Progress Indicator Animation

Summary
All together, we came into contact with 50+ experts and stakeholders out of more than 18 different countries from all continents. Their contributions shaped our project into what it is now and the various backgrounds of our experts and their sometimes contradictory opinions elevated our project to a real-world application that aroused interests in farmers, mycologists and experts from the industry alike. For example, we discussed the optimisation of our proof of concept with Professor Dr. Mark Varrelmann from the Institute for sugar beet research (IfZ) at the University of Göttingen. He suggested us to extent our proof of concept and test our system for a filamentous fungus. As a result, we also tested our system in Aspergillus niger to closer resemble a crop pathogenic fungus. Furthermore, we were able to improve biosafety and biosecurity aspects of our project according to the legal situation, guidelines and suggestions for improvements through experts. By doing so, we made sure our Troygenics do not pose any harm for the environment or the consumer.

	General Experts
	Project
	Forwardfarm

General

First contact 02.07
Skype Conference 04.07
Extended Phone call 23.08
Visit at Bayer Forward Farm in Rommerskirchen 26.09
Dr. Patrick Beuters has been one of the most supportive experts during the course of our project. We first approached him at the beginning of July but remained in contact ever since. Since he is responsible for the Fungicide Consulting at Bayer CropScience in Germany and has helped us a lot to receive insights into the economic role of fungicides as well as their overall importance.
During our first Skype talk we described our project, our plans as well as our situation. Dr. Beuters then gave us insights into the current situation regarding fungicides and crop protection.
He explained, that he would consider plant damaging fungi to be the biggest threat to wheat plants we have to face in Europe. In South America or Africa for example insects are having a larger impact. In Central Europe the harvest loss for wheat through crop damaging fungi is estimated to be between 30 – 60 %.
Fungal diseases like powdery mildew (Erysiphe graminis) and leaf blotches (Septoria tritici) are can pose severe threats to cereal plants. And pathogens like Ramularia for barley can have serious impacts on harvests. Furthermore, many fungicides, like Chlorothalonil, are being banned next year due to increasingly stricter regulations and currently, there are no suitable alternatives available.
The demand for alternative approaches to fungicides has grown immensely over the last years. Unfortunately, alternatives to fungicides, like biologicals, do not show the same efficiency as fungicides. Because of this the need for alternative approaches is ever increasing.
One approach used for the farming of grapes, potatoes and general organic food are fungicides based on copper particles. However, these can have negative side effects on insects and other ground-dwelling animals and accumulate in the soil. Luckily as they are not taken up by the plant, they do not pose a threat to the consumer. Currently, agriculture for grapes or potatoes is not feasible in Europe without the application of copper particles. Besides that, copper is a limited resource and there are many other important usages of copper on an industrial scale, for example electronics.
Our Troygenic would be almost impossible to apply under current laws and regulations for genetic engineering in Europe. Furthermore, the application of CRISPR outside the laboratory is forbidden in Europe, while it is allowed in, for example, the USA. This also means, that CRISPR cannot be used to increase the resilience of plants against fungi in Europe.
Like many other stakeholders, Bayer is investing in research regarding alternatives to fungicides and intensively tries to make biologicals more applicable and a better alternative to common reagents. Of course, this requires close cooperation with farmers, agricultural societies and other stakeholders.
In our discussion, we also talked about potential ideas for a cooperation with Bayer. Dr. Beuters forwarded us to a broad range of experts from varies fields, contacts within Bayer and a broad network of farmers.
In addition to that, he sent us more research materials to deepen our understanding for our topic even further.
Dr. Patrick Beuters explained to us how fungicides are generally applied by farmers. Through his suggestions, we decided to put more thought into adjusting our production system, to make our Troygenics applicable with commonly used methods of applying fungicides. By emulating these methods, our Troygenics would be applicable with the tools farmers already possess. Thereby, the transition from using fungicides to using our Troygenics would facilitate their use by farmers. Since the most common fungicides are used in concentrated liquid form, we adapted our production process to receive a product comparable to the emulsion’s concentrations or concentrations in water that are used to formulate fungicides. After this initial idea, we decided to focus on altering the formulation of our product to resemble exactly the formulation of the most common fungicides. The most widely used fungicides are azoles, since they are broadly applicable.
Furthermore, we asked Dr. Beuters for consulting, regarding which kind of fungicide our systems comes close to in its function. Because our Troygenic effects pathogenic fungi directly and therefore prohibits the growth of fungi but also stops the spreading of already existing infections, its mode of action is comparable to the one of most azoles. As a result, Dr. Beuters further consulted us about azoles and their mode of action as well as their formulations and methods of application. In the end, he even gave us access to two fungicides that we used for our proof of concept, some test on self-grown wheat plants and analyses of fungicides. He also sent us the respective data sheets, manuals and security information as well as further details about safety measures for farmers. He highlighted the importance of the “German Plant Protection Act” (deutsches Pflanzenschutzgesetz) and the “Ordinance on Plant Protection” (Pflanzenschutzsachkundeverordnung).
We also called Dr. Beuters to ask for some information to optimize our modelling regarding the development of resistances against fungicides.
He pointed out, that since the risk and speed of resistances occurrence is difficult to estimate for a novel mode of action, the risk of resistance development is not easy to include into the development for new fungicides. The threat of resistance development can partially be taken into account for the breeding of resistant plants as an alternative measure to counteract harvest loss. Dr. Beuters forwarded us to a larger set of experts regarding questions for our modelling and data to imbed into it.
In general, the risk of resistances being developed is strongly influenced by a sheer endless number of factors. Furthermore, there are many different fungicides with very different modes of action. Farmers try to avoid the progression of resistance development with multiple measures, for example switching fungicides or the order of their usage. Others use mixtures of different reagents. The chambers of agriculture have many suggestions for the usage of fungicides, but at the end farmers are responsible for their own decisions on how to use them, as long as they stick to the law.
Besides his extensive support he also gave us the chance to visit the Bayer Forward Farm in Rommerskirchen, a testing area to educate people about agriculture, common methods and recent technological advances. It also resembles a specialised facility for people to get in touch with farmers and stakeholders.

First Contact: 05.09
We approached Professor James Brown as an expert for fungal diseases of crops and other plants to receive an evaluation of our project as well as our concept for a potential application.
Prof. Brown gave us advice on various points of our project, which he depicted as an interesting approach that should quite certainly be a completely novel approach.
First of all, he portrayed that fungicides, although they can be unspecific and have off-target effects, are generally quite specific in their mode of action. Moreover, off-target effects are rather rare and usually weak or hard to detect. Besides this, we would have to put thought into how our system could have similar side effects, he explained. Also, he pointed out that specificity towards a certain pathogen, as well as a broader specificity can both be advantageous. For our system we would have to argue why it would be beneficial to have such a specific mode of action and off-target effects would be important enough to be worth avoiding.
Also, he pointed out, that the process of safety testing for fungicides is a rigorous, expensive and time-consuming one. During this process, the fungicides are extensively tested for potential negative impacts for the environment and for humans. This procedure takes nine to ten years after the discovery of the fungicide molecule. These tests are also an important reason, why fungicides generally have very few off-target effects if they are allowed to be commercially used. The patent life of fungicides lasts 17 years. Hence, the companies have only seven to eight years to significantly profit from the sales of a new fungicide while it is still in patent. This period can only be extended by patenting the manufacturing process or keeping important features of the product secret. The process for testing medical pharmaceuticals is quite analogous, although the period of safety testing and is two years shorter, he added. If our Troygenics would be aimed for a commercial use we should put more thought into product marketing and a strategy for IP protection to make sure to make a profit on the system, he suggested.
In his view, the short patent life of fungicides is also contributing to the problem of resistance because to make a quick return on their investment, companies have to sell as much of their product as fast as possible. The same effect applies to medical antibiotics, which results in them not being attractive investments for pharma companies anymore. For a commercial application a way to extend the patent life of the Troygenics would thereby be beneficial to maximise the potential profits.
If we knew of any possibility that fungi could evolve resistance to our Troygenics this would also have to influence our marketing strategy, Prof. Brown mentioned.
Regarding mathematic models he named us two further contacts to come in touch with.
One thing Prof. Brown especially pointed out, was that non-specific effects of fungicides are actually often advantageous and thereby desired by farmers because this substitutes multiple treatments with different pesticides. This way a crop can be sprayed just once and still be well protected from multiple diseases. Prof. Brown proposed, that the most important limitation for disease management on the fields is the number of opportunities over the course of a year, when the ground is dry enough for the usage of a tractor but not to dry, so plants are not getting damaged because of the drought. Only then, it is possible to spray against crop diseases of local importance. The conditions to do this are really fairly specific. There should be no rain at the moment and no rain expected for the next 12 to 14 hours at wind speed forces of 1-3. For crops in greenhouses, there is more flexibility, but the number of sprays is aimed to be minimized because of two reasons. Firstly, the cost of spraying, the staff and the fuel should be minimized. And secondly, crops with fewer treatments of reagents like fungicides are more attractive for supermarkets. Prof. Brown also gave a distinct for this development: During the 34 years of his career the average number of sprays applied per year to blackcurrants, an important crop where he lives, dropped from 37 to one.
Moreover, there has been concerns about negative, non-specific effects of fungicides on soil microbiota but far to less research on the subject. Prof. Brown portrayed, that he would consider a larger effect of the fungicides very unlikely. The amount of fungicides sprayed per unit area is very low. Furthermore, fungicides are usually applied when the crop canopy is well-formed and the great majority of the spray lands on the plant.
While there are some advantageous in having a very specific targeted method to fight certain crop pathogens, there are also define disadvantageous. It is important to know when such a system would be beneficial and when it would not be, Prof. Brown concluded.
The biggest current concern, regarding the off-target effects of fungicides is that triazole fungicides, which are targeting ERG11 (CYP51) in the ergosterol synthesis pathway in fungi, can have off-target effects on at least one enzyme involved in the hormone synthesis in mammals. These effects are still very small though and far below the level detectable by epidemiological analysis. The EU legislation still requires strictly no off-target effects on the mammalian reproductive system, by whatever method of testing.
This behaviour has made the companies, that develop new fungicides, very nervous, since the increasing threat of losing huge amounts of invested money because of unknown off-target effects. Thereby, a system like ours would have to prove that it does not have any off-target biological and biochemical processes, especially regarding the mammalian reproductive system in any way.
Since the interest in fungicide analogues is quite high, a range of methods have been suggested. But these are most often not as effective as the fungicides and for example require multiple applications on the field. Also transporting and application cost would increase.
Further very important characteristics of our method would be its efficacy and therefore the likely cost of the application. Additionally, a reinfection of the plant after the application should be expected. Also, it would be important to know how long our Troygenics would take to degrade and thereby how many applications would be necessary to effectively protect the crop.

First Contact 19.09 We reached out to Dra. Francismar Corrêa Marcelino-Guimaraes as a contact at The Brazilian Agricultural Research Corporation (Embrapa) to ask her about the most important fungal crop pathogens in Brazil and South America, especially the Asian Soybean Rust. Dra. Marcelino-Guimaraes confirmed the Asian Soybean Rust to be the most important crop pathogen in Brazil and depicted the current situation: All strategies to control this pathogen have failed and the resistance to fungicides is continuously increasing. Moreover, genetic resistance, referring to the resistance genes from Soybean, are not effective against all variants of the pathogen that occur on the field. The fungus that is causing the Asians Soybean Rust (Phakopsora pachyrhizi) is a biotrophic pathogen. Because of this, it can not be grown in artificial culture media. As a result, the transformation is very complicated and there is no protocol available. Dra. Marcelino-Guimaraes is also part of an International Consortium with the objective of obtaining a reference genome of this pathogen. The genome should be public this year (2019) with a set of all 22000 predicted genes. The Asian Soybean Rust is the only crop disease, single pathogen targeted fungicides are being used for because of the massive impact of this crop harming fungus in Asia, Africa and South America. This makes this fungal pathogen an exceptional good target for the application of our Troygenics. Dra. Marcelino-Guimaraes even pointed out, that we are taking part in really important research for the scientific community. She also forwarded us to colleagues of her and named us further sources of information about the impact of the Asian Soybean Rust and the current state of research.

