We sought to develop a modular system for plant disease detection that could be used on-site. The system is based on toehold switches that regulate the expression of a reporter gene in a cell-free expression system. The toehold switch allows translational activity only in the presence of a specific trigger sequence.
The toehold switches are in a sensor plasmid composed of five parts. We wanted to optimize the plasmid so that every part could be identical between plasmids designed to detect different pathogens, apart from the exchangeable toehold switch. The system’s modularity also lends itself to future modifications of any of the parts in the plasmid, if the detection kit is to be used in environments where these standard parts are not the most optimal.
Here we describe the rationale for selecting the parts and methods used to build our system. We visualized our system to be affordable and for it to be used on-site and with minimal equipment, which was the main driver behind most of our decisions.
We chose a cell-free expression (CFE) system because it allows for safer operating for the end-user. By harvesting the molecular machinery needed for protein synthesis and lyophilizing it on paper, the system can be easily transported, stored and used without the risk of releasing GMOs (Arce et al, 2021; Pardee et al, 2016).
CFE systems have been documented to work well in producing proteins in small volumes. They have for example been proven to express enough β-galactosidase to produce enough colorimetric substrate to be seen by the naked eye. The small volumes also drive down the cost. (Arce et al, 2021; Ma et al, 2018; Sun et al, 2013)
Cell-free systems can be constructed in multiple ways, but they all share three main components: the protein components of transcription and translation, smaller functional molecules, such as NTPs and amino acids, and reaction buffers designed to maximize protein yield.
To drive down the cost of making a CFE system, we opted to use maltodextrin and hexametaphosphate as the energy source instead of the 3-phosphoglyceric acid. Maltodextrin was previously proven to work well in cell extract-based CFE systems (Arce et al, 2021). Otherwise we did not seek to optimize the reaction buffers and the composition of the reaction setup in the context of the smaller functional molecules.
There are multiple ways of harvesting the molecular machinery needed for protein synthesis. The traditional and most used way is to lyse the cells and harvest crude protein extract. PURE (Protein synthesis Using Recombinant Elements) was developed in 2001 by Shimizu et al and the method has been developed since. In this method, the 36 necessary proteins are His-tagged and then purified by nickel affinity chromatography. The ribosomes are also purified separately. In 2019, Lavickova & Maerkl developed OnePot PURE, a method in which all the proteins can be produced in a single culture, which simplified the production and made it more accessible for smaller laboratories. However, we landed on using cell lysates, because the PURE systems still require an upfront investment in acquiring the production plasmids for the proteins. Thus, we reasoned that cell lysates would be even more accessible for smaller laboratories so our method could be replicated in less equipped laboratories. Using cell lysates also has the benefit that we don’t need to purify ribosomes separately. We chose E. coli as the source of our protein production machinery. It’s widely researched and used in cell-free applications and in laboratories in general (Kim et al, 1996; Takeda & Kainosho, 2012; Sun et al, 2013).
We chose the BL21-Gold-dLac (DE3) strain developed by Didovyk et al. in 2017. This is a modification of the widely used BL21 (DE3) strain, specifically suitable for protein expression. It carries λDE3 lysogen, which lets us produce T7 RNA polymerase for lysates to promote more efficient production of our toehold constructs in the final reaction. The lac-operon was deleted from the genome, so the lysate doesn’t contain β-galactosidase despite IPTG induction, which lets us use it as a reporter.
To get the necessary proteins from cell cultures, the cells must be lysed. The most established method with cell-free systems is bead-beating, which has been proven to work for the production of CFE systems (Arce et al, 2021; Sun et al, 2013). However, bead-beating requires special single-use tubes, which will be costly in the long run. For this reason, we wanted to use two different and cheaper methods as alternatives for bead-beating. We chose sonication as one alternative because it’s widely used in other applications when disruption of the cell is necessary. Sonication requires a bigger up-front investment, but the equipment is not single-use, which lowers the per-use cost. This also lines up with our sustainability value as less waste is created.
The second method we sought to test reduces these costs even further. Autolysis is a relatively new method, introduced by Didovyk et al in 2017. This method requires a strain that carries the gene R from phage λ. This gene produces an intracellular endolysin. When the cells are exposed to freeze-thaw cycles, the inner membrane is disrupted and the endolysin gains access to the periplasm and can break down the cell wall, which causes cell lysis. This method is an attractive option because it doesn’t require any equipment to carry out the cell lysis, which makes it more accessible for smaller laboratories.
