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Team:KU Leuven/Project/E.coligy
From 2013.igem.org
Secret garden
Congratulations! You've found our secret garden! Follow the instructions below and win a great prize at the World jamboree!
- A video shows that two of our team members are having great fun at our favourite company. Do you know the name of the second member that appears in the video?
- For one of our models we had to do very extensive computations. To prevent our own computers from overheating and to keep the temperature in our iGEM room at a normal level, we used a supercomputer. Which centre maintains this supercomputer? (Dutch abbreviation)
- We organised a symposium with a debate, some seminars and 2 iGEM project presentations. An iGEM team came all the way from the Netherlands to present their project. What is the name of their city?
Now put all of these in this URL:https://2013.igem.org/Team:KU_Leuven/(firstname)(abbreviation)(city), (loose the brackets and put everything in lowercase) and follow the very last instruction to get your special jamboree prize!
Ecology
Modeling
Ultimately our project aims to reduce crop loss due to aphid infestations. For practical reasons and difficulties with regulations, it is impossible for us to conduct a field experiment for our BanAphids during this summer. We therefore attempted to predict the effect of our pheromones on the environment and the ecosystem through a series of modelling steps. On this page you will get a thorough explanation of these steps, software and results.
Wetlab
To be able to perform behavioural experiments with aphids and aphid predators such as ladybugs and the green lacewing, we were invited to perform these experiments at the companies Biobest, a worldwide leader in biological pollination and sustainable crop management, and pcfruit, whose mission is to research prospects of new crop protection methods. On this page you will find more information about these experiments.
E. coligy: Plants with BanAphids
Essentially, our project aims to offer plants a beneficial protection mechanism against aphids. Our BanAphids, the genetically modified E. coli strain, will provide plants with a protection mechanism by the production of two substances, methyl salicylate (MeS) and E-beta-farnesene (EBF). a phytohormone and a pheromone. MeS will activate plant defence systems and attract natural predators of aphids. Furthermore, EBF will repel aphids off of the plant and will attract natural predators of aphids. These two substances are naturally used in the communication between plants, predators and aphids. Therefore our BanAphids will blend into the ecosystem and minimise the disruption of the communication between plants, predators and aphids. Below, you will find more information concerning the effects of MeS and EBF on plants, predators and aphids.
BanAphid's products
Proposed functions of methyl salicylate
When aphids feed on plants, plants will react by emitting herbivore-induced plant volatiles, and these mediate relationships between plants and insects through the attraction of natural enemies and the repulsion of herbivores (Vlot et al, 2008). Aphid feeding will specifically induce a significantly higher production of MeS than any other herbivore-induced plant volatile (Blande et al, 2010). MeS is a volatile phytohormone that is a product of the salicylic acid pathway. Since it is a volatile, MeS can induce defence systems in neighbouring plants as well as itself (Heidel and Baldwin, 2004). In addition to the activation of plant defence systems, MeS has been shown to have another function, it is a potent attractor of natural and effective aphid predators, this includes the seven-spotted ladybug (Coccinella septumpunctata) (Zhu and Park, 2005). Therefore, with the information we have gathered, we expect the BanAphids to be able to attract aphid's natural predators and activate plant defence mechanisms in order to fend off current and future aphid infestations.
Figure 1ǀ Winged aphid, aphid population getting ready to migrate.
Figure 2ǀ Temporal changes in aphid host plant acceptance with plants treated with volatile methyl salicylate. Bars show median aphid settling + standard deviation.