First contact 09.09 Phone call 10.09 Video conference 13.09 Paul Zabel works at a Research Associate for the German Aerospace Center (DLR) in the Space Segment Systems Analysis department and has been part of a unique pilot experiment, the EDEN ISS project. The objective of this project, founded by the European Commission under the Horizon 2020 research programme, was to test a special laboratory suited for plant breeding and food production on future space missions. To test this laboratory under extreme conditions Paul Zabel spent one year in Antarctica near the AWI's Neumayer Station III to cultivate vegetables and herbs.
We approached him to receive an evaluation on the impact of fungi under these special conditions, as well as the precautions that have to be taken to prevent plant damaging fungi.
After we called Paul Zabel, we organised a video conference to discuss the role of fungi during his expedition and some aspects of our project.
There have been two shifts on the EDEN ISS since the start of the project, one in 2018 and one in 2019. A season lasts from February to November. Paul Zabel spent the very first season at the South Pole for this project.
In both years, there have been problems with fungi on the station. During the first months of its use the fungi display a faster growth and can form biofilms in the nutrient solutions of the plants. Under these conditions, fungi can start to grow on moist parts of pipes and uneven surfaces. Over time the growth of the fungi reaches a point of self-regulation. During both years, chlorine cleaner was used to counteract this problem with only moderate success. In the next years, the usage of such methods is planned to be reduced to a minimum. Samples from plant surfaces and the water have been taken and are being analyzed by the Astrobiology Group of the Institute of Aerospace Medicine in Cologne. Still the general burden of fungi on vegetables produced in the Antarctic has shown to be 1000 – 10000 times smaller than the one of store bought vegetables. Analyzed contamination samples at the arctic greenhouse rather contained spores of fungi than bacteria.
For the plants, these fungi do not pose severe threats, only the optics of some plants are affected by the fungi. But beyond that, pipes and filters can become clogged through the biofilms and can cause technical issues. Also, the plants are growing in special cubes out of mineral wool that can become overgrown by fungi. This can potentially lead to the stem of the plants to soften up. As a result, the stems of healthy plants can break and the plant dies. Still this issue only affects a small number of plants per year.
At the moment, the seeds are not being sterilized or treated in any special way, since most of the surroundings are clean surfaces, also the cubes of mineral wool are being heat-treated before use.
It is still under discussion, how extensive the sterilization process for later space missions would have to be. Since, on the International Space Station (ISS), everything is mandatory being sterilized and there are still fungal contaminations occurring in space, Zabel reports. Besides that, growing plants can not be sterilized. In the Antarctic, people are visiting the greenhouse with protective clothes, are not allowed to touch anything and have to follow certain rules like “no food” as an “intermediate solution”. This approach could be adapted for space missions later on. Cleaning and sterilization procedures are time-consuming and expensive and will be avoided if possible. In addition to this, vapors of cleaning agents would have to be filtered out in space.
The question arises, if a sterilization of a garden in space would even be possible. For example, on the ISS, four salads have been grown, sterilized with chlorine solution and water and eaten. But it is a rather minor effort to sterilize four salads. The greenhouse Pail Zabel worked in has a base area of 12,5 m2 and would be almost impossible to completely sanitize. Moreover, the water consumption of the sterilization process is quite high. To sterilize 1,5 kg of rocket (salad) around 60-70 l of water are needed, although the salads shelf life expands for about one or two weeks treated this way. So, it has to be evaluated, if the sterilization process would be worth all of this and if the approach sterilizing everything on space missions could actually be continued on this level. After all, the vegetables grown in space could still be eaten without any sterilization process. Maybe the rules on this have to be loosened a little bit, Paul Zabel concludes.
Tests have been conducted with ozone lead into the nutrient solution to lower the growth of fungi. But this has led to the binding and flocculation of nutrients like Iron or Calcium, making them inaccessible for the plants. For future mission in the Antarctic, further measures are already planned. Like tests regarding the sterilization of the seeds and the usage of hydrogen peroxide solution with silver ions added. Also, biological approaches are planned to promote a natural balance and the usage of other strains of fungi to keep the harmful ones at bay could also be a potential measure.
One thing Paul Zabel also pointed out, was that the need for the sterilization of the plants would considerably limit the selection of the plants, since the plant surface has to be accessible for the fungicide. While a salad is quite easy to sterilize, this is almost impossible for crops like carrots or potatoes because they need to be in direct contact with the nutrient solution. Research on soil-free cultivation methods of these plants is being conducted but showed to be rather challenging. Keeping the current situation of plant breeding in space in mind, there could actually be an application of our Troygenics for future space missions. Since the ultimate goal of a colony on, for example, Mars would be the total self-sufficiency of the colony, the supply on reagents to fight plant pathogens would be extremely limited. A possibility to produce targeted reagents, that only work on the fungi that are harming the plant without harming any of the beneficial fungi for the plant or the plant itself could be a huge benefit. Furthermore, our Troygenics could be modified, adapted and produced in a laboratory in space using E. coli. As a result, the colony could operate independent from supplies from earth. Since the dependency on the own harvest would be way more important in a self-sufficient space colony and the loss of plants through pathogens, fungi, or even radiation in space should be limited to an absolute minimum, our system could contribute to ensure the protection of crops under these conditions. Avoiding technical difficulties, like clogged pipes or filters would be another important issue, since repair parts are often not available.
The ability to manufacture tools or measures to solve problems, like the Troygenics, to assure the self-sufficiency of the colony in space is extremely important, Paul Zabel resumes.

First Contact 05.09 Skype Conference 06.09 Gopaljee Jha is doing his PhD. At the National Institute of Plant Genome Research (NIPGR) in New Delhi and focused his Research on plant microbe interactions.
After we presented our project and our situation, we discussed several aspects of our work.
He suggested us to alternatively think about modifying mycoviruses for our project, which could be a focus of later research.
He pointed out, that special bacteria, called Burkholderia, are able to express proteins which can destroy the cell walls of fungi. As far as they were researched, this worked in every fungus that was tested.
Additionally, he mentioned that fungi are also capable of quorum sensing, like bacteria. Because of this, he expected hormones to be a better possibility to induce endocytosis. At this point, we already decided to use mating factors for this purpose and have been affirmed by this advice.
Gopaljee Jha underlined, that the cell walls of yeasts and fungi differ in many ways and tests with yeast are thereby not representative for the functionality in fungi. This was reflected in our proof of concept, by introducing Aspergilli as filamentous fungi as a second organism to test our system in.
In general, Gopaljee Jha assessed specificity through endocytosis to be difficult to achieve, since many fungi are very closely related.
As far as he knew, there is no gene that is expressed by all pathogenic fungi during infection but expected that we should be able to find some via RNA analysis if we put enough effort into it.
Regarding the legal situation of genetic engineering in India, Gopaljee Jha stated, that because India is located between the USA and the EU its market is influenced by both. As a result, transgenic organisms would be almost impossible to set free under legal conditions. To some extent, specially breed variations of crops should be possible, though. At least as long as they are interspecific.

First contact 25.06
Phone call 19.08
We contacted Prof. Dr. Karl-Heinz Kogel, head of the institute for phytopathology at the University of Giessen (Germany), to gather additional information on fungal crop pathogens, their impact and their ecological and economic value.
After we explained what the iGEM competition is and our project we discussed the different parts of our work.
Prof. Kogel pointed out, that our project has to be planned with a lot of foresight, since the current political and social opinion on genetic engineering would prevent such an application in the near future. With the legal situation regarding this topic in the EU he did not expect a system like our Troygenics to be applicable anytime soon.
Besides that, he still liked our approach and considered it a creative idea. Although, he also mentioned, that most comparable approaches do not try to solve this many problems at once, which makes our project susceptible to more problems in direct comparison.
He was especially interested in our approach of an induced endocytic uptake into the pathogenic fungi via specific ligands on the surface of the Troygenics.
As an additional, similar approach he proposed the idea of using only dsRNA instead of the Troygenics and gene silencing instead of CRISPR/ Cas. While this concept would elute some of the problems of our system, like the required uptake of, in comparison, larger particles into the target cell, it would also establish new ones. On one hand this would prevent GMOs to form on the fields. This would make it more realistic to implement and is closer to the current praxis. On the other hand, this would require the use of siRNAs which are distinctly more instable and way larger amounts of them would be needed to achieve the same effect. Moreover, this alternative approach would lack the shuttle our concept provides. Finally, the biological production of the Troygenics via E. coli would be a clear advantage in direct comparison.
Although, this was a great suggestion, we still considered our approach as a better solution to generate a broader range of potential applications.
Furthermore, Prof. Kogel gave us additional information about pathogenic crop fungi and their impact on agriculture. As a further potential target, that would be important to come by, he named Fusarium graminearum as an important pest of wheat and corn plants all around the world. He even mentioned some genes of certain pathways to target for our system. In this case, the pathogen usually is treated with azole-based pesticides, which deactivate proteins essential for the infection of the plant.
F. graminearum has become a substantial threat to the food security of the named crops.
Regarding Puccinia graminis, a devastating wheat pathogen, that managed to develop multiresistant strains and is threatening the food supply in parts of Africa and that is spreading continuously, he confirmed the danger of the pathogen and added, that its impact is going to increase dramatically because of the progressing climate change.
Prof. Kogel also confirmed, that problems due to fungicide resistance are already a relevant threat to our food security and supply. A far range of papers are published about this topic.
At the end, we asked Prof. Kogel about methods to distinguish between different spores of fungi and to determine their species without sequencing parts of their genome. Prof. Kogel explained, that the morphology of the spores of different fungal species are quite easy to distinguish with a closer investigation via microscopy.

First Contact 21.09 We reached out to Wei Xiong as a member of the International Maize and Wheat Improvement Center (CIMMYT) in China after we read about his former research, investigating in the influences of weather conditions on harvest in Eastern Europe.
We asked him about how changes of the weather and the influence of climate change is estimated to impact the crop production in Europe. While his work only not considered the influence of crop pathogens, but he stated, recent research estimated that the magnitude of the effects such diseases have on the global food production is on the same level than the effects of climate disaster. Currently, global research efforts are aiming for overall estimations on the global scale impacts diseases and pests are going to have on key crops worldwide. For this historical record, predictions for the global climate, simulations and remote sensing are used to calculate risks that have to be expected. These new insights can help to effectively react towards the increasing influences of more extreme weather conditions or crop pathogens. For example, observation data showed a significant linkage between the occurrence of specific pathogens and global warming and presented implications of this linkage for plant breeding and food production.
We also asked Wei Xiong for advice for our modelling of fungicide resistance development. He stated, that it is generally possible to create such a mathematic model to calculate these results as long as you have enough mathematic data to establish such a model. There are algorithms to estimate the risk of specific pathogens based on, for example, remote sensing and weather information. After all, resistance to fungicides is more or less an ecosystem process, it depends on a broad range of environmental factors, like the soil, the weather or the type of fungi.
He and his research group are still working on modelling the impact and mutation of rust fungi and mainly focused on interactions between the pathogen and its environment. They fed these data into a statistical model they created to estimate the development of rust pathogens. By incorporating said algorithm into a wheat mechanical model effect of rust pathogens could be assessed. They used the NWHEAT model, which was embodied in DSSAT. This model worked well to simulate this development.
Moreover, other models like for example Stics could still be introduced.
In the end, we also asked Wei Xiong about the most important fungal crop pathogens in China and East Asia. He replied, that the most influential pathogenic fungi to mention here would be a set of rust pathogens, like for example leaf rusts or stem rust.

