Abstract

Our project, KILL XYL will be a cure against the disease caused by the plant pathogen Xylella fastidiosa. This disease currently causes the loss of thousands of hectares of European crops and no extensive cure exists against it.

KILL XYL has a two step approach. Since the symptoms of disease are only observed when xylem vessels are highly colonized by the bacterium and photosynthesis is blocked, we first detect the symptoms of the disease using a drone equipped with a camera.

We will then treat the disease with a combination of three strategies. To directly attack the bacterium, we developed specific phage-like particles that inject toxins. To disrupt the biofilm, which is the principal cause of plants death, a particular fatty acid that quenches the quorum sensing activity of the bacterium and an enzyme that degrades the biofilm will be used.

Motivation

Distribution map of Xylella fastidiosa worldwide.

Xylella fastidiosa is a bacterium that infects the xylem tissues of a wide range of plants. This bacterium has been widespread in the Americas for many years and occurs also in Asia. The bacterium is associated with several diseases of crops of economic significance, and has a wide range of host plants comprising 312 species.

X. fastidiosa is transmitted from plant to plant by xylem sucking insects. The bacterium can persist in symptomless uncultivated plants, from which insects may acquire the bacterium and pass it to crops. Symptoms of disease are only observed when xylem vessels are highly colonized by the bacterium. The xylem transports water and soluble mineral, nutrients from the roots throughout the plant, and used to replace water lost during transpiration and photosynthesis. Xylem vessels are interconnected by bordered pits, which allow the passage of xylem sap, but block the passage of larger objects (such as bacterium) by a membrane.

The control of the movement of potential hosts and insect vectors and the eradication of infected material is considered the most effective method of limiting the spread of the disease in the European Union. But there is no method yet to cure the trees.

KILL XYL concept

KILL XYL project.

At Aix-Marseille University we thought about a solution that enclose many aspect of the cure.

First, we wanted to improve the detection of the disease, to do so we wanted to use a NDVI camera that will help us to see if the plant do photosynthesis or not. If the plant is heavily affected by Xylella fastidiosa, it support a hydric stress that stop the photosynthesis.

Secondly, we want to get rid of the bacterium. Phages are natural predators of bacteria. They can also be used to transfect DNA into a bacterial cell. Phages has also the advantage of being specific to a strain and to be modulable. As we wanted to be eco-friendly, we create phage-likes particles, that aren't able to spread. Thus we have nanobots specific to Xylella fastidiosa, capable to inject toxic genes into the bacterium.

The main cause of the plants death, is the hydric stress induced by the accumulation of biofilm into the xylem vessels. To disrupt the biofilm we thought about different solutions. The first one is to stop the bacterium producing any extra poly-saccharide. This could be achieved by quenching the quorum sensing of the bacterium with the help of a little fatty acid called : 2-cis-decenoic acid. Secondly, we wanted to destroy the exo-polysaccharids. An enzyme coming from a bacteriophage could fulfill the use by the hydrolysis of polysaccharides.

Hence, KILL XYLL simply detects, disrupt and kill Xylella fastidiosa.

Module 1: Detection of the disease

Module 2 : Engineering bacteriophages

Phage-like particles process.

Bacteriophages play a special role in nanoscale cargo-delivery developments, because they can be regarded as naturally occurring nanomaterials. Viral nanoparticles (VNPs), in particular bacteriophages, are attractive options for cargo-delivery as they are biocompatible, biodegradable, and non-infectious to mammals.

Phage systems, like M13, have been employed in biotechnological applications, most prominently in the identification and maturation of medically-relevant binding molecules through phage display. The application of phages in materials and nanotechnology is mainly due to their nanoscale size and simple life cycles. We choose to use those application in our advantage in order to target Xylella fastidiosa and other pathogenic bacteria.

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Module 3 : Quorum sensing approach

Xylella fastidiosa uses quorum sensing, as an inter-bacterial communication system, to regulate its biofilm production. The quorum sensing is based on the emission of specific fatty acid, there are fatty acids antagonistic to its quorum sensing, this disturbance is called quorum quenching. 2-cis-decenoic acid is one of these fatty acids whom has quorum quenching effect to Xylella fastidiosa. This action of quenching will prevent the formation of biofilm, dependent of the quorum sensing and therefore will have both action preventive and curative to the symptoms caused by the biofilm on the plants. We want to produce this fatty acids from E. coli to inoculated the infected trees. Thus saving the plants from Xylella fastidiosa.

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Module 4 : Disrupting the biofilm

Extracellular polysaccharide (EPS) is a major virulence factor of Xylella Fastidiosa, the causative agent of Olive Quick Decline (OQD) associated with binding of water, ions and nutrients; keeping them in close contact with the bacteria; and protection against the recognition by plant cell defense mechanisms. Some bacteriophages carry coat proteins that can degrade bacterial polysaccharides. The Depolymerase binds to the capsular EPS and degrades the polymer until the phage reaches the cell surface, where it binds to an outer membrane receptor and injects nucleic acid to initiate the lytic cycle. To disrupt the biofilm we centered our research on this enzyme, which we called deps to cleave the extracellular polysaccharides of Xylella Fastidiosa freeing up the tension cause by EPS in the xylem vessels. I hope that curing the plant from the hydric stress caused by the bacteria.

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InterLab

We used plasmids containing different promoter from the 2017 distribution kit and transfect into E.coli. Before measuring fluorescence expression, we used LUDOX and fluorescein to calibrate the TECAN. We prepared a dilution series of fluorescein in 4 replicates and measure the fluorescence in a 96 well plate in a plate reader. By measuring these in all standard modes, we generated a standard curve of fluorescence for fluorescein concentration. We used this to correct cell based readings to an equivalent fluorescein concentration and convert this into a concentration of GFP.

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Modelisation