Team:Groningen/Poster

RootPatch: Protecting Plants, Conserving Biodiversity

Presented by Team Groningen 2020

Jelle Molenkamp¹, Julius Fülleborn¹, Kim van Maldegem¹, Ilya Ivanov¹, Sietse Couperus¹, Fennie van der Graaf¹, Reuben Rajadhyaksha¹, Andreea Stan¹, Ronja Hulst¹, Arne Hellwege¹, Alex Gajda¹, Job de Lange¹, Sucharita Sarkar¹, Jaya Gajjar¹, Rianne Prins², Sonja Billerbeck², Jan Kok³

¹iGEM Student Team Member, ²iGEM Team Instructor, ³iGEM Team Primary PI

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Abstract

Crops all over the world are in danger of attack by plant-parasitic nematodes (PPNs). PPNs feed on the roots of crop-plants and thereby consume the energy that the plant needs to develop normally. Especially, potato plants are terrorized by the potato cyst nematodes Globodera pallida and Globodera rostochiensis. According to current estimations, these two parasites are responsible for an annual loss of 460 million Euros in Europe alone due to reduced potato yields. Current methods to fight these parasites off are either insufficient or detrimental to the soil biodiversity. New and sustainable ways of potato crop protection against potato cyst nematodes are, therefore, urgently needed. We, the iGEM Groningen 2020 team, have developed a novel way to fight potato cyst nematodes with RootPatch, a community of bacteria that coats the roots and protects the potato plants from the nematodes. By producing neuropeptides acting specifically on the nervous system of the potato cyst nematodes, RootPatch repels the nematodes from the plants without having any effect on other organisms in the soil.

Team iGEM Groningen 2020 (from left to right). Top row: Reuben Rajadhyaksha; Fennie van der Graaf; Alex Gajda; Kim van Maldegem; Ronja Hulst. Middle row: Jaya Gajjar, Ilya Ivanov, Sucharita Sarkar, Jelle Molenkamp, Andreea Stan. Front row: Job de Lange; Julius Fülleborn. Not on the group picture: Sietse Couperus; Arne Hellwege; Rianne Prins (instructor); Sonja Billerbeck (instructor); Jan Kok (primary PI).

Inspiration

The inspiration for our iGEM project came from the father of our team member Kim. He is a local potato farmer and explained that small nematodes (which he called "cysteaaltjes") are responsible for crop losses in the potato field. After looking deeper into the problem, we realized that it is a single nematode species, called Globodera, which is responsible for losses of his crop. Globodera pallida especially appeared to be detrimental for the potato crop, not only here in the area but in many different places around the world. Moreover, we realized that many different crops around the world are affected by similar parasitic nematodes and that so far, no sustainable and fully effective solution has been developed.

Our team member Kim with her father Pieter van Maldegem.

Problem

Crops all around the world are negatively affected by plant parasitic nematodes (PPNs). PPNs feed on the roots of the crop plants and ultimately impair their growth. While this is not always directly visible in the crop, one can observe the effect on the crop yield at the end of the season. According to estimations, PPNs are responsible for an annual crop loss of 80 to 118 billion USD (Nicol et al., 2011).

Potato field affected by G. pallida. Plants on the left were treated by conventional methods (fumigation) whereas plants on the right were not treated.

Illustration of plant parastic nematodes (PPNs) feeding on the plant root system. Artist: Kathy Merrifield.

In Europe alone, PPNs are responsible for the loss of 460 million Euro worth of potatoes per year. G. pallida is the biggest contributor to this and so far no method has been developed to combat this nematode in a sustainable way. There are currently five approaches to deal with G. pallida, which all have considerable drawbacks (see Figure below). Concerned with these issues, we developed an innovative technology to compensate the current drawbacks.

Five current approaches to fight off G. pallida and their drawbacks.

References

Nicol, J., Turner, S., Coyne, D., den Nijs, L., Sue, H., & Maafi, Z. (2011). Current Nematode Threats to World Agriculture. In Genomics and Molecular Genetics of Plant-Nematode Interactions (pp. 21–43). https://doi.org/10.1007/978-94-007-0434-3_2

Our Aims

At an early stage in our project we set 4 key aims. We envisioned RootPatch to be a safe and easy-to-use solution. But at the same time, it should be designed to be nematode-specific but with broad applicability.

