apply granulated fertilizer at the beginning of the season. No additional equipment would therefore be needed to apply RootPatch on a large scale. We suggest using talc as the natural carrier. Talc powder is suitable for long-term storage and has been used previously for creating an inoculant for Bacillus cereus, a species close to Bacillus mycoides (Martinez-Alvarez et al., 2016). To give nutritional support, carboxymethyl cellulose, calcium carbonate and glucose will be added to the inoculant. The nutritional environment of the root system will trigger the spore germination.
Liquid formulation
As an alternative to the solid granulated formulation, we suggest to use a liquid formulation. Liquid inoculations are easier to produce and often at lower costs than solid inoculants (Lobo et al., 2019). However, their shelf life is often worse because the bacteria are more susceptible to abiotic stresses such as nutrient depletion (He et al., 2015). Farmers are more accustomed to the application of granulate at the moment of tuber planting. If experiments would point out that re-application of RootPatch is necessary at a given moment in the season, only a liquid formulation would then be possible.
Required Protocols
References
Cabrefiga, J., Francés, J., Montesinos, E., Bonaterra, A., 2014. Improvement of a dry formulation of Pseudomonas fluorescens EPS62e for fire blight disease biocontrol by a combination of culture osmoadaptation with a freeze‐drying lyoprotectant. J. Appl. Microbiol. 117, 1122–1131.https://doi.org/10.1111/jam.12582
He, Y., Peng, Y., Wu, Z., Han, Y., & Dang, Y. (2015). Survivability of Pseudomonas putida Rs-198 in liquid formulations and evaluation of its growth-promoting abilities on cotton. Journal of Animal and Plant Sciences, 25(3), 180–189.
Lobo, C. B., Juárez Tomás, M. S., Viruel, E., Ferrero, M. A., & Lucca, M. E. (2019). Development of low-cost formulations of plant growth-promoting bacteria to be used as inoculants in beneficial agricultural technologies. Microbiological Research, 219(November 2018), 12–25. https://doi.org/10.1016/j.micres.2018.10.012
Martínez-Álvarez, J.C., Castro-Martínez, C., Sánchez-Peña, P., Gutiérrez-Dorado, R., Maldonado-Mendoza, I.E., 2016. Development of a powder formulation based on Bacillus cereus sensu lato strain B25 spores for biological control of Fusarium verticillioides in maize plants. World J. Microbiol. Biotechnol. 32, 75. https://doi.org/10. 1007/s11274-015-2000-5
Safety Mechanism
A very important aspect of RootPatch is safety. After all, RootPatch is a layer of genetically modified bacteria present in nature. What would happen if these bacteria spread and possibly affect other organisms outside the potato field? It is impossible to provide valid answers at the moment; all we can do is to make sure that we never get into such a situation. To ensure this, we aim to develop molecular mechanisms that will make the bacteria dependent on the plant. Whenever a bacterium leaves the root environment of the potato plant, it will not survive. We contemplate using one of the two possible methods to achieve this goal. One is to make our host bacterium auxotrophic for the amino acid tryptophan. The other is to make it dependent on solanine. To improve the efficiency of the safety mechanism, we might even look at combining both approaches. This “potato” will explain how these approaches work.
Tryptophan auxotrophy
Root exudates are mixtures of organic compounds secreted by the root systems of plants; they have the ability to attract different bacterial communities. Tryptophan is one of the most abundant organic molecules in the potato root exudate according to mass spectrometry studies when compared to the exudates of other plants (Ochola et al, 2020). Transcriptomics studies of differential gene expression of Bacillus mycoides in response to potato root exudate showed downregulation of tryptophan biosynthesis genes (Yi et al, 2018). These findings suggest that tryptophan is one of the molecules that might be used by the potato plant to attract bacterial communities and that Bacillus mycoides may take advantage of that. By designing a tryptophan auxotrophic Bacillus mycoides, one might be able to still attract the mutant to the potato roots while at the same time restricting its spreading into the environment to a significant degree, as it would die from a shortage of the now essential amino acid.
Figure 1: The trp operon. To make Bacillus mycoides auxotrophic, we plan to knock-out the trpE gene necessary for the tryptophan synthesis.
In order to create a tryptophan auxotroph, we plan to knock-out the trpE gene, the first structural gene of the trp operon that is responsible for tryptophan synthesis (Figure 1) (Band et al., 1984). In its absence, Bacillus mycoides will rely on the tryptophan secreted by the roots for survival. To delete trpE, we intend to use the plasmid pYCR described earlier. We designed guide (g)RNAs that target the trpE gene and together with the Cas9 protein inactivate it. More information about the gRNAs and the cloning strategy can be found here.
One of the crucial steps for inactivating genes is the efficiency with which this is done by CRISPR-Cas9 in combination with gRNA; this can be quantified by on-target and off-target scores. We have selected gRNAs using a criterium that maximizes on-target activity while minimizing off-target activity. If our first choice of gRNAs turns out to be inefficient, we suggest alternatives that will expectantly introduce the required deletion.
