Probing the molecular origins of the species-selectivity of microtubule-directed fungicides

Septoria leaf blotch is a key disease affecting wheat and barley production, especially in Northern Europe. Septoria is currently controlled using antifungal sprays that target the fungal microtubules whilst leaving wheat and human microtubules unaffected. Inhibiting microtubules prevents the fungal cells from dividing and ultimately kills them, whilst leaving wheat and human cells unaffected. The approach works, but resistance is an increasing problem. We need urgently to understand how resistance arises and we need to develop new and better antifungals that are effective against the resistant strains of the Septoria fungus. Syngenta is trying to do this, and would ideally like to be able to test possible new agents on isolated microtubules from Septoria, comparing their response with that of microtubules from wheat and from humans. Ideally, Syngenta would like to have purified microtubules from resistant Septoria cells, to compare them with those from non-resistant cells and understand why the microtubules from the resistant cells are not affected by the fungicide. Nothing like this is currently possible - mammalian microtubules can be purified fairly readily, but no one has so far been able to purify Septoria microtubules, or wheat microtubules. The lack of tests based on purified microtubules at present limits the rate at which new antifungals can be designed and developed at Syngenta and elsewhere.

The Cross lab is expert at purifying microtubule proteins. For example, we have recently succeeded in purifying microtubule protein from yeast cells. In this project we will collaborate with Syngenta to isolate microtubule protein from Septoria cells, wheat cells and human cells, and develop miniaturised tests so that possible antifungal compounds can be checked to make sure they target the Septoria microtubules whilst leaving wheat and human microtubules unaffected. Syngenta will grow Septoria cells for us in industrial quantities, and we will process them to obtain tubulin using the techniques we have already developed for purifying yeast tubulin. We will also make tubulin from wheat seedlings, and from cultured human cells. To obtain large quantities of microtubule protein from all these sources, we will combine our own methods with newly-available techniques for highly efficient purification of microtubule protein.

We will compare the actions of different antifungals on our collection of purified microtubules from Septoria, resistant Septoria, wheat and humans, using existing measures of microtubule stability, but also by looking at the microtubules directly by light microscopy. We can then formulate ideas about how the fungicides actually work at the molecular level, and what it is that is different, at the molecular level, between the resistant and non-resistant Septoria. We will test theses ideas by engineering yeast tubulin to make it susceptible or resistant to our antifungal agents. This is the ultimate benchmark of our understanding - if we can prove that we know how to engineer the microtubule protein from yeast so that it has the same susceptibility to antifungals as the Septoria protein, than we can truly say we understand how the antifungal fits into the microtubule protein and controls its behaviour. This information can then be fed back into the workflow for the development of new antifungals.

Septoria infection affects virtually all wheat grown in the UK. Application of fungicides is estimated to boost yields by ~20% and therefore without fungicide use UK cereal growers would suffer an economic loss of between £380 and £465 million each year (figures sourced from a Rothamsted on-line report). If ultimately successful, the potential impact of this research would therefore be significant for the UK economy. The new techniques and knowledge that we develop will be transferred to Syngenta, thereby accelerating the discovery and development of new MT-directed antifungals within the project lifetime.

Septoria leaf blotch is a key disease affecting intensive wheat and barley production, especially in Northern Europe. Septoria is currently controlled using species-selective antifungals. Resistance is however developing. We need urgently to understand the molecular basis of resistance and to develop new and better antifungals that are effective against the resistant strains. To do this, it would be very helpful to have in hand purified microtubule protein (tubulin) from resistant and non-resistant Septoria strains and from target and off-target species, to be able to dissect the molecular basis of susceptibility and resistance to candidate agents. The lack of in vitro assays at present limits both our understanding of the inhibition mechanism and the corresponding practical rate at which new antifungals can be designed and developed.

The Cross lab has expertise in tubulin purification and microtubule biochemistry. In this project we will collaborate with Syngenta to isolate microtubule protein from wild type Septoria cells, resistant Septoria cells, wheat seedlings and human (HeLa) tissue culture cells, and develop microscale assays so that potential new antifungals can be assayed in vitro for activity. Experiments with new and existing antifungals will allow us to identify candidate sequence features in the tubulins that confer susceptibility/ resistance to particular antifungals - some of these will be local to the fungicide binding site, and some will be allosteric. We will test the power of these sequence signatures in conferring fungicide susceptibility/resistance using protein engineering of S. cerevisae tubulin. Techniques and knowledge will be transferred to Syngenta on the lifetime of this project.

Without fungicide use UK cereal growers would suffer an economic loss of between £380 and £465 million each year (figures sourced from a Rothamsted on-line report). If ultimately successful, this research could therefore have significant impact.

Prospective impacts from our work are relevant to two of BBSRC's three strategic priorities, BASIC BIOSCIENCE UNDERPINNING HEALTH and GLOBAL FOOD SECURITY and to one of BBSRC's three enabling themes, that of PARTNERSHIPS. Our work is an industry academic partnership between the Cross lab at Warwick Medical School and Syngenta's fungicide biochemistry department at Jealott's Hill.

Our work will firstly have COMMERCIAL, ECONOMIC AND INDUSTRIAL impact on the research practices of companies developing new fungicides, especially and immediately on our industrial partner Syngenta who will benefit from the faster and more effective research methods that we will develop over the course of the project. As we develop better and faster tests for the actions of fungicides on their target protein, we will also harness these tests to improve basic scientific knowledge of the mechanisms of fungicide action on its target protein. This new knowledge will also feed into the workflow for the design and development of new fungicides at Syngenta via our partnership and later to other researchers in industry. The methods we develop will reduce the time required to screen new compounds and better identify those with the desired activity profile against target and non-target species. This will improve the economic competitiveness of our partner with respect to its fungicide biochemistry business by enhancing their capacity to innovate new fungicides. Improving the capacity of Syngenta and others to develop safe and effective species-selective fungicides rapidly may potentially also have an ENVIRONMENTAL impact, because the more potent and specific the antifungals, the less potential there is for potentially environmentally damaging activity against off-target species.

Other companies developing anti-tubulin drugs for use in human and animal health care would also benefit from the new techniques developed in the project with the same economic benefits and potential for wealth creation in development of new drugs and treatments.

The new screening tests that we will develop will in the longer term also be useful more widely in the discovery of new pesticides, herbicides and therapeutics across the agribusiness and pharma sectors. This is because our tests and the new basic knowledge we acquire by harnessing them will accelerate the discovery process for new compounds in all of these areas. By this route, we will also have impacts on human and animal HEALTH AND WELLBEING and on TECHNOLOGY development. The new technology and new basic insights that we develop will be transferred promptly to the industrial partner, and later to other researchers, via publication and via a Microtubule Chemical Biology website that we will develop.

Finally we also anticipate having an impact on the PUBLIC AWARENESS OF SCIENCE.
The potential economic and health benefits arising from our research are obvious even with no technical or scientific background and can therefore be an accessible topic via which to engage with the general public and with school children through public presentations and through a non-technical section on our proposed website, explaining the role that scientific research plays in the economy and health care. Last year the Cross lab ran an exhibit at the Royal Society Summer Exhibition, which receives national coverage and provides a highly effective forum for improving public understanding across the generations. We will do this again.