Understanding the role of GSTF1 in MHR

Multiple herbicide resistance (MHR) in grass weeds is a growing global problem with serious implications to sustainable arable agriculture. In contrast to target site-based resistance, MHR confers resistance to all classes of selective graminicides irrespective of mode of action. Since the first identification of an MHR population of black-grass (Alopecurus myosuroides) in the UK in 1982, this type of resistance is now well established in wild oat (Avena fatua) and annual rye-grass (Lolium rigidum) weed populations around the world. In extreme cases, MHR in competing grasses can result in total crop loss. In the UK alone, herbicide-resistant black-grass has been reported at 2000 sites, with 80% of these populations tolerating multiple herbicides. MHR can render newly developed herbicides ineffective and is now a major hurdle to the development of the next generation of crop protection agents. In recent work at Durham we have identified a key protein AmGSTF1 involved in the onset of the MHR response in black grass. Although, from sequence analysis, a GST, this protein does not appear to have a metabolic function and the molecular basis of its action remains unclear. However, in preliminary studies we have shown that the MHR response is inhibitable using as lead structures small molecules of the benzoxadiazole class. The aim of this project is to build on this approach and use a combination of chemical probes and fundamental enzymology to ascertain how the MHR response is regulated and whether orthologous proteins exist in other weed species. In the first goal of the project we will explore the effects of inhibition of AmGSTF1 on markers of stress responses such as peroxidase activity. In particular we will probe the hypothesis that this resistance protein controls the regulation of a signal transduction pathway and look for up-regulation of putative signalling molecules using the range of high-resolution LC-MSn techniques available in the department. Molecules identified by this approach will be independently synthesised and fed back to the plants to see if they mirror the MHR responses observed through the analyses of the markers of xenobiotic metabolism. The second goal of this four year project will be to see if related resistance pathways exist in other plants. We will achieve this by chemically synthesising arrays of probes based both on the CNBF lead inhibitor and also on other known GST inhibitors drawn from the pharmaceutical arena particularly focusing on those associated with multi-drug resistance in cancer chemotherapy. Each inhibitor will be modified to incorporate a second (reactive) functionality that can facilitate the isolation and purification of the target protein either through affinity chromatography or through covalent coupling via 'click' ligation to a biotin tag. Once isolated each protein will be sequenced, over expressed, purified and characterised following the precedents established in our work with AmGSTF1.