21ENGBIO: Engineering targeted activation of fungicides at the plant-pathogen interface

A secure food supply sufficient to feed the world requires the use of chemical fungicides to protect crops from fungi and other micro-organisms. Fungicides and the industry that produces them are economically critical, but intensive use of fungicides can pose a risk to the environment. The fungi that damage crops can quickly evolve resistance to individual chemicals and this drives the 'ratcheting-up' of doses sprayed onto fields as the fungi further adapt. This process quickly reaches a stage where even the highest doses of fungicide become ineffective. The constant development of new fungicidal chemicals is therefore required to keep pace with the phenomenon of resistance and to minimise the amount of fungicide released into the environment.

Discovery of a new chemical that is toxic to fungi does not mean it is immediately suitable as a commercial product. A 'lead compound' must be optimised for potency and pass rigorous safety tests as well as have suitable characteristics for being distributed onto the field and into crops. Modifications to the lead compound that make it more effective against fungi can make it less effective in some other aspect such as its solubility. Similar problems are encountered by the pharmaceutical industry during new drug development. One solution to this problem is the design of 'pro-drugs' that have a chemical structure that is soluble, less toxic and suitable for delivery to the target site within the human body. The pro-drug then becomes converted to the drug form; ideally at the precise site where it is needed. We propose to adapt this concept to agricultural fungicides by engineering a biological system that can activate 'pro-fungicides' exactly where they are needed at interfaces between plant cells and attacking fungi.

We have discovered that it is possible to send an enzyme of our choice (beta-glucuronidase) exclusively to the site on a plant cell surface where a fungus is attempting an attack. Beta-glucuronidase is exploited by some pro-drugs in the human body to form an active drug through the release of glucuronic acid. When attached to the pro-drug the glucuronic acid chemical group supports the solubility of the compound and can reduce the pro-drug activity compared to the drug form. We will optimise our beta-glucuronidase system and produce a pro-fungicide with an attached glucuronic acid group. This will be used in 'proof-of-concept' experiments with the model plant species Arabidopsis thaliana. We will measure the predicted improvement in the ability of the pro-fungicide to penetrate into the plant and we will also measure the efficiency of our synthetic beta-glucuronidase in converting pro-fungicide to fungicide at the site it is needed. We will combine expertise from multiple areas of science (biology, chemistry and physics) to achieve this. This example of engineered biology will be used to establish new industrial partnerships to 'unlock' the full potential of abandoned or challenging lead compounds using our pro-fungicide strategy. We also aim to inspire a new generation of ideas that utilise our growing understanding of how the plant immune system physically interacts with disease-causing microbes.

We will engineer the biology of the plant immune system to activate pro-fungicides at the interface between plant and microbe. Despite over a million tonnes of agricultural fungicide being applied per year it is estimated that 30% of potential global crop yield is still lost to fungal disease. Resistance to fungicides quickly builds in pathogen populations and continually pressurises the development of new products from novel lead compounds. Optimisation and deployment of these lead compounds is limited by multiple factors including bioavailability and ecotoxicity. The pharmaceutical industry has met analogous challenges using pro-drug strategies where a compound is processed within the body at the site of action to produce an active drug form. The pro-drug is optimal for delivery while the released drug has unimpeded affinity for its target. We have designed and tested a prototype transgenic system in which an enzyme (beta-glucuronidase) suitable for pro-drug activation is trafficked to the precise site of fungal invasion. This is a bio-inspired concept that has been enabled by our research into the mechanisms of basal plant resistance and the targeting of exocytosis to sites of pathogen contact. This project will optimise our design and produce a cognate 'pro-fungicide' for use in proof-of-concept experiments. To do this we will use synthetic chemistry to glucuronidate an agricultural fungicide (tebuconazole) and apply cutting-edge Raman microscopy to quantify pro-fungicide availability and conversion to its fungicidal form in the plant. This high-risk high-gain bio-engineering project could unlock the potential of abandoned and sub-optimal lead compounds within the fungicide industry and significantly expand the options available to industrial agrochemical research.