Team:KU Leuven/Project/E.coligy

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  <h3>Ecology</h3>
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    <h3>Modelling</h3> </a>
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      <p align="justify">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.</p>
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      <p align="justify">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.</p>
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  <h3>E. coligy: Plants with BanAphids</h3>
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   <p align = "justify">Essentially, our project aims to offer plants a beneficial protection mechanism against aphids. Our <B>BanAphids</B>, the genetically modified <I>E. coli</I> 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 <B>BanAphids</B> 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.</p>
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   <p><B>Aphid infested Rose plant</B> (Mauro Mandrioli)</p>
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  <h3>BanAphid's products</h3>
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    <a href="#Methyl Salicylate"><h4>Methyl Salicylate</h4></a>
    <a href="#MeS effect on aphids"><p>Aphids</p></a>
    <a href="#MeS effect on predators"><p>Predators</p></a>
    <a href="#MeS effect on plants"><p>Plants</p></a>
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    <a href="#E-beta-farnesene"><h4>E-beta-farnesene</h4></a>
    <a href="#EBF effect on aphids"><p>Aphids</p></a>
    <a href="#EBF effect on predators"><p>Predators</p></a>
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    <a href="#Economical impact"><h4>Economical impact</h4></a>
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   <a href="#References"><h4>References</h4></a>
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	      <h3>Proposed functions of methyl salicylate</h3>
		  <p align="justify">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 <I>(Vlot et al., 2008)</I>. Aphid feeding will specifically induce a significantly higher production of MeS than any other herbivore-induced plant volatile <I>(Blande et al., 2010)</I>. 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 <I>(Heidel and Baldwin, 2004)</I>. 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 <I>(Coccinella septumpunctata)</I> <I>(Zhu and Park, 2005)</I>. Therefore, with the information we have gathered, we expect the <B>BanAphids</B> to be able to attract aphid's natural predators and activate plant defence mechanisms in order to fend off current and future aphid infestations. 
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    <p><B>Winged aphid getting ready to migrate</B> (Vilhjalmur Ingi Vilhjalmsson)</p>
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    <p><B>Changes in aphid acceptance with MeS treated plants</B> Bars show median aphid settling + standard deviation, plant stages are categorised by the number of leaves. (Ninkovic <i>et al.</i>, 2003) </p>
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    <h3>Aphids</h3>
    <p align="justify">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.
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It is commonly known that when insects damage plants, these plants respond by emitting a range of volatile organic compounds (VOCs) <I>(Blande et al., 2010)</I>. Damage caused by chewing herbivores releases a different profile of VOCs than damage by aphid feeding (piercing sucking insects) <I>(Leitner et al., 2005)</I>. <I>Blande et al.</I> 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 aphid infested plants  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. MeS comprises almost two-thirds of the total emission, even after 21 days of aphid feeding, compared to a negligible emission from control plants <I>(Blande et al., 2010)</I>.  
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MeS is an important compound of a plant’s defence mechanisms acting both as an aphid repellent <I>(Glinwood and Pettersson, 2000)</I> and an attractor of natural predators and parasitoids of aphids <I>(Zhu and Park, 2005)</I>. In field trials, MeS successfully reduced initial colonisation of cereals by bird cherry-oat aphids <I>(Rhopalosiphum padi)</I> migrants <I>(Pettersson et al., 1994)</I>. <I>R. padi</I> alternate between a winter host, bird cherry Prunus padus tree and a summer host, cereals, by means of winged aphid migrants<I>(Pettersson et al., 1994)</I>. With this and following studies, it has been shown that the principal mode of action against aphids is repellency, which is what we want to achieve with MeS and EBF. The behavioural response of aphids to these substances is increased mobility, reduced 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 <I>(Glinwood and Pettersson, 1999)</I>. 
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A further study showed that application of MeS significantly reduced the mean aphid numbers in cereal crops by 25-50% <I>(Ninkovic et al., 2003)</I>. The immigration and settling of <I>R. padi</I> 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 <I>R. padi</I> on barley decreases with increasing plant age, demonstrating yet again the dynamicity of aphid behavioural responses <I>(Ninkovic et al., 2003)</I>. 
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    <p align="justify">Predatory and parasitoid insects have a specialized sensory nervous system to detect their prey <I>(Hatano et al., 2008)</I>. 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 <I>(Vet and Dicke, 1992)</I>. In response to an aphid attack, plants modify their volatile emissions and these changes are detected by the natural predators of aphids <I>(Du et al., 1998)</I>. 
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Host selection occurs in three phases: habitat localization, host localization and host acceptance <I>(Vinson, 1976)</I>. 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 MeS <I>(Zu & Park, 2005)</I>. 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 <I>(Birkett et al., 2000)</I>. 