Aphids
Aphids are small, soft-bodied insects that feed on sap from leaves, twigs, or roots. Adult aphids exist in two forms, winged or wingless. They are most common in spring and summer. Under ideal temperatures, many aphid species can complete their life cycle in less than 2 weeks, and because of their prolific reproductive capacity, enormous populations of aphids can build up in a short time. It is commonly known that when insects damage plants, these plants respond by emitting a range of volatile organic compounds (VOCs) (Blande et al, 2010). Damage caused by chewing herbivores releases a different profile of VOCs than damage by aphid feeding (piercing sucking insects) (Leitner et al, 2005). Blande et al. identified MeS as the most distinctive indicator of aphid feeding in the VOC emission profile. A significant increase in MeS VOC emission was detected from plants after aphid feeding as well as a significant time effect, meaning that MeS emission intensity increased with the length of the aphid infestation. The effect of aphid feeding on a plant’s MeS induction is immense even though the feeding pressure due to aphids must exceed a threshold level before inducing volatile emissions. It comprises almost two-thirds of the total emission even after 21 days of aphid feeding, compared to a negligible emission from control plants (Blande et al. 2010). MeS is an important compound of a plant’s defence mechanisms acting both as an aphid repellent (Glinwood and Pettersson 2000) and an attractor of natural predators and parasitoids of the aphid (Zhu and Park 2005). In field trials, methyl salicylate successfully reduced initial colonisation of cereals by Rhopalosiphum padi migrants (Pettersson et al. 1994). R. padi alternate between a winter host, bird cherry Prunus padus tree and summer hosts, cereals by means of winged aphid migrants. With this and a following study, it has been shown that the principal mode of action against aphids is repellency, which is what we want to achieve with MeS and beta-farnesene. The behavioural response to this is increased mobility, overall reducing reproductive efficiency and increased mortality of adults. An aphid’s response however, appears to be dynamic, losing their negative response to MeS after three or four days of adult life (Glinwood and Pettersson 1999). A further study showed that application of MeS significantly reduced mean aphid numbers in cereal crops by 25-50%; both at high and low aphid attach rates (V. Ninkovic et al. 2003). The immigration and settling of R. padi in barley fields was delayed due to MeS application as well as a significant reduction in maximum aphid densities. Preference tests however showed that the effect of MeS on settling of R. padi on barley decreases with increasing plant age, demonstrating yet again the dynamicity of aphid behavioural responses.
Predators - prey localisation
Predatory and parasitoid insects have a specialized sensory nervous system to detect their prey (Hatano et al, 2008). They use volatiles produced by the herbivores, reliable but at low concentration, or those produced by the plant to locate their prey. These latter are easily detectable, but are less reliable. To overcome the reliablility-detecability problem predators and parasitoids focus on the responses on stimuli created by specific interactions between the herbivore and its plant (Vet & Dicke, 1992). In response to an aphid attack, plants modify their volatile emissions and these changes are detected by the natural predators of aphids (Du et al, 1998).
Host selection occurs in three phases: habitat localization, host localization and host acceptance (Vinson, 1976). Aphid natural enemies must first locate the aphid habitat, the host plant where aphids are present. Therefore, plant-derived volatiles are used, since evidence of aphid damage is acquired. One of the important herbivore-induced plant volatiles that are used by predators is methyl salicylate (MeS) (Zu & Park, 2005). The feeding of aphids on the plant induces the de novo production of salicylic acid (one of the main components in plant defence systems) which can then be metabolised into MeS (Birkett et al, 2000).
Following habitat localization, natural enemies use short-range physical (colour, shape, movement of aphid) and chemical cues to search for a suitable herbivore on the host plant (Hatano et al, 2008). One of the chemical cues used by natural enemies of aphids is honeydew. Mostly, the natural enemies need physical contact with honeydew to change their behavior (Ide et al, 2007). In addition to aphid honeydew, the aphid alarm pheromone, (E)-β-Farnesene (EBF) is also an important semiochemical in aphid localization. It is secreted from the cornicle of many aphid species (Franscis et al, 2005) to alert surrounding aphids of the presence of natural enemies (Kunert et al, 2005). Detection of these short-range chemical cues does not lead to the aphid directly, but only indicates its presence, improving prey detection of the predators and parasitoids.
