The molecular basis of multiple herbicide resistance in grass weeds

The current demands on global cereal production for food and biofuels production have placed renewed emphasis on the need for science and technologies to support sustainable and high yielding arable agriculture. A key component of efficient cereal production is the careful use of chemical control to suppress competing weeds. However, over the last 40 years the intensive use of herbicides has selected for populations of grass weeds which are resistant to selective graminicides (grass-killers) used in major cereals like wheat. In 1982 a new form of resistance was determined in a black-grass population in the UK which extended to all classes of herbicides licensed for its control in wheat and barley. Since then, incidences of multiple herbicide resistance (MHR) have become more frequent and have also developed in many other pernicious grass weeds. In extreme cases MHR weeds can devastate crops and can only be controlled by non-sustainable practices like deep ploughing. Recently, we have shown that a glutathione transferase (GST) termed AmGSTF1 is the causative agent of MHR in black-grass. Expression of the AmGSTF1 protein in other plants causes them to adopt the same unusual changes in their antioxidant defences seen in MHR black-grass. MHR plants behave as it they are being oxidatively stressed and this is n turn results in them becoming more resistant to chemicals including herbicides. Normally GSTs function to metabolize herbicides so a regulatory role for a family member in co-ordinating MHR was unexpected. Using our knowledge of medicinal chemistry, we have identified a class of chemicals which selectively bind to and inhibit AmGSTF1. If these synergists are co-applied with herbicides they can restore chemical control in MHR weeds and so this discovery is potentially an extremely important discovery in crop protection. We now propose to work with the agrochemical company Syngenta, which has its herbicide discovery centre located in the UK, to determine how AmGSTF1 causes MHR in black-grass by using these chemical inhibitors to disrupt its function. We also want to understand how the inhibitors bind selectively to AmGSTF1 so we can rationally design new synergists and establish if these compounds also work in a similar way in other grass weeds. Finally, there may be a positive aspect to MHR which we want to study. It is unlikely that the MHR response has evolved just to counteract herbicides. Instead, we suggest that AmGSTF1 signaling is an important part to the natural antioxidant stress response system of cereals and grass weeds. To test this possibility we will express the amgstf1 gene in other plants and determine whether or not the resulting changes in their biochemistry make them more resistant to adverse environmental conditions. At the conclusion of our studies we intend to understand the fundamental biology behind how AmGSTF1 functions to regulate MHR in grass weeds, have developed novel chemistries to block its activity and restore herbicide sensitivity and determined a potential new route to engineering stress tolerance in crops. Finally, our project may shed new insights into the mechanisms underlying multiple resistance to drugs and pesticides which have developed in animals and microbes.

The acquisition of multiple herbicide resistance (MHR) in weeds such as black-grass (Alopecurus myosuroides) renders these plants tolerant to all classes of graminicides used in selective weed control in wheat and has been a growing problem over the last 25 years both in the UK and internationally. Building on earlier work, we have established that MHR in black-grass appears to be a specific type of stress response involving the upregulation of thiol and flavonoid antioxidants which is mediated by the aberrant up-regulation of a phi (F) class glutathione transferase, termed AmGSTF1. Thus expression of amGSTF1 in Arabidopsis caused a similar set of changes in thiol and flavonoid metabolism to that determined in MHR black-grass and this was associated with acquired tolerance to three classes of herbicides. Using a chemical intervention approach, we have shown that substituted benzoxadiazoles selectively inhibit AmGSTF1 and in so doing render MHR black-grass herbicide susceptible again. In a three year industrial partnership programme with agrochemicals company Syngenta we propose to study the primary mechanisms of AmGSTF1 signalling in black-grass using the inhibitors as functional probes to investigate binding partners and/or substrates of the enzyme. Using directed synthesis, we will generate a series of inhibitor chemistries to establish structure-activity relationships with respect to disrupting AmGSTF1 activity and counteracting herbicide resistance. These studies will be informed by determining inhibitor binding mechanism and protein structure such that new synergist chemistries can be rationally designed. AmGSTF1 function will then be assessed in transgenic plants and as we postulate its associated signaling activity is linked to responses to abiotic stress we will test these plants for alterations in their antioxidant metabolism and tolerance to adverse environmental conditions.