Solanine dependency
The most abundant organic compound in potato root exudate is solanine, a glycoalkaloid specifically synthesized by potato plants (Koroney et al., 2016; Ochola et al.,2020). We will use this molecule in another approach to make the bacterium dependent on the potato plant root environment. We designed a system that uses solanine as a dependency molecule in the YpcG/YcpF toxin-antitoxin system described in Bacillus subtilis by Holberger and colleagues in 2012.
Figure 2: The proposed system for making for Bacillus mycoides dependent on solanine. A solanine-responsive promoter/operator will bind to solanine and allow the expression of YpcF. YpcG, which shows DNase activity, is continuously expressed in the genome and can be inactivated by YpcF, thereby preventing the death of Bacillus mycoides. This mechanism will allow the bacterium to only survive in an environment rich in solanine.
The toxin, YpcG, exhibits DNase activity and will be continuously expressed in the host bacterium (Eibaz et al., 2015). The antitoxin YpcF inactivates YpcG and our aim is to place its gene under the controlled expression of a promoter responsive to the presence of solanine (see Figure 2) (Holberger et al., 2012). By using a toxin with DNase activity, we expect to minimize the spread of recombinant bacteria in the environment, away from the potato plant. More information about, among others, the strategies for cloning can be found here.
For this experimental setup to work, the expression of the toxin and antitoxin should be well regulated to prevent one of the two being constantly more abundant. Also, the toxin should be stable and should not be removed too quickly from the bacterial population over time. By integrating the toxin gene into the genome and that of the antitoxin in a plasmid, we aim to minimize this risk.
So far, no promoter has been described that is responsive for solanine. Only a few bacteria species seem to use solanine as a source of carbon. For all of these, the solanine degrading enzymes are constitutively expressed. Because Bacillus mycoides is attracted by potato root exudate (which contains a relatively high solanine concentration), we presume that it is already equipped with a solanine-responsive promoter/operator. To find this promoter, we have designed a transcriptomics experiment that will help pinpoint solanine-responsive genes to identify and use a solanine-responsive promoter/operator to drive the toxin-antitoxin containment system.
Combining tryptophan auxotrophy & solanine dependency
To ensure that RootPatch is well contained in the designated root environment, we suggest combining the described kill switch strategies. Thus, Bacillus mycoides will be dependent on both tryptophan and solanine in the potato root exudate. To accomplish this, the antitoxin gene should be placed under the control of the solanine-inducible promoter and this cassette should be integrated in the trpE gene, knocking this gene out. By this strategy the resulting bacterium will be both auxotrophic for tryptophan and in need for solanine to suppress the DNase activity of the toxin YpcG.
Required Protocols
References
Band, L., H. Shimotsu, and D. J. Henner. 1984. Nucleotide sequence of the Bacillus subtilis trpE and trpD genes. Gene 27:55-65
Elbaz, M., & Ben-Yehuda, S. (2015). Following the fate of bacterial cells experiencing sudden chromosome loss. MBio, 6(3), 1–11. https://doi.org/10.1128/mBio.00092-15
Holberger, L. E., Garza-Sánchez, F., Lamoureux, J., Low, D. A., & Hayes, C. S. (2012). A novel family of toxin/antitoxin proteins in Bacillus species. FEBS Letters, 586(2), 132–136. https://doi.org/10.1016/j.febslet.2011.12.020
Koroney, A. S., Plasson, C., Pawlak, B., Sidikou, R., Driouich, A., Menu-Bouaouiche, L., & Vicré-Gibouin, M. (2016). Root exudate of solanum tuberosum is enriched in galactose-containing molecules and impacts the growth of pectobacterium atrosepticum. Annals of Botany, 118(4), 797–808.https://doi.org/10.1093/aob/mcw128
Ochola, J., Cortada, L., Ng’ang’a, M., Hassanali, A., Coyne, D., & Torto, B. (2020). Mediation of Potato–Potato Cyst Nematode, G. rostochiensis Interaction by Specific Root Exudate Compounds. Frontiers in Plant Science, 11(June). https://doi.org/10.3389/fpls.2020.00649
What if RootPatch fails?
Ultimately, to prove the concept of RootPatch, we will need to perform greenhouse experiments. We could grow potato plants with RootPatch and test its efficiency against nematode infiltration.
But what if the suggested approach, of having a bacterium producing the neuropeptides, does not suffice or is not safe enough? Or even if we achieve proper results with RootPatch, what if the market would not accept a GM microbial solution as, in fact, several of our stakeholders have suggested?
Together with stakeholders we brainstormed possible alternative solutions that still utilize neuropeptides, but are not dependent on GM bacteria to be released in the soil. This “potato” summarizes these alternative solutions that came up during our conversations and describes the various advantages and disadvantages of each.
Production of NLPs in a bioreactor
One of the alternative, albeit very labor intensive, option is the in vitro production of NLPs and their subsequent application via a liquid solution to the field. Escherichia coli could be used as a potential expression host in such a setup because of the relatively low costs of growing it on a large scale (Cardoso et al., 2020; Baolei et al., 2016). After the production step, the NLP should be purified to get rid of the GM Escherichia coli. However, to maintain a constant NLP environment around the roots, this would require regular application of the neuropeptide solution to the soil, which would be labor intensive and would demand an extremely high quantity of neuropeptides on a yearly basis.
