Glutathione transferase (AmGSTF1) as a biomarker of multiple herbicide resistance in Alopecurus myosuroides (black-grass)

Abstract

Modern agriculture manages competitive weed species through the intensive use of herbicides to ensure crop productivity. Resistance to aryloxyphenoxypropionate and sulphonylurea herbicides in the problem weed black-grass (Alopecurus myosuroides) is now a major problem in the United Kingdom, affecting approximately 80% of cropland. Increasing numbers of black-grass populations now exhibit multiple herbicide resistance (MHR), based on an enhanced expression of the biosystem responsible for herbicide detoxification termed the “Xenome”. The xenome is composed of cytochrome P450s (CYPs), glutathione transferases (GSTs) and glycosyltransferases (UGTs) and transporter proteins from the ABC and MATE families. In the current work, specific xenome components as biomarkers of MHR were identified for use as diagnostic markers of metabolic resistance. The identification of potential biomarkers was carried out using a combination of targeted and non-targeted “omic” approaches. De novo next generation sequencing (NGS) was carried out on MHR and WTS (wild-type susceptible) black-grass, enabling a virtual transcriptome of xenome and associated genes to be assembled. Eight xenome unigenes were identified as being differentially expressed namely a CYP, GSTU6, GSTF1, an oxophytodienoic acid reductase (OPR1), UGTZ, a thiol methyltransferase (TMT) and ABC and MATE transporters. To examine the potential of these expressed RNAs as biomarkers of resistance, real-time qPCR was used on characterised populations of resistant black-grass from around the UK. The results indicated several sequences as potentially functional transcriptional biomarkers of MHR. Of the MHR-associated genes identified, GSTF1 was particularly interesting as the respective protein was known previously for constitutive up-regulation in populations showing metabolic resistance. To characterise the role of this protein in greater detail, a polyclonal antiserum was raised to the recombinant GSTF1 from MHR “Peldon”. The anti-AmGSTF1-serum reacted with three polypeptides of 25kDa, 24kDa and 22kDa in crude extracts of MHR black-grass. In Peldon, the two upper polypeptides were up-regulated relative to WTS plants. When tested blind against a panel of ten populations the antiserum proved diagnostic for these polypeptides in MHR populations using either immunoblotting, or ELISA assay. In contrast, WTS or plants showing target site resistance (TSR) mechanisms did not show elevated GSTF1 expression. The antiserum also potentially identified orthologous polypeptides in MHR Lolium rigidum and Avena fatua indicating GSTF1 is diagnostic of metabolic resistance in other grasses. Intriguingly, the relative abundance of the GSTF1 polypeptides of differing molecular mass varied between populations, suggesting the presence of multiple component isoenzymes. Based on the results of the PhD, a prototype lateral flow device using immunodiagnostic detection of GSTF1 has been developed with industrial partners. It is undergoing field evaluation for the determination of MHR in black-grass populations. The project has also identified the potential use of other xenome genes as DNA biomarkers of MHR for diagnostic applications in managing herbicide resistance in black-grass