Human Practices are the aspects of iGEM projects that open up the remarkable achievements of the iGEM teams all around the world to the public and reward them with new insights and ideas to their projects.<br>
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All together, we came into contact with 50+ experts and stakeholders out of more than 18 different countries from all continents.
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To develop our Troygenics as a completely novel approach on the transformation of eukaryotic pathogens, a lot of bottlenecks had to be overcome.<br>
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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.
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We approached experts from numerous fields of research to implement their interdisciplinary suggestions into our project. Integrating their advice and contrary points of view made a huge difference for the realization of our project.<br>
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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.
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Since our main application was aimed for fighting crop damaging fungi, we got into contact with experts from various fields including agriculture, industry, politics, biosafety, legislation and a large number of researchers.<br>
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Additionally, reaching out to farmers, visiting them on their farms, having discussions and optimising the application of our Troygenics accordingly added new facets to our iGEM project and made it more applicable for the real world in the long run.<br>
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Human Practices have been one of the major driving forces throughout our project and implementing their suggestions into our work has significantly leaped our project forward multiple times.<br>
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Mrs. Roos helped us to understand the process of decision which kind of wheat fits best under certain circumstances, helped us to decide which kind of wheat strain we would need for our proof-of-concept and ultimately got us access to the strains for using them in our project. </button>
Mrs. Roos helped us to understand the process of decision which kind of wheat fits best under certain circumstances, helped us to decide which kind of wheat strain we would need for our proof-of-concept and ultimately got us access to the strains for using them in our project. </button>
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<div class="content conLeft"> <div> First contact 07.08
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<div class="content conLeft"> <div> First contact 07.08<br>
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Multiple contacts via e-mail and phone for organizational purposes
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Multiple contacts via e-mail and phone for organizational purposes<br>
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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.
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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.<br>
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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.
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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.<br>
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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.
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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.<br>
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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.
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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.<br>
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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.
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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.<br>
<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Reinhard Fischer</b><br>
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Professor at the Institute for Applied Biosciences, Department of Microbiology <br>
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Karlsruhe Institute of Technology – KIT <br>
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Karlsruhe, Germany </div>
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<hr>
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We contacted Professor Reinhard Fischer as an expert for Aspergillus. He furthered our understanding of the processes we had to manipulate to induce endocytosis. Besides that, he affirmed our proceeding for our proof of concept and supported our research to realize an uptake of our Troygenics by the targeted fungi.
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First contact 16.08<br>
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Skype conference 04.09.<br>
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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.<br>
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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<br>
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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.<br>
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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.<br>
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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.<br>
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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.
<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Richard Oliver</b><br>
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Professor of Agriculture<br>
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Centre for Crop Disease Management (CCDM) at Curtin University<br>
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Curtin, Australia</div>
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Prof. Richard Oliver helped us to improve our project in various ways. He advised us on how to induce endocytosis reliably and how to design our CeDIS in a way to minimalize the chance of pathogens overcoming it by mutation. This was a major influence on our modeling project. Beyond that, we worked together to optimize our concept of effective appliance of our system and the verification of the functionality our system. Last but not least, he helped us to improve the purification process of the Troygenics from the E. coli cultivation broth to optimize their applicability. </button>
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First contact 14.08
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Skype conference 15.08<br>
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We first got in touch with Prof. Richard Oliver through the forwarding of Professor Peter Langridge of the Wheat Initiative. As Professor of Agriculture at the CCDM, the Centre for Crop Disease Management at Curtin University (Australia), Prof. Richard Oliver had accumulated some knowledge we could use to optimize our project.<br>
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Regarding the bottleneck of endocytosis that many experts had mentioned before, he pointed out that researchers had thought about using endocytic uptake in fungi before and that there had been a focus in research on this since.<br>
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He considered our approach as feasible, since fungi can take up larger molecules from their surroundings. Binding one of these molecules to our Troygenic could thereby, theoretically, induce its endocytic uptake. For comparison, Prof. Richard Oliver mentioned the cholera toxin, which, in a similar manner, consists out of two parts that induce the uptake by the cell and respectively its toxicity.
