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The agricultural landscape plays a critical role in contributing to Australia’s social, economic and environmental sustainability. In particular, the grains industry accounts for the second largest agricultural industry in Australia. Unsurprisingly, however, as grains such as wheat and barley remains an important staple in many countries and has applications in various other industries, not just used in flour to make bread, cakes and other sweets, but also to produce alcohol, paper, pharmaceutical drug capsules and soap (Trade Finance Global, 2022). In fact, Australia generates 25 million tonnes of wheat per year, accounting for 3.5% of the global wheat production, but represents around 15% of the world’s global wheat trade (Australian Export Grains Innovation Centre, 2022). Around 70% of the wheat produced in Australia is intended to be exported to more than 50 countries, generating revenue amounting to nearly 4 billion U.S. dollars (Australian Bureau of Statistics, 2021).
Aside from facts and statistics, this year’s UNSW iGEM team, and those that have come before and will in the future, relish in enjoying the fruits of the wheat industry’s labours. With a land so multicultural and diverse, it is nothing but a pleasure to share the commonalities brought by wheat. Imagine, sitting around the lunch table, one friend bringing the iconic Australian spread Vegemite on toast, another bringing their famous Asian-inspired noodle stir-fry, with a third bringing some classic Italian spaghetti bolognese, and to top it all off, a nice cold wheat-based beer. We cannot help but applaud the exhaustive and intensive efforts of the Australian farmers that work endlessly to bring these goods to our doors and our plates.
Which is why it is disheartening and worrying to learn of a serious threat faced by the wheat industry, that is, cereal rusts. Most shockingly, when engaging in conversations with the general public, a problematically large number have never heard of this issue and typically believe it to involve the rusting of metallic structures.
On the contrary, cereal rusts present a damaging and widespread disease caused by the fungus Puccinia spp. that threaten the yields of several varieties of wheat and other horticultural crops, not only in Australia but around the world. Here, infected wheat plants may produce a shrivelled grain, reducing food production by an astounding 10% (Australian Bureau of Agricultural and Resource Economics, 2018). In doing so, rust costs the wheat industry 350 million U.S. dollars due to this loss in production as well as the implementation of desultory fungicidal treatment and prevention plans (The University of Sydney, 2022). Not only would this be a significant loss to the Australian economy by impacting one of its major exports, but is a troubling cry for help for the tens of thousands of integral grain farmers whose livelihoods and quality of life are put on the line without any backing or certainty for the future. Similarly, this loss for the wheat industry possesses implications for the various other industries that rely on the use of wheat, such as those mentioned before. Thus, a cascading collapse in the Australian economy is foreseeable if cereal rust is left unchecked.
This issue does not restrict itself to industry alone, but extends far beyond the social reaches and delves into the lives of the common people. Seeing as the vast majority of food and beverages involving wheat that is consumed in Australia are sourced from and dependent upon domestic farming production, an alarming concern for the risk of national food shortages is ostensibly within the near future. As such, consumer prices are likely to rise, which may only be a minor hassle to some, but are a weighty encumbrance to those most susceptible to food insecurity, including unemployed people, single-parent households, low-income earners and Indigenous Australians (Australian Institute of Family Affairs, 2011). In fact, the rates of food insecurity are highest in remote communities and an increase in price would lead to a further struggle to provide wheat products for those in rural areas.
Evidently, cereal rust is a problem that can no longer be ignored and is not one that will be settled on its own. Indeed, it is only going to get bigger as our dependency on wheat production also intensifies. In saying that, wheat production forecasts estimate around 32 million tonnes to be produced in the next year or so, significantly higher than what has been seen before (Grain Central, 2021). It appears this can be attributed to the continuing wet weather resultant from climate change being ideal conditions for wheat production, which, however, also ensures optimal growth and spread of cereal rusts, leading to greater incidences (NSW Department of Primary Industries, 2020).
Over recent times, novel aggressive strains of rusts have emerged, forcing the use of fungicides as an essential control measure. Here, fungicides contain active chemical compounds that inhibit or eradicate the growth of fungi. Some prominently used antifungal drugs include quinone outside inhibitors (QoIs) and demethylation inhibitors (DMIs), which target essential metabolic pathways unique to the fungus (Carmona et al., 2020). Though, using fungicides presents several problems involving harmful environmental consequences that may poison the surrounding ecosystems and microbiomes, as well as the costly and labour-intensive application required. Interestingly, the spreading of fungicides beyond their intended area of usage may mean they seep into deeper soil layers where a clinically relevant human fungal disease Aspergillus sp. resides, possibly generating antifungal resistance in these organisms and posing a threat to human health (Sugui et al., 2014). One other major problem resides in the limited dedication and investment towards the research regarding the correct application of fungicides in controlling cereal rusts, such as optimal application time and dose, effectiveness of the active molecule and surveillance of the development of resistance to the fungicide. The latter of which is quite evident due to the high degree of variability by the Puccinia pathogen, meaning they can adapt and evolve to infect different wheat cultivars (Carmona et al., 2020). In fact, the Australian Bureau of Agricultural and Resource Economics and Sciences (ABARES) predicts that an outbreak of a newer and more virulent wheat rust strain called Ug99 could cost the Australian agricultural industry an accumulative 1.4 billion U.S. dollars in the coming years (Grain Central, 2018).
