1. CONFIDENTIAL
Unraveling the MDR mechanism in
Zymoseptoria tritici French isolates
UMR BIOGER-CPP (confidential)
Selim OMRANE 10/06/2013

2. CONFIDENTIAL
Context
Rising of the multi-drug resistance in Zymoseptoria tritici population
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.02

3. CONFIDENTIAL
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

4. CONFIDENTIAL
Rising of the MDR in Z. tritici
Shift 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

5. 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
.05
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10

6. CONFIDENTIAL
Overview of MDR mechanisms
Qualitative or quantitative trait ?
.06
Target Target
Regulator *
Sensing
ReceptorEfflux
transport
DiffusionPleiotropic
Transport?
Signaling
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
Target
over-expression
Binding
Affinity
Target
mutation
Penetration/Extrusion
Metabolization
Degradation

7. 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
.07
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10

8. CONFIDENTIAL
MDR efflux type transporters
Both MFS and ABC multigenic families involved in the mdr
.08
Selim OMRANE / The MDR mechanism in Z. tritici JOUR / MOIS / ANNEE

9. CONFIDENTIAL
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
.09
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10

10. CONFIDENTIAL
Confirmation of the efflux mechanism
Intracellular radio-labbeled fungicide accumulation assay
_01
.010
Selim OMRANE / The MDR mechanism in Z. tritici 13/0610

11. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
[14C] prochloraz accumulation and modulator effect
Intracellular radio-labbeled fungicide assay
.011

12. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
What about other modulators ?
Intracellular radio-labbeled fungicide assay
.012

13. CONFIDENTIAL
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

14. CONFIDENTIAL
Identification of the O/E transporter
whole Transcriptomic analysis and O/E fraction screening
13/06/10Selim OMRANE / The MDR mechanism in Z. tritici
_02
.014

15. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.015
Critical path method
“Whole” transcriptomic analysis
abandon
abandon

16. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.016
Go term classification of the up-regulated transcriptomic fraction
Gene ontology and contrasts

17. CONFIDENTIAL
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

18. 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
Quantitative expression of ID 110044 BK norm
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
MDR6 MDR7 SEQ1 SEN
Strains
2^(-deltaC(t))
ID 110044 BK norm
Quantitative expression of ID 102686 (Bk norm)
0
0,005
0,01
0,015
0,02
0,025
0,03
MDR6 MDR7 SEQ1 SEN
Strains
2^(-deltaC(t))
ID 102686 Bk norm
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 strains
MgMFS1 is
The candidate

19. 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

20. CONFIDENTIAL
Identification of the mdr mutation
Bulk segregant analysis & next generation sequencing
13/06/10Selim OMRANE / The MDR mechanism in Z. tritici
_03
.020

21. CONFIDENTIAL
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 >1050
mg/L
TriS
Strain
MIC

22. 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

23. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.023
Spot test phenotyping of MDR6XSEQ2 & MDR7XSEQ1 progenies
Progenies phenotyping & segregation

24. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/0610
Bulks constitution in liquid test after OD measurement
.024
Tolnaftate
Tolnaftate & Amitryptiline
ni/bulk=50
16.66%
ni/bulk=60
18.4%
Identification of the MDR mutation

25. 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
Convention
MDR freq = 1
SEQ freq = 0
Bulk R and Bulk S oscillating
between both parental 1 and 0 freq.
5 months

26. 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
Chr7
Chr7
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

27. 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
-0,1
0,1
0,3
0,5
0,7
0,9
1,1
458
1878
892
1551
1135
1977
1110
2017
220
972
22
2443
530
2276
175
610
266
803
2047
2144
690
719
1890
1945
1650
896
1599
1625
1177
1281
1253
1668
886
2059
1460
522
2049
1632
878
1925
1397
1180
464
1842
1822
1981
1340
1788
1874
601
2412
2258
1348
2263
1747
566
K32ctg ID
Freqs(orDvalues)
MDR6 Freq R Bulk Freq
S Bulk FSeq D value
1135
7,8 Kb 46,77
Cyp51
0
102
2276
66
22
156.6
997
1110
1977
The distortion in R co-segregating SNPs concerns the same region in MDR6 and MDR7

28. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.028
MDR7XSEQ1 frequencies plotting along chromosome 7
MDR co-segregating gene cluster

29. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.029
Genetic co-segregation & expression co-segregation
In-bulk MgMFS1 expression pattern
Quantitative expression of ID 110044 BK norm
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
MDR6 MDR7 SEQ1 SEN
Strains
2^(-deltaC(t))
ID 110044 BK norm
MgMFS1 O/E in both
MDR6&7 RNA seq data
High Bulk
Low Bulk
MgMFS1 co-segregates
in expression as well !