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We investigated the alternative CeDIS idea of introducing siRNAs into our system, but after discussing it we discarded the idea. We did not expect this approach to work in all species of fungi and moreover, this would not allow us to create a lab application, that we are aiming for with our Troygenics.
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Regarding other aspects of our CeDIS, Prof. Richard Oliver advised us to increase the number of genetical targets to prevent the targeted fungi from circumventing our Troygenics through mutation. He suggested to use three different targets to assure the applicability of our system. As a result, of this recommendation we also focused our modeling on the question of the optimal amount of gRNAs for our system.
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Moreover, Prof. Richard Oliver gave us insights into the combination of methods like fungicides, analogous reagents and plant breeding to protect harvests. He advised us to come up with a similar concept, to assure the integrated functionality of our approach. To proceed with this idea, he also named us a broad range of further experts and associations to get in contact with.
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As a potential formulation he suggested using clay nanoparticles that could be attached to our Troygenics and applied to the field. He pointed out that, since they are cost-effective and widely applicable materials, they could be of great use for our project. Contrary to this, we discussed emulating the usage and formulation of currently used fungicides to keep the amount of necessary adaptation to apply our system minimal. However, designing a clay nanoparticle-based system to achieve a slow release system of the Troygenics could open whole new possibilities and potential new applications for our project.<br>
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Another addition he made addressed our proof of concept and the verification of the functionality of our system. To investigate the effects our Troygenics would have on plants, we bred wheat plants ourselves to apply our system on them in a controlled environment. In that case we should be able to evaluate the effects of our applied Troygenics based on the visible changes of the plants. For this experiment Prof. Richard Oliver advised us to use specially altered strains of Arabidopsis thaliana with fluorescent reporter genes for the detection of physical damage to the plant. Using these special strains would improve the reliability and the quantification of the potential damage to the plant and would be more precise than an examination of regular wheat plants by eye. He also gave us some lists where we should be able to find said strains and experts to contact to gain access to them.<br>
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Prof. Richard Oliver further advised us to watch out for microscopic defense responses of the plant after our tests. If these should occur the composition of the mixture containing our Troygenic probably has to be reconsidered.<br>
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A further optimization we designed with Prof. Richard Oliver and implemented into our work was the purification process for the application on the plants. Since our Troygenics are produced in E. coli, just concentrating the Troygenics after discarding cells and debris and using the concentrated supernatant would be an obvious approach. What we did not take into consideration up to that point was that many cultivation media like LB are containing components from bacteria that could cause a PAMP (Pathogen-associated molecular pattern) mediated immune reaction in the plant. Because of this, applying a direct concentration of the supernatant would probably trigger immune reactions of the plant and would thereby become useless for our application. This was one of the reasons why we decided to purify our Troygenics prior to continue working with them. We also thought about that when improving the purification protocol.
<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Susanne Zeilinger-Migsich</b><br>
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Deputy Head of the Department of Microbiology <br>
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University of Innsbruck <br>
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Innsbruck, Austria </div>
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Prof. Dr. Susanne Zeilinger-Migsich evaluated our concept of inducing endocytosis in our targeted fungi and gave us some great advice. She specifically suggested to have a closer look on mating pheromones to induce the specific uptake of our Troygenics. We implemented this for the proof-of-concept in S. cerevisiae cells.<br>
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Furthermore, she helped us to get in touch with other important experts
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First contact 29.07<br>
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Phone call 06.08<br>
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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.<br>
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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.<br>
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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.<br>
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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. <br>
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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.<br>
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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).<br>
<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Robert Park</b><br>
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Judith and David Coffey Chair of Sustainable Agriculture and
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Director of Cereal Rust Research at the University of Sydney Plant Breeding Institute
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University of Sydney<br>
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Sydney, Australia</div>
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Prof. Robert Park shared his extensive experience regarding rust fungi with us and affirmed the idea of using mating factors to induce endocytosis in yeast. Moreover, he helped us to decide to use Aspergillus for our proof of concept to characterize our system. Furthermore, he gave us great insights into different models regarding pathogenic fungi over the course of the last decades and pointed out potential struggles with modeling their gaining of resistances.