We wanted to use a method that could be easily modified to detect various pathogens. We chose toehold switches as our detection method because they can detect RNA and can be programmed to recognize virtually any sequence, which made them an ideal choice for our project. Toehold switches are specifically designed mRNA sequences that have the ribosome binding site and start codon in a stem-loop followed by a reporter gene. They are sequestered in the secondary structure, which hinders the translation of the reporter gene. The toehold switch has a specific binding site to its trigger sequence, which extends to the base of the stem-loop. When the trigger binds, it unwinds the lower part of the stem-loop, leaving only a weak secondary structure intact. This remaining structure is designed to be weak, so ribosome binding unwinds the structure, allowing translation to occur (Green et al, 2017). Toehold switches have been proven to work in detecting different pathogens (Pardee et al, 2016; Ma et al, 2018; Arce et al, 2021). This mechanism is illustrated in Figure 1.
The toehold switches were first introduced by Green et al. in 2014 and have been optimized since. In 2016, Pardee et al optimized them to have a greater dynamic range, which would be optimal for detection purposes. They reduced the size of the loop which has the RBS in A-series switches and removed the refolding domain to yield the B-series switches. We decided to use both of them and try to find an optimal switch structure for us. The B-series should have a lower background signal, ideal for bioassays, but the A-series has a higher output in the presence of the trigger, which makes it more likely to produce a signal intense enough to be seen with the naked eye. These properties are caused by the presence or absence of the refolding domain, which stabilizes the active form.
Toehold switches contain a linker sequence immediately downstream of the stem-loop. This linker sequence is typically 21-nt (Green et al, 2014), but we chose to replicate the 30-nt linker present in the 27B toehold switch (Pardee et al, 2016), because it was proven to work well. However, we switched this linker sequence later in our project to the more common 21-nt sequence, as the extra nucleotides are not necessary and toeholds containing the shorter sequence have also been proven to work well (Ma et al, 2018; Arce et al, 2021).
We also collaborated with TrigGate, who sought to develop a program to create optimized toehold designs to see if we could get better sensors with their designs. We also gave them feedback on their program and had discussions about modeling used to score screen designs in silico as well as provided measurement data about their toehold switches.
More detailed information on how we selected the best toehold switch sensors for our project can be found in our modeling page.
As mentioned previously, our end goal was to create a library of detection methods for multiple pathogens. We sought to create a single plasmid blueprint, which could easily be modified according to the pathogen to be detected. That’s why we wanted to create a modular assembly-inspired method, in which every part has its unique overhang that can be used to assemble the plasmid. Our plasmids consist of five parts: the plasmid backbone, promoter, toehold switch, reporter, and terminator. We decided to use Golden Gate assembly as it’s ideal for creating modular constructs (Casini et al, 2015). We sought to optimize all these parts, except the terminator.
As the plasmid backbone, we decided to use pOdd-1 from the iGEM distribution kit. It can be amplified in E. coli, which makes it ideal for laboratory use. The plasmid also has an RFP with flanking BsaI sites, making it ideal for Golden Gate assembly, as we can insert our parts and identify the colonies with the correctly assembled plasmid.
As the promoter, we wanted to compare the conventional T7 promoter with the T7max promoter introduced by Deich et al in 2021. We wanted to use the T7 RNAP in our cell-free system because of its high fidelity. The T7max promoter sequence was suggested to work efficiently in cell-free systems. To optimize our system, we wanted to compare the two and pick an optimal promoter sequence.
In our system, the toehold switches are the only parts that have to be designed according to the pathogen to be detected. They can be designed with NUPACK (Wolfe et al, 2017; Wolfe et al, 2015; Zadeh et al, 2011; Dirks et al, 2004) or other algorithms, for example, the program developed by team TAU.
The reporter protein is an important consideration because it determines the signal detection method. We decided to test out β-galactosidase, mScarlet, and mScarlet-I. We chose β-galactosidase because its expression can be detected via a colorimetric signal, ideal for low-cost applications. There are numerous colorimetric substrates for these reactions but we decided to use ONPG as our substrate. We also wanted to test out two red fluorescent proteins. mScarlet and mScarlet-I can clearly be seen with the naked eye if the concentration is high enough. We sought to test whether our cell-free reactions could produce enough protein to be seen without equipment. Using fluorescent proteins has a couple of advantages: mScarlet-I has a maturation time of 36 minutes so we wanted to test if it could be detected quicker than β-galactosidase.
We did not see the need for the optimization of the terminator. We decided to use the standard T7 terminator sequence in all of our constructs.