First contact 04.10
Phone call 16.10
We approached Prof. Haberlah-Korr because of her extensive knowledge about plant protection and ecology and wanted to ask her about the impact fungicides have on ecosystems.
Prof. Haberlah-Korr pointed out, that the impact of the fungicide depends massively on the kind of fungicide that is used as well as its mode of action. Besides that, fungicides can have negative side effects regardless of if they are biological or chemical reagents. Biological alternatives like copper particles are widely used. These particles can accumulate in the soil but are not harmful for humans in the doses they are used in.
A very broad range of fungicides is used with very different modes of action. Some of them can have insecticidal off-target effects or can have negative impacts on soil or water systems.
Despite the public opinion, fungicides do not have a massive negative influence if they are used properly. Besides that, their usage can be justified in many cases, since our level of food production would not be feasible without them.
Many fungicides work quite specific and do not pose a threat through off-target effects. Others, like many azoles, have a broader mode of action and can even show endocrine effects in mammals. Recently, many fungicides have been revaluated because of stricter regulatory limits, but still for example endocrine effects are difficult to measure.
The Federal Office of Consumer Protection and Food Safety monitors the effects of different fungicides and their potential hazards.
Meanwhile, farmers often try to avoid fungicides by more carefully choosing their crop strains or are using resistant plants.

First contact 02.08 Skype conference 13.08 We first came into contact with Prof. Peter Langridge after approaching the Wheat Initiative, a global initiative that establishes, coordinates and strategically organizes research in developing and developed countries on an international level. Moreover, the Wheat Initiative provides a platform for communication between the research community, the funders, policy makers and stake holders. They also support activities and events for this cause. After we got in touch via e-mail, we eventually had a Skype conference and discussed our project, as well as how the Wheat Initiative could support us and who in the Wheat Initiative could help us best. Further, we described our project and our current progress to Prof. Peter Langridge, he highlighted, that the uptake into the fungus resembles a problem in most similar approaches, because fungi are quite complex. Also, he considered our project a good idea, since there is an enormous economic interest in new alternative approaches like our system to fight crop pathogens. Moreover, there is a growing interest in “non-chemical” alternatives that are supposed to lower the negative impact on the ecologic systems. At the Wheat Initiative they are also researching what happens if you would have to farm wheat without using any chemical compounds like pesticides. Furthermore, he pointed out, that the regulations on actually applying systems like our Troygenics in the field can be quite challenging and time-consuming. For example, in the US, there are some regulated and some unregulated forms of CRISPR/Cas and new measures would have to be tested on their classification. Prof. Peter Langridge further pointed out, that the Wheat Initiative is a great platform to get into contact with more experts and also forwarded us to a whole range of expert from all around the world. At the end, he listed some programs and events to reach out to, which might help us gaining attention, like the 1st International Wheat Congress in Sasketchewan, Canada, some workshops about molecular measures in plant breeding at the end of September as well as some Newsletter we could present our project in.

First Contact 05.09
We reached out to Dr. Singh as a contact at the International Maize and Wheat Improvement Center (CIMMYT), an international non-profit agricultural research and training organization that is connecting scientists and research programs worldwide to advance in crop protection for the two most important cereal grains in the world: maize and wheat.
Dr. Singh pointed out, that the International Maize and Wheat Improvement Center has mainly set its focus on keeping damages through fungal pathogens under control by using host resistances of the plants. Furthermore, fungicides are rarely used by smaller farmers in Asia or Africa. Keeping this in mind, the usage of our Troygenics would be quite complicated to realise in those African and Asian regions, since there is no infrastructure to distribute our system in an easy way. Also, our Troygenics are designed in a way, that they could be applied with the tools that are commonly used by farmers to apply fungicides, but without them being used, distributing the respective tools would be an additional problem.
Another point he criticized, was that the targeting and neutralizing of one single fungal pathogen would not avert the threat of fungal damages for the crop. Because of the high variety of fungal pathogens in a country or across countries. To effectively protect the crops a broader applicability would probably be necessary, he concludes. Beyond that, in his opinion this method would have to be implemented into the host plant but like our system, this would be genetic engineering and Dr. Singh did not expect such a measure, based on transgenic organisms, to be applicable in the real world, since the legal situation would clearly prevent such a system from being accessible for farmers.

First contact 02.10 We came in touch with Åsmund Asdal because of his position at the Svalbard Global Seed Vault in Spitsbergen in one of the northernmost parts of Norway. The Svalbard Global Seed Vault is a secure seed bank for collections of seeds from all around the world. For our project, we wanted to find out if problems with fungi occur even under these extreme conditions.
The staff at the Svalbard Global Seed Vault did not have problems with fungi and also did not take any measures to additionally sterilize the stored seeds. The facility is solely for the storage of the seed, nations send in as backups. The staff of the vault is not responsible for the sterilization of the seeds. Besides that, the sent in boxes are never being opened by the staff of the vault.
To research the resilience of seed borne fungi disease, the Nordic Gene bank started a seed storage experiment in 1986 in a remote coal mine near Longyearbyen. The objective of this experiment is the monitoring of the longevity of seeds as well as the seed born plant pathogens on the seeds themselves.
The experiment was planned for 100 years. After the first 30 years, the first results were published in “Seed longevity and survival of seed borne diseases after 30 years conservation in permafrost - Report from the 100-year storage experiment” in 2019.
After 30 years under permafrost conditions, the level of disease on the seeds stayed almost the same. Moreover, all initial seed born plant pathogens have still been observable.
The experiment of the Nordic Gene bank convincingly portrays the extreme resilience of fungal pathogens.

First contact 13.08 Phone call 15.08
We approached Prof. Dr. Gabi Krczal because as the director of AlPlanta, the Institute of plant research (Neustadt an der Weinstrasse), and former head of department of integrated plant protection in Mainz and former leader of the “Center of green genetic engineering” in Neustadt we really valued her evaluation of our project. During a phone call, we discussed different parts of our novel approach.
Prof. Krczal considered our system as a sensible approach to reach our goal of transforming pathogenic fungi. Besides that, she also mentioned a lot of things that have to taken into consideration for the successful commercialization of our system. For example, the price of our system should not dramatically exceed the price of similar, commonly used reagents, unless we would pose some drastic advantages. Moreover, these kinds of reagents would have to undergo tedious testing processes to be used in agriculture. The legal standards in agriculture are high, even higher than the ones applied in the testing of new pharmaceuticals. To receive an official approval for a new reagent of this kind an investment of about ten million Euro would be considered as normal expenses. Of course, the process of approval would also include sophisticated legal assessments and since using genetic engineering is seen rather critically in the EU it would be hard to realize. Regarding agricultural genetic engineering Prof. Krczal stated that the overall perception of this topic is rather a negative one. But, although the public opinion is mostly against using these methods, genetically altered animal feed is still allowed in Germany. In general, the development of this topic can be described as kind of stagnated in Germany, Prof. Krczal depicts the situation. For example, more than half of the European countries positioned themselves in favor of these new methods while Germany has abstained from the vote. The government of the Netherlands repeatedly tried to permit using genetically engineered products in the EU but was not successful. Because technologies like CRISPR have such a bad reputation in the EU, countries like Germany could encounter problems if they try to import products that have been altered at any point using these techniques. Because of this, the USA already signalized, that they would get the WTO involved to open up the German market for selling these products. Regarding our project, Prof. Krczal stated that it would be an important advantage if our Troygenics would be applicable together with commonly used methods for similar reagents. She hinted that some fungi growing into or inside the plants could pose a technical problem to our system, as they can be hard to reach for substances applied to the outside of the plant. Upon discussing the specifically of our Troygenics in laboratory environments, Prof. Krczal confirmed that they could be used for specifically fight contaminations in cultivations or the detection of pathogenic fungi. To easily validate that the system works, Prof. Krczal also advised us to use reporter genes to assure an easy detection of successful integration of our system into the targeted organism. Beyond that, Prof. Krczal named us some politicians to reach out to, who are dealing with the regulations of genetically modified organisms on a national level.

First contact at German iGEM Meetup in Düsseldorf 05.07 Mail contact 25.07 We first came into contact with Prof. Dr. Lutz Schmitt at the iGEM Meetup Germany 2019 in Düsseldorf and contacted him again a few weeks later to refer on his statements. Prof. Schmitt focussed on the discrepancy between the public perception between the usage of genetic engineering in pharmaceutical and agricultural biotechnology. While using these methods for medical purposes is widely accepted by the society, genetical engineering, that is used to improve food production is even considered unethical by parts of the society. Prof. Schmitt reasoned this with the higher willingness of people to take uncommon approaches to maintain their health. In these kind of situations any measures that assured the survival of the patient would be taken into consideration. On the other hand, regarding agriculture, a strict rejection of genetic engineering and the associated methods can be observed in the society. Since the people in Europe are not threatened by shortages of our food supply the necessity of improving the food production is much lower in the first place. Thereby, progression in the use of said methods is being impended by a lack urgency to improve the food production. Besides that, genetic engineering has become a topic that is often used for political elections or similar agendas and has become kind of a bad issue for many people. For our project, this is especially important for the possible implications of our system. Since we are developing our Troygenics as a platform system, that can be adapted to a broad range of pathogens through minor changes of the system itself, we have to be aware of the opportunities it could resemble for medical and agricultural applications but should anticipate the opposition against its use for food production by many people. To avoid this, we should focus even more on explaining our security precautions and have to point out the possibilities our system could represent.

First Contact: 19.10 Skype Conference 02.10 We approached Tessa Alexanian as a member of the iGEM Human Practice Committee to talk about the value of Human Practice for iGEM and beyond. Moreover, we received a general evaluation of our Human Practice.
We talked about the possible risks from an accidental environmental release as well as rights around the world with regards to the release of GMOs, which is something we worked on in our team team before. Afterwards, we talked about different approaches to regulating scientific research and to transfer it to the field. This gave us a great overview about important topics of Human Practice and gave us examples on how Human Practices can be used.
Furthermore, we talked about judging in general and the interaction with judges at the Giant Jamboree. Tessa also explained to us, that integrating Human Practices into your project is often a question of how your work as changed by the interactions with experts etc.. Human Practice efforts should fit into your overall story, she concluded.

