Team:Wageningen UR/Description

Xylencer

Project Description

Group Photo

Xylencer

Silencing Xylella fastidiosa

It all started in the summer of 2013, in the Apulia region of Italy. Olive farmers and their families noticed that some of their olive trees started to dry out. They couldn’t understand why, as it wasn’t a particularly dry year. The olive trees had seen much worse during the countless generations they had been in the family. By the next summer, whole olive groves had died out. Consequently, olive farmers went out of business, rendering them unable to care for their families. Local scientists started to investigate the devastation and discovered that the olive trees were suffering from an infectious plant disease, Xylella fastidiosa. The bacterium X. fastidiosa was found deep under the bark of infected olive trees, slowly depriving them of water, therefore killing them from the inside out. Though new to European farmers, X. fastidiosa is notorious in North and South America for killing a wide variety in plants. The pathogen has been plaguing Californian vineyards for over 100 years [1] and it has brought the Brazilian citrus industry to a grinding halt [2].

Olive trees infected with Xylella fastidiosa
Figure 1: An olive grove in Italy, decimated by X. fastidiosa

Despite the long history of the disease, there is still no effective cure for X. fastidiosa [3]. Currently, the only control measures are spraying large amounts of pesticides, burning diseased plants and preventatively removing susceptible plants near areas of infection [4]. Also, these methods negatively impact other ecosystems and the bee population [5]. However, the disease is still spreading through Europe [6], not only infecting olive trees, but also other crops like cherry and ornamental plants like lavender. The devastation caused by this disease inspired our project: Xylencer, which uses bacteriophages to silence Xylella fastidiosa once and for all.

  • The Biology of X. fastidiosa arrow_downward

    X. fastidiosa is a gram-negative bacterium that lives in the xylem, part of the plants vascular system, as can be seen in figure 2 [1]. Here, it grows until it clogs up the vessels, preventing waterflow and eventually killing the plant, visible in figure 2 [7]. It is spread from plant to plant by xylem-feeding insects such as glassy-winged sharpshooters and spittlebugs. Symptoms of X. fastidiosa infections normally show late in the infection process, making early detection of the disease especially difficult. Many infected plants do not show any symptoms but can still spread the disease [8]. Treatment of X. fastidiosa infection is currently impossible [5].

    For a more indepth information on X. fastidiosa visit our background page.

    Figure 2: The disease cycle of X. fastidiosa. The pathogen can be spread from an infected tree to healthy by traveling via an insect vector.
  • Bacteriophages arrow_downward

    Bacteriophages, also called phages, are viruses that specifically target bacteria. Lytic phages, used in Xylencer, kill the bacterium by lysis [15]. Phages cannot infect plants or animals. Figure 3 describes the lytic cycle of phages. Phages infect the bacteria and inject their genome. This genome is transcribed and translated, leading to the formation of new phage particles. Upon lysis of the host cells, these particles are released. As phages target bacteria they can be used to treat bacterial diseases. Phage therapy has also been applied in agriculture, although there are several drawbacks [16].

    Figure 3: the life cycle of phages, from infection until the release of new phage particles.

Xylencer

Xylencer is a modular enhanced bacteriophage therapy for X. fastidiosa, that utilizes genetically engineered phages, based on the naturally occurring phages against X. fastidiosa to cure infected plants [9;10]. The use of bacteriophages therapy for plants is very promising, as bacteriophages are highly specific and effective. Phages are often known to be specific to only a handful of bacterial strains. However, on-field phage therapy is still held back by multiple limitations. To overcome these limitations, Xylencer focusses on four main pillars of the problem: detection, delivery, remediation and spreading.

Detection

Infection doesn’t have deadly consequences for all plant species, with over two thirds of X. fastidiosa’s known hosts not showing any symptom of infection [8]. It is therefore difficult to pinpoint locations of X. fastidiosa infection, requiring sampling of individual plants. To simplify the process, the detection of X. fastidiosa has a major role in our project. We have built an automated in-situ detection device that can detect the presence of X. fastidiosa in their insect vectors. This device constitutes a plant mimic, on which the insect vectors can feed. X. fastidiosa is detected in the insects spit and will give a color-based output as an easy indication for farmers that plants are diseased.