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<div class="content conLeft"> <div> First contact 13.08
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First contact 20.08
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Skype conference 26.08<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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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. <br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
Research Professor at the Dept. of Cellular and Molecular Biology <br>
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Centro de Investigaciones Biológicas (C.S.I.C.) <br>
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Madrid, Spain </div>
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<hr>
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Prof. Espeso Fernández supported the progress of our research by helping us to comprehend the biosafety levels of Aspergillus niger and Aspergillus nidulans and furthered our knowledge about endocytotic uptake in fungi. After his suggestions, we discarded saccharides as potential ligands for our Troygenics.
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First contact 22.08<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. George Diallinas</b><br>
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Professor of Biology<br>
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National and Kapodistrian University of Athens - Department of Biology - Aspergillus Genetics Lab<br>
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Athen, Greek</div>
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<hr style="color:#f1f2f3">
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We approached Prof. George Diallinas as a Professor for transport proteins in Aspergillus to ask him some questions regarding our proof of concept concerning methods to induce endocytosis in Aspergilli. He gave us the valuable hint to use Aspergillus-specific amino acid transporters to induce endocytosis, like many viruses do.
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<div class="content conLeft"> <div> First contact 22.08<br>
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Skype conference 27. 08<br>
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Mail with helpful papers attached: 30.8.<br>
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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. <br>
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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.<br>
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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.<br>
Lara Petersen helped us in many ways regarding the optimal handling procedures for S. cerevisiae and providing us with different yeast strains and established protocols for transformation and fluorescence microscopy.
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First contact: June<br>
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Advise on fluorescence microscopy: August<br>
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Supporting performed fluorescence microscopy experiments during September<br>
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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.<br>
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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.
<button class="collapsible colLeft"> <div id="HP"> <b style="color:#39F;">Prof. Dr. Matthias Hahn</b><br>
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Professor of Phytopathology<br>
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University of Kaiserslautern<br>
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Kaiserslautern, Germany</div>
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<hr style="color:#f1f2f3">
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As a plant pathologist, Prof. Hahn could give us great insights into the metabolic workings of fungi and the interaction between plant and pathogen. We discussed and pondered different methods to induce endocytosis and received advice to verify said methods, for example how to implement reporter genes into our concept.<br>
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He also forwarded us to further experts and supported our research with a range of papers.<br>
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<div class="content conLeft"> <div> First contact 13.08
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First contact 25.06
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Skype conference 19.08<br>
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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. <br>
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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.<br>
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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.<br>
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Overall, he highlighted the need of novel approaches to fight such pathogens and the increasing demand of the industry for them.<br>
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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.<br>
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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.<br>
<button class="collapsible colRight"> <div id="HP"> <b style="color:#39F;">Dr. Alexander Lichius</b><br>
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Department of Microbiology <br>
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University of Innsbruck<br>
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Innsbruck, Austria</div>
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<hr>
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Dr. Alexander Lichius helped us to affirm some of our decisions for our concept and forwarded us to experts of his local research community.
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<div class="content conRight"> <div>
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First contact 01.08<br>
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Phone call 02.08<br>
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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.<br>
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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.<br>
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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.<br>
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Additionally, Dr. Lichius named us a list of important, potentially human-pathogenic fungi, that we could target or use as examples for our outlook.<br>
Dr. Fabio Gsaller helped us to improve and adapt our concept for Aspergillus niger. His evaluations and suggestions improved our planning of the according experiments and his experience with transformations of fungi helped us to plan our project accordingly. Additionally, he forwarded us to further important experts and provided us with information we integrated in our project.<br>
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.<br>
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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.<br>
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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.<br>
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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. <br>
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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.<br>
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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.<br>
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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. <br>
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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.<br>
Bayer AG - Research & Development, Crop Science <br>
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Monheim, Germany </div>
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Dr. Ruth Meissner helped us to evaluate a larger set of factors for our modeling and gave us her suggestions regarding the application of our system, how we could optimise our concept in general and make it competitive or even beneficial in comparison to common fungicides.<br>
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First Contact 26.08<br>
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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.<br>
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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.<br>
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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.<br>
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A similar approach is used for fungicides based on copper particles, they have to be applied almost constantly.<br>
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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.<br>
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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.<br>
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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. <br>
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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.