Project

First contact 25.06
Skype conference 23.07
We approached apl. Prof. Dr. Ulrich Schaffrath from the Department of Plant Physiology at RWTH Aachen University to gather further information about pathogenic fungi and their impact on different aspects of society. Besides that, we hoped for an evaluation of our project and some advice for the optimization of our early concepts. We discussed our project and some questions during a Skype conference.
The working group of Prof. Schaffrath is doing research on pathogenic fungi that damage crops like cereals, for example wheat, barley and rice. Asian Soybean Rust is of especially high interest because it has the largest economic impact. A part of his research group is also working on the transformation of fungi. Prof. Schaffrath pointed out, that fungicide resistance has been an underrated problem for quite some time now. Meanwhile, resistances have become an even bigger problem and new strategies to fight them are urgently searched for. For example, the plant pathogen stripe rust diminishes the area of chlorophyll of the plant and thereby reduces its yield. Because they need lower temperatures, they only spread around the UK but meanwhile a new strain emerged that also thrives under the warmer climate in countries like Germany. Furthermore, the wheat stem rust strain Ug99 had overcome all resistances implemented into plants that have been used against it and poses a threat to whole harvests in all affected areas. A new counter strategy for the fight against pathogenic fungi, is the usage of a mixture between different reagents and coordinated usage of different fungicides at different times. Research has shown, that only a single nucleotide mutation is required to potentially gain resistance in pathogens against a fungicide. Because of this, resistances can emerge after one or two years of fungicide usage. Because only one base pair has to change to overcome such a pesticide, pathogens like wheat blast cannot be treated effectively with fungicides, which has led to huge problems in Brazil and other parts of the world. These pathogens make the necessity of new approaches even more urgent. Regarding the realisation of our system, Prof. Schaffrath described the legal situation in Germany and the EU as the biggest barrier. For the induced uptake via endocytosis he indicated that finding the right surface ligands will be one of the hardest tasks to accomplish, since many pathogens are not well researched. Moreover, many pathogenic fungi are quite hard to study in the lab. The wheat stem rust (Puccinia graminis f.sp tritici) for example is an obligatory biotrophic organism which cannot be grown in a petri dish. After we focussed on P. graminis at the beginning of our project, we shifted our attention on other pathogens after our discussion with Prof. Schaffrath.
While achieving specificity through the surface ligands, can be difficult to achieve for some species, accomplishing specificity through the Cas 13a is more likely to be successful, since many fungal genomes are characterised quite well. We decided to make this part the most important one to assure the specificity of our Troygenics, additional to the other measures like the specific ligands. Another approach we discussed, was the idea to use mycoviruses as alternatives to our Troygenics. Prof. Schaffrath mentioned to not be an expert in this field, but we still managed to evaluate this topic. Since mycoviruses are not transmitted easily between fungi but rather through the fusion of hyphae and asexual reproduction their uptake would be rather difficult to achieve. If the mycoviruses would be sprayed onto the fields like fungicides or the Troygenics, the uptake would be estimated to be rather low in comparison.
Another topic we talked about was, if the usage of fungicides or the usage of pathogen resistant plants would be more effective to which he replied, that both are important measures of agriculture and are equally useful and necessary. Resistance genes interacting with pathogen proteins by protein-protein-interaction can be overcome by the pathogen quite easily by point mutations. Therefore, plant breeders began stacking resistance genes to lower the risk of these preventive measures losing their effect. Because plant breeding requires a lot of time, fungicides are often needed as a faster counter measure against new pathogens. However, fungicides might impact the environment and therefore must be used carefully and new, more precise versions of fungicides have to be developed. Furthermore, to lower the negative impact on the environment modern pesticides are applied at very low concentrations to fit the changing legal situation. The optimal solution to this would be perfectly working resistant plants, but for now, fungicides are still required. A well-managed mixture of both measures should be the optimal approach nowadays. We also talked about genetic engineering and its public perception and concluded, that the public discussion is often held on an emotional level. While many scientists are in favour of genetic engineering, it has also become a topic often misleadingly used for politics. The unease about genetic engineering should be evaluated critically, as people often disapprove genetically altered food while openly accepting new medications based on these methods. Moreover, there are products accepted by the society that have been produced using genetically altered organisms, like recombinant proteins, which are used in the production of cheese. However, these proteins do not require to be declared as genetic engineering. To preserve our abundant food supply, genetic engineering would represent an important tool.
For our proof of concept, we wanted to know the most common strain of wheat in our area to conduct our experiments under realistic conditions. According to Prof. Schaffrath, there is no such thing as the one most common strain of wheat for Germany. Instead, the optimal strain varies locally and from year to year as well as regarding to the location in comparison to, for example its distance to the coast. He also advised us on contacting a plant breeding companies like KWS to get access to wheat seeds, which we implemented later on during our project.
As further important crop pathogens he named Phytophthora infestans, the potato blight, and Asian Soybean Rust but also some bacteria and crop damaging insects. Besides that, he explained that fungi can have a very negative influence on trees as well. There are fungi that can destabilize trees and endanger whole species. Further, trees in cities have to be cut down regularly due to these fungi infections. Regarding pathogenic fungi for humans, he explained the problem is taking a similar course as antibiotic resistant bacteria. He suspected a higher acceptance of our system as in a medical application opposed to an agricultural tool.

First contact 13.08 Phone call 15.08
We approached Prof. Dr. Gabi Krczal because as the director of AlPlanta, the Institute of plant research (Neustadt an der Weinstrasse), and former head of department of integrated plant protection in Mainz and former leader of the “Center of green genetic engineering” in Neustadt we really valued her evaluation of our project. During a phone call, we discussed different parts of our novel approach.
Prof. Krczal considered our system as a sensible approach to reach our goal of transforming pathogenic fungi. Besides that, she also mentioned a lot of things that have to taken into consideration for the successful commercialization of our system. For example, the price of our system should not dramatically exceed the price of similar, commonly used reagents, unless we would pose some drastic advantages. Moreover, these kinds of reagents would have to undergo tedious testing processes to be used in agriculture. The legal standards in agriculture are high, even higher than the ones applied in the testing of new pharmaceuticals. To receive an official approval for a new reagent of this kind an investment of about ten million Euro would be considered as normal expenses. Of course, the process of approval would also include sophisticated legal assessments and since using genetic engineering is seen rather critically in the EU it would be hard to realize. Regarding agricultural genetic engineering Prof. Krczal stated that the overall perception of this topic is rather a negative one. But, although the public opinion is mostly against using these methods, genetically altered animal feed is still allowed in Germany. In general, the development of this topic can be described as kind of stagnated in Germany, Prof. Krczal depicts the situation. For example, more than half of the European countries positioned themselves in favor of these new methods while Germany has abstained from the vote. The government of the Netherlands repeatedly tried to permit using genetically engineered products in the EU but was not successful. Because technologies like CRISPR have such a bad reputation in the EU, countries like Germany could encounter problems if they try to import products that have been altered at any point using these techniques. Because of this, the USA already signalized, that they would get the WTO involved to open up the German market for selling these products. Regarding our project, Prof. Krczal stated that it would be an important advantage if our Troygenics would be applicable together with commonly used methods for similar reagents. She hinted that some fungi growing into or inside the plants could pose a technical problem to our system, as they can be hard to reach for substances applied to the outside of the plant. Upon discussing the specifically of our Troygenics in laboratory environments, Prof. Krczal confirmed that they could be used for specifically fight contaminations in cultivations or the detection of pathogenic fungi. To easily validate that the system works, Prof. Krczal also advised us to use reporter genes to assure an easy detection of successful integration of our system into the targeted organism. Beyond that, Prof. Krczal named us some politicians to reach out to, who are dealing with the regulations of genetically modified organisms on a national level.

Phone call: 30.8.19 and 17.09.19
We repeatedly talked to Prof. Holger Deising, positioned at the Martin-Luther-Universität Halle-Wittenberg. He advised us to have a look at another fungus: Colletotrichum graminicola. It is a fungus pathogenic for corn and has previously been used to test RNAi systems.
We were curious, whether he thought our system could work in this fungus and he stated that we could just try it out. Following up on this exciting example we discussed all our subsystems and thought about which one we could test for C. graminicola. Upon learning from Prof. Deising, that endocytic uptake is not an issue for using our system in this fungus, we thought it might be interesting if our CeDIS would work.
Discussing this possibility, Prof. Deising mentioned that we could come and visit him in his lab to transform the fungus with our CeDIS, actually enabling us to test it in a real-world corn pathogen.
During our first talk, we discussed the basics of using our system in this fungus: which genes would we want to target, and which promoters should we use to express it.
Since we wanted to use inducible or repressible promoters to be able to properly estimate the efficiency of our CeDIS, allowing us to distinguish whether the fungus just did not grow or got targeted by it, Prof. Deising suggested using an iron-dependent promotor. He also stated that, for this initial test, the genes would not have to be essential: there were some genes he stated he was sure they would be expressed in the conditions we would grow them in. While we did talk to him two more times and discussed our design adapted to C. graminicola, we sadly did not get to visit him in his lab due to the limited time within the iGEM-competition.

First contact 19.09 Video conference 29.07
Prof. Dr. Russel Cox leads a research group that focusses on the biosynthesis of natural products by fungi using methods of Synthetic Biology. We discussed our project during a Skype conference and evaluated some ideas we had together.
He assured us the importance of projects like ours, since these are essential to face the growing demand for food in the future and to secure the food supply. Besides that, the danger of fungi gaining resistance to fungicides becomes more and more threatening and the impact of fungi on food production or the health care system can be expected to increase drastically in the future due to climate change.
He also affirmed that fungi are of great importance for the Industry of Biotechnology but still have an enormous unused potential. Although they are used on an industrial scale many fungi are still not well established. For this, a new method to accelerate the transformation and selection steps would be an important tool.
The importance of fungi for the industry is increasing drastically in recent times. They are being used in fermentation processes on an industrial scale in sectors like medicine and food production, for example for penicillin.
Since he and his research group are investigating in metabolites, we discussed the usage of toxic metabolites to fight pathogenic fungi. Prof. Dr. Cox estimated, that 4-5 different complex genes would have to be expressed to efficiently work for fungi. Through our discussion he affirmed our plan to use a Cas System for our system, since it would be easier to apply for fungi.
Regarding our lab application, Prof. Dr. Cox mentioned, that most substances can be produced with fungi, although this often requires a huge amount of work. The problem of these processes rather concerns the slow growth rate of fungi, the difficult transformation and the specific integration. Because of this CRISPR is often regarded as the easier choice. Also, research with CRISPR as transformation method for fungi is being conducted. An important goal for improving the work with fungi would be a faster method for transformation.
For our proof of concept, he advised us on thinking about yeast and filamentous fungi in different ways because filamentous fungi are considerably more complex. Filamentous fungi are also becoming increasingly important for industrial processes.
Prof. Dr. Cox especially underlined the fact that our system has to be really specific. A system that would target fungi in general would be disastrous for the ecosystem.

First contact 16.08
Dr. Primrose Boynton is conducting research on Saccharomyces yeasts and their interactions with their environment at the Max Planck Institute for Evolutionary Biology in Plön (Northern Germany).
We approached her to receive information about yeast strains and their phylogenetic relationship for our proof of concept. Over the course of our project she advised us on several aspects of our work. To demonstrate the specificity of our Troygenics, we came up with some experiments to compare the reaction of Troygenics targeted on Saccharomyces cerevisiae on said strain of yeast and its closest relative Saccharomyces paradoxus.
Based on her given information we wanted to use the Saccharomyces paradoxus strains 5696 and N44. Besides giving us access to the strains, Dr. Boynton advised us on how to best work in the lab with those strains, for example, by sharing her experience on the cultivation media.
At a later point of our project, she advised us on how to avoiding flocculation of the yeast strains during cultivation. Since the flocculation of yeast cultures massively complicates the determination of the optical density it can resemble a problem. Beyond that, the flocculation of yeast stopped us from cultivating them in our microfluidic chips. Dr. Boynton pointed out, that the level of Calcium in the media is known to be the most important factor for the tendency of yeasts to form flocs. Furthermore, she recommended to ad small amounts of EDTA to the cultivation media to prevent this effect. When yeast cultures are floculating, the comparison and quantification of the growth and thereby the influence of our Troygenics would be almost impossible.
Additionally, floculating cultures can hardly be analyzed with microfluidic devices.