PRECISION

In contrast to the pesticides, we wanted RootPatch to avoid affecting harmless soil organisms. Therefore, we aimed to make RootPatch a targeted solution.

SAFETY

Because we are working with a genetically modified organism, RootPatch should be contained, preventing its uncontrolled spread to other areas in the environment.

VERSATILITY

There are numerous PPNs that cause damage to many other crops. Our solution should not only target G. pallida, but should be applicable with minimal adjustments to other PPNs as well.

EASY-TO-USE

We care about our future users, the farmers. RootPatch should be an easy-to-use solution so it does not take a lot of the farmer’s time and resources.

Our Solution

RootPatch is a sustainable solution circumventing all the problems encountered with the current methods to fight plant parasitic nematodes (PPNs). By employing neuropeptides and the implemented safety mechanisms, RootPatch is not only precise and effective, it is also easy-to-use, safe, and applicable to other parasitic nematodes.

Grow & Maintain

RootPatch is a bacterial layer consisting of Bacillus mycoides, a natural soil isolate that is abundant in the potato plant rhizosphere. After application to the seed potato, RootPatch will grow along with the roots of the plant, maintaining itself with the nutrients from the plant. Moreover, RootPatch is intended to form a biofilm on the roots, giving it structural strength to survive throughout the season.

The bacterium B. mycoides bacterium in the RootPatch is engineered to produce a compound called neuropeptide-like protein 14a (NLP14a). This peptide is known to influence the chemotactic behavior of G. pallida (Warnock et al., 2017). In a normal situation this nematode is attracted by the exudate of the potato plant. By letting B. mycoides create an environment rich in the repellant NLP14a, G. pallida will avoid the plant and will not enter the root system of the plant, thereby keeping the plant healthy and safe from nematode attacks.

Avoid

Contain

To prevent the spread of our genetically modified bacterium in the soil, we designed molecular mechanisms to contain it in the rhizosphere of the potato plant. The potato rhizosphere is characterized as being particulary rich in tryptophan and solanine. By making the survival of our B. mycoides dependent on these compounds, it will not spread to other areas in the environment and will stay where it should exert its effect.

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

Engineering

Due to the COVID-19 pandemic, we were unable to perform experiments in the laboratory this year. However, we still developed extensive wet lab plans so that they can be executed in the future. Below we briefly describe the engineering strategies of RootPatch.

Chassis choice

As a chassis for RootPatch, we chose B. mycoides M2E15. This is a soil bacterium endemic to the potato rhizosphere. It has been studied for its beneficial effect on the growth of the potato plant. Moreover, it has been genetically engineered before and shows potential as an efficient protein production and secretion host.

Cloning strategy

Introducing the genes of interest in the chromosome of B. mycoides will ensure transmission of this gene in the next bacterial generation without the use of a selection method. To accomplish this we employed a CRISPR-Cas based cloning strategy using pYCR, a plasmid that has proven to be effective for cloning in B. mycoides (Yi et al., 2018).

Using the pYCR plasmid (left), the genome of Bacillus mycoides M2E15 will be modified to produce the neuropeptide-like proteins under the control of the pta promoter (right). By exploiting a secretion signal at the N-terminus, the peptide will be secreted into the soil.

Engineering components

Three components need to be coupled and integrated into the genome of B. mycoides M2E15 to ensure NLP production: the pta promoter, a secretion signal (usp45) and the nlp14a gene

Kill switch cloning strategy

Because safety is a major concern before GMOs can be used in agriculture, we implemented safety mechanisms based on the amino acid tryptophan and the glycoalkaloid solanine, both rhizosphere-specific molecules. Both compounds are abundant in the potato root exudate and by making Bacillus mycoides dependent on both, we can contain the bacterium in the root environment.

To make the bacterium dependent on tryptophan, an auxotroph is made by knocking out trpE, an essential gene in tryptophan synthesis. Solanine dependency is implemented by utilizing the YpcG/YpcF toxin-antitoxin system (Holberger et al., 2012). The toxin will be constitutively expressed whereas the antitoxin will only be produced when solanine is present.