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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.<br>
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Dr. Ruth Meissner estimated, that an application for agriculture would be easier to realize than one for human healthcare.<br>
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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.
We had a conference call with Andreas Mehl und Jürgen Derpmann from the Fungicide Resistance Action Committee (FRAC). We discussed the likelihood of fungi gaining resistances against fungicides and whether our system would be beneficial to help decrease the likelihood of resistance development. Additionally, we discussed important factors for modeling the gain of fungicide resistance on crops.<br>
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<div class="content conLeft"> <div> 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. <br>
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These concepts involve optimizing the spraying sequence and composition of batch fungicides. They also work on improving the general application process of fungicides.<br>
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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.<br>
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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.
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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. <br>
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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. <br>
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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: <br>
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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. <br>
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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. <br>
Catherine Sirven helped us to understand the progress of regulation, fungicides have to go through and the problems our Troygenics could possibly encounter. She also furthered our knowledge regarding the specificity and how it would affect its applicability and process of admission. Moreover, she helped us to optimize our modelling by naming us some of the factors that have to be taken into account. </button>
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First contact 27.08<br>
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Phone call 28.08
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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.<br>
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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.<br>
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The market of fungicides is extremely well regulated, so delays in the regulation procedure of a completely novel approach could also be expected.<br>
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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. <br>
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Besides that, a general tendency against the use of fungicides can be observed, which would represent an argument in favor of our alternative approach.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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Last but not least, she forwarded us to experts and institutes to evaluate our project and to ask about further information for our modelling.<br>
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Catherine Sirven highlighted, that her opinions and suggestions are her own and do not engage Bayer as a company.<br>
Professor at School of Biology and Environmental Science<br>
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University College Dublin (UCD)<br>
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Dublin, Ireland</div>
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Prof. Fiona Doohan evaluated our system and gave us advice on some biosafety aspects regarding our application. She also hinted us towards a well-researched fungus that could be used as an example organism in our modeling. Besides that, she furthered our knowledge about the historical impact of fungi and named us additional pathogens to target.<br>
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<div class="content conLeft"> <div> First contact 14.08<br>
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Skype call 13.09<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
emeritus researcher at the National Agricultural Research Institute (INIA) of Uruguay <br>
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Montevideo, Uruguay </div>
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Prof. Silvia Germán evaluated the applicability of our system and helped us to further our understanding about the joint usage of fungicides and the breeding of resistant plants. Moreover, she advised us on optimizing our proof of concept by using additional closely related fungi and suggested us to increase the number of genetic targets of our CeDIS. This also inspired us to investigate the optimal number of gene targets through our modeling. </button>
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First contact 14.08<br>
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Skype conference 03.09<br>
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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.<br>
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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”.<br> Beyond that, getting lawyers and lawmakers included could additionally benefit the process of admission.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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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”.<br>
We had a chance to personally visit Michael Kleimann on his farm to discuss our project and his opinion on the topic. He advised us on several optimizations on our system to make it more applicable for farmers and more competitive towards commonly used fungicides.<br>
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<div class="content conLeft"> <div> First contact 10.09.<br>
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Phone call 11.09.<br>
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Visit at his farm and interview 17.09.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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In general, new approaches like ours have to be affordable and commercially competitive. They also should be appliable with the commonly used methods.<br>
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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.<br>
We talked to Susanne Günther multiple times and discussed our project. As a philosopher, editor and farmer her opinion has been especially interesting for us. She evaluated our project and strongly furthered our understanding of the relationship between the consumers, farmers and genetic engineering.<br>
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Susanne Günther advised us on taking the stability of our particles under the circumstances of agriculture into consideration. Something we have put a lot of thought into afterwards.<br>
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First contact at German iGEM Meetup in Düsseldorf 05.07<br>
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Mail contact 25.07<br>
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Skype conference 05.08<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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In Addition to this, the spores of fungi can also have negative impact of pigs or turkeys.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
Kristina Waldschmidt extended our knowledge about crop pathogens by reports on their influence in sub-Saharan Africa. This also improved our understanding of the interaction between crop damaging fungi, the different ways of plant cultivation and styles of artificial irrigation. She also highlighted the focus on increasing our systems resistance against higher doses of UV-rays to adapt it to such conditions.