Phone call 21.06
We had an extensive phone call with Prof. Dr. Mark Varrelmann from the Institute for sugar beet research (IfZ) at the University of Göttingen. As the Research group leader for Phytomedicine he was able to give us a lot of insights into the impact crop damaging fungi have on our agriculture and food production in general. He also gave us an overview about the current situation of research for these kinds of fungi and the latest ideas and methods to fight these pathogens.
We discussed our early concept of our project and asked him to evaluate its functionality. According to him our project is feasible even as a completely novel approach, but we have to take some bottlenecks into consideration. All in all, he estimates our project to be very sophisticated and challenging.
The most important problems have advised us to focus on were the Endocytosis into the fungus, the surface ligands for the uptake of our Troygenics and the adaptability to a wider range of fungi, since fungi are an extremely diverse group of organisms.
The Endocytosis is problematic because the cell walls of fungi are considerably more complex than cell membranes of bacteria and resemble a quite selective barrier between the fungus and its surroundings. Often there is little research conducted on the cell walls of certain species of fungi. Dr. Varrelmann considered the endocytosis uptake of larger particles, like our Troygenics as an especially challenging task to achieve.
Furthermore, he estimated, that the specific surface ligands our system is using to initiate specific uptake into the targeted crop pathogen are limited by the current level of research on this field.
Often the molecular composition as well as the function of some fungal cell wall structures is lesser understood than it would be necessary for our project. So, to realise our project for a larger range of targets, we would have to put some thoughts into how phytopathogenic surface proteins could be identified, since they have not been characterized yet. He highlighted, that a thorough knowledge about surface proteins would be necessary to realise our system as an application for a special fungus. He also added, that genome databases often only contain a small amount of information about phytopathogens.
Most importantly, Dr. Varrelmann advised us to extend our proof of concept with yeast by an additional filamentous fungus to reduce the difference to a, mostly filamentous, pathogenic fungi. Therefore, he suggested Aspergillus niger for this addition, as it represents a well-studied member of the filamentous fungi and has a broad application in the biotechnology industry. Since we are aiming to create a new lab application based on our system to transform fungi and the potential targeting of pathogenic fungi for humans, like some Aspergilli species, a model organism closer to a pathogenic fungus is crucial for out project. Therefore, we decided to integrate Dr. Varrelmanns suggestion into our project.
Last, but not least, Dr. Varrelmann introduced us to several different fungal crop pathogens of economic importance and lead us to further research about this. Moreover, he put us into contact with other molecular mycologists that helped us to optimise our project.

First Contact 30.08
Phone call 02.09
We contacted Dr. Olaf Kniemeyer in the framework of our research regarding Aspergillus niger and Aspergillus nidulans. Moreover, we hoped to receive access to a strain of A. nidulans for our project, since we planned to use this strain to verify the specificity of our Troygenics with our proof of concept.
After we depicted the iGEM competition, our project and the situation we were in we discussed potential methods to induce endocytosis in Aspergilli.
Dr. Olaf Kniemeyer explained, that A. niger is mostly used for applications in biotechnological processes, while A. nidulans is rather used to conduct research on the genetics of Aspergilli.
As a method to generate an uptake through the fungal cell wall he mentioned the possible usage of amphotericin B which enables an endosomal uptake. For our project this would not be applicable though, because as a polyene it intercalates with the cell membrane to induce said uptake by opening small holes in the cell membrane. By doing so it would also damage liver and kidney tissue, which would make it difficult to apply for therapeutically uses. Since, Aspergilli are able to cause aspergilloses some of the have human-pathogenic characteristics. Around 75% of aspergillosis are caused by Aspergillus fumigatus, but A. niger can also be found responsible for this disease in less common cases. Industrial production strains are considered as bsl 1, like A. nidulans.
Regarding A. nidulans, we asked him for access to a wild type strain and thought of the different steps that have to be taken to receive access to these trains. We later received a lab strain of Aspergillus nidulans per post.
Dr. Kniemeyer depicted, that common methods to work with these fungi in the lab are fully functional. If we would want to establish our Troygenic as a lab application, the referring method would have to be at least faster, cheaper or more efficient that these commonly used methods.
As further important human-pathogenic fungi, that could be targeted with our system, Dr. Kniemeyer named especially A. fumingatus and some other fungi. In the USA he named some Cryptococcus strains. These fungi would resemble important possible targets our Troygenic could be constructed for.

First contact 07.08
Multiple contacts via e-mail and phone for organizational purposes
We reached out to Mrs. Annika Roos who is the marketing consultant for wheats in our part of the country at KWS Lochow GmbH, a leading distributor of seeds for agriculture in Germany and beyond.
For our proof-of-concept, we were planning to cultivate wheat under lab conditions to conduct some tests on the plants. To recreate conditions as close to the regional agriculture as possible, we wanted to find out which strain of wheat is the most common and resilient, so we could use it for our project.
Annika Roos taught us, that there is no such thing as the the most used wheat strain in Germany. Due to differing weather conditions, soil composition or general demands on wheat strains, the choice of the optimal wheat strain has to be taken by every farmer, each year independently and often relies on complex consultancy to optimize said choice.
After this clarification, Mrs. Roos helped us to make a decision on which wheat strains would fit our needs best and which factors and characteristics have to be taken into consideration for this decision. One of these factors would be whether we should use pickeled or un-pickeled seeds.
Ultimately, Mrs. Roos got us access to the strains we decided to use. We received three different strains, each one stained and un-stained. We got the strains “KWS Talent”, “KWS Emerick” and “KWS Fontas” who differ in the quality of the harvest, the yield and their susceptibility to weather or potential fungicides respectively.

First contact 16.08
Skype conference 04.09.
We contacted Professor Fischer as an expert for the molecular biology of Aspergillus and other fungi and asked him for an evaluation of our proof of concept. During a Skype conference we received a chance to discuss different aspects of our project.
Like we heard before, he pointed out, that the legal situation especially in Germany and the EU would make it really difficult to make a system like the Troygenics commercially usable
Regarding our proof of concept, he affirmed that inducing endocytosis in Aspergillus niger or Aspergillus nidulans would assure the applicability in the respective other fungus. Since we based our proof of concept partly on A. niger, this was an important information for the further testing of our system, because the genetics of A. nidulans is better understood than the one of A. niger. Therefore, including research on A. nidulans has made it easier for us to find a method to induce endocytosis in A. niger.
We further discussed our current status in our approach. Professor Fischer suggested to test the possibility of inducing endocytosis using peptide transporters of the cell. We implemented this in our endocytosis-design for A. niger. We utilized a proline transporter as a target to enable endocytosis of our Troygenics.
He also acknowledged our approach of targeting essential genes of the fungi to prevent the bypassing of our system through mutation as well as our approach of blasting the gRNAs we use to target the fungus, making sure that they do not appear in any other organisms. By doing so, we disabled our Cas-system from accidentally damaging other organisms and causing unwanted off-target effects. Professor Fischer also named us some potential target genes.
He also mentioned the general current discussion regarding genetic engineering in fungi and the respective legal situation. He also mentioned different seminars and discussions in the near future that cover this topic.

First contact 29.07
Phone call 06.08
We approached Prof. Dr. Susanne Zeilinger-Migsich as a Professor for Microbiology and asked her to review some aspects of our project. During a phone call she gave us advice on endocytosis and our lab application.
As a mycologist, she pointed out that fungi have a cell wall that would be a particular obstacle to overcome. Also, the structure and composition of the cell wall can differ from fungus to fungus. Yet, she thought an endocytotic uptake like it was planned for our project would be possible. Depending on the structure and state of the cell wall certain substances can also be adsorbed but would not be taken up into the cell.
She mentioned that filamentous fungi can communicate via pheromones. By doing this, male and female fungi are able to recognize each other, while asexual fungi can be more complicated. The receptors for such signals are really sensitive and only tiny amounts of the receptor-bound substance are taken up via endocytosis. A substance like this could be sufficient to generate an uptake of our Troygenics, Prof. Zeilinger-Migsich estimated. After this, we focused on researching these kinds of molecules.
For S. cerevisiae we used the Mating factor alpha as a promising pheromone that does induce endocytosis. We lateron showed experimentally that the uptake initiated by it is specific for S. cerevisiae.
We also asked for further information that might help to estimate how helpful our system might be for lab work. We wanted to know more about fungal metabolites that are hard to produce, and whether our system could maybe present an improvement for the productivity of these. Prof. Dr. Zeilinger-Migisch explained, that genetic engineering or specialized signaling would be necessary to achieve the production of many substances, because most gene clusters for secondary metabolites are turned off in most fungi used in lab applications. On the other hand, substances that are being produced on a commercial scale are produced with already existing high-performance strains, specialized on products like penicillin or cephalosporin. So, the most relevant future achievements would be finding new interesting metabolites by switching on silent gene clusters.
At the end, she additionally helped us to get in touch with experts on yeast and the medical view on fungi from Innsbruck (Austria) as well as with experts for Aspergillus at the Institute of Technology in Karlsruhe (Germany).

First contact 13.08 First contact 20.08 Skype conference 26.08
We came into contact with Prof. Robert Park after a forwarding of one of the other experts of the wheat initiative we discussed our project with. Prof. Park is conducting research about cereal rust and sustainable agriculture.
Regarding our project, we discussed different aspects of our work as well as the problems we had encountered. As we considered the endocytosis as the most important bottleneck of our project, he suggested to use mating proteins to initiate endocytosis. Since we had been using them for yeasts, he stated that these could not only work for those, but that they also exist in rust, even though they do not use sexual recombination. There stil would be further research necessary to determine if this would a valid approach. He also suggested further literature to read about this. Since there is only little published on rust, like he pointed out himself, this has been very helpful.
He also named a set of further potential pathogens for our system to target. For example, the pathogens Ustilago and Tilletia that resemble important pathogens for maize and wheat. These two are also known for the research on their mating but, as pathogens, must be handled according to higher biosafety levels. Despite the fact that they do usually live as parasites, these fungi can be grown in artificial media. Furthermore, he suggested some additional fungi for the testing of our system, that rather resembled the crop pathogens we wanted to fight. Since they were rather difficult to obtain, we have not been able to get access to these strains for our project int this short amount of time.
Prof. Robert Park also gave us general information about his research on rust pathogens and explained, that Mildew or Septoria are having comebacks lately because of their insensitivities to many commonly used reagents.
Together, we discussed the suitability of different fungi for our proof of concept but ultimately decided to stick with Aspergillus for the characterization and later adapt the system to fungi, closer related to the actual crop-damaging fungi. This discussion also settled our choice of using Aspergillus and not adding further fungi with a closer resemblance of actual crop pathogens, mostly due to time constraints.
As an addition, Prof. Park pointed out, that some fungi are dikaryotic and getting the Cas into both nuclei would probably decrease the effectiveness of the system, since our Cas do not has to enter the nucleus, this would not apply to our system.
For our modeling Prof. Robert Park evaluated the parameters we planned to take into consideration and underlined, that the actual rate of mutations in rust fungi is really hard to estimate, since the number of random mutations can vary significantly and depends on a lot of different factors. Moreover, many essential genes would be unsuitable for a targeting due to the rapid evolution of rust fungi, he stated. He also gave us an overview about different approaches of models, that have been developed regarding pathogenic fungi.
For our project, he highlighted the importance of the specificity to assure the biosafety of our application. It is really important to reassure people, that the new application does not damage things unintentionally. The public perception is extremely important for these kinds of approaches.
At the end, Prof. Park underlined once more, that while more research has to be conducted on rust fungi prior, projects like ours are going to have an important impact in the future since the knowledge about these pathogens increases constantly. He pointed out, that our system cold really have a positive impact. We just need some further research.

