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CONFIDENTIAL
Unraveling the MDR mechanism in Zymoseptoria tritici French isolates
UMR BIOGER-CPP (confidential)
Selim OMRANE 10/06/2013
CONFIDENTIAL
Context
Rising of the multi-drug resistance in Zymoseptoria tritici population
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.02
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Rising of the MDR in Z. tritici populations
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
Resistance spectrum vs. population structure over time scale
.03
What is the issue? A-S. Walker
CONFIDENTIAL
Rising of the MDR in Z. triticiShift in the resistance factors and cross resistance
.04
Phenylpyrroles
1.1
0.46
0.85
1.57
1.32
1.16
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
A-S. Walker
CONFIDENTIAL
Context
Rising of multi-drug resistance in Zymoseptoria tritici population
Since 2007 the spectrum of the DMI resistance showed a shift toward the resistance=> Low frequencies but large spreading=> Important reduction in DMI efficacy and cross resistance to other MoA
Mechanisms of multi-drug resistance in Zymospetria tritici
.05Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
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Overview of MDR mechanismsQualitative or quantitative trait ?
.06
Target Target
Regulator *
Sensing
ReceptorEffluxtransport
DiffusionPleiotropic Transport?
Signaling
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
Target over-expression
BindingAffinity
Target mutation
Penetration/Extrusion
MetabolizationDegradation
CONFIDENTIAL
Context
Strategies to unravel the MDR mechanism in Zymoseptoria tritici
Birth of the MDR consortium and project (since 2010). Focusing on the Efflux based mechanism
.07Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
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MDR efflux type transportersBoth MFS and ABC multigenic families involved in the mdr
.08Selim OMRANE / The MDR mechanism in Z. tritici JOUR / MOIS / ANNEE
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Objectives
1. Confirmation of the fungicide efflux mechanism
2. Identification of the over-expressed transporter
3. Identification of the mdr mutation
4. Validation of the mdr mutation
Diagnostic test for MDR detection in field population
Exploring fitness of MDR phenotypes
.09Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
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Confirmation of the efflux mechanismIntracellular radio-labbeled fungicide accumulation assay
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.010Selim OMRANE / The MDR mechanism in Z. tritici 13/0610
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Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
[14C] prochloraz accumulation and modulator effect Intracellular radio-labbeled fungicide assay
.011
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Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
What about other modulators ? Intracellular radio-labbeled fungicide assay
.012
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Efflux mechanism conclusions