With the vitality of the wheat industry ingrained into Australian culture, it is imperative to put cereal rusts at the forefront of the intertwined fields pertaining to agricultural, microbiological and synthetic biology research to solve such a rife and dire concern. Not only will this be central in stabilising a fragile and fluctuating economy, but will provide the necessary requirements for a well-fed and nourish society.
Immediately in the journey to design a solution to cereal rusts, we sought to understand the social landscape surrounding the fraught reality of rusts and the potential for synthetic biology as a remedy. It was challenging at first to immerse ourselves in a world foreign to us, something we have heard very little, if anything, about, yet has lived in our own country among us for all these years. In doing so, we aspired to engage with a variety of key stakeholders, including farmers, traditional land owners, agricultural businesses, governments and academics, and incorporate their attitudes within our own to develop a solution that is human-centric. As such, in our approach to synthesise our human practices framework, which would feed significantly into other aspects including wet lab and dry lab, we enacted a human-centred design approach based on three fundamental principles.
Firstly, we wanted to understand and address the core problems contributing to cereal rust, not just the symptoms. In empathising with those affected, we felt it necessary to collate the perspectives of disparate user groups to enhance and deepen our understanding of the extent of cereal rust. By interacting with and listening to the counsel of farmers, traditional land owners, government and non-government bodies, agricultural companies and social scientists, we broaden our approach to encompass the needs that were required of a synthetic biology solution. We approached this using a people-centred lens. We understood from the beginning that this problem affects people and that our solution would be lived by people. In turn, we considered the history, culture and environment of the community as well as their beliefs towards synthetic biology.
Secondly, we focused not only on isolated components of our solution, but on its entirety as a living and functioning machine. We knew that for a problem that exists within an environment that is interconnected rather than in isolation, we must design a solution that befits such a paradigm. Here, with extensive discussions with academics in the fields of microbiology, environmental sciences, protein biology and the social sciences integrated into our approach, we proposed a viable and safe solution to be implemented in order to minimise its risks to the environment and to the wider population, one that involves an alternative approach to traditional fungicides. Here, we marvelled at the idea of using a peptide specific to the cereal rust pathogen to inhibit its spread without causing harm to the surrounding species, delivered by a genetically modified (GM) bacterial vector sourced from the natural microbiome of the soil to minimise potential disruptions.
Lastly, considering the proposed model to which we aimed to implement, in combination with our continued talks with experts and consulting with the literature, we understood the value of constantly improving and modifying certain aspects of our solution. With each iteration of our solution, we refined it so as to be considered usable and good for the world. We explored the potential risks our solution may present and engaged ways to mitigate them in order to put the wider public at ease and gain acceptance. Looking onward, we have planned the next steps that the future holds for our solution, long before it can be used in any practical way.
Taken together, this framework we established provided a grounded and well-considered approach in tackling the diverse perspectives on cereal rusts and synthetic biology as responsible alternative solution for the world.
Having limited knowledge of cereal rusts and the social landscape it occupies, we would not have been as successful in understanding the nuances of the problem and attempting to design a well-considered solution without implementing the guidance and perspectives of vital stakeholders. Learning from consultations with five key stakeholder groups, involving academics from the field of the social sciences, traditional land owners, academics within the realm of microbiology, molecular biology and environmental sciences, governments and agricultural companies and farmers. Unfortunately, we were challenged when engaging with farmers as many were hesitant to be contacted in light of recent disastrous flooding events that added extra stress on their already busy lifestyles. However, farmers remain at the forefront of the user groups we desire to cater to and will be an integral stakeholder to gain guidance from in the future. Ultimately, we followed our human-centred approach to listen to the diverse yet pertinent voices of those impacted by and integral in our research and design of a solution relating to cereal rust.
Being the first stakeholders to engage in discussions with, social scientists and ethicists aided largely in framing our human-centred approach and informing the delicate and sophisticated conversations had with other user groups. Here, they guided us along the complicated tightrope when indulging in the world of synthetic biology and reminded us of perceptions society holds in light of recent scientific interventions, such as the introduction of the cane toads as a biological control of the sugarcane beetle. These discussions were integral in informing how we communicate our solution by listening to what the root problem is and involving user groups in a meaningful way.
The UNSW iGEM team, and the greater UNSW community, acknowledge that Aboriginal and Torres Strait Islander peoples are the traditional owners holding native title over the land. As such, we would like to pay our respects to elders of the community, past, present and those emerging. Their knowledge, customs and traditions are of paramount importance in informing us of the history of the land as a way to direct the future of our society. Here, their sacred and spiritual connection to the land has existed for more than 60,000 years (Australian Institute of Aboriginal and Torres Strait Islander Studies, 2019). The land where Australian farmers grow their crops all are grown on Indigenous land, therefore a central component of the UNSW iGEM team is to listen and incorporate the Aboriginal people’s values and strategies into our project. Subsequently, traditional owners play an important role in the management and long-term sustainability in the soil that Australian wheat grows on.
Integral to the design of our solution were the discussions with a range of academics in various fields which all hold a stake within the greater realm of cereal rust infections. By actively engaging with the expertise of these academics, we were able to synthesise our knowledge of a thought-out and theoretically safe proposed solution to be implemented in addressing the exacerbating and consequential actions of cereal rust. With the knowledge gained from these conversations, our team could evaluate the viability of our synthetic biology solution and effectively reiterate the earlier prototypes and brainstorming ideas, all part of our human-centred approach principles.