First contact 22.08
We approached Prof. Eduardo Antonio Espeso Fernández to gather further information about methods to induce endocytosis in Aspergillus and to receive an experienced opinion on the biosafety level of Aspergilli.
Prof. Espeso Fernández is working with Aspergillus nidulans, which has been used as a model organism for decades. He was interested in our project, because of its therapeutic application in fungi.
We asked him for methods of transformation for Aspergillus nidulans and Aspergillus niger and differences between them. Prof. Espeso Fernández stated, that both fungi can be transformed, although his expertise rather concerns A. nidulans.
Since we were using A. niger as a part of our proof of concept, although A, nidulans is better understood genetically, we asked about the biosafety levels about the two fungi and if it would be sensible to also use A. nidulans or research A. nidulans for methods appliable for both fungi. While A. niger is considered GRAS (generally accepted as safe) A. nidulans is not, but there are no special needs to handle the strain, Prof. Espeso Fernández pointed out. Both fungi have been used as a model in the past but for the use in biotechnological processes, A. niger would be preferable. He also considered A. niger a reasonable choice, since we wanted to use a GRAS organism for our project.
Prof. Espeso Fernández displayed, that an uptake of larger particles would maybe be hard to achieve, because Aspergilli usually secretes enzymes to search for nutrients. Carbon sources and amino acids are taken up through more or less specific transporters in the cell wall. Disaccharides and Polysaccharides are processed in the extracellular medium to be taken up afterwards. As a result, using a certain sugar or similar nutrient as a ligand to generate a specific uptake is not expected to work. Prof. Espeso Fernández explained that endocytosis is not used for the uptake of nutrients but for the recycling of transporters and signalling machinery of the plasma membrane. A “recognizable cargo” like a nitrogen-source compound could be taken up via induced endocytosis. An example for a similar process would be the urea transporter of some fungi. If this transporter is localized in the plasma membrane and urea as a nitrogen source is depleted in the surrounding medium, the transporter would be initialized as soon as a rich nitrogen source, for example ammonium, is added to the medium. In that case the urea or amino acid-particles could be internalized via endocytosis.
These comments on our project helped us to progress in our research and to discard saccharides as potential ligands for our Troygenics to induce endocytosis.

First contact 22.08
Skype conference 27. 08
Mail with helpful papers attached: 30.8.
In August, we started extensive research on receptor specific endocytosis in Aspergillus niger. Prof. Reinhard Fisher forwarded us to Prof. George Diallinas. At the end of August we had a very informative skype conference. Prof. Diallinas enlightened us about the different Aspergillus-strains and recommended that we should proof our concept in related strains like Aspergillus niduland. He stated, that principles, that work in nidulans will work in niger, too. A disadvantage of niger Diallinas pointed out was, that this strain lacks a sexual cycle so the usage of mating pheromones, that worked properly for S. cerevisiae, was no possible option.
Together with Prof. Diallinas we worked out it would be a promising option to use a virus-like approach. He told us that many viruses use cell-specific transporters to get actively internalized by the target cell. Although he stated that he has little expertise on mycoviruses, he suggested us to target PrnB, an aspergillus-specific proline transporter, with a short proline-peptide fused to our Troygenics.
On the 30th of August, Prof. Diallinas forwarded us some very useful and interesting papers on viruses and how they exploit cellular transporters to get inside their target cell which helped us to design an endocytosis-inducing fusion-protein for A. niger.

First contact: June
Advise on fluorescence microscopy: August
Supporting performed fluorescence microscopy experiments during September
As soon as we decided to work with S. cerevisiae as a model organism we approached Lara Petersen to get some helpful advise regarding S. cerevisiae. During a first meeting in June we explained our project to her and received some advise regarding our experimental design for S. cerevisiae. Lateron in August we got in touch with Lara again to talk about the planned fluorescence microscopy experiments.
As soon as the neccessary preparations were done, we received an introduction into the usage of the provided fluorescence microscope. During September Lara supported us by giving helpful tips to improve the performed fluorescence microscopy experiments and being constantly reachable in the case of further questions.

First contact 13.08 First contact 25.06 Skype conference 19.08
We established contact to Prof. Dr. Matthias Hahn to receive an evaluation of our project and to gather further information on plant pathogen interaction. Prof. Hahn send us some interesting papers and even parts of his lectures. Later, we also had a Skype conference to ask more complex questions.
As a phytopathologist, we mainly asked him about the uptake of the Troygenics by the pathogenic fungi. In his opinion, the specific, endocytotic uptake resembled the most critical part of the project, because the cell walls of fungi are quite hard to overcome.
While our concept was considered a good idea, we also discussed other approaches. For example, we discussed methods using mycoviruses for transformation through the fusion of hyphen or the interaction between the plant and the fungus through RNAi and exosomes. Though these methods show some benefits they did not featured the same rate of uptake or specificity as our concept.
Overall, he highlighted the need of novel approaches to fight such pathogens and the increasing demand of the industry for them.
We also talked about early ideas for our modelling with Prof. Hahn. In his opinion, predicting the chance of resistances forming is a really difficult thing to do because for natural mutations to occur only one nucleotide has to be altered and every mutation can be dependent on context for itself. Furthermore, we agreed upon the difficulty of testing our hypothesis empirical in the short timeframe of iGEM.
At last, he gave us extended information on metabolic functions of fungi and advised us on implementing a reporter gene into our system, since a validation of our methods via fluorescence would be most convenient to verify.

First contact 01.08
Phone call 02.08
We came into contact with Dr. Alexander Lichius after reaching out to experts on the Mycology Tyrol Homepage, an interactive online platform for the local mycology research in Tyrol. During a phone call we had a chance to describe our project, our current status, plans and problems. He helped us to get in touch with further experts and advised us on general ideas.
Dr. Alexander Lichius also stated, that a system that works in Aspergillus nidulans should also work in other Aspergilli, like Aspergillus niger, although he forwarded us to his colleges to receive conformation for this. This information was important for our research, since the genetics of A. nidulans are way better understood than the ones of A. niger, that we used for our proof of concept, since it was easier to obtain and better understood regarding methods for its cultivation.
He also affirmed our choice of A. niger as an addition for our proof of concept, because of its degree of relationship to other Aspergilli that have proven to be pathogenic for humans.
Additionally, Dr. Lichius named us a list of important, potentially human-pathogenic fungi, that we could target or use as examples for our outlook.

First contact 02.08
Phone call 06.08
We got forwarded to Fabio Gsaller PhD. by Dr. Alexander Lichius. Both work at the Institute of Molecular Biology at the Medical University of Innsbruck (Austria). Dr. Gsaller has long-standing experience in working with Aspergilli and could advise us on the genetic manipulation of Aspergillus.
During a phone call, we discussed our plans for our project. Although, Dr. Gsaller did not use Cas13a, he had worked with CRISPR/ Cas9 and successfully used the system in Aspergillus fumigatus. While transformation for fungi are quite hard to achieve, it is posible and the protocols for the methods already exist.
He helped us by providing important information on the procedures to transform A. niger. The fungi have to grow for three or four days after the transformation until the colonies become visible. After this, the colonies have to be transferred to new plates multiple times to be sure that they were actually single colonies and overcame the selection marker. Because of this, a transformation process requires two to four weeks, an amount of time that has to be taken into consideration – especially in a competition like iGEM.
As Dr. Gsaller had experience with the transformation of Aspergillus fumigatus and Aspergillus nidulans, he offered to check out our protocols for Aspergillus niger if we would like him to.
He further advised us which lab strain to choose best to and named some scientists we should get into contact with to receive further expertise.
When we discussed our project, he stated that the largest bottleneck within our concept might be endocytosis. Fungi have a cell wall that can be hard to overcome and many therapeutically approaches fail on this task. However, Dr. Gsaller still assumed that the endocytic uptake might work in A. niger if it also worked in closely related strains like A. nidulans.
He thought that our aim should be to create a system where only the gRNAs had to be integrated to adapt the system to a fungus, sincea cas system on its own does not seem to have an impact on Aspergilli.
Moreover, he has sent us further information about A. niger and other fungi that are pathogenic for humans and informed us about their impact. This was important information for evaluating the possible applications of our Troygenics to fight pathogens for humans.

First Contact 26.08
We have been forwarded to Dr. Ruth Meissner by Dr. Patrick Beuters and approached her to ask for some suggestions regarding our modeling and for a general evaluation of our project and its applicability. We extensively discussed different aspects of our project.
Dr. Meissner has 17 years of experience in the fungicide research and is heading the cereals screening lab at Bayer Crop Science at the moment.
She pointed out, that the majority of modern plant protection agents are being taken up by the plant. Since most commercially important fungi grow inside the plant, this is necessary to fight the pathogen effectively. If our Troygenic would not be able to be taken up, the application would only be protective from the outside and the number of treatments would have to be increased dramatically up to one application every few days. Fungicides that work on contact, like our system, work based on several multisides. These are considered critical regarding their toxicity but are broadly applicable to many pathogens. Those agents are often very cheap because using them in the necessary amounts is only feasible if their prices are low enough.
A similar approach is used for fungicides based on copper particles, they have to be applied almost constantly.
One other point of criticism she illustrated, was that plants are constantly growing. This would also has to be implemented in the application. Usually, plants receive treatment when they are still small. Wheat plants, for example, are treated at a height of 10 – 15 cm but have to grow protected up to a height of approximately 50 cm.
Dr. Ruth Meissner would not expect a system based on RNAi to work. Besides the transgenic approach, the uptake by the plant would not be efficient enough, she stated. Since we have heard from this approach before, this affirmed us in our decision to built our Troygenics.
Another economical problem would be the general specificity of the agent. A system that is not specific enough could have side effects while a more specific one could target only parts of the fungal population that pose a threat to the crop would not be feasible, except for some diseases like the Asian Soybean Rust. A system to effectively target a smaller, related group of fungi would probably most beneficial, she estimates.
For our modeling, we asked her a set of questions regarding the different factors that have to be taken into account. She explained, that the amount of fungi per field can vary massively, depending on the season and the time. The extend of the fungus would have to be measured microscopically. Also, it would have to be clarified what defines as a fungus. For example, if the fungi in the soil or the spores in the air also have to be considered. It has to be kept in mind though, that the calculation of the amount of fungi for a modeling would also be flawed, because if the plant would be 100% infested by the fungi, there would be no plant material left. In agriculture, different fungicides are used as a mixture, while some agents with special mode of actions are only used once. By doing so, the development of resistances can be slowed down.
Dr. Ruth Meissner estimated, that an application for agriculture would be easier to realize than one for human healthcare.
For the usage of our system, she suggested to use it in combination with further agents, like for example fungicides. By doing so, different agents with different modes of action could help to slow down the development of resistances.

