1) Researchers generated a new antiserum specific for the protein AmGSTF1, which is a potential biomarker for herbicide resistance in black-grass weed. 2) The antiserum reacted strongly with protein extracts from a herbicide-resistant black-grass population, detecting three polypeptide bands, but only weakly with a susceptible population. 3) Testing on additional populations validated the antiserum in differentiating resistant from susceptible or intermediately resistant populations, suggesting it could be developed into a diagnostic tool.

1. Molecular diagnostics for herbicide resistance in
Alopecurus myosuroides (Black-grass)1 2 3 1,3,4
Rebecca Stafford , Rick Mumford , Stephen Moss , Robert Edwards
1 2 3
Contact Information- University of York, Biology Dept, York, YO10 5DD, UK, The Food and Environment Research Agency (FERA), Sand Hutton, York YO41 1LZ, UK, Rothamsted Research,
4
Hertfordshire, AL5 2JQ, UK. University of Newcastle, School of Agriculture, Newcastle, NE1 7RU, UK.
Identification of Biomarkers: New anti-AmGSTF1-serum
 One potential biomarker of MHR in black-grass is the phi class glutathione transferase AmGSTF1. Previously the
antibody utilised in screening was raised to ZmGSTI-II.
 Essential to generate a new antiserum which was more specific for potential use as a diagnostic kit therefore,
synthesised from the cDNA encoding AmGSTF1 from MHR “Peldon” black-grass plants.
 Having cloned the cDNA it was expressed in E.coli as a strep II tagged protein for affinity purification, prior to
rabbit immunization.
 Test antisera samples were then tested for their immuno-selectivity on crude black-grass protein extracts and
resolved by SDS-PAGE prior to western blotting.
Results
 The anti-AmGSTF1-serum reacted with three polypeptides of Mr 25kDa, 24kDa and 22kDa respectively in crude
extracts from Peldon Black-grass. In contrast, the AmGSTF1 polypeptides were only weakly detected in WTS
black-grass.(Fig 3.)
 Subsequently to validate the new antiserum, the antibody was then used to probe extracts from 10 different black-
grass populations, demonstrating different resistance. susceptibility traits to graminicides. (Fig 4.)

What is AmGSTF1?
 In 1999, it was identified that the independent
MHR black-grass population biotype “Peldon”
constitutively expressed a protein which
belonged to a plant specific phi class
glutathione transferase which plays a role in the
detoxification of xenobiotics, this protein was
4&5
later termed AmGSTF1 .
 AmGSTF1 has high glutathione peroxidase
(GPOX) activity reducing toxic organic
5
hydroperoxidases to less reactive alcohols
 Although poorly understood, in common with
multiple-drug resistance (MDR) in tumors,
MHR is associated with an enhanced ability to
6
detoxify xenobiotics .
Introduction
Over the last 40 years there has been a dramatic increase in the frequency and diversity of
herbicide-resistant weed biotypes, which poses a substantial threat to the sustainability of
agriculture both locally and globally. Modern agriculture couples the management of
invasive weed species with enhancing crop yields through the intensive use of herbicides.
Many of the most problematic weeds including black-grass, have now evolved resistance
to multiple herbicides with various modes of action. This project focusses on identifying
markers of ultiple erbicide esistant ( ) in the weed black-grass.M H R MHR
Modern agriculture couples the management of invasive weed species with enhancing crop
yields through the intensive use of herbicides. Unfortunately, as a result of continuous use
and a lack of variety in the modes of action (MoA) the frequency and diversity of weed
biotypes expressing evolved resistance to one or more herbicides has dramatically
increased1. The growing persistence of grass weeds amongst arable crops has indicated a
1-3
strong negative impact on the sustainability of agriculture both at local and global level ,
with crop yield losses of up to 50%.
Currently, the greatest concern facing agriculturalists globally is MHR, which is currently
found in many of the most problematic weeds including black-grass (Alopecurus
myosuroides) . The data surrounding the resistant mechanisms a plant utilises to survive is
vast, although gaining a more coherent understanding is vital in order to maintain and
optimize crop yields for future generations.
Conclusions
 Recognition of the AmGSTF1 polypeptides was only determined
in plants displaying MHR, rather than WTS or TSR populations.
 Relative abundance of the AmGSTF1 polypeptides of differing
relative molecular mass varied between MHR populations
suggesting the presence of multiple component isoenzymes.
 Need to purify the polypeptide further due to detection of an
upper band at 60kDa.
Future Work
 Following its primary validation as a useful biomarker of MHR,
work on developing anti-AmGSTF1-serum based diagnostic
devices for the identification of resistant weed populations in the
field is currently in progress.
Figure 4. Western blot analysis depicting the results of extracts from ten different black-grass
populations following exposure to anti-AmGSTF1-Serum. Polypeptides of 25,24 and 22kDa detected.
Figure 3. Western blot analysis depicting the result of the
immuno-selectivity on crude black-grass protein extracts .
In MHR polypeptides of 25,24 and 22kDa detected strongly.
References
1.Powles, S.B., et al. Annu Rev Plant Biol, 2010. 61: p.317-47
2.Gressel, J., et al. Pest Management Science, 2009. 65 (11): p.1164-1173
3.Yuan, J.S., et al. Trends in Plant Science, 2007. 12 (1): p.6-13
4.Cummins, I., et al. Plant Biotechnology Journal, 2009. 7 (8): p.807-820
5.Cummins, I., et al. Plant Journal, 1999. 18 (3):p. 285-292
6.Cummins, I., et al. PNAS, 2013. 15 (110):p.5812-7
7.Pohl, F., Cummins., Pohl, E., and Edwards, R. unpublished
Metabolomics
 Metabolomic profiling
 LC/MS
 GC/MS
Proteomics
 Protein Identification
 LC/MS
Transcriptomics
 Gene expression
 Microarrays
Identification of Biomarkers
 Use in diagnostic kits
 POC (point-of-care test)
 Microarrays
Aims
 This project focusses on identifying biomarkers of MHR in the weed black-grass which have the potential to be developed into molecular research tools
to study the evolution of MHR in the field.
 The approach adopted was that of “omics,” to identify biomarkers which could be developed into a series of portable diagnostic platforms based on
DNA, immuno or small molecule recognition technologies.
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Figure 2. AmGSTF1 crystal structure
AmGSTF1
LrGSTF1
HvGSTF1
TaGSTF4
OsGSTF1
OsGSTF8
HvGSTF8
OsGSTF7
HvGSTF13
TaGSTF1
HvGSTF5
TaGSTF3
ZmGSTF1
OsGSTF2
Figure 1. Dendrogram depicting AmGSTF1 and
other closely related phi class glutathione transferases