1. Amitryptyline induces [14C] prochloraz accumulation in the MDR strains
2. Chlorpromazine showed the same effect on [14C] prochloraz intracellular accumulation
3. Verapamil discriminate between both MDR6 and 7
4. Fungicide accumulation is much more affected by reversal agents in the MDR strains than in the sensitive ones.
Efflux mechanism is involved in MDR phenotypes
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.013
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Identification of the O/E transporterwhole Transcriptomic analysis and O/E fraction screening
13/06/10Selim OMRANE / The MDR mechanism in Z. tritici
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.014
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Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
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Critical path method “Whole” transcriptomic analysis
abandon
abandon
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Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
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Go term classification of the up-regulated transcriptomic fraction Gene ontology and contrasts
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Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.017
Differential expression in the 3 contrasts A, B & C Transporter gene candidates
Contrast (C)
Contrast (A)
Contrast (B)
MFS; tetracycline antiporter activity; FC = 3,98; chr_3
Ca2+ Channel; non selective cation channel; FC = 3; chr_9
MFS; monocaboxylate transport activity; FC = 3; chr_13
ABC-C; MRP transport activity; FC = 2,6; chr_2
ABC-C; MRP transport activity; FC = 2; chr_6
(A): MDR6&7 vs SEN&SEQ(B): MDR6 vs SEN&SEQ(C): MDR7 vs SEN&SEQ
MFS; tetracycline antiporter activity; FC = 2,8; chr_1
MFS; drug activity; FC = 2,74; chr_2
MFS; tetracycline antiporter Activity; FC = 5,64; chr_7
MFS; phtalate transport activity; FC = 2,72; chr_11
ABC-B; MDR transporter activity; FC = 1,78; chr_2
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.018
qPCR analysis of the in silico RNAseq results
Confirmation of the transcriptomic analysis
MFS tetracycline H+ antiporter O/E only in MDR7 RNA seq data
MFS multi-drug O/E only in MDR6 RNA seq data
MgMFS1 Over-expressed
+/-80 times
In MDR strainsMgMFS1 is The candidate
CONFIDENTIAL
Over-expressed transporter conclusions
1. Contrast A,B and C revealed different categories that were properly annotated in the different gene ontology databases
2. Transport category showed many upregulated transporters annotated as involved in drug transport and that need confirmation
3. MgMFS1 up-regulated in both MDR6 and MDR7 is our first candidate
MgMFS1 has to be further functionally investigated
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.019
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Identification of the mdr mutationBulk segregant analysis & next generation sequencing
13/06/10Selim OMRANE / The MDR mechanism in Z. tritici
_03
.020
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Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.021
MIC vs. triazole population resistance distribution (Walker & Leroux, 2010) Tolnaftate discriminate the MDRs
TriLR, TriMR
MDR
1 >105
0mg/L
TriS
Str
ain
MIC
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.022
Phenotyping the MDR6XMDR7 cross Preliminary results
Tetrad 100%
Resistant to
[TOL]= 5mg/L
141 offsprings
100% resistant
to [TOL]= 5mg/L
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.023
Spot test phenotyping of MDR6XSEQ2 & MDR7XSEQ1 progenies Progenies phenotyping & segregation
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/0610
Bulks constitution in liquid test after OD measurement
.024
TolnaftateTolnaftate & Amitryptiline
ni/bulk=5016.66%
ni/bulk=6018.4%
Identification of the MDR mutation
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
Illumina Sequencing Flowchart
.025
NGS procedure & results
2 R/S bulks + MDR6 + MDR7 = 6 DNA extractions/samples
6 DNA Qualities check
[MDR7, R7, S7 aligned to Refseq SEQ1]
[MDR6, R6, S6 aligned to Contigs SEQ2]
BWA alignment GATK genome analysis
Illumina HiSeq2000 sequencing
(PE 2X100bp; 2Gb/sample)
Variant Call
GATK unified genotyper & snpEff
Freq plotting
ConventionMDR freq = 1SEQ freq = 0 Bulk R and Bulk S oscillating between both parental 1 and 0 freq.
5 months
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.026
MDR7, R7 and S7 frequencies plotting along Refseq SEQ1 : Chr 7 case SNPs frequency analysis: MDR7XSEQ1cross
Chr7Chr7
Chr7
Co-segregating zone
Left arm of chromosome 7
=> less than 100 Kb => 66Kb
Raw sequencing freq.Freq MDR7=1; R bulk>or= 0.5; S bulk <0.5
Chr7Freq MDR7=1; R bulk>or= 0.5; S bulk <0.5 and D value > or = 0.4
GQ value : Genotype value
Bulk S => 0
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.027
MDR6, R6 and S7 frequency plotting along Refseq SEQ2 : Ctg1135 case SNPs frequency analysis: MDR6XSEQ2 cross
1135
7,8 Kb 46,77
Cyp51
0
102
2276
66
22
156.6
997
11101977
The distortion in R co-segregating SNPs concerns the same region in MDR6 and MDR7
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.028
MDR7XSEQ1 frequencies plotting along chromosome 7 MDR co-segregating gene cluster
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.029
Genetic co-segregation & expression co-segregationIn-bulk MgMFS1 expression pattern
MgMFS1 O/E in both MDR6&7 RNA seq data
High Bulk
Low Bulk
MgMFS1 co-segregates in expression as well !