On August 30 we had a phone call with two specialized for resistance development in fungi against fungicides: Dr. Andreas Mehl and Dr. Jürgen Derpmann. They are both positioned at the FRAC and have been working on developing concepts to decrease the number of fungi that gain resistances against fungicides.
These concepts involve optimizing the spraying sequence and composition of batch fungicides. They also work on improving the general application process of fungicides.
They told us that the fungi are pretty fast at becoming less sensitive towards fungicides. Often, the first resistances appear within the time span needed, to develop a new one. The estimated time it takes to develop a new fungicide is about 8-10 years and costs several hundred million dollars. Even though the process is well thought out, the first fungi usually develop resistances after two years.
Derpmann and Mehl taught us, that fungi, as well as fungicides, are usually considered either high risk or low risk when it comes to developing resistances. The usual expectancy is that it is more likely that fungi overcome fungicides with a really specific mode-of-action. We also learnt that fungi becoming resistant against fungicides usually do so via shifting type, meaning that the dose required to fight them increases over time, rather than having one disruptive mutation that allows the fungi to grow normally, no matter the dose of the fungicide. This is due to the fact that most mechanisms responsible for fungi gaining resistances are cumulative and influence the fitness of the fungi since the mutations required would be in genes also beneficial for survival.
Based on that, we discussed possible ways to decrease the likelihood of fungi gaining resistance against our Troygenics. Firstly, we would have to make sure that, the mutations that have to take place would be in genes that are beneficial, or ideally essential for the fungus.
Moreover, Derpmann and Mehl were concerned that, due to the high specificity of our system, we would only target subpopulations of fungi, enabling other ones to just keep growing. They underlined their concern with an example of how different the development of resistances can go:
The fungus Botrittis is a pathogen for many different plants, raspberries and salad among them. There have been reports from other experts that cultivated salad and raspberries next to each other. When testing the resistance of those fungi, he realized that those on the strain on the raspberries had developed a resistance, while the one on the salad was still sensitive for the same fungicides. To make sure that we could target two closely related species like that, we should use gRNAs that would be relevant to all of the ones we want to target.
While we agreed that this would be feasible for a real-world approach, we agreed upon that this would not be to relevant for a proof-of-concept and that it would be more important to prove that specificity was possible.

First contact 27.08
Phone call 28.08 We approached Catherine Sirven to receive further information for our modelling and discussed multiple aspects of our project as well as potential problems regarding its regulation.
Catherine Sirven pointed out, that the regulation could become a problem for our system. Since around 90% of the cost for fungicides are needed for the development and admission of the reagent, fungicides are usually targeted towards a broader applicability. Since our Troygenics work specifically towards a special pathogenic fungus, the newly adapted version of the Troygenic would have to be reconsidered every time it is altered. Which means the development of new adapted Troygenics would be really expensive and potentially time-consuming. In general, fungicides with only one target are the exception. One example for this would be the agents against Asian Soybean Rust (ASR). This pathogen poses a severe threat to soybean harvests in Africa or South America by destroying up to 80-90% of them. For a very specific system like our Troygenics, pathogens like Asian Soybean Rust or Puccinia graminis would be great targets to justify the effort needed for its development.
The market of fungicides is extremely well regulated, so delays in the regulation procedure of a completely novel approach could also be expected.
Because of the use of GMOs, our system would probably be hardly applicable in the EU but should be legal in the USA or Brazil.
Besides that, a general tendency against the use of fungicides can be observed, which would represent an argument in favor of our alternative approach.
Fungi are having an enormous impact on agriculture, Sirven explains. Moreover, they had far-reaching influences over the course of history, like famines, diseases or severe intoxications caused by mycotoxins.
Catherine Sirven explained to us, that farmers are using models to calculate when to use fungicides and when a fungi contamination risk in field is the highest. These models mostly depend on historical data and local weather conditions and work quite well. They are helping to decrease fungicide usage and have sustainable practices.
The most important factors, that have to be taken into consideration are the weather, the growth stadium of the plant, the time of appliance, the occurrence of the fungi over the year or the growth rate.
Still there are experiments based on the artificial induced development of resistances of fungi, followed by a screening of the sequencing of the random generated resistant fungi.
Moreover, bioinformatic tools could be used to estimate the position of certain codons. Studies have shown, that fungi can gain resistances in less than three years, depending on the extent of the fungicide usage. In comparison to the many years of testing that are required for most fungicides, this also explains the demand for alternative measures.
Last but not least, she forwarded us to experts and institutes to evaluate our project and to ask about further information for our modelling.
Catherine Sirven highlighted, that her opinions and suggestions are her own and do not engage Bayer as a company.

First contact 14.08
Skype call 13.09
We approached Prof. Fiona Doohan because she was suggested to us due to of her broad experience in Molecular Biology, Plant Pathology and Environmental Science. At the Universal College Dublin (UCD) she is part of the UCD Centre for Plant Science, the UCD Institute of Food and Health, the UCD Earth institute and the UCD School of Biology and Environmental Science, which makes her a great expert to evaluate our project. We discussed most of our plans and the concept of our project during a Skype conference.
In the opinion of Prof. Fiona Doohan a system like ours might be applicable if it would not cause any problems. She focused on the safety requirements needed to implement a system like ours. Mostly, Prof. Fiona Doohan highlighted that we would have to assure that fungi targeted by our Troygenics would not be able to survive or reproduce while carrying our system if we are aiming for an application in agriculture. Additionally, sophisticated tests would be needed to ensure the specificity of our system: not only because the specificity for the respective target has to be distinctly assured but also because it could be partially beneficial to target a group of related pathogens rather than only one specific species. For example, the crop pathogen fusarium occurs in a small range of different species and eliminating one of multiple occurring pathogens in one field could potentially even strengthen the untargeted ones in its prevalence. In this case a slightly broader specificity would actually improve the efficiency of our system as a precise tool against crop damaging fungi. For this, the Troygenic and its uptake would not only have to be specific enough, they would also have to be widely applicable enough to work with an optimal efficiency to protect the crop. Of course, this exact specificity would have to be attested countless times to preclude the possibility of unwanted off-target effects. This, broadening the possible application our system has really extended our understanding of the necessity of precise targeting of the pathogen and made the problem we want to solve more multifaceted.
Besides that, she postulated that she would not expect the admission of such a system within the EU, as the strict legal situation towards the usage of GMOs and the comparatively high level of regulations complicates this process. Moreover, of course our Troygenics would have to be tested in a smaller, closed system before they could be used by farmers on a larger scale.
We also asked Prof. Fiona Doohan about historical records of fungal crop pathogen, for example their influences on harvest losses. While fungi are suspected to have a major influence on crop losses and famines during former times, there are almost no records highlighting fungi as the main cause. That is mostly due to the fact that people did not recognize the fungi as the disease and thereby the cause of the plant damages. But there are records of influences of these fungi during the last couple of centuries. One example is ergotism: a poisoning through the ingestion of wheats that have been infected by a special fungus (Claviceps purpurea ). These toxins can lead to a broad range of symptoms including convulsive and gangrenous ones. There are even historians that postulate that the illness and deformations caused by these fungal toxins lead to the accusation of infected people being witches. The Salem witchcraft accusations as well as the Salem witch trails in the USA during the 17th century can be seen as a rather modern example for this kind of influence.
Prof. Fiona Doohan introduced us to pathogenic crop fungi that produce mycotoxins which can be dangerous in very low quantities and can accumulate in infected wheat. Because of this, the infection rates and the occurrences of these fungi have been very precisely monitored and recorded. This data could be a great foundation to build our modeling on.
For our list of important pathogens to target, she highlighted two fungi that have a severe impact on the food production in Ireland: Septoria tritici, a wheat rust disease, and the “potato famine disease” which, especially in Ireland, had a great historical impact on the food supply. She further named us additional sources and statistics to extend said list.

First contact 14.08
Skype conference 03.09
We had a Skype conference with Prof. Silvia Germán, an emeritus researcher at the National Agricultural Research Institute (INIA) of Uruguay. She has worked together with plant breeders for wheat and barley and focused her research on obtaining pathogen resistances in plants.
She considered our project as a promising approach but also highlighted, that we could expect a quite negative public perception of our system, since we are using GMOs. As a result, we should put even more effort into explaining how our system works and how we assure its safety, highlighting the implemented safety measures. Additionally, she suggested that we carefully think about our wording. For example, many people would rather support a project using “genetic engineering” than using “GMOs”.
Beyond that, getting lawyers and lawmakers included could additionally benefit the process of admission.
We described our proof of concept to her, to depict our concept of validating the specificity our system must demonstrate before it could become a commercial product. Prof. Silvia Germán suggested to include a further, closely related fungus to the tests. Doing so we could see that we can directly target the specifically transformed fungus and spare its close relative. That way the precision of the Troygenics could be presented more effectively. We wanted to implement this by showing that our Troygenics could decrease the growth rate of S. cerevisiae while not harming its close relative Saccharomyces paradoxus. However, due to limited time we could not perform those experiments.
Moreover, she stated that the search for fungicide alternatives has become a really important issue and that creative approaches, like the Troygenics, would definitely be welcomed as an additional option.
Beyond that, Prof. Silvia Germán named us some bigger, nearby events, like conventions and discussions on this topic that underlined the broad interest in it. She also forwarded us to a range of experts and associations, for example some further experts of the Global Rust Initiative, the CIMMYT (International Maize and Wheat Improvement Center) and the John Innes Center in Great Britain.
Prof. Silvia Germán also mentioned that rust pathogens are quite susceptive to fungicides but are still having a major impact on crop yields all over the world. They start new cycles of infection all ten days, causing them to be a considerable threat to harvests. However, this also causes them to be good diseases to study when looking at the interaction between the fungicides and the resistance development in plants. Furthermore, she pointed out, that per definition, a resistance in a plant is defined as an intermediate resistance. Which means a pathogen grows slower on a resistant plant than on an unmodified plant. Resistant plants are basically “rusting slower than other plants”, Prof. Silvia Germán described. This way fungicides have more time to operate. In general, it is important to rely on multiple lines of defense that act jointly to protect the crops. This gives the fungicides a better chance of stopping the fungus from damaging the plant in an early stage. For this method, it has been shown, that targeting many minor genes is harder to overcome by the pathogen than damaging one major gene. We included this question, searching for the optimal amount of targeted genes into our project and tried to answer it with our modeling part.
If our system works efficiently and safely, Prof. Silvia Germán would expect farmers to use it if it comes at a reasonable prize. The most important barriers are the legal situation and the consumer acceptance regarding the genetic engineering. For example, in South America, the admission of such a system might be much easier to realize than in Germany or Uruguay.
As a conclusion, she once again highlighted the importance of the problem we are tackling: “Since pathogens evolve fast, they are immediately endangering our food supply”.

First contact 10.09.
Phone call 11.09.
Visit at his farm and interview 17.09.
We came into contact with Michael Kleimann trough a regional farmers' association and explained our project and situation via mail and phone calls. Eventually, we visited Mr. Kleimann at his farm to have a personal interview.
After a very informative guided tour through his farm, Michael Kleimann underlined the fact that there is a major interest in alternatives to fungicides. Moreover, crop damaging fungi are definitely a problem for farmers in Germany. Mildew for example has become a huge problem in almost every year and leads to crop loss and an increased use of fungicides. Other harmful fungi for wheat are Puccinia striiformis and Septoria. For these, general appliable fungicides, like Tebuconazol, are often used. Especially for Septoria a way of targeting a specific fungus would be a really important tool to fight these fungi. Here our Troygenics could be very helpful.
Other important influences are the weather, like periods of drought. Also crop rotation is used to generate healthier plants and more attention is paid on choosing the date of seed to prevent the occurrence of fungi.
Since, 90% of the agents are taken up by the plant after one hour the substances ca not be washed of by rain afterwards. Even if our Troygenics would be washed off the plant, they could potentially still work because they could be taken up by the roots.
For now, the plant protection agents work quite well but because of recent efforts to re-test many pesticides, around 75% of these agents could be reconsidered in their status and could be banned. Fungicides like Tebuconazol, that have been used reliably and are cost-effective would be inaccessible to use. This also increases the danger of the development of resistance against fungicides. Because of this the urge to find alternatives to these agents has become even more prevalent and makes approaches like our Troygenics even more important.
A system like ours would hardly be executable. The regulations on genetic engineering are still way too harsh and many farmers are not well-informed about this topic, Kleimann said. Since the public opinion on GMOs is mostly negative, and the political view on this topic is often strengthening this opinion, farmers are often pressured to avoid these kinds of modern technologies, even if they would be widely beneficial. As an analogy Mr. Kleimann invoked a similar situation some years ago, were sewage sludge had been used as fertilizers. After some hardly degradable substances have been found inside the sludge, the demand on it broke in completely. If there would be a similar case of a sudden problem with a working system of genetic engineering, people would disgust this the same way, he prophesized.
In general, new approaches like ours have to be affordable and commercially competitive. They also should be appliable with the commonly used methods.
Furthermore, he advised us that our system has to be appliable preventive and as a fast reaction and should work in combination with other agents.