Schematic representation of tryptophan auxotrophy (left) and solanine dependency (right). To make the bacterium auxotroph, the trpE gene will be knocked out. For the solanine dependency, a solanine dependent promoter will control the expression of the antitoxin YpcF in the cell. When present, this antitoxin will counteract the YpcG toxin that is constitutively expressed from the genome. Thus, only in a solanine rich environment will the bacterium be able to survive.

References

Yi Y, Li Z, Song C, Kuipers OP. (2018). Exploring plant-microbe interactions of the rhizobacteria Bacillus subtilis and Bacillus mycoides by use of the CRISPR-Cas9 system. Environ Microbiol. 2018;20(12):4245–60.

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

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

Model

To investigate the functioning of RootPatch in the absence of lab experiments, we developed two models that aim to assess the stability of RootPatch in the soil and its effect on nematode populations.

Bacteria model

The RootPatch population model is used to examine what factors are the biggest determinants of the survival of RootPatch. This information can be used to develop and apply RootPatch in such a way that it maximizes the probability of survival. The key takeaway messages from this model are:

• RootPatch is the most sensitive to the water activity in the soil
• RootPatch is most vulnerable to environmental conditions in the first 50 days after application
• Competition between bacteria can be detrimental for the survival of RootPatch

Sensitivity plot of minimum water activity (Aw_min) and temperature (T_min) throughout the potato season. Both values are characteristics of the bacteria. In the beginning of the season, the sensitivity towards water activity is the highest during the phase in which RootPatch moves towards a stable population size.

Bifurcation plot of the effect of competition on the engineered bacteria in RootPatch. The higher the strength of the competition on the bacteria, the lower the population size of RootPatch around the roots.

Nematode model

The nematode model gives insights in the population dynamics of Globodera pallida and the effectiveness of our intervention strategy. The most important accomplishment of this model is that it can predict how strong the repelling RootPatch efficiency must be to provide adequate root protection for the given environmental conditions.

Graph showing what RootPatch strength is required to protect the plant. The model allows calculating this threshold for all environmental conditions. The recovery rate is the rate at which the nematodes clear the NLP from their system and regain their normal function.

To improve the model it is most important to experimentally determine the recovery rate of the nematodes and the repulsive effect of a given concentration of NLP. This can help determine if an engineered strain secretes enough NLP to protect the plant.

Conclusion

Both models show that drought and temperature are of great influence influences on the functionality of RootPatch and the nematodes, encouraging us to think about ways to engineer RootPatch to have a better drought-tolerance. Since the water activity and temperature in the summer are the worst for RootPatch but optimal for the nematodes, the plant will be most susceptible during this period. If reapplication of RootPatch is necessary, we would advise to do this after rainfall or irrigation since the models show that then the bacteria thrive and nematode mobility is reduced.

Implementation

In order to successfully implement RootPatch into the current market, we had to consider many aspects. Among others, the design of a bioformulation strategy and a safety mechanism were particularly relevant.

Storage

RootPatch will be produced as a powdered bioformulation of Bacillus mycoides. Powdered formulations are easier to store and to transport than liquid formulations. We propose adding talc powder as a natural carrier to prolong shelf life (> 1 year) in addition to carboxymethyl cellulose and glucose for bacterial nutritional support.

Application

RootPatch powder is added to the soil together with the planted potato tuber. The bacterial spores will germinate upon entering the nutrient-rich environment of the growing potato plant. RootPatch will form a biofilm on the growing roots. This biofilm sustains itself throughout the season so thatr eapplication is not required. As indicated by the farmers, this application strategy would save a lot of effort because their equipment already has all the necessary features to realize such an application.

Safety

To make sure that RootPatch stays on the roots, we will implement mechanisms for its biocontainment. To achieve this, the survival of Bacillus mycoides will be directly linked to the presence of tryptophan and/or solanine, both abundantly present in the potato root secretions. While a trp-auxotrophic Bacillus mycoides strain is easier to engineer, solanine dependency could provide a more reliable safety mechanism. To identify solanine-dependent promoters in Bacillus mycoides, we plan to do a transcriptomics experiment screening for promoters that get activated in presence of this compound.