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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 <I>(Hatano et al., 2008)</I>. 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 <I>(Ide et al., 2007)</I>. In addition to aphid honeydew, an aphid's alarm pheromone, EBF, is also an important semiochemical in aphid localization. It is secreted from the cornicle of many aphid species <I>(Franscis et al., 2005)</I> to alert surrounding aphids of the presence of natural enemies <I>(Kunert et al., 2005)</I>. 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. 
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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 <I>(Hatano et al., 2008)</I>. More information about host localisation can be found below in E-β-farnesene, effect on predators.
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    <p><B>Ladybug has found an aphid.</B> (John Flannery)</p>
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    <p><b>Aphids attacked by a parasitic wasp larva transform into a mummy and die</b></p>
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    <p><B>Plant communication via MeS</B> (Taiz and Zeigner, 2010)</p>
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    <h3>Plants - defence systems</h3>
    <p align="justify">Since plants are sessile organisms, they have no chance of escaping attacks from herbivores and must use other strategies to defend themselves <I>(Mithöfer and Boland, 2012)</I>. A plant’s defence mechanisms can be divided into two broad categories: constitutive and induced defences <I>(Pieterse et al., 2009)</I>. Constitutive defences are always present in the plant, while induced defences are produced in response to damage by herbivores or other organisms. Many physical defences and large quantitative defences (eg. Cellulose, tannins) are constitutive, they are usually distributed in permanent woody tissue and are thus difficult to mobilise. Induced defences include toxic chemicals, pathogen-degrading enzymes and physiological changes <I>(Mithöfer and Boland, 2012)</I>. Contrary to constitutive defences, induced defences are only produced when needed <I>(Wu and Baldwin, 2009)</I>. Therefore, the induced defences are less costly for the plant, especially when the presence of herbivores is variable <I>(Karban et al., 1997)</I>. 
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Biotrophic pathogens, like aphids, colonise living plant tissue and establish a long-term feeding relationship with their hosts, instead of killing the host tissue and extracting the nutrients from the dead host cells like necrotrophs <I>(Jones and Dangl, 2006)</I>. Plant resistance to biotrophic pathogens is classically thought to be mediated through salicylic acid signalling <I>(Loake and Grant, 2007)</I>. 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 <B>systemic acquired resistance</B> (SAR), which is a "whole-plant" resistance response that occurs following an earlier localised exposure to a pathogen <I>(Mauch-Mani and Métraux, 1998)</I>. SAR is analogous to the innate immune system found in animals. 
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The resistance observed following induction of SAR is effective against a wide range of pathogens and is linked to an accumulation of endogenous SA. SA modifications such as methylation and amino acid conjugation provide biological specificity in plant defence responses <I>(Loake and Grant, 2007)</I>. 
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MeS, a volatile ester, is normally absent in plants but is dramatically induced upon pathogen infection, this is an example of an induced defence mechanism. It acts as a mobile inducer of SAR by carrying this ‘under attack’ signal to neighbouring plants. The following hydrolysis of MeS to SA is catalysed by a methyl esterase in it’s immediate surrounding and activates the plant defences.</p>
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    <h3>Plants - optimal defence theory</h3>
    <p align="justify">The distribution of the induced defence chemicals is predicted by the optimal defence theory <I>(McKey, 1974)</I>.  The optimal defence theory is based on three factors: cost of defence, value of the plant part and risk of attack. First, the plant’s chemical defence has a cost, since the energy spent on defence cannot be used for other functions, such as reproduction and growth. Second, lost plant tissue has a fitness impact, but the impact is dependent on the value of the plant part. Generally, terminal leaves and reproductive parts, seeds in particular, have greater value than the other plant parts. Therefore, these plant parts contain more defences. Finally, not all plant parts are equally likely to be attacked. Plants invest heavily in effective defences in parts that are easily found by herbivores. Plant parts that are more likely to be located by herbivores are parts that have been available longer and parts that consist of more apparent tissues. Hence, the distribution of the defensive compounds within plant parts is highly organized. The optimal defence hypothesis predicts that the plants will allocate more energy towards defence when the benefits of protection outweigh the costs, specifically in situations where there is high herbivore pressure <I>(Zangerl and Bazzaz, 1992)</I>. </p>
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    <img src="https://static.igem.org/mediawiki/2013/d/dc/Whitefly_SA_reduction.png" alt="effect on plantgrowth"/>
    <p><B>Effects of whitefly infestations and SA on pepper growth</B> (Yang <i>et al</i>, 2011)</p>
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    <p><B>Plant communication via MeS</B> (Taiz and Zeigner, 2010)</p>
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    <h3>Plants - Above vs below ground herbivory </h3>
    <p align="justify">Induced resistances are mainly regulated by salicylic acid (SA)-, jasmonic acid (JA)- and ethylene (ET)-dependent signalling pathways, which are interconnected by complex signalling networks and cross-talk phenomena (Pieterse et al, 2009). Because most systemic responses are mediated by long-distance signals they can cross the aboveground-belowground border, meaning that aerial parts of the plant with its herbivores can change the resistance status of the roots and vice versa (Heil and Ton, 2008). The resulting patterns are highly complex and difficult to predict (Yang et al, 2011).