First contact at German iGEM Meetup in Düsseldorf 05.07
Mail contact 25.07
Skype conference 05.08
We first came into contact with Susanne Günther at the iGEM Meetup Germany 2019 in Düsseldorf. After she took part in a panel discussion about genetic engineering, we approached her to discuss aspects of our project. Later we contacted her again and exchanged some ideas via a video conference.
Fungicides are used depending on how much of them are needed in each situation. The requirement relies heavily on the weather, the crop and the time of the year.
During the last years, many fungicides or other spraying agents have been banned due to their unspecific effects. This has become a problem for farmers that have to rely on fewer options to protect their crops.
Especially fusaria are a problem in agriculture because they not only damage the plants but also produce mycotoxins that can harm animals and humans. To minimize this, farmers use fungicides to assure the quality of their products rather than the fight against the pathogen. For organic produced crops, copper- and sulfur- components are used instead of fungicides. But the use of these substances equals an ecological disaster, Günther said. Some of the substances already have been declared as not suitable for organic farming.
In Addition to this, the spores of fungi can also have negative impact of pigs or turkeys.
For our project, Mrs. Günther advised us on doing some research on the stability of our protein particles, since this would be an important factor that could limit the applicability of our system. If weather conditions or factors like UV-radiation on the field would decrease the durability of our particle, this would be an important aspect to take into consideration.
Also, the specificity is not the sole problem here. If there are many different variations of one harmful fungus, like fusarium, an approach that is targeting a specific kind of closely related fungi could be more applicable for farmers.
Susanne Günther also told us that farmers are indeed interested in topics like GMOs. To further their understanding of this often complicated topic, they take part in educational events and ask consultants on this topic.
She told us she would expect farmers to be open minded towards genetic engineering but since people would stop buying their crops if they would use it, farmers have to stick to conventional methods. Because genetic engineering does not present any direct advantages for consumers, they also do not have a reason to pressure farmers to use GMOs.

First Contact 19.09 Phone call 23.09 To gain insights into the process of farming in dry areas, like the ones in sub-equatorial Africa, we got in touch with Kristina Waldschmidt, a Farmer from Witvlei in Namibia to discuss our project. While due to the climate, fungi as a threat for crops have far less of an impact in these regions, they are still influencing the food production. Especially, constant artificial irrigation can lead to fungi growing on the fields. Parallel crop farming of plants like tomatoes, cabbages or cucumbers are often affected by fungi, since they are more demanding in regards to water usage. Without the use of reagents, harvest losses would be expected, Waldschmidt forecasts. A system like our Troygenics could thereby still help to secure the food supply in these regions. She also suggested that making the Troygenics resilient to higher doses of UV radiation would be an important step to ensure an effective application of our system in countries like Namibia. However, the farmers in Namibia have become skeptical towards genetic engineering in the course of the last years but would still be open minded to use such a system as long as it poses a real advantage over conventional methods Waldschmidt predicts.

02.09 first contact
11.09 extensive phone call
We approached Phillip Krainbring from northern Germany. As a farmer for wheat, corn, sugar beets and more, his opinion on our project has been particularly interesting for us. He also reports regularly on topics in agriculture and engages in educating people about the current situation farmers have to face.
We had an extensive phone call to talk about our project and the impact of fungal crop pathogens on agriculture.
Phillip Krainbring confirmed us that crop damaging fungi are having a large impact on agriculture and crop production, although the importance varies from year to year. The threat of fungal infections is also tied to weather conditions and depends on the location. Farms closer to the shore are more susceptible and thereby more dependent on fungicides.
Fungicides are partially used as prevention, but most farmers try to avoid this.
Regarding wheat there is an estimated harvest loss of 30 – 60 % per year in Germany due to pathogenic fungi. Fungi like the oilseed rape pathogen Sclerotinia sclerotiorum destroy around 50 % of the infected harvest.
The decision when to use fungicides is made after direct, personal inspection of the fields by the farmer. The fungicides are then applied 2 to 3 times per harvest according to the situation. Some crops are requiring a more extensive use of fungicides than others. Because farmers have to prove they took action in order to protect their crops in case of larger losses, additional appliances are the common practice.
Besides the fungi and droughts there are also some other smaller pathogenic influences like insects or viruses.
Another problem farmer face is that more and more pesticides are being banned due to stricter regulations and increasing concerns towards them by the industry and public.
For example, staining the grain is prohibited for sugar beets and oilseed rape starting this year in Germany. To avoid a break-in in crop production, preventative use of fungicides becomes an important measure.
Moreover, by prohibiting more and more pesticides agriculture relies on a smaller range of different fungicidal compounds which increases the danger of resistance development. “Even if these problems are manageable at the moment, they will become important in the future.”, Phillip Krainbring predicts.
Due to this, there is an overall interest in alternatives to fungicides.
To avoid resistance gain, common approaches are the reduction of fungicides to a minimum, a more careful selection of more robust wheat strains and the precise improvement of the nutritional value of the soil, since stronger and healthier plants result in a smaller risk of fungal infections. Also, there are some experimental approaches based on the use of microorganisms.
Fungicides are remaining as an emergency solution.
In former times fungicides have been used regularly, but as a result of the negative opinion towards pesticides in the society they have become a measure to react if most of the other approaches have failed.
However, this also limits the actions farmers can take to save their crops.
“The larger the toolbox to protect your own crops, the better you can react to the situation.”, Phillip Krainbringsaid.
The negative opinion the society has on topics like GMOs or pesticides has also dramatically increased. As a result, questions regarding which companies are influencing the crop production or if glyphosate has been used in the process have become more frequent. But people are also openly showing their disregard towards pesticides. If a farmer is encountered while he is applying pesticides to his fields some people are covering their faces, insult the farmer, or in some cases, block the tractor to stop the farmer from proceeding his work. Furthermore, insults via social media occur from time to time.
In contrast to the radical, negative opinion on GMOs the society has, the farmers think about this in a more pragmatic way. In Phillip Krainbrings opinion they would be open towards new possible tools for their profession, even if they are based on genetic engineering. Of course, these new tools would have to compete with commonly used methods, but if the price and the applicability of the new measures would be comparable to the common practices they would be used in the same way.
Moreover, only a few farmers sell their products directly, so the important part here would be how the crops are sold later on without damaging the appearance of the farmers.
In the end, farmers are mostly pressured to avoid using GMOs by the public opinion and politics. This antipathy can be expected to increase in the future.
In the end, every farmer has to take responsibility for their own work, even though there are ways to receive advice on pesticide use, consulting or information on how to optimise production.
-> Phillip Krainbringgave us insights and opinions about his profession from his point of view. Since farmers are the ones most affected by the problems we want to solve, his opinion has been really important to our understanding of this topic. Furthermore, he gave us the chance to integrate the views of farmers into our project and compare them with the ones from the society, politicians and scientists.

First contact at German iGEM Meetup in Düsseldorf 05.07
Mail contact 25.07
Skype conference 01.08
We first met Prof. Dr. Peter Westhoff at the German iGEM Meetup in Düsseldorf, where he functioned as the moderator of a panel discussion about genetic engineering and its perception and value for the society. Some weeks later we contacted him again to receive an evaluation of our project as well as some input about our concept of biosafety, as this was one of the main topics of the panel discussion.
At first, we explained our process of thought regarding our concept of biosafety. As far as he could think of, Prof. Westhoff had no distinct complains about our concept and liked how we implemented multiple precautionary measures to assure the safety and specificity of our system.
Moreover, he pointed out some similar approaches in current research like the uptake of RNAi by nematodes.
Prof. Westhoff assumed we had good chances of achieving specificity by using gRNAs of well-known fungi as a significant amount of prior research had been conducted. Although, he expected the assurance of specificity by using promotors to be clearly more unpredictable.
He validated our concept of biosafety for our potential application as a biological fungicide alternative.

Forward farm

On the 26th of September 2019, we visited the Bayer Forward Farm in Rommerskirchen

We visited the Bayer Forward Farm in Rommerskirchen, the “Damianshof” accompanied by Bernd Olligs (farmer and owner of the farm), Dr Patrick Beuters (Product Development Manager for fungicides in cereals, sugar beet and potatoes) and Karl Eschenbacher (Head of Bayer Forward Farming).
The farm in Rommerskirchen is one of two locations in Germany where Bayer established a Forward Farm: An experimental testing area for novel approaches to solve problems in the field of agriculture. Thereby, the farm’s purpose is also to educate the public about farming and emerging technologies. The engagement with the public and the promotion of face to face interaction is one of the foundations the project is based on.
During our visit, we received extensive first-hand insights into agriculture from different points of views. The different experts who accompanied us each represented a different important aspect of modern agriculture.

Dr Patrick Beuters

He is in the department of Market Development Manager for fungicides in cereals, sugar beet and potatoes.

Dr Patrick Beuters gave us insights into the importance of fungicides for agriculture. As a consultant for fungicides for cereals, sugar beet and potatoes he has an extensive overview about all their important products and modes of actions. His explanations regarding the history and recent change in usage of fungicides as well as the latest interests in research completed the depictions of Karl Eschenbacher and Bernd Olligs very well. At the same time, he stressed the need to investigate alternatives to meet future challenges. Furthermore, he showed us the extend of the recent interests of the industry for new alternatives, e.g. biologics. According to Dr Beuters, it is important to work closely with farmers to ensure proper use of fungicides and effective food safety.

Karl Eschenbacher

He is Head of Bayer Forward Farming.

Karl Eschenbacher is the Supervisor Head of Bayer Forward Farming in Germany. He stressed the importance of a close cooperation between industry, farmer and consumer. A lack of understanding can easily lead to problems and misconceptions according to him. He explained how valuable open communication about agricultural measures are to educate people about the necessity of certain methods and applications, e.g. the use of pesticides. To reduce prejudices towards agriculture, engagement and education with the public are among the greatest tools to achieve a well-understood, fair-regulated, sustainable and safe food production. Considering we live in a time, where technological advancements are achieved quickly and should be easily accessible and understood by the public, engagement with everyday people is often sparse. An open conversation about such topics and new approaches to communicate the subject in a target group specific manner can really make a difference. To sum up, Karl Eschenbacher clarified how important well-informed consumers are to achieve an integrated agriculture.

Bernd Olligs

Is the farmer that is taking care of the farm Damianshof, where the forward farm is located

Bernd Olligs is the owner of the farm. He gave us great first-hand insight into the work as a farmer. He showed us how full of meaningful decisions his work is and underlined his statements with stories and anecdotes from his work. His depictions of the life as a farmer made us realise how underestimated and unappreciated the work of farmers is in modern society and how complex the issues can be that they are confronted by. They have to assure a maximum yield while minimizing the costs and the impact on the environment while planning years far ahead. This is a task unmatched in modern society. During our visit, Bernd Olligs also described the importance of improving the personal contact between farmers and consumers to create a broader understanding of the reasons that drive their decisions and our dependency on their experience.

References

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