We also needed to optimize the junction sites between the parts in our assembly. We mostly stuck with the iGEM standards, but we needed to make two changes. In the standard AATG junction site between the toehold (RBS part) and cds, the ATG is a part of the cds, but the first A is not part of either part. Because translation starts from the toehold’s sequence, we needed to use scarless assembly between the toehold switch and the cds to prevent frame-shifting. The junction site still remains the same as in the standard, as the last nucleotide of the toehold switch is an A. The second change was in the junction site between the cds and the terminator. In the standard, this site has the sequence GCTT, which shares three consecutive nucleotides with the CGCT. We decided to change the sequence to GGTT to make sure of the correct assembly of our constructs.
Although toehold switches can detect different sequences, their limit of detection is quite high. Pardee et al measured their designs to be able to detect RNA concentrations as low as 30 nM. Virus concentrations can vary in different parts of the plant, depending on the virus and where it primarily infects the plant. After the infection, the viral particles are transported within the plant host (Hong & Ju, 2017). While more research is needed to confirm if our system could detect the virus from plant samples, we strongly believe that the viral genomic material needs to be amplified.
We wanted to use an isothermal amplification because it would require less sophisticated equipment to use. NASBA (Nucleic Acid Sequence Based Amplification) was chosen, because it is a simple isothermal amplification method, which can be used for DNA or RNA (Compton, 1991), making it an ideal method for our purposes of creating a universal protocol for different pathogens. It has also been proven to work well with toehold switch-based detecting methods (Pardee et al, 2016; Arce et al, 2021).
To showcase the global relevance of our detection system, we created a library of toehold switches designed to detect various pathogens. As our system is designed to be modular and the toehold switch can be programmed to detect virtually any sequence, the system can be easily modified to detect the desired pathogens. To create this library, we collaborated with the National Resources Institute of Finland as well as other research centers and iGEM teams to discover globally and locally relevant pathogens. We created toehold switches for each of them and selected two best-ranking A- and B-series switches for this library to streamline the creation of a kit capable of detecting any of these pathogens. Anyone wishing to create such a kit can combine them with other parts of the sensor plasmid and verify their viability. Here we discuss the importance of these pathogens and why we chose them as part of our library.
Below we have provided a short introduction to each pathogen in the library. The pathogens were chosen for the library for different reasons. Potato Virus Y and Tobacco Mosaic Virus are viruses that are related to our partnership with Patras, read more from the Partnership page. Some of the viruses, such as Wheat Dwarf Virus, Tomato Brown Rugose Virus and Cucumber Green Mottle Mosaic Virus were included in the library after our discussions with professionals (read more in Integrated Human Practices). We included some viruses targeting high-value crops, such as Pepper mild mottle virus and Papaya Mosaic Virus after realizing that higher value plants might be a better target for our detection system than field plants. As the main goal of our toehold switch library is to showcase the modularity and potential global impact of our project, we included viruses that infect plants around the world.
Due to restrictions with time and computing power, we decided to only focus on virus pathogens within our library. The library could be further extended by adding other kinds of pathogens, such as bacteria.
Wheat dwarf virus
Wheat Dwarf Virus (WDV) is a virus that induces Wheat dwarf disease (WDD) on small-grain cereals, such as wheat, oat, rye and many wild grasses. The disease causes dwarfing of the plants, as well as yellowing and streaking of leaves (Abt et al, 2019). The severe symptoms of WDD can induce up to 90 % of yield losses (Lindblad & Waern, 2002). WDV is transmitted between plants by leafhoppers from the genus Psammotettix. (Abt et al, 2019). The genome of the virus consists of a single-stranded circular DNA virus, which encodes four proteins: the coat protein, movement protein and two replication-related proteins.
WDV infection is controlled mostly by targeting the vector, the leafhopper that is responsible for carrying the virus. The chemical control should be done in the early phase of the growth season, as young plants are most vulnerable against WDV. (Vieraskasvit.fi)
Cucumber green mottle mosaic virus
Cucumber green mottle mosaic virus (CGMMV) belongs to the genus Tobamovirus and infects many cucurbits, such as cucumber, watermelon, zucchini and pumpkin. Many cucurbitaceous weeds have been identified as reservoir hosts for CGMMV, and can also transfer the virus to cucurbit fruits. CGMMV is a widespread virus that occurs mostly in Europe and Asia, but also in North America, Australia and Africa. The virus is a positive-sense RNA virus, containing a genome of 6.4 kb. (Webster & Jones, 2018)
The symptoms of CGMMV infection include green mottling on the young leaves, mosaic development, foliage symptoms, and even premature death of the plant. Especially young plants are especially affected by CGMMV (Webster and Jones, 2018). CGMMV can cause marketable yield losses due to poor quality of the fruits, even up to 50 % in watermelon. (Dombrovsky et al, 2017). Yield losses can occur even if the foliage is asymptomatic, and the outbreaks of the virus are a problem especially in greenhouse grown cucumbers. (Dombrovsky et al, 2017).