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.030
MDR7XSEQ1 frequencies plotting along chromosome 7
CDS5’UTR 3’UTR
MgMFS1 genetic polymorphism
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.031
MgMFS1 zonal amplification in SEQ1 vs MDR6&7 strainsMgMFS1 genetic polymorphism
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.032
MDR6 or 7 sequences vs SEQ1 5’UTR sequenceMgMFS1 5’UTR polymorphism
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
SEQ1 MDR
---CAAC
****
1
2 3
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.033
Insertion blast and chromosome distribution in SEQ1 backgroundRetro-transposon evidences
LTR1
LTR2
Mycgr3 chr_18:163000-170000 5'pad=0 3'pad=0 strand=+LTR1 Score = 941 bits (509), Expect = 0.0 Identities = 511/512 (99%), Gaps = 0/512 (0%)
LTR2 Score = 929 bits (503), Expect = 0.0 Identities = 509/512 (99%), Gaps = 0/512 (0%)
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.034
Insertion blast and chromosome distribution in SEQ1 backgroundRetro-transposon evidences
Mycgr3 chr_18:163000-170000 5'pad=0 3'pad=0 strand=+
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.035
Role of the insertion in the MDR backgroundPhenotypic vs genotypic correlation
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.036
Distribuion of the Z4 amplicons vs. phenotype (A-S. Walker & C. Duplaix data)Dispersion of MgMFS1 insertion in field pop
Pop phenotypes
100
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.037
MDR7XSEQ1 genotype vs. phenotype and HRM profile vs. Insertion (Z4)Progeny SNP-IN/DELS confirmation
geno ≠ pheno
geno = pheno
CONFIDENTIAL
mdr mutation identification conclusions
1. Bulk sequence analysis revealed that MDR phenotypes co-segregate with chr7’s left arm
2. The identified 50 kb region harbors a gene cluster (14 genes) including MgMFS1.
3. Only one SNP in the N termini region of MgMFS1 in the MDR7 background.
4. High level of polymorphism in the MgMFS1 5’UTR and 3’UTR in MDR6 and 7 background.
5. The promoter insertion (514 bp) exhibits an enhancer motives.
6. Good correlation between the MDR phenotype, the presence of the insertion and the over-expression of MgMFS1.
7. Subset of progeny genotyping revealed a possible implication of a second mutation in the MDR resistance
MgMFS1 has to be further functionally investigated
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
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CONFIDENTIAL
Validation of the mdr mutationExpression patterns and MgMFS1 knockout analysis
13/06/10Selim OMRANE / The MDR mechanism in Z. tritici
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.039
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
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MgMFS1 in SEQ1 background
Fungicide gene regulation in SEQ1 vs. MDR backgrounds MgMFS1 expression pattern
MgMFS1 in MDR6/7 vs SEQ1 backgrounds
Rel
ativ
e e
xpre
ssi o
n
Abs
o lu
te e
x pre
ssio
n
MgMFS1 expression is fungicide dependent in the SEQ1 background
MgMFS1 expression is fungicide independent in the MDR background
(0.01mg/L) (0.01mg/L)
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.041
MgMFS1 KO in MDR6 strain (& in SEQ1 strain) Knockout mutants creation
MUTANT MDR6/7_KO
3’UTR
44nt
5’UTR
hph
MDR6/7
3’UTR
44nt
5’UTR
SEQ1
REFseq MgMFS1
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
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Lab strain vs. field strain Transformation yield
SEQ1 transformants 15 days on MMII MDR6 transformants 25 days on MMII
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
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Distribution of the MIC doses of the used molecules (Walker and Leroux 2010) Fungicide screening with discriminating doses
TriS
TriLR TriMR
MDR TriS MDR
TriS MDRTriS MDR