Major stakeholders in our technology would be government and agricultural manufacturing companies. We have considered how our technology affects them and how they are able to benefit from it. With the government and companies, they need to consider the safety and public perception of GMOs. Public perception of GMOs need to be considered as it is ultimately their lives being directly affected. There has been a history of fear against GMOs such as the activist group Greenpeace advocating against the usage of GMOs in agriculture due to concerns of monoculture susceptibility and corporate monopoly. Consequently, from the government and companies’ viewpoints, these fears must be tackled through education and policy implementation. In doing so, we needed to hear the opinions of these stakeholders to explore how the government can protect against corporate monopolies, and what sort of policies need to be implemented to protect the economic rights of farmers and land owners.
With companies, we needed to figure out how they will introduce GMOs to their consumers and find out if people are willing to accept it. We also wanted to know the impact our technology will have on consumers financially to see if we can not only benefit their health but their wallets as well. One major manufacturing company of grains and other wheat-based products in particular, Sanitarium, holds a firm non-GM position after responding to consumers’ concerns in 1999, being the first food manufacturer in Australia to take this step. To this day, Sanitarium is committed to avoiding the use of genetically modified material to keep with consumer expectations. Though they keep this stance, the company is not opposed to the use of synthetic biology and recognise the improvements in food production and resistance to diseases it can offer. Largely, Sanitarium is in full support for the responsible introduction of new technology, but strongly emphasised the importance of keeping consumers informed at every step of the process.
In terms of both governments and companies, we hoped to be able design a sustainable model of including GMO crops into our agricultural rotation. They need to work together to develop policies that balance the health advantage of utilising GMOs with the risk of monocultures, unknown side effects and negative public perception. Through collaborating with these stakeholders now and in the future, solutions to these major problems can be developed and our technology more efficiently implemented to help society.
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Following on from the dialogues with key user groups, we ensured their voices were heard and integrated all throughout the design, building, testing and proposed implementation of our solution. Informed by these exchanges which we regarded in the highest esteem, we worked assiduously to align our values with those shared by the pertinent stakeholders. Again, we were highly driven by our human-centred design in approaching the discourse surrounding cereal rust.
From the beginning of our iGEM journey, we were dedicated to contributing to the growing body of work that resides within the highly understudied yet still significantly troublesome problem of cereal rust. As an issue being exacerbated by external dynamic circumstances and fuelled by the problematic human intervention of inadequate control measures, we sought to shift the current paradigm to better protect the invested stakeholders, including the wider public, from the damaging effects that are foreseeable in the coming years.
We approached CSIRO social scientist Doctor Carter very early on, who cautioned us to gain more clarity of the application of our technology. Though we were still at the dawn of the design of our solution, she made it explicit for us the importance of articulating the specifics, would such a solution lead to higher crop yield? Given that wheat is such a cash crop, Doctor Carter reminded us that there is not much capacity to take a risk. In doing so, we would need to be sure our solution would work as farmers’ livelihood depended on the viability of their crops. By advising us to add more context to our application, we doubled-down on constructing a thorough planning of our proposed implementation, one that recognised the trade-offs of integrating synthetic biology to solve an agricultural, and by extension, an environmental sustainability problem.
As a team, we initially discussed the logistics regarding an RNAi approach. Though we had the encouragement of Doctor Wang from the CSIRO, we ultimately shifted our focus to one that involves designing a peptide capable of neutralising integral fungal target protein. Here, this opened the door to a wide array of support available to our team, enabling us to harness the expertise of our own project supervisor, whose research focuses on the application of synthetic biology for the engineering of proteins at UNSW. Doctor Dominic Glover offered significant guidance throughout all stages of our project, with his expertise on peptide engineering engaging us to think about the limitations posed by RNAi technology. With the difficulties involved in delivering RNA through the plant cell wall and the challenges surrounding its stability in environmental conditions, the benefits peptides presented were too surmountable. Based on our continuous discussions with Doctor Glover, the approach we thought was best involved targeting a surface receptor on the fungal cell as a means to successfully inhibit the signalling pathways within the fungus and, hence, hinder its growth and invasion in the wheat plant.
In considering the minute details of protein engineering and designing a peptide to inhibit a specific protein produced by the fungus, Doctor Lenardon encouraged us to take a step back and consider the biology of the fungus as a whole. Here, by delving into this intricate life cycle, Doctor Lenardon pondered whether targeting when the step of the reproductive cycle when fungal spores land on the wheat plant and protrude their filamentous hyphae to invade the plant. In doing so, it would involve finding proteins involved in the polymerisation process of the fungal filaments. In a similar notion, Associate Professor Böcking also recommended approaching the problem by first understanding how the fungus invades, and then identifying any crucial enzymes involved in its pathogenesis. Enzymes present good targets for peptide inhibitors as noted by Associate Professor Böcking, and suggested that targeting metabolic pathways unique to the fungus or ones in the plant cell that benefit fungal growth but are not required by the host to survive are avenues he would pursue. Integrating his advice into our design and modelling, he provided us with information of key databases containing relevant resources to study and break down the functioning of biological systems, namely the KEGG PATHWAY database.
