A Molecular Basis for Multiple Herbicide Resistance in Black

A Molecular Basis for Multiple
Herbicide Resistance in Black-grass
Rob Edwards
School of Agriculture, Food and Rural
Development
Newcastle University
Overview
• The plant xenome
• Multiple herbicide
resistance (MHR)
• The role of GSTF1 in
MHR
• The omics of MHR
• Molecular diagnostics
Biotransformations of biologically active
metabolites in plants
OMe
OH
HO
O
OMe
O
HO
O
HO
O
OH
O
O
O
Tolerant species (maize)
Glutathione
OH
Cl
N
N
H3C
H3C
C N
H
OH
N
NHCH2CH3
OH
Sensitive species (peas, weeds)
HO
O
OH
O HO
HO
MeO
OH
O
O
OH
OH
HO
Plants meet Chemicals- Xenome
The biosystem
responsible for
detecting, detoxifying
& transporting
xenobiotics (in plants)
The Xenome
OXIDOREDUCTASE
HYDROLASE
R-X
R
1
R
X
1
UGT
R-OH
R-OGlc
2
GST
GST
MT
2
RE-EXPORT &
INCORPORATION
4
R-SG
3
2
R-OGlcMal
3
R-conjugates
HYDROLYSIS
Vacuole
Xenome interests
•
•
•
•
Metabolic pathway discovery
Herbicide selectivity
Engineered herbicide tolerance
Natural product biotransformations for
biotechnological applications
• Chemical biology
• Enzymology- GSTs, UGTs, esterases, CYPs
• Regulation (safening and metabolic resistance)
Herbicide resistance in grass
weeds in the UK
• Now widespread in black-grass and rye-grass
• These weeds affect 1.2 million Ha in the UK
• Responsible for major yield losses in wheat
Source: Defra
The Growth of Multiple Herbicide Resistance in the UK
Percentage of wheat area treated with 4
different herbicide modes of action during growing season (excluding fops and dims)
2008
2010
2012
64
8
Control strategies
suggest multiple
resistance may be
most prevalent in
Oxon, Northants, Notts
& Bucks
6
26
11
7
12
14
62
35
28
23
1
17
11
12
20
22
45
37
19
6
21
13
15
37
< 5% area treated
9
29
37
33
4
9
7
18
6
5-10% area treated
29
> 10% area treated
Numbers are % area treated with fops & dims in each county. Counties with high usage in 2008
generally show reduced usage in 2012 and potentially higher incidence of resistance
12
Genetic perturbation of the xenome –Multiple
Herbicide Resistance in black-grass
• First described in 1982 by
Steve Moss group in Peldon,
Essex
• Results in a loss of control by
all classes of existing
graminicides
• Distinct from more common
target site based resistance
caused by point mutations
• NTSR/ Enhanced Metabolic
Resistance/ MHR is
(allegedly) caused by an
upregulation in the Xenome
Xenome components associated with MHR
HYDROLASE
R-X
R
1
R
1
X
R-OH
2
GST
2
RE-EXPORT &
INCORPORATION
R-SG
R-OGlc
MT
2
R-OGlcMal
4
R-conjugates
HYDROLYSIS
Vacuole
GSTs as upregulated xenome
components
OH
O
NH2
CH3
O
O
O
O
Cl
NH
CH3
GST
O
O
N
GSH
Cl
O
S
O
N
HN
O
OH
Fenoxaprop
Fenoxaprop-SG conjuga
AmGSTF1 discovery
MHR – Multiple herbicide resistant biotype
TSR – Target site resistant biotype
WTS – Wild-type sensitive biotype
20-fold elevated GSTF1 expression
Cummins et al. (1999), The Plant Journal, 18, 285
WTS
MHR
MHR
TSR
MHR
WTS
MHR
MHR
In 1999 the Edwards lab identified AmGSTF1,
constitutively expressed in MHR black-grass but not
in herbicide sensitive black-grass or target site
resistant biotypes.