30. 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

31. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.031
MgMFS1 zonal amplification in SEQ1 vs MDR6&7 strains
MgMFS1 genetic polymorphism

32. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.032
MDR6 or 7 sequences vs SEQ1 5’UTR sequence
MgMFS1 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

33. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.033
Insertion blast and chromosome distribution in SEQ1 background
Retro-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%)

34. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.034
Insertion blast and chromosome distribution in SEQ1 background
Retro-transposon evidences
Mycgr3 chr_18:163000-170000 5'pad=0 3'pad=0 strand=+

35. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.035
Role of the insertion in the MDR background
Phenotypic vs genotypic correlation

36. 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

37. 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

38. 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
.038

39. CONFIDENTIAL
Validation of the mdr mutation
Expression patterns and MgMFS1 knockout analysis
13/06/10Selim OMRANE / The MDR mechanism in Z. tritici
_04
.039

40. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.040
MgMFS1 in SEQ1 background
Fungicide gene regulation in SEQ1 vs. MDR backgrounds
MgMFS1 expression pattern
MgMFS1 in MDR6/7 vs SEQ1 backgrounds
MgMFS1 expression is fungicide
dependent in the SEQ1 background
MgMFS1 expression is fungicide
independent in the MDR background
(0.01mg/L) (0.01mg/L)

41. 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
hph
MDR6/7
SEQ1
REFseq MgMFS1

42. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.042
Lab strain vs. field strain
Transformation yield
SEQ1 transformants 15 days on MMII MDR6 transformants 25 days on MMII

43. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.043
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/L
5 >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

44. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.044
KO quality check : Tolnaftate phenotyping & PCR amplification
Functional 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

45. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.045
Tolnaftate phenotype effect at 2dpt
Transformants phenotyping
MDR6 T1 MDR6 T1SEQ1 T1
0
1

46. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.046
Tolnaftate effect on germ tube elongation (Mean)
Transformants phenotyping
SEQ1_T
SEQ1_unT
MDR6_Tect
MDR6_T

47. CONFIDENTIAL
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.047
Tolnaftate and unrelated chemicals effect on growth rate
Transformants phenotyping

48. CONFIDENTIAL
= 6.8 mg/L
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.048
Boscalid have at least 2 resistance mechanims
Transformants phenotyping
7X time less Boscalid inhibit KO MFS1 growth
Scaliet et al., (2012)
CTRL
MDR6 MIC

49. CONFIDENTIAL
6 & 10dpt
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.049
Prothioconazole effect on macroscopic spot test and EC50
Transformants phenotyping
= 2
= 1
= 0.5
= 0
EC50 (mg/L)
0.065

50. 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

51. CONFIDENTIAL
Work Plan Timeline
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.051
Phase 2
2011
Phase 3
2012
Phase 4
2013
MDR confirmation
Identification 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
ActivitiesOutcomes
• 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

52. CONFIDENTIAL
Many thanks
Selim OMRANE / The MDR mechanism in Z. tritici 13/06/10
.052
Sponsors & Collaborators
ARVALIS
MAUMENÉ Claude
COULEAUD Gilles
WUR-PRI
KEMA Gert H.J.
AOUINI Lamia
Industrial partners
Team
AMAR
DEBIEU Danièle
FILLINGER Sabine
WALKER Anne-Sophie
SGHYER Hind
AUDEON Colette
LANEN Catherine
BACH Jocelyne
AUCLAIR Christiane
AZEDDINE Saad
LALEVE Anaïs
DUPLAIX Clementine
IGNACE AMANDINE
Advices, & Computational support
A. GENISSEL
M-H. LEBRUN
J-M. PRADIER
A. SIMON
E. MARCHEGIANI
J. VALET