the mdr project

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

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CONFIDENTIAL

Rising of the MDR in Z. tritici populations


Resistance spectrum vs. population structure over time scale

.03

What is the issue? A-S. Walker

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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


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

CONFIDENTIAL

Overview of MDR mechanismsQualitative or quantitative trait ?

.06

Target Target

Regulator *

Sensing

ReceptorEffluxtransport

DiffusionPleiotropic Transport?

Signaling


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


CONFIDENTIAL

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


CONFIDENTIAL

Confirmation of the efflux mechanismIntracellular radio-labbeled fungicide accumulation assay

_01

.010Selim OMRANE / The MDR mechanism in Z. tritici 13/0610

CONFIDENTIAL


[14C] prochloraz accumulation and modulator effect Intracellular radio-labbeled fungicide assay

.011

CONFIDENTIAL


What about other modulators ? Intracellular radio-labbeled fungicide assay

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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


.013

CONFIDENTIAL

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

CONFIDENTIAL


.015

Critical path method “Whole” transcriptomic analysis

abandon

abandon

CONFIDENTIAL


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Go term classification of the up-regulated transcriptomic fraction Gene ontology and contrasts

CONFIDENTIAL


.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


.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


.019

CONFIDENTIAL

Identification of the mdr mutationBulk segregant analysis & next generation sequencing


_03

.020

CONFIDENTIAL


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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


.022

Phenotyping the MDR6XMDR7 cross Preliminary results

Tetrad 100%

Resistant to

[TOL]= 5mg/L

141 offsprings

100% resistant

to [TOL]= 5mg/L

CONFIDENTIAL


.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


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


.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


.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


.028

MDR7XSEQ1 frequencies plotting along chromosome 7 MDR co-segregating gene cluster

CONFIDENTIAL


.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


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MDR7XSEQ1 frequencies plotting along chromosome 7

CDS5’UTR 3’UTR

MgMFS1 genetic polymorphism

CONFIDENTIAL


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MgMFS1 zonal amplification in SEQ1 vs MDR6&7 strainsMgMFS1 genetic polymorphism

CONFIDENTIAL


.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


.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


.034

Insertion blast and chromosome distribution in SEQ1 backgroundRetro-transposon evidences

Mycgr3 chr_18:163000-170000 5'pad=0 3'pad=0 strand=+

CONFIDENTIAL


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Role of the insertion in the MDR backgroundPhenotypic vs genotypic correlation

CONFIDENTIAL


.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


.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


.038

CONFIDENTIAL

Validation of the mdr mutationExpression patterns and MgMFS1 knockout analysis


_04

.039

CONFIDENTIAL


.040

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


.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


.042

Lab strain vs. field strain Transformation yield

SEQ1 transformants 15 days on MMII MDR6 transformants 25 days on MMII

CONFIDENTIAL


.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/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


.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


.045

Tolnaftate phenotype effect at 2dpt Transformants phenotyping

MDR6 T1 MDR6 T1SEQ1 T1

0

1

CONFIDENTIAL


.046

Tolnaftate effect on germ tube elongation (Mean) Transformants phenotyping

SEQ1_T

SEQ1_unT

MDR6_Tect

MDR6_T

CONFIDENTIAL


.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


.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


.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


.050

CONFIDENTIAL

Work Plan Timeline


.051

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


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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

the mdr project

Technology