MHR-linked amgstf1 gene confers a resistance
phenotype
Metabolite profiles
Physical phenotypes
Soil
Amount present (nmol per gram
FW)
Agar
Transgenic Arabidopsis
1400
1200
1000
800
600
WT
400
amgstf1
200
0
Glutathione
Major
anthocyanin
Major
flavonoid
Vector – negative control
Line 8 – mid AmGSTF1-expressor
Line 12 – high AmGSTF1-expressor
Cummins and Wortley et al. (2013), PNAS, 110, 5812
Amount present (nmol per
gram FW)
Black-grass
500
450
400
350
300
250
200
150
100
50
0
Sens
MHR
Glutathione
Major
Major flavonoid
anthocyanin
Functional similarity with GSTP1 in
MDR
Some cancer cell lines with a multi-drug resistance (MDR) phenotype highly express the
GST isoform, GSTP1.
Evolutionarily distinct from AmGSTF1 but promotes MDR through protein interactions and
catalytic detoxification of drug compounds.
Drug detoxification via GSH
conjugation
Kinase regulation via proteinprotein interaction
GSTP1
Antioxidant enzyme regulation
via protein-protein interactions
e.g. peroxiredoxin
Intersubunit communication
and interaction with
xenobiotics via Cys47
AmGSTF1 inhibition
NBD-Cl
Mean enzyme specific
activity (%)
Inhibitoryprofiles
profile of
Inhibitory
ofAmGSTF1
AmGSTF1
withwith
NBD-Cl
andtreated
LrGSTF1
CNBF
100
50
IC50
AmGSTF1
LrGSTF1
0
DMSO
GSTP1-inhibiting pharmacophore
6.91 M
6.58 M
10 -7
10 -6
10 -5
[NBD-Cl],
[CNBF],
MM
IC50 = 6.91 µM
AmGSTF1 cys120 residue was covalently bound
to NBD-Cl.
Is cys120 of AmGSTF1 playing a similar role in
MHR as the cys47 residue of GSTP1 in MDR?
Cummins and Wortley et al. (2013), PNAS, 110, 5812
10 -4
Cys120 – an activity switch?
AmGSTF1 and C120V treated with 1µM or
100µM NBD-Cl
Specific activity(nmol s-1 mg-1)
30.0
• C120V mutant generated by
overlap PCR.
25.0
8%
20.0
15.0
AmGSTF1
10.0
C120V
47 %
5.0
72 %
>99 %
0.0
DMSO
NBD-Cl (1µM)
•Recombinant AmGSTF1 and
C120V mutant independently
expressed as Strep II tag fusions.
• In vitro inhibition of activity
towards CDNB.
NBD-Cl (100µM)
Compound
Specific activities of AmGSTF1 and C120V mutant after a 10 min
incubation with NBD-Cl. % are % inhibition vs. DMSO control.
1. C120V mutant (incapable of being alkylated in this position by NBD-Cl) is dramatically less inhibited
by NBD-Cl than AmGSTF1.
2. C120V mutant has a 15 % higher specific activity toward CDNB than AmGSTF1 (see DMSO control).
These results suggest Cys120 must interact with the enzyme active site
AmGSTF1 structure and inhibition
• GSTF1 structure solved
at resolution of 2 A
• Presence of conserved
GST fold and flexible
loop structure
• Loop (cys120) interacts
at active site
• Alkylation at Cys120
projects into active site
NBD-Cl as a herbicide synergist
WT- susceptible
Peldon- MHR
•
•
•
•
A- Formulation
B- NBD-Cl
C- Herbicide
D- herbicide + NBD-Cl
Functional analysis of AmGSTF1
Stable transgenic lines:
Strep-AmGSTF1, StrepC120V, Strep-S12A or
strep tag vector
+/- herbicide
Physical
phenotype
MHR
Arabidopsis?