Future Directions

This section describes our efforts to communicate and get feedback from the various stakeholders relevant to our project. We identified 5 key stakeholder groups, which are presented below.

• Developed comprehensive experimental planning
• Designed and implemented computational models
• Engaged in educational activities

• Performed extensive human practices
• Built up team communication and bonded

But the project is far from being over. Certain aspects need experimental evidence and the models can not predict everything. Given enough time, we would focus our efforts on:

• Studying biofilm formation and robustness of RootPatch as well as secretion and stability of NLP14a
• Testing our proposed biocontainment strategy
• Exploring the potential of NLPs for protection against plant parasitic nematodes
• Applying the experimental findings to improve our models
• Supervising iGEM Groningen 2021 in developing their project

Human Practices

This section describes our efforts to communicate and get feedback from the various stakeholders relevant to our project. We identified 5 key stakeholder groups, which are presented below.

Potato Farmers

Farmers are the end users of RootPatch and are, therefore, central to our project. During our visits to several local farms we learned their passion for producing potatoes but also the severity of the Globodera pallida problem in the Netherlands. Their insights helped us to make RootPatch more user-friendly.

Crop Protection/Crop Development

During our project we contacted a number of crop protection companies. They mainly focus on developing crop protection products or resistant cultivars. We were told that there is no 100% effective solution on the market and that the adapting Globodera pallida populations can counteract the resistance. Their feedback helped us to improve multiple aspects of our project.

Potato Processing

It was important for us to get in contact with potato selling/processing companies to get an idea how they experience GMO solutions. We found out that the consumers are reluctant to eat GMO-associated foods in general, especially if the crop itself is genetically engineered. This helped us shift away from engineering the potato plant.

Policy Makers

To uncover all possible safety issues in our project, we spoke to the National Institute for Public Health and the Environment (RIVM) of the Netherlands. We talked about all the current guidelines and exposed the safety risks of RootPatch. This helped us to get an understanding of what needs to be done before RootPatch can enter the market.

Experts

In order to develop RootPatch we contacted many experts in different fields, from plant science to theoretical biology. We discussed potential pitfalls and what could be done to improve the project.

Education

We spent a lot of time and effort in educating the public about science areas linked to synthetic biology. In our main education project, we developed an online lesson series about the principles and ethics of genetic modifications in human embryos. Apart from that, we were also active in on-site education (as much as the COVID-19 pandemic allowed us to) as well as in writing blogs and newspaper articles to promote our project to the broader public.

Lesson series

We have developed an online lesson series about engineering of human embryo-DNA using CRISPR-Cas. The lesson series is implemented in a national discussion on human genome editing, called DNA-dialoog (English: DNA-dialogue), which encourages a two-way dialogue with the whole society. The lesson series is available online in both dutch and english, making it available for everyone who is interested.

The landing page of our Lesson series "Engineering Life". At the bottom: course-cards leading to Lesson 1 and Lesson 2 of the lesson series.

We incorporated a questionnaire from one of our partners, who is collecting opinions about changing human embryo-DNA. The results of the questionnaire will be used by the Dutch government to create policies on the future use of genome editing techniques in our society. To bring science communication to a higher level, three of our team members are Graphical Design students. They designed the videos and animations for our lesson series as well as our project promotional video and project presentation video.

The course interface of Lesson 1 of our online lesson series "Engineering Life". On the left hand side of this page, the learners can navigate through the different Chapters and follow their course progress.

Outreach activities

Apart from developing the lesson series, we actively taught school kids about scientific topics in an event called Ontdekdag (Discovery-day in English). In addition, we communicated our project to the broader public via blogs and newspaper articles.

Teaching school kids during the Ontdekdag.

Our project, presented in the University newspaper UKrant and in the newsletter of the Nederlandse Aardappel Organisatie (Dutch Potato Organisation).

Acknowledgements

• Jan Spoelder - for his help with the experimental design

• dr. Sander van Doorn - for the modelling guidance

• prof. dr. Oscar Kuipers - for helping us choose our host organism and giving ethical advice for our lesson series

• Niels Alberts - for the support and guidance during the development of our lesson series

• Sjefke Allefs - for his involvement and professional support

Sponsors

We would like to thank all of our partner institutions and sponsors for their input in our project.