Conflictingly, as part of our ongoing deliberations with Doctor Lenardon, she highly advised against using online databases such as the National Centre for Biotechnology Information (NCBI), noting that they were not very useful in studying fungal genomes like those from Puccinia spp. Something that we learned from her is that the genes which are annotated in these databases are mainly studied due to their homology with other, well-studied organisms. As such, the fungal protein targets we selected from them, such as translation initiation factors, are not the best to target due to their similarity to other eukaryotes like humans or other fungal organisms, and this overlap may lead to serious off-target effects. Instead, Doctor Lenardon urged us to consult with the published literature, in combination with the genome databases, as they may contain characterisation of the structure, functions and localisation of the fungal protein.
After an intensive search in the literature for the fungal target proteins, we compiled a list which summarised key features of the proteins, including their studied function, structure and notes on the advantages and disadvantages of targeting such a protein, for example, if it was well conserved between the cereal rust pathogenic strains. When brought to the attention of Doctor Lenardon, she provided an in-depth analysis of why and why not we should choose each target from her perspective and years of experience studying pathogenic fungi. One fungal target we were considering targeting was a hexose transporter involved in the uptake of hexose from the plant cell to the fungus to fuel its growth (Chang et al., 2020). However, Doctor Lenardon prompted us to ask whether inhibiting this process would not just be compensated by another nutrient transporter on the fungus. In addition to expressing her concern of redundancy, she raised others pertaining to the similarities of a hexose transporter, a vital protein that may be conserved among the domains of life, as well as the issues in expressing and purifying a transmembrane protein in Escherichia coli in the lab. As part of our solution, in consultations with Doctor Lenardon and Professor Park, we agreed that targeting only one fungal protein would not significantly affect the virulence of the pathogen. As such, we developed a strategy to hit multiple proteins contributing to the virulence of the fungus. The targets include two effector proteins, PstSCR1 and Pst_12806, which are proteins secreted by the fungus to alter plant cell functioning and have been shown to reduce fungal growth and damage to the wheat plant when knocked down individually in previous experimentations (Dagvadorj et al., 2017; Xu et al., 2019). Another added benefit to studying effector proteins, as pointed out by Doctor Lenardon, was the idea that they are significantly easier to express in and extract from E. coli cells, paving the way for our experimental testing.
Though it is easy to target any fungal target associated with the virulence of the cereal rust pathogen, our talks with Professor Tanaka aided in ensuring we targeted the “right” proteins, such that our peptide does not become ineffective as a result of the pathogen’s natural evolution processes. Here, Professor Tanaka cogitated the importance of the mutation rate and population size in predicting the development of resistance by the fungus. By researching the available information about the mutation rate of Puccinia spp., we considered how many different mutations in the target proteins can confer resistance, which can provide a sort of timeframe as to how long it would take for resistance to evolve. Though, our research and modelling shows the highly conserved nature of the PstSCR1 and Pst_12806 fungal proteins , suggesting that this should pose too great a threat to the effectiveness of our peptides (Dagvadorj et al., 2017; Xu et al., 2019). The other key factor, population size, may, however, present an issue. Professor Tanaka warned us that in a large population, there could be multiple mutants even if the mutation rate per individual is low. “Many of these mutants could be lost by chance but you want to avoid lineages that rise in frequency”. Thankfully, Professor Tanaka provided a resolution to restrict the effect of population size, by introducing regular population bottlenecks, that is, an event that drastically reduces the size of a population. Experimentally, this can be performed through the use of serial dilution, however in the environment, we may need to plan a mechanism by which to accomplish such a feat (National Geographic, 2022).
By approaching from a protein biology perspective, our talks with Professor Park enlightened us in understanding the evolved processes plants experience to sense the presence of fungal effector proteins. When detecting these effector proteins, the plant then switches a range of immune response pathways on. The important mediators that coordinate this phenomenon are, as Professor Park educated us, pathogen recognition receptors such as nucleotide-binding leucine-rich repeat-containing (NLR) receptor proteins. Likewise, the promise of antifungal defensins, which are antimicrobial peptides, reveals a conceptually potential avenue (Sher Khan et al., 2019).
Not only are we presented with an approach to alter the plant immune system by blocking the pathogen-host interactions or overexpressing defensins in the plant cell, but in order to neutralise the fungal proteins, we could also build on the integral advice imparted by Associate Professor Böcking, which involved the use of the random non-standard peptides integrated discovery (RaPID) system, as well as several other modelling methods. This system will enable us to design peptide ligands for the fungal proteins of interest with limited prior knowledge of protein-protein interactions from the plant receptor proteins, for example, and the fungus (Goto & Suga, 2021). When consulting with the literature, we were able to find proteins present in the plant that have been shown to, in some way or another, interact with the selected fungal proteins. Here, our research showed that Pst_12806 has been involved in interacting with a domain of the TaISP protein, a component found in the membrane of chloroplasts of wheat plant cells (Xu et al., 2019). Equally, PstSCR1 has been widely tested using the BAK1/SERK3 receptor signalling pathway, and so we investigated this as a potential basis for our peptide design (Dagvadorj et al., 2017). After consulting with Doctor Glover, analysis of our modelling revealed that the TaISP protein from wheat and SERK3B from the model plant organism, Nicotiana benthamiana, showed predicted binding and instructed us to proceed with these two plant proteins as a sort of basis for the design of our peptide. As such, our dry lab team concentrated their efforts to generate a collection of potentially better binding peptides than that shown by the natural plant proteins.