Delivery

Currently, the best methods for the application of phage therapy involve spraying the phages on leaves of the plant. However, this method is inefficient as phages are very fragile entities, easily degraded by environmental factors like sunlight and heat [11]. Additionally, this application method does not guarantee high phage concentrations in the xylem, where the battle against X. fastidiosa takes place. Therefore, we aim to overcome these limitations in delivery with the introduction of the Phage Delivery Bacterium (PDB).

This is a non-pathogenic bacterium carrying the repressed Xylencer phage genome on a plasmid. The PDB protects the phage whilst allowing for easy application into the tree. Inside the plant, the phages are delivered to the areas of infection. The PDB is equipped with machinery to sense X. fastidiosa's presence. Upon detection of X. fastidiosa, the repression of the phage genome by a continuously expressed CRISPR-Cas system will be alleviated. This will result in phage, eventually lysing the PDB, releasing a high concentration of phages in the infected area. PDBs that do not encounter X. fastidiosa within a set time frame, will be killed off by a kill switched linked to a molecular timer, limiting the spread of genetic material in the environment.

Remediation

Unfortunately, even though phage therapy is promising as a method to combat bacterial-based plant diseases, complete eradication of X. fastidiosa is not guaranteed. For that reason, Xylencer combines two remediation methods to eradicate X. fastidiosa. First and foremost, the use of lytic phages will efficiently kill these pathogenic bacteria. However, often a few bacteria escape phage infection [12]. Plants are unable to fight off X. fastidiosa as it camouflages itself from the immune system of the plant [13]. To further improve the effectivity of our phage therapy, we aim to artificially trigger plant immune responses. The phages are engineered to encode for immune response-activating bacterial peptides which are being released upon phage mediated cell lysis of X. fastidiosa and consequently active a plant's immune response. Thus, we forge an alliance with plant's own immune system to better combat X. fastidiosa [14].

Spread

In order to prevent reinfection by asymptomatic hosts, the cure needs to be re-administered time and time again. Manual application of therapy to all possible hosts is near impossible. To break this cycle of reinfection we propose to equip the natural phages with the same spreading mechanism as X. fastidiosa, by fusing the adhesion protein, used for insect transmission, to the capsid of the phage. This would allow the phages to reach the same areas as the pathogen, without the need for manual application. Additionally, this allows our phages to combat X. fastidiosa not only in the plants but also in the insect vectors. Different adhesion proteins were tested for optimal binding capacity. This will allow them to bind to, and be spread by, the insect vector. This way, phage therapy does not have to be manually applied to each tree and asymptomatic non-crop plants do not evade treatment.

Overview

In short, Xylencer’ s modular technologies will detect X. fastidiosa in the insects, efficiently deliver the bacteriophages to the infected areas of the plant using our PDB, ally with the plant by alerting it to the presence of X. fastidiosa and improve coverage by spreading the phages using the insect vector. The use of our therapy aims to reduce the use of pesticides, by providing an efficient alternative that is easy to apply and most importantly, helps to protect these valuable trees.

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Figure 4 : An Overview of the Xylencer project. Left to right: X. fastidiosa is detected by the plant mimick, treatment is then applied by injecting the phage delivery bacterium (PDB) into the xylem. Upon detection of X. fastidiosa the phage is expressed and lyses the phage delivery bacterium, releasing both phages and immune activating compounds. The immune activating compounds trigger the plant's immune stystem and it in turn helps to combat X. fastidiosa. Part of the phages are taken up by the insect vector and are spread to an untreated field, tranfering the therapy and curing the field.
  • Inspiration arrow_downward

    At the start of our iGEM season in March we brainstormed about a wide variety of projects, ranging from biomining to biocomputing. At our very first meeting a teammember suggested to tackle the plant disease targeting olives. This idea stayed with us in the following weeks. We continued to be drawn to this project because of immense scale of the problem. Solving this could be coupled to bacteriophage therapy, another often reoccurring idea within the group. The combination of these ideas inspired us to start Xylencer.