Leaves and roots are separated from each other by great distances, nevertheless roots are implicated in aboveground plant-herbivore dynamics (Kaplan et al, 2008). Because roots are surrounded by soil and therefore less susceptible to herbivory, they are an ideal storage site for chemicals used in aboveground defences. This provides a direct link between belowground and aboveground resistance (van der Putten et al, 2001). In addition to providing a safe storing site for defence molecules, roots have also been implicated in tolerance to aboveground herbivores. The roots provide also a temporary storage site for primary metabolic products that would otherwise be vulnerable to the aboveground consumers through an elevation in the sink strength. These resources can then be re-allocated at a later time for aboveground growth and reproduction (Schwachtje et al, 2006). 

The production of leaf defences belowground gives root herbivores the chance of interfering with the production and translocation and hence benefit aboveground herbivores by reducing plant resistance (Kaplan et al, 2008). Similarly, if aboveground herbivores elicit a tolerance response whereby plants allocate valued nutritional resources belowground, this storage effect may benefit root herbivores (Schwachtje et al, 2006). In conclusion, herbivore-induced facilitation may be an important feature that links the dynamics of above- and belowground communities (Yang et al, 2011).</p>


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	      <h3>Proposed functions of E-beta-farnesene</h3>
		  <p align="justify">Aphid populations are regulated by natural enemies including ladybugs <I>(Minks and Harrewijn, 1978)</I>. For many species of aphids, avoidance of these enemies involves the release of an alarm pheromone E-beta-farnesene (EBF) <I>(Xiangyu et al., 2002)</I>. EBF is released from the aphid’s cornicles when they are attacked <I>(Dixon, 1998)</I>. Therefore, EBF functions as a direct repellent of aphids. In addition, it also and acts as an attractant of their natural enemies <I>(Hatano et al., 2008)</I>. Therefore, we expect the <B>BanAphids</B> to be able to repel aphids and on top of that, attract their natural enemies to make sure the aphids are thoroughly removed.
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    <p><B>Green peach aphid</B> (Nick Monaghan)</p>
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    <h3>Aphids</h3>
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Methyl salicylate (MeS), 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).

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    <h3>Predators - prey localisation</h3>
    <p align="justify"> After localising the aphid’s habitat by the use of herbivore-induced plant volatiles (see above), predators must find the exact location of the aphids on the plant. For this purpose, predators use short-range physical (colour, shape, movement of aphid) and chemical cues <I>(Hatano et al., 2008)</I>. 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 <I>(Ide et al., 2007)</I>. In addition to aphid honeydew, the aphid alarm pheromone EBF is also an important semiochemical in aphid localisation. It is secreted from the cornicle of many aphid species <I>(Franscis et al, 2005)</I> to alert surrounding aphids of the presence of natural enemies <I>(Kunert et al., 2005)</I>. Predators have specific neuronal receptors to detect pheromones emitted by their prey. Various aphid natural enemies, including Adalia bipunctata, showed fast and pronounced orientation behaviour toward the EBF source in an olfactometer bioassay <I>(Francis et al., 2004)</I>.  </p>
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    <img src="https://static.igem.org/mediawiki/2013/e/e7/Adalia.png" alt="Adalia"/>
    <p><B>Adalia bipunctata</B> (Jon Law)</p>
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