From an environmental microbiologist perspective, Professor Thomas pointed out to us that the release of self-replicative genetically modified material into the environment remains a major concern in the current social climate. As such, he suggested that instead of implementing a completely novel peptide inhibitory solution, we could potentially look into microorganisms within the soil microbiome that out-compete the fungus. By finding bioactive molecules produced by these bacteria to modulate fungal growth, this approach could complement what we planned to do or even avoid the de novo design of our peptide. Though, Professor Thomas stated this approach does require an intensive search and cultivation of soil microbiomes which extends beyond the safety, time and resource constraints of our project. Nevertheless, this approach may yield results not only beneficial for cereal rust infections, but for other plant and animal diseases as well.
Now that we had an idea beginning to form, we explored ways in which we could test the binding of a peptide inhibitor to our selected fungal targets. Ideally, we would have liked to use the Puccinia fungus itself, though we were warned by Professor Park of the dangers of handling such a pathogen, given that it is spore-forming and fast spreading posing a significant safety threat to our team and the broader surrounding community. Another interesting piece of information communicated by Professor Park was that rusts are highly adapted to their hosts and cannot grow on artificial media or even dead plants. These presented significant challenges in approaching to build and test our solution in the lab. Though, Professor Park suggested the use of other fungal organisms also used in studying the rust pathogenesis, including Fusarium spp. and Rhizoctonia spp. However, these organisms also possess spore-forming features and are difficult to isolate in the lab given the time constraints and resources available to us. Though it may be difficult to find a well-researched model organism that isn’t a safety concern, Doctor Lenardon recommended the use the yeast, Saccharomyces cerevisiae, as a better model organism than bacteria due to the post-translational modifications present in fungi but not in bacterial cells. Ultimately, it was the decision made by the team and our supervisor Doctor Glover to proceed with the use of the workhorse organism, E. coli, as it fell in line with our expertise and provided added benefits such as their rapid growth and expression of recombinant proteins and cost-effectiveness. Hopefully, the safe use of the fungal organisms in the next phases of our project would become a reality, though because the biological mechanisms behind cereal rusts are still largely unknown, adding the additional layer of testing an understudied fungal protein and organism simultaneously would prove too great for our team to overcome.
Our team engaged in extensive conversations regarding the specifics of testing the interaction of our designed peptide and the fungal proteins. After conversing with Doctor Lenardon, we were considering coating the wells of a standard well plate with the fungal effectors followed by the addition of the peptide. Then an indirect enzyme-linked immunosorbent assay (ELISA) could be performed to measure the binding ability of the peptide. However, an ELISA relies on the use of antibodies that are known to bind to the proteins we would be using, but we could not find one available to us. To overcome this, Doctor Lenardon suggested we use a small biotin tag attached to the protein due to the plentiful supply of commercial antibodies with the ability to bind to this tag. Though, due to a constriction with the funds available, we could not justify purchasing these antibodies when a cheaper and possibly more efficient way of testing protein interactions could be accomplished.
Instead, we followed the advice suggested by Doctor Glover, who provided guidance on the design of the plasmid vector, the purification technique of our proteins, and the experimental method we would use to test protein-peptide interactions. When imagining how we would clone our DNA sequences, of which had been codon optimised to be expressed in E. coli, Doctor Glover provided us with the readily available pET-19b plasmid as well as the overhang sequences needed to be included on both sides of the sequence encoding the fungal protein and peptides for us to incorporate into the plasmid via a Gibson assembly procedure. Preemptively thinking of the method of purification, Doctor Glover referred us to use a nickel-NTA chromatography column. In doing so, we would need to build in a sequence encoding a polyhistidine tag, called a 6xHis-tag, known to bind to the beads on the column. There was some hesitation expressed by our team that the extra histidine residues may affect the folding of the protein, and hence hinder its binding potential, though Doctor Glover eased these concerns when he verified our predicted models of the proteins were not severely disturbed by this addition.
A widely used fluorescent technique endorsed by Doctor Glover to study the bi-molecular interactions within cells involved Förster resonant energy transfer, or FRET. Part of the building of our fungal proteins and designed peptides involved the fusion with a fluorescent protein tag to give off a signal when the fungal target and our peptide come into close proximity. However, we had to do so without resulting in a significant change in the structure. By consulting with the three-dimensional models of the proteins and under the direction of Doctor Glover, we decided to conjugate the fluorescent tags (either mCerulean3 or mVenus) to the following parts of the proteins (the fungal protein or the peptide, respectively):
Pair 1:
mCerulean3 to the N-terminus of the fungal protein PstSCR1
mVenus to the N-terminus of the peptide based on SERK3B
Pair 2:
mCerulean3 to the C-terminus of the fungal protein Pst_12806
mVenus to the N-terminus of the peptide based on TaISP
These well-studied fluorescent proteins were chosen because not only of their availability in our lab, but also because they are an optimal fluorescent pair for FRET due to the excitation wavelength of one being the same (or close to) the emission wavelength of the other, as explained by Doctor Glover.