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  • References arrow_downward
    1. Sicard, A., Zeilinger, A. R., Vanhove, M., Schartel, T. E., Beal, D. J., Daugherty, M. P., & Almeida, R. P. (2018). Xylella fastidiosa: Insights into an emerging plant pathogen. Annual review of phytopathology, 56, 181-202. https://doi.org/10.1146/annurev-phyto-080417-045849
    2. Bové, J. M., & Ayres, A. J. (2007). Etiology of three recent diseases of citrus in Sao Paulo State: sudden death, variegated chlorosis and huanglongbing. IUBMB life, 59(4‐5), 346-354.
    3. EFSA Panel on Plant Health (EFSA PLH Panel), Bragard, C., Dehnen‐Schmutz, K., Di Serio, F., Gonthier, P., Jacques, M. A., ... & Milonas, P. (2019). Effectiveness of in planta control measures for Xylella fastidiosa. EFSA Journal, 17(5), e05666. https://doi.org/10.2903/j.efsa.2019.5666
    4. Commission Implementing Decision (EU) 2015/789 of 18 May 2015 as regards measures to prevent the introduction into and the spread within the Union of Xylella fastidiosa (Wells et al.) (notified under document C(2015) 3415)
    5. Sanchez-Bayo, F., & Goka, K. (2014). Pesticide residues and bees–a risk assessment. PloS one, 9(4), e94482.
    6. Update of a database of host plants of Xylella fastidiosa: 20 November 2015. (2016). EFSA Journal, 14(2). https://doi.org/10.2903/j.efsa.2016.4378
    7. Newman, K. L., Almeida, R. P. P., Purcell, A. H., & Lindow, S. E. (2003). Use of a green fluorescent strain for analysis of Xylella fastidiosa colonization of Vitis vinifera. Applied and Environmental Microbiology, 69(12), 7319–7327. https://doi.org/10.1128/aem.69.12.7319-7327.2003
    8. Chatterjee, S., Almeida, R. P. P., & Lindow, S. (2008). Living in two worlds: the plant and insect lifestyles of Xylella fastidiosa. Annu. Rev. Phytopathol., 46, 243-271.
    9. Ahern, S. J., Das, M., Bhowmick, T. S., Young, R., & Gonzalez, C. F. (2014). Characterization of novel virulent broad-host-range phages of Xylella fastidiosa and Xanthomonas. Journal of Bacteriology, 196(2), 459–471. https://doi.org/10.1128/JB.01080-13
    10. Das, M., Bhowmick, T. S., Ahern, S. J., Young, R., & Gonzalez, C. F. (2015). Control of Pierce’s Disease by Phage. PLOS ONE, 10(6), e0128902. https://doi.org/10.1371/journal.pone.0128902
    11. Jones, J. B., Jackson, L. E., Balogh, B., Obradovic, A., Iriarte, F. B., & Momol, M. T. (2007). Bacteriophages for plant disease control. Annu. Rev. Phytopathol., 45, 245-262.
    12. Stern, A., & Sorek, R. (2011). The phage‐host arms race: shaping the evolution of microbes. Bioessays, 33(1), 43-51.
    13. Rapicavoli, J. N., Blanco-Ulate, B., Muszyński, A., Figueroa-Balderas, R., Morales-Cruz, A., Azadi, P., … Roper, M. C. (2018). Lipopolysaccharide O-antigen delays plant innate immune recognition of Xylella fastidiosa. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-02861-5
    14. Baccari, C., Antonova, E., & Lindow, S. (2018). Biological Control of Pierce’s Disease of Grape by an Endophytic Bacterium. Phytopathology, 109(2), 248–256. https://doi.org/10.1094/phyto-07-18-0245-fi
    15. Sulakvelidze, A., Alavidze, Z., & Morris, J. G. (2001). Bacteriophage therapy. Antimicrobial agents and chemotherapy, 45(3), 649-659.
    16. Monk, A. B., Rees, C. D., Barrow, P., Hagens, S., & Harper, D. R. (2010). Bacteriophage applications: where are we now?. Letters in applied microbiology, 51(4), 363-369.