Once an aphid is located, natural enemies have to recognize it as a potential prey before they attack it. For host recognition, non-volatile chemical cues are important, in particular contact with the cornicle wax on the surface of the aphids. Predators use their antennae or their mouthparts to recognize the prey. Parasitoids use probing to assess host quality before oviposition (Hatano et al, 2008).
Figure 3ǀ This ladybug has found an aphid.
Figure 4ǀ Aphids attacked by a parasitic wasp larva transform into a "mummy" and die.
Figure 5ǀ Induced-resistance systems in plants
Plants - defence systems
Plant resistance to biotrophic pathogens is classically thought to be mediated through SA signalling. Salicylic acid (SA), a phenolic phytohormone, is involved in many functions such as mediating in plant defence against pathogens. SA induces the production of pathogenesis-related (PR) proteins and is involved in the systemic acquired resistance (SAR), which is a "whole-plant" resistance response that occurs following an earlier localised exposure to a pathogen. SAR is analogous to the innate immune system found in animals.
The resistance observed following induction of SAR is effective against a wide range of pathogens and the activation of SAR requires the accumulation of endogenous SA. SA modifications such as methylation and amino acid conjugation provide biological specificity in plant defence responses (Loake et al. 2007).
Methyl salicylate (MeSA), a volatile ester, is normally absent in plants but is dramatically induced upon pathogen infection. It acts as a mobile or volatile inducer of SAR by carrying this ‘under attack’ signal to neighbouring plants, following hydrolysis by methyl esterase in it’s immediate surrounding. MeSA is synthesised by SA carboxyl methyltransferase (SAMT).
Plants - optimal defence theory
Predatory and parasitoid insects have a specialized sensory nervous system to detect their prey (Hatano et al, 2008). They use volatiles produced by the herbivores, reliable but at low concentration, or those produced by the plant to locate their prey. These latter are easily detectable, but are less reliable. To overcome the reliablility-detecability problem predators and parasitoids focus on the responses on stimuli
Figure 3ǀ This ladybug has found an aphid.
Figure 4ǀ Aphids attacked by a parasitic wasp larva transform into a "mummy" and die.
Figure 1ǀ Induced-resistance systems in plants
Aphids
Methyl salicylate (MeSA), a volatile ester, is normally absent in plants but is dramatically induced upon pathogen infection. It acts as a mobile or volatile inducer of SAR by carrying this ‘under attack’ signal to neighbouring plants, following hydrolysis by methyl esterase in it’s immediate surrounding. MeSA is synthesised by SA carboxyl methyltransferase (SAMT).
Rapid resynchronization
The synchronized oscillation is an inherent property of this system. Because of the small space-scale, the diffusion distributes the molecules almost evenly throughout the colony, as long as most partake in the production. This colony-wide concentration of the diffusible molecules ascertains the rapid resynchronization of subpopulations that do not follow the others. If for instance, the unsynchronized subpopulation produces the other colony-wide molecule, that concentration does not reach any significant level because of the rapid diffusion at that scale. This can be explained by using an approximation of the diffusion coefficient of a typical quorum sensing molecule; The diffusion coefficient D of N-(3-Oxododecanoyl)-L-homoserine lactone equals 4.9 x 102 µm2s-1(Stewart, 2003). The equation (Einstein, 1905) gives the average displacement of molecules because of diffusion in 3 dimensions after time t. In this example the average displacement after 1 second would be 54 µm, which is about equal to 50 times the size of one cell. This affirms the statement that the production of one cell is rapidly dispersed throughout its environment. Next, this also implies all cells have an approximately equal concentration, since the distance between cells in moderate density cultures is approximately 16 µm (= 2 x 108 cells/ml, Park et al., 2003). This distance corresponds with one third of the diffusion length after one second, if we assume the production is evenly distributed among the population. This colony-wide concentration holds the key to a rapid resynchronization as explained below.
Figure xǀ Text