In the process of trying to express these proteins in E. coli, our team ran into several setbacks. Echoed in our minds were the warnings Doctor Lenardon offered earlier on when she informed us of the difficulties of handling fungal proteins, and even plant-based proteins, in bacteria. It was not helpful that due to the lack of previous experimentation on both the fungal proteins and our novel peptides, we had to optimise much of the expression and purification. As such, Gustave Severin, a PhD candidate under the supervision of Doctor Glover, suggested we alter the induction temperature to include 16oC, 30oC and 37oC, and the IPTG inducer concentration to include 0.1 mM, 0.4 mM and 1 mM. Additionally, because we were not generating usable amounts of the proteins, Gustave Severin also opted for the use of larger scale expression systems, increasing the volume of the cell culture to hopefully generate enough sample to be tested for FRET. Similarly, we switched our protocol for purifying the protein from using a manual gravity flow chromatography column to engaging a more automated approach involving the Äkta start machinery to improve our protein purification yields and mitigate the problems involved in the former technique, such as the column drying out when left too long to stand.
Another aspect we wanted to incorporate into our testing was the impact of our peptide within the wheat plant cell. Although time constraints limited our ability to do so, we were advised by Professor Thomas that a type 3 secretion system, a ‘needle-like’ projection responsible for injecting active molecules into a host cell, present in several bacterial species remained a highly utilised approach in his field of research as a delivery means. Professor Thomas mentioned two species he would use to overcome the barrier of the plant cell wall involved Agrobacterium tumefaciens or Pseudomonas syringae, who are both reasonably genetically amenable to deliver a desired peptide of interest directly into the plant, given that a signal sequence is integrated into our peptide. Though this is not possible to be accomplished at this point in time, in future studies testing our peptides safety and biological activity, this approach would prove instrumental.
Having considered the technical details surrounding our solution, the next step in our human-centred design approach was to consider the integrated system involving the safety and ethical implications of implementing such a technology. These concerns are felt by all the pertinent user groups, as the words of Doctor Carter still ring in our ears, there is no capacity to fail with such high economic and social demands on the line. To ensure a well reflected approach was pursued, our team integrated expert advice to guide the direction of our solution to one that falls in line with the ideals of the stakeholders.
Unlike other crop diseases, cereal rusts deserve special attention with regard to the timing when fungicides are applied and the frequency at which they are reapplied. Being the most destructive disease of wheat, agriculturalists and governments should ensure that the use of such fungicides lessen the experienced losses of crops, particularly in wheat varieties most susceptible to disease. Professor Thomas reminded us that in the current social and environmental context, using fungicides demands extensive analysis that maintains the sustainability of the environment and avoids unnecessary damage, whilst guaranteeing profitability. As such, we aimed to incorporate such extensive testing before implementing our solution into the environment. As noted by Professor Thomas, we should test our solution on various stages of the disease to determine its role in the disease initiation and progression. The question posed by Professor Thomas, “does the peptide provide protection or reversion?”, motivated our attitudes in clarifying these specifics.
Beyond the use of the peptides, our team sought to develop a means to deliver our active molecule to the site of application in the wheat plant. Naturally, our team thought to use E. coli as the bacterial vector, however as Doctor Lenardon pointed out, it would not be the wisest idea seeing as farmers would be against spraying it on their crops. Instead, following on from Professor Thomas' advice, we decided that, in the future stages of our project, we would choose a bacterial species that is naturally found in the microbiome of the wheat plant or its surrounding soil. Here, we collated a list of potential candidates that could be tested, including Pantoea agglomerans, Brevundimonas canariensis sp. nov., Burkholderia gladioli, or Flavobacterium odoratum, to name a few germane bacterial species (Chen et al., 2022). With these species in mind, we are brought a step towards implementation, though we would need to develop a risk-averse and thorough guide to minimise the disruption to the natural ecosystem, as outlined below:
Performing long-term observation after introducing our peptide and delivery system to the Puccinia spp. pathogens when infecting the wheat plant within a controlled and contained environment simulating soil conditions.
Performing careful observation after introducing our peptide and delivery system to the Puccinia spp. pathogens in a diseased wheat plant in its natural soil environment.
Only then can we allow the successful release of our solution for widespread use under continual observations.
When considering that our proposed solution to cereal rust involves the release of genetically modified materials into the ecosystem, we must incorporate a bio-containment system to reduce the potential for unintentional invasion outside the site of application. Here, we plan to include a kill switch in our design as per the expertise from Doctor Wang. A kill switch provides a mechanism to cause our genetically modified bacteria to die without the presence of a certain molecule, of which we chose reactive oxygen species (ROS) due to their accumulation during cereal rust infection. The presence of ROS may switch on the expression of our MazEF toxin/antitoxin system under the control of the ROS-inducible OxyR transcription factor interacting with TrxC promoter (Zheng et al., 1998; Engelberg-Kulka et al., 2005). The promoter, which would be present upstream of the MazE antitoxin, could control the survival of the bacterial vector only when in contact with the wheat plant. If there are not enough ROS molecules, such as when the bacterium escapes the site of application, then the promoter would not be turned on and MazE would not neutralise the MazF toxin, effectively killing the vector.
Our conversations with Doctor Carter provided insights into the end users of our solution. In discussing the weight of the voices held by farming user groups, she made the clear distinction between small-scale farmers using wheat crops to sustain their livelihood and family as compared to larger, commercial scale farming. Here, Doctor Carter noted there might be tight contractual agreements with buyers which can limit the access to or the ability to invest in our technology.