Biochemical
phenotype
 Antioxidant enzyme
activities
 Flavonoid profile
 AmGSTF1 – positive control
 C120V mutant
 S12A mutant – catalytically retarded
Substrate
Strep II tag
isolation
 Associated ligands
 Protein partners
Recombinant protein
Strep-AmGSTF1
Strep-C120V
Strep-S12A
CDNB
25.4 ± 0.8
27.8 ± 1.6
5.5 ± 0.5
CuOOH
19.6 ± 1.4
24.1 ± 0.8
5.1 ± 0.1
Herbicide resistance – new tools for
mitigation
• Identification of AmGSTF1 reveals a regulatory
‘Achilles Heel’ of MHR in black-grass
• AmGSTF1 mediates a non-catalytic protein
signaling function (protein-protein/ ligand
binding)
• Native AmGSTF1 activates MHR,
inhibition/alkylation suppresses MHR
• AmGSTF1 orthologs are also present in other
grass weeds (Lolium) presenting MHR
MHR Diagnostics: Biomarker discovery through Omics
Proteomics
Transcriptomics
Protein id
LC/MS
Gene expression, iRNA
Microarrays
Discovery
Biomarkers
Genomics
Metabolomics
DNA/RNA seq, Methylation
Polymorphism
Metabolic profiling
NGS platforms
LC/MS, GC/MS
RNAseq
New technologies deliver fast, inexpensive
and accurate genome information
- de novo genome sequencing
- transcriptome sequencing
- metagenomics
- ancient genomes
Platforms used:
454 Titanium (Roche)
Ion Proton (Ion Torrent)
Illumina
RNAseq: setup and data
Populations:
Herbicide sensitive (Rothamsted)
Multiple herbicide resistant (Peldon)
21-24 °C
16 h light - 8 h dark
Three biological replicates
Instrument: Ion Torrent PGM
Contig assembly performed with MIRA (Fios Genomics)
Alocuperus myosuroides transcriptome
Total bp sequenced
2648000000
Number of total reads
17040484
Reads post QC control
15408722
Total assembly length
129824532
No. contigs >100 bp
383149
No. contigs >500 bp
46791
Average contig size
339
Number of Uniprot unigenes
17403
Zoomed in Region - Complete Transcriptome Map of MHR,SUS,TSR – 32
plant samples
The MHR Transcriptome
Gene Ontology: Molecular Function
thiol S-methyltransferase activity
geranylgeranyl-diphosphate geranylgeranyltransferase activity
2-fold up-reg genes
oxidoreductase activity, acting on other nitrogenous…
uroporphyrin-III C-methyltransferase activity
Reference RNAseq library
nitrate reductase activity
glycine dehydrogenase (decarboxylating) activity
Cellular Component
geranyltranstransferase activity
glutathione transferase activity
chlorophyll binding
tetrapyrrole binding
iron ion binding
heme binding
anion binding
nucleic acid binding
ATP binding
adenyl nucleotide binding
purine ribonucleoside binding
purine ribonucleotide binding
nucleoside binding
nucleoside-triphosphatase activity
hydrolase activity, acting on acid anhydrides, in phosphorus-…
RNA binding
0
0.05
0.1
0.15
% of genes
0.2
0.25
0.3
Metabolomics of MHR
Autosampl
Magnet
Chiller
Cryoprob
e
Non-targeted profiling comparison
CoEMS York
Omics team at Fera
Search for metabolic signature of
resistance
Comparison with transcriptome data
LC-HRMS PCA analysis
PCA of pre sprayed plants:
MHR - Peldon
TSR
PCA of post sprayed plants – Day 13
MHR - Peldon
Susceptible
TSR
From omics to multiplex molecular diagnostic
for resistance testing
LAMP
reaction
Clondiag
LFD
Quo vadis
• The use of molecular diagnostics as a research tool to
study the evolution of MHR in the field (BBSRC Lola
grant (CoIs Neve, Freckleton, Childs, Norris)
• The use of the technology to evaluate the effectiveness
of new control/ prevention strategies
• The development of new chemical intervention and
biologic strategies to disrupt GSTF1
• The molecular role of GSTF1 and other MHR proteins
in signaling and resistance phenotype
• The physiological role of ‘MHR signaling’ in plant stress
responses
Collaborators
Zhesi He
Federico Sabbadin
David Wortley
Bekki Stafford
Catherine Tetard
Jones
Melissa BrazierHicks
Ian Cummins
Lesley Edwards
Patrick Steel
Ehmke Pohl
Stefanie Freitag-Pohl
Chris Coxon
Hannah Straker
Jonathan Sellars
Rick Mumford
Neil Boonham
Mike Dickinson
Rachel Glover
Rob Stones
Ian Adams
Dave Hughes
Deepak Kaundun
Sarah-Jane Hutchings