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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. 7 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
Transcript
Page 1: Stafford poster frankfurt

Molecular diagnostics for herbicide resistance in Alopecurus myosuroides (Black-grass)

1 2 3 1,3,4Rebecca Stafford , Rick Mumford , Stephen Moss , Robert Edwards

1 2 3Contact 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, 4Hertfordshire, 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&5later termed AmGSTF1 .

AmGSTF1 has high glutathione peroxidase (GPOX) activity reducing toxic organic

5hydroperoxidases to less reactive alcohols

Although poorly understood, in common with multiple-drug resistance (MDR) in tumors, MHR is associated with an enhanced ability to

6detoxify xenobiotics .

IntroductionOver 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

1-3strong 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.

References1.Powles, S.B., et al. Annu Rev Plant Biol, 2010. 61: p.317-472.Gressel, J., et al. Pest Management Science, 2009. 65 (11): p.1164-11733.Yuan, J.S., et al. Trends in Plant Science, 2007. 12 (1): p.6-134.Cummins, I., et al. Plant Biotechnology Journal, 2009. 7 (8): p.807-820

5.Cummins, I., et al. Plant Journal, 1999. 18 (3):p. 285-2926.Cummins, I., et al. PNAS, 2013. 15 (110):p.5812-77.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.

7Figure 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

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