The most important prevention method for cucumber green mottle virus is virus-free seeds. Seeds can be treated with for example heat in order to reduce the infection level, but the virus cannot be completely eliminated by these measures. The virus is quite stable, and can survive on different surfaces. Therefore, it is important to dispose of all the infected material, including plastics, crop debris and weeds, and disinfect the equipment used (Reingold et al, 2015). There are some chemical agents that can be used against the virus, such as oxidizing agents, however the effect on the environment is a problem. There are also some other commercially available chemicals that are not as harmful to the environment. There are also cultural measures that should be done after CGMMV-infected cucurbits have been present on a field. (Dombrovsky et al, 2017)
Tomato brown rugose fruit virus
(Text written by our team for Farmer’s Handbook, a collaboration between us, team Patras and Tec Cem)
Tomato Brown Rugose Fruit Virus (ToBRFV) is a plant virus causing a disease affecting the fruits of tomatoes. It was first found in Jordan and Israel in 2014. ToBRFV is able to overcome a particular resistance gene found in tomatoes, thereby causing an infection. The disease has spread throughout the Middle East, Europe, America, and China. (Kabas et al, 2022)
At its worst, the infection leads to a non-marketable product due to yellow spots and brown and necrotic areas in the tomato fruit. The disease can be transmitted via mechanical contacts, which is the main pathway for the virus to infect the plant. It can also spread via contaminated fruits or seeds over long distances and bumblebees in a greenhouse. (Kabas et al, 2022)
Pepper mild mottle virus
Pepper mild mottle virus (PMMoV) belongs to the genus Tobamovirus in the family Virgoviridae, and is currently a major pathogen destructing pepper plants and other solanaceous crops leading to reduced crop yield and quality (Guan et al, 2018). The genome of the virus is a positive-sense, single-stranded RNA, and it was originally isolated from Sicilian-grown Capsicum annum. The virus has spread globally and affects major pepper-planting areas.
The symptoms of infected peppers include small white mottles and systemic infection, however, the symptoms can be mild or undetectable. Unnoticed infection allows virus replication and spreading. The virions of PMMoV are transmitted to healthy plants by contact, and they remain stable in the environment for a long time, even after removing the infected crops. Additionally, the virus has been described to be extremely resistant to physical and chemical agents (Kitajima et al, 2018).
Plum pox virus D
Plum pox virus (PPV) is the causal agent of a disease referred to as Sharka, that affects many types of ornamental as well as stone fruits, including plum, peach and apricot. The virus was first detected in Europe, but has spread worldwide and been introduced in all the continents. PPV-Dideron (PPV-D) is the most widespread strain of the virus. (Maejima et al, 2020) The symptoms of PPV infection are found first in the leaves of the tree during spring. The symptoms include chlorotic spots, rings, vein clearing on the leaves. The infection also affects the quality of the fruits. Infected plums and apricots, for example, show browning of the flesh. PPV spreads via grafting of trees by aphid vectors. (Dunez et al, 1994)
The most effective control methods include targeting the vectors with aphicides and destroying the infected trees in the tree plantations as early as possible (Kegler and Hartmann, 1998).
Soybean dwarf virus
The soybean dwarf virus (SbDV) is a significant virus affecting the soybean yields considerably in Japan, where it was firstly reported. The yield losses caused by SbDVc reported from Japan go up to 80 % decrease in yield. There is a possibility that SbDV could become significant in the United States as well. The symptoms of the disease caused by SbDV include stunting, yellowing and leaf puckering. It can even lead to a reduced amount of seeds if the infection happens at the seedling stage. (Hill and Whitham, 2014)
As other members of the Luteoviridae, SbDV is transmitted by aphids (Harrison et al, 2005). Some strains of SDV cause damage in common beans and in legume pastures. The prevention methods include altering the planting date, which might enable crops to avoid the aphid vector. There are also some chemical control methods against the aphid vectors.