mg/L5 >10 1 2 > 5
mg/L
mg/Lmg/L
0.03 0.15 1 0.3 0.4 0.5
TriLR TriMR
TriLR TriMRTriLR TriMR
1
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.044
KO quality check : Tolnaftate phenotyping & PCR amplificationFunctional analysis
[Tolnaftate]mg/L
0
0.1
1
10
+/- 1300 bp
+/- 2100 bp
hph
TOL Phenotyped
69 MDR6T + 8 SEQ1T
TOL Sensitive
29 MDR6T + 2 SEQ1T
Locus integration
10 MDR6T out of 12
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
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Tolnaftate phenotype effect at 2dpt Transformants phenotyping
MDR6 T1 MDR6 T1SEQ1 T1
0
1
CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
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Tolnaftate effect on germ tube elongation (Mean) Transformants phenotyping
SEQ1_T
SEQ1_unT
MDR6_Tect
MDR6_T
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Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.047
Tolnaftate and unrelated chemicals effect on growth rate Transformants phenotyping
Rat
io O
D60
0 1
1dpt-3dpt [
trea
ted/
untr
eate
d]
CONFIDENTIAL
= 6.8 mg/L
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.048
Boscalid have at least 2 resistance mechanimsTransformants phenotyping
7X time less Boscalid inhibit KO MFS1 growthScaliet et al., (2012)
CTRL
MDR6 MIC
CONFIDENTIAL
6 & 10dpt
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.049
Prothioconazole effect on macroscopic spot test and EC50Transformants phenotyping
= 2
= 1
= 0.5
= 0
EC50 (mg/L)
0.065
CONFIDENTIAL
Functional validation conclusions
1. MgMFS1 pattern of expression is independent from fungicide in MDR background.
2. MgMFS1 KO strains showed increased sensitivity to tolnaftate.
3. MgMFS1 KO strains showed as well increased sensitivity to terbinafine (data not shown) and to boscalid.
4. MgMFS1 KO strains are insensitive to trifloxystrobine (data not shown) and to chlorothalonil.
5. MgMFS1 KO strains show increased sensitivity superior to equal to some reported SDH point mutations effect on boscalid resistance.
6. MgMFS1 KO strains showed reduced resistance to prothioconazole among 4 tested DMIs (data not shown)
EC50 of DMIs and other MoA have to be accurately determined
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.050
CONFIDENTIAL
Work Plan Timeline
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
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Phase 2 2011
Phase 3 2012
Phase 4 2013
MDR confirmationIdentification of o/e transporter
Identification of mdr mutation
Gene validation
• Prochloraz efflux• Growth tests• Analysis of
MDR6xMDR7 progeny
• RNA Seq• Data mining• qPCR confirmation
• Progeny phenotyping• Bulk sequencing• Data mining
• in silico analysis of insertion
• Population screening for insertion and o/e
• Knockout
Activ
ities
Out
com
es
• MDR is a fungicide based efflux mech. In MDR6/7
• mdr6 and mdr7 closely linked
• 10 transporter candidates
• Confirmed by qPCR• MFS1 strongly o/e in
both MDR strains
• Monogenic inheritance • MFS1 co-segregates
with MDR phenotype• Insertion in MFS1
promoter
• Enhancer in the insertion • MFS1 expression co-
segregates in bulks• Strong pop vs. insertion
correlation• Positive KO phenotypes
Phase 1 2010
CONFIDENTIAL
Many thanks
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
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Sponsors & Collaborators
ARVALISMAUMENÉ Claude COULEAUD Gilles
WUR-PRIKEMA Gert H.J. AOUINI Lamia
Industrial partners
Team
AMARDEBIEU Danièle FILLINGER Sabine WALKER Anne-Sophie SGHYER Hind AUDEON Colette LANEN Catherine BACH Jocelyne AUCLAIR Christiane AZEDDINE Saad LALEVE Anaïs DUPLAIX ClementineIGNACE AMANDINE
Advices, & Computational support A. GENISSELM-H. LEBRUNJ-M. PRADIERA. SIMONE. MARCHEGIANIJ. VALET
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