Encapsulation of the NLPs
Building further on the bioreactor initiative above, the purified neuropeptides could be encapsulated and applied in this form to the field. The advantage of encapsulation is that it could be done in such a way that the neuropeptides are slowly and constantly released. This would significantly lower the number of re-applications to the potato fields. Many different studies have examined encapsulation methods for the release of various agrochemicals and some of these show really promising results (Adisa et al., 2019; Gonzales et al., 2015; Jarosiewicz et al., 2003; Sampathkumar et al., 2020). Although the number of applications per season is lowered, there is still a need for re-application, which makes this method, again, higher in cost and more labor-intensive than when RootPatch could be used.
Using the potato plant as the production chassis
Another alternative strategy for using NLPs as a protective agent for potato plants is to let the plants produce the peptides. Plants are very good at secreting peptides, which in some cases even doesn't rely on the usage of signal sequences (Wang et al., 2017). Plants are already being explored as an alternative for protein production in the industry but because of problems with purification, due to many other compounds secreted by plants, they are not yet used commercially (Schillberg et al., 2019). However, for RootPatch, this would not matter. Besides, the potato plant, Solanum tuberosum, has already been genetically modified using CRISPR-Cas9 (Butler et al., 2016; Ma J. et al., 2017).
There are some disadvantages to creating a genetically modified potato plant. During our conversations with stakeholders, it became clear that a GM crop is not a solution that is considered safe by the consumers. The Innate potato is a good example for this. This GM potato plant was approved by the US FDA but never found a market since consumers were reluctant to eat something that was genetically modified (Gunther, 2013). Also, there are over three thousand different potato cultivars. To get the approval for the genetic modification of RootPatch for every different cultivar would be an immense challenge. By having a bacterium that coats the roots and that can protect every potato cultivar, deregulation would only be necessary once.
Required Protocols
References
Adisa, I. O., Pullagurala, V. L. R., Peralta-Videa, J. R., Dimkpa, C. O., Elmer, W. H., Gardea-Torresdey, J. L., & White, J. C. (2019). Recent advances in nano-enabled fertilizers and pesticides: A critical review of mechanisms of action. Environmental Science: Nano, 6(7), 2002–2030. https://doi.org/10.1039/c9en00265k
Butler, N. M., Baltes, N. J., Voytas, D. F., & Douches, D. S. (2016). Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases. Frontiers in plant science, 7, 1045. https://doi.org/10.3389/fpls.2016.01045
Cardoso, V. M., Campani, G., Santos, M. P., Silva, G. G., Pires, M. C., Gonçalves, V. M., … Zangirolami, T. C. (2020). Cost analysis based on bioreactor cultivation conditions: Production of a soluble recombinant protein using Escherichia coli BL21(DE3). Biotechnology Reports, 26, e00441. https://doi.org/10.1016/j.btre.2020.e00441
González, M. E., Cea, M., Medina, J., González, A., Diez, M. C., Cartes, P., … Navia, R. (2015). Evaluation of biodegradable polymers as encapsulating agents for the development of a urea controlled-release fertilizer using biochar as support material. Science of the Total Environment, 505, 446–453. https://doi.org/10.1016/j.scitotenv.2014.10.014
Gunther, M. (2013). McDonald’s GMO dilemma: why fries are causing such a fuss. Guardian News and Media Limited.
Jarosiewicz, A., & Tomaszewska, M. (2003). Controlled-release NPK fertilizer encapsulated by polymeric membranes. Journal of Agricultural and Food Chemistry, 51(2), 413–417. https://doi.org/10.1021/jf020800o
Jia, B., & Jeon, C. O. (2016). High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. Open biology, 6(8), 160196. https://doi.org/10.1098/rsob.160196
Ma, J., Xiang, H., Donnelly, D. J., Meng, F.-R., Xu, H., Durnford, D., et al. (2017). Genome editing in potato plants by agrobacterium-mediated transient expression of transcription activator-like effector nucleases. Plant Biotechnol. Rep. 11, 249–258. https://doi.org/10.1007/s11816-017-0448-5
Sampathkumar, K., Tan, K. X., & Loo, S. C. J. (2020). Developing Nano-Delivery Systems for Agriculture and Food Applications with Nature-Derived Polymers. IScience, 23(5), 101055. https://doi.org/10.1016/j.isci.2020.101055
Schillberg, S., Raven, N., Spiegel, H., Rasche, S., & Buntru, M. (2019). Critical analysis of the commercial potential of plants for the production of recombinant proteins. Frontiers in Plant Science, 10(June). https://doi.org/10.3389/fpls.2019.00720
Wang, X., Chung, K. P., Lin, W., & Jiang, L. (2017). Protein secretion in plants: Conventional and unconventional pathways and new techniques. Journal of Experimental Botany, 69(1), 21–37. https://doi.org/10.1093/jxb/erx262