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<div class="content conLeft"> <div> First Contact 19.09
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Phone call 23.09
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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.
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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.
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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.
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Without the use of reagents, harvest losses would be expected, Waldschmidt forecasts.
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A system like our Troygenics could thereby still help to secure the food supply in these regions.
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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.
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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.
Theo Többer incused our view on the relation between agriculture, fungi and fungicides. His point of view also has shed new lights on our understanding of the opinion farmers have about genetic engineering. </div>
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First contact 27.03<br>
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Phone call 10.04<br>
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Visit at his farm and interview 23.08<br>
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Beside some phone calls, we visited the farmer Theo Többer at his farm for an interview and asked him some questions regarding our project.<br>
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According to Mr. Többer, fungi are still a major problem in agriculture. Especially mildew is threatening the plants at the early stages of their growth. Fungicides are applied 2 – 3 times for wheat plants each growing season, which equals an optimal amount, Mr. Többer said.<br>
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For him, the most important selection criteria are the recommendations of the distributors and the broad applicability of the spraying agents. Also, an important thing to take into consideration is the public opinion on pesticides. Mr. Többer is using herbicides, fungicides and insecticides and none of them are well regarded by the public opinion. If e. g. insecticides are used, people are worried that innocent insects could get harmed.<br>
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Mr. Többer expects agricultural losses to increase since, besides fungal infections, extreme weather conditions are making the potential agricultural losses unpredictable. To him the most important threats to his harvests are extreme weather conditions like droughts or storms and crop damaging fungi. Also, long periods of rain can keep him from using praying agents.<br>
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For Mr. Többer, genetic engineering already plays an important role in agriculture, but because the European Union is significantly restricting these technologies, he is not expecting its role to change that much in the next few years. <br>
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Furthermore, genetic modifications have to be controlled and monitored. If the agents are checked, evaluated and their advantages prevail, Mr. Többer would not see any problem with the use of genetic engineering for agriculture. In general, he expects more possibilities than potential dangers from these new technologies<br>.
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Mr. Többer showed us, that many farmers would be really open minded towards the use of genetic engineering in agriculture. Of course, their security and applicability has to be ensured before but there is no general reluctance in this topic as it is often portrayed.<br>
We approached Phillip Krainbring, a farmer for wheat, corn, sugar beets and more. His opinion and insights furthered our understanding of farming and the impact of fungi and fungicides on it. Furthermore, he gave us a chance to actively integrate the views of farmers into our project and compare them with the ones from the society, politicians and scientists.<br>
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<div class="content conLeft"> <div>02.09 first contact<br>
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11.09 extensive phone call<br>
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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.<br>
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We had an extensive phone call to talk about our project and the impact of fungal crop pathogens on agriculture.<br>
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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.<br>
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Fungicides are partially used as prevention, but most farmers try to avoid this.<br>
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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.<br>
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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.<br>
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Besides the fungi and droughts there are also some other smaller pathogenic influences like insects or viruses.<br>
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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.<br>
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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.<br>
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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.<br>
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Due to this, there is an overall interest in alternatives to fungicides.<br>
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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.<br>
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Fungicides are remaining as an emergency solution. <br>
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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.<br>
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However, this also limits the actions farmers can take to save their crops.<br>
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“The larger the toolbox to protect your own crops, the better you can react to the situation.”, Phillip Krainbringsaid.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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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.<br>
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-> 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.<br>
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Revision as of 00:11, 22 October 2019
Progress Indicator Animation
Human Practices
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.