Most importantly, it is important we recognise that our synthetic biology approach is a “silver bullet” in dealing with cereal rust, because it is evident that the other drivers exacerbate the problem. In reality, in order to solve an issue of this magnitude, we would need a multipronged approach, as Professor Park referred to it as. Echoing his words, Doctor Carter revealed that it is often about balancing the many prevention and control methods and that each technology acts in a complementary action. In saying this, these conversations informed us to think realistically and meaningfully about the problem, instructing us to remember that our solution was one that was supposed to be good and responsible for the world in whatever capacity it could.
An important aspect of proposing a solution that is good and responsible for the world involves communicating it honestly and effectively to key user groups to gain insights into how it fits into their vision of a better future. With her experiences with such situations, Doctor Carter informed us that people often do not want to hear that a novel technology is exactly what is needed. Here, she warns us that we should not approach stakeholders trying to convert them to a new synthetic biology solution as the problem generally does not reside with improving scientific literacy.
Instead, we should continue engaging our human-centric approach by first trying to understand the problem and the issues presented to the user groups because of it. As Doctor Carter notes, people make decisions based on their immediate emotional reactions in relation to the social norms imposed. As such, we should gently weave in our technology over time, by suggesting it has a role in treating cereal rust. Most importantly, it is our responsibility to ask stakeholders their opinions on the role synthetic biology has in helping the issue. This generally results in a greater acceptance as people feel as though their voices are heard and integrated in the improvement of the solution. The design of our human-centred approach has guided the way in which we connect with people, effectively closing the loop through the acknowledgement and engagement with affected user groups, their needs and their values.
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With such an intricate and delicate problem we are trying to resolve, our aim was to design a well-thought-out approach involving the use of novel fungicidal peptide candidates through the integration of stakeholder insights, all in the name of synthesising a good and responsible solution.
Our first and foremost value involved the design of peptide candidates with the potential to inhibit the growth of the cereal rust fungus, thereby increasing the productivity and profitability of yields of cereal farmers across Australia and overseas.
Our aim for this project was to be able to develop an alternative solution to chemical pesticides. Using pesticides comes with a plethora of issues such as resistance and environmental damage, but with our solution we hope to be able to overcome them. Having a bacterial vector being able to produce its own bioactive molecules saves on the time and costs as well as maintaining an environmentally friendly solution.
Australia spends around 180 million U.S. dollars annually on pesticides, which could be diverted to more research and development towards not only agriculture, but to all facets of scientific and social endeavours (Reliance Chemicals Australia, 2021). Even though currently genetically modified products are more expensive, the costs are gained back through less pesticide usage and labour requirements. With more research in this area, costs can further be lowered in the future as production gets more efficient.
Maintaining a connection with the various stakeholders has proven invaluable to our team this year. Our conversations with experts from the fields of academia to governments and companies to traditional land owners reminded us of the vitalness of the need for dialogue between science and the people meant to benefit off it. Naturally, as these are the people affected by cereal rust, they will be the most important in voicing their concerns for the increasing threat of this widespread problem. Ultimately, their opinions will guide how we approach resolving the issue.
This integral value encompasses the inclusion of our stakeholders in voicing their concerns of our approach at various design, building, testing, implementation and communication phases in order to develop a respectful and responsible solution. The discussions we conducted with stakeholders enabled us with a more defined and established understanding of the trade-offs that may be experienced with the introduction of our technology. Thus, we developed a risk assessment table as discussed in the following section, which highlighted the foreseeable issues that may arise from knowledge imparted by our key experts. Whilst weighing the benefits that may also emerge from the technology as well as the methods to mitigate the presented risks, we believe that our solution, with the best of intentions, is responsible and good for the world.
Traditional approaches to managing cereal rusts are far from perfect. In fact, pesticides have a negative impact on human and environmental health, frequently leaching into water sources, poisoning surrounding wildlife and even our drinking water. Some are volatile, spreading beyond their intended area of usage, and poisoning even more land. The direct impact to human health has been linked to cancer, Alzheimer’s disease and an array of birth defects. Thus, we hope to be able to create a safer alternative.
With our solution, we can cater to specific targets of the cereal rust fungus not found in humans and other organisms, therefore allowing precision targeting of plant diseases. In future, we would like to test the capability of the kill switch that we also plan to implement within our solution for an added level of protection against undesired consequences related to the escape of genetically modified materials in the environment. Though, in proceeding with our solution, we must always be cautious as there is no room for failure with an issue so delicate and wide spread.
A synthetic biology approach to cereal rust is a logical progression, seeing the application of genetic engineering in other, more well-studied plant diseases, such as in Bt. cotton. As such, this avenue should be explored for an equally damaging and widespread problem that has been left for too long now. Though, this topic still remains controversial, with perspectives ranging from fully devoted to strong opposition for its development. Our team believes that although synthetic biology possesses the ability to result in devastating negative impacts, the intention and careful planning of the proposed implementation of the technology can greatly reduce its risks and greatly benefit the world.