Sweet potato leaf curl virus
Sweet potato leaf curl virus (SPLCV) infection causes mainly upward curling of the leaves and swelling of veins in young sweet potato plants (Kim et al, 2015). Mature plants often become symptomless, but despite this, SPLCV can cause up to a 30 % yield decrease (Clark and Hoy, 2006). SPLCV is particularly relevant in Asia, where most of the world’s sweet potatoes are grown. SPLCV is transmitted by a whitefly as well as by grafting, and potentially even via seed transmission. (Kim et al, 2015)
Control measures for SPLCV include mainly chemical controls against the vectors, but this has also proven quite difficult to the resistant vector strains (Jackson et al, 2014).
Tomato chlorosis virus
The tomato chlorosis virus (ToCV) belongs to the genus Crinivirus and causes a disease mainly in tomatoes, but also some other economically important vegetable crops. The symptoms of the disease often referred to as “yellow leaf disorder” include yellowing and interveinal thickening of leaves. The symptoms develop first in the lower leaves of the plant. The older leaves can start to bronzen and develop necrosis. Like other viruses in the Crinivirus genus, ToCV is transmitted by whiteflies. (Fiallo-Olivé & Navas-Castillo, 2019)
The control measures of ToCV include cultivatory control measures, such as limiting the accessibility of alternate host plants, and chemical controls targeting the vector. There are also some wild tomato species that have resistance against ToCV, but there are no commercially available tolerant tomato varieties as of 2019. (Fiallo-Olivé & Navas-Castillo, 2019)
Papaya mosaic virus
The Papaya mosaic virus (PapMV) belongs to the genus Potexvirus in the family Flexiviridae, and among papaya ringspot virus and papaya leaf distortion mosaic virus, threatens the production of papaya (Carica papaya L.) (Huo et al, 2015). PapMV was reported in Florida for the first time, and has spread to other countries such as Bolivia, Peru, Venezuela, and Mexico. (Varun et al, 2017). The genome of PapMV is positive sense single-stranded RNA, with length of 6.6 kb, and the virions are filamentous in length of 530 nm (Noa-Carrazana et al, 2006). Symptoms of PapMV infection in papaya plants include downward curling and irregularities in leaves as well as mottling or mosaic vein
Tobacco mosaic virus
(Text by Team Patras 2022, read more on our partnership page.)
TMV has an RNA genome that is single-stranded and linear, with a length of approximately 6400 bases. Due to extensive studies on TMVs’ structure, the virus has been established as one of the best-investigated models of macromolecular organization in biology (Creager et al, 1999). In addition, plant biology benefits greatly from TMV, which has led the way in elaborating functional host-virus interactions, including mechanisms of cell-to-cell movement through plasmodesmata and RNP transport from nucleus to cytosol (Scholthof et al, 2011).
Tobacco mosaic virus enters plant cells only through mechanical wounds that allow either transient opening of the plasma membrane or pinocytosis. TMV begins to disassemble within 3 min of entry, and degradation of coat protein (CP) from the capsid is associated with translation of viral RNA. The viral infection causes the disease by preventing chloroplast development, resulting in stunted plants whose leaves have a characteristic mosaic pattern of light and dark green. (Creager et al, 1999)
Given that the virus has been studied to an extent there has been several studies regarding defense mechanisms and substances that can be used, including biocontrol agents.
Potato virus Y
(Text by Team Patras 2022, read more on our partnership page.)
PVY is a type of member of the genus Potyvirus in the family Potyviridae comprising viruses with a single-stranded, positive-sense RNA genome of approximately 9.7 kb. It infects a wide range of hosts (over 40 aphid species) and with a worldwide distribution. The PVY genome has a poly(A) tail at the 3-terminus and a covalently linked VPg protein at the 5 terminus; both terminal structures are involved in genome protection and replication as well as regulation of genome expression. (Scholthof et al, 2011)
Regarding potatoes, the virus can cause several different diseases that can be divided into foliar and tuber diseases. Symptoms differ, depending on potato cultivar, strain of PVY, and environmental conditions, in addition to whether the type of infection is primary (current season) or secondary (tuber-borne). (Karasev & Gray, 2013)
Due to the lack of effective resistance to PVY isolates, which cause necrotic symptoms on leaves and tubers in cultivated varieties, and the transfer of isolates from plant to plant via daughter tubers, the control strategy to reduce the incidence of PVY is mainly based on certification of seed production. However, there are no effective means to manage the risks of epidemics caused by emerging necrotic variants. (Scholthof et al, 2011)
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