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.
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.
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 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 14.08
Skype conference 15.08
We first got in touch with Prof. Richard Oliver through the forwarding of Professor Peter Langridge of the Wheat Initiative. As Professor of Agriculture at the CCDM, the Centre for Crop Disease Management at Curtin University (Australia), Prof. Richard Oliver had accumulated some knowledge we could use to optimize our project.
Regarding the bottleneck of endocytosis that many experts had mentioned before, he pointed out that researchers had thought about using endocytic uptake in fungi before and that there had been a focus in research on this since.
He considered our approach as feasible, since fungi can take up larger molecules from their surroundings. Binding one of these molecules to our Troygenic could thereby, theoretically, induce its endocytic uptake. For comparison, Prof. Richard Oliver mentioned the cholera toxin, which, in a similar manner, consists out of two parts that induce the uptake by the cell and respectively its toxicity.
We investigated the alternative CeDIS idea of introducing siRNAs into our system, but after discussing it we discarded the idea. We did not expect this approach to work in all species of fungi and moreover, this would not allow us to create a lab application, that we are aiming for with our Troygenics.
Regarding other aspects of our CeDIS, Prof. Richard Oliver advised us to increase the number of genetical targets to prevent the targeted fungi from circumventing our Troygenics through mutation. He suggested to use three different targets to assure the applicability of our system. As a result, of this recommendation we also focused our modeling on the question of the optimal amount of gRNAs for our system.
Moreover, Prof. Richard Oliver gave us insights into the combination of methods like fungicides, analogous reagents and plant breeding to protect harvests. He advised us to come up with a similar concept, to assure the integrated functionality of our approach. To proceed with this idea, he also named us a broad range of further experts and associations to get in contact with.
As a potential formulation he suggested using clay nanoparticles that could be attached to our Troygenics and applied to the field. He pointed out that, since they are cost-effective and widely applicable materials, they could be of great use for our project. Contrary to this, we discussed emulating the usage and formulation of currently used fungicides to keep the amount of necessary adaptation to apply our system minimal. However, designing a clay nanoparticle-based system to achieve a slow release system of the Troygenics could open whole new possibilities and potential new applications for our project.
Another addition he made addressed our proof of concept and the verification of the functionality of our system. To investigate the effects our Troygenics would have on plants, we bred wheat plants ourselves to apply our system on them in a controlled environment. In that case we should be able to evaluate the effects of our applied Troygenics based on the visible changes of the plants. For this experiment Prof. Richard Oliver advised us to use specially altered strains of Arabidopsis thaliana with fluorescent reporter genes for the detection of physical damage to the plant. Using these special strains would improve the reliability and the quantification of the potential damage to the plant and would be more precise than an examination of regular wheat plants by eye. He also gave us some lists where we should be able to find said strains and experts to contact to gain access to them.
Prof. Richard Oliver further advised us to watch out for microscopic defense responses of the plant after our tests. If these should occur the composition of the mixture containing our Troygenic probably has to be reconsidered.
A further optimization we designed with Prof. Richard Oliver and implemented into our work was the purification process for the application on the plants. Since our Troygenics are produced in E. coli, just concentrating the Troygenics after discarding cells and debris and using the concentrated supernatant would be an obvious approach. What we did not take into consideration up to that point was that many cultivation media like LB are containing components from bacteria that could cause a PAMP (Pathogen-associated molecular pattern) mediated immune reaction in the plant. Because of this, applying a direct concentration of the supernatant would probably trigger immune reactions of the plant and would thereby become useless for our application. This was one of the reasons why we decided to purify our Troygenics prior to continue working with them. We also thought about that when improving the purification protocol.
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
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 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 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.
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.
Phage Assembly
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Endocytosis
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Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
CeDIS
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lab Application
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