Benefits experienced by our technology |
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| Improving the quality of life of producers, particularly small-scale farmers whose livelihood depends on their crop yields |
Higher yields enabling the resolution of food shortages and improving food security for those most at risk |
Funnelling of the money saved into further research of other understudied plant diseases |
Decrease in the reliance on toxic agricultural chemical fungicides |
Translational capability of our technology into solving other plant and animal diseases |
Easy use as compared to the current approaches in managing cereal rust, such as the laborious breeding programs to create resistant varieties of wheat |
Risks involved with out solution |
Mitigating these concerns |
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Off-target effects impacting the diversity and functioning of plants, insects and microbial ecosystems |
Thorough modelling and testing of our peptide candidates and impact of bacterial vector on the ecosystem prior to its practical release |
Genetic pollution of modified genes into the wild as well as the potential effects of the run-off of our biologically active molecule |
Creation of a kill switch to prevent the escape of genetically modified material beyond the site of application |
Disrupting the natural processes of evolution, leading to unforeseen complications or consequences |
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Development of resistance against our biological peptide candidates |
Use of multiple peptides designed for different targets of the fungus to prevent resistance |
Effects on human health and safety for consumption |
Thorough modelling and testing of our peptide candidates and impact of bacterial vector prior to its practical release |
Consumer rights and choices on “modified/contaminated” cereal products, coupled with the strict and occasionally inconsistent global regulations and legislations, and the commercialisation and privatisation of the technology restricting the use by all user groups |
Consultations with policy makers, in line with the continual involvement of user groups in every step of the design and implementation of our solution |
Ultimately, following a human-centric approach which prioritises the values corresponding to those of the key stakeholders, a synthetic biology approach, although quite new in relation to cereal rust management, is necessary in the current climate. With the issue only going to worsen, we need the emergence of a solution which is good and responsible before it is left too late to be explored.
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In order to close the loop and ensure our solution aligns with the desires and needs of our stakeholders, we felt it necessary to evaluate the values of our project. These values instructed the synthesis of a good and responsible solution, and involve: (1) creating a novel alternative to the harmful and inadequate control measures put in place to manage cereal rust; (2) meaningfully engaging with key user groups affected by the implications of cereal rust; (3) responding to concerns expressed against the implementation of our solution; and (4) integrating our proposed solution with limited disturbances to the existing ecological and social dynamics.
Cereal rust is not as simple as just killing a pathogen. The complex social and ecological drivers behind the issue remain a challenge that even we, as a team, are unsure if we could solve it alone. In saying that, our solution provides a step in the right direction by reducing the need for harmful agricultural chemicals that further perpetuate the problem of cereal rust by the emergence of more dangerous, resistant strains that can cause devastating losses to the social and economic landscapes. Our solution, which relies on the application of synthetic biology to engineer novel inhibitory peptide candidates, can contribute to a growing body of efforts towards solving the problematic disease, rather than acting alone as a “silver bullet”. Therefore, this aligns with the values imparted by several stakeholders, including Doctor Carter, Professor Kearnes and Professor Park, among others.
Engaging in fruitful conversations with affected stakeholders allowed us to understand the social, ecological and economic contexts surrounding cereal rust. The deeper we pursued, the more we realised how close to home this problem hit. In a land with a rich history and culture, we sought to prioritise the voices from traditional land owners, who have been protecting the land from destruction for longer than we realise. As such, it was vital that we heard their voices on such a devastating disease brought from foreign bodies to their lands. Here, Joshua Gilbert reminded us that engaging with traditional land owners in projects like the ones regarding cereal rust are of the utmost importance in order to not disturb the land’s steep history and culture. By engaging with Gilbert and other stakeholders, we garnered a greater appreciation of the problem.
However, we hope to expand the reach of our engagement to cereal farmers around Australia, one of the most important user groups, who can provide a more detailed account of the issue, enabling us to better understand the needs required of a solution. This will form the central goal for the future of our project.
In light of the conversations we had with a wide range of user groups, we have responsively integrated their concerns and voices all throughout the design and proposed implementation of our project. Here, our team feels this has been significant in constantly improving upon the earlier prototypes of our solution. Again, the future of our project will heavily depend on integrating the insights from the perspective of cereal farmers to ensure we create a solution that is ethically and socially responsible.
Agriculture is one of the earliest sciences that benefited humanity. Yet, with the current problems impeding on its functioning, we believe that synthetic biology is the next step in its evolution. Following on from dialogues with academics and the wider public, we discerned an innate disconnect in the tenor of the GMO conversation, one that needs to be resolved before we can continue any further with our work. This disconnect, as Professor Kearnes points out, is the product of the exclusion of stakeholders in understanding a problem and defining a solution for it. As Doctor Carter made us realise, these processes result in feeble outcomes. As such, our team was called to action to close the loop and encourage meaningful conversations to resolve such a disparaging disconnect.
Due to intense use of fungicides in the past, the risks associated with traditional approaches, such as resistance development and harmful off-target effects, must be taken into account as a priority to develop sustainable cereal rust control strategies. By designing a proposed implementation of our solution, we are broadening the available alternatives for governments and other user groups to utilise to tackle cereal rust and take a leap forward in the advancement of science in agriculture. Therefore, it is extremely important that we begin to engage a holistic or integrated approach that incorporates our solution, including:
Furthermore, with the advice from Professor Thomas and Doctor Wang to incorporate a bacterial delivery vector with a kill switch involving the MazEF antitoxin/toxin system, we have blended a potential biocontainment system. However, though our team did not have the opportunity to test such a system, we are hopeful that the next steps of our project explore its applications in light of ecological and soil microbial relationships. All this is to ensure a positive and lasting impact from our solution.
This year, our team has had a rewarding engagement with the intricate and dynamic interactions at play when designing and planning to implement a synthetic biology solution in society. By approaching our human-centred design, it has become increasingly apparent that good and responsible outcomes may be evolved when there are proper channels of communication between the science and the user groups.
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