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Genetic Manipulation of Susceptibility to Fusarium Head BlightFusarium Head Blight
Michael LawtonMichael LawtonBiotech Center, Rutgers University, NJ
2009 National Fusarium Head Blighy ForumDec 7th-9th, 2009, Orlando, FL
2009 National Fusarium Head Blighy ForumDec 7th-9th, 2009, Orlando, FL
Fusarium Head Blight
• Few sources of effective wheat or barley germplasm• Few sources of effective wheat or barley germplasm conferring resistant to FHB in wheat or barley. This makes crop improvement difficult.
• Transgenics are an alternative but gene assays in transgenic wheat or barley is slow and expensive and has only a moderate capacity. OK for testing single genes, but not efficient enough for de novo gene discovery.
• Approach: Use higher capacity model plants to assay genes for their ability to confer resistance to Fusarium.
A pipeline for gene discovery and deployment
Select Select potentialpotentialantifungalantifungal
Isolate Isolate selected selected genesgenes
PhyscomitrellaPhyscomitrella::Create gene Create gene knockouts andknockouts and
PhyscomitrellaPhyscomitrella::Assay plants for Assay plants for FHB resistanceFHB resistance
WheatWheat: assay : assay selected genes forselected genes forFHB resistanceFHB resistance
WheatWheat: Stable: StableTransgenicsTransgenicsgg
genesgenes genesgenes oc outs a doc outs a doverexpressorsoverexpressors
FHB resistanceFHB resistance FHB resistanceFHB resistance gg
d
Transient assay in
TransformedFHB ResistantBased on genetic,
molecular data from wheat and other species
Clone and sequence genes
Mutate or express genes in
PhyscomitrellaExpose mutant
plants to FHB, DON
assay in wheat using
VIGS(Scofield Lab)
FHB-ResistantWheat, Barley (Dahleen Lab)
Physcomitrella patens
Grows like yeast but is a multicellular plantGrows like yeast but is a multicellular plant
Haploid gametophyte dominates life cycleHaploid gametophyte dominates life cycle
Genome size: 511Mbp; 27 chromosomesGenome size: 511Mbp; 27 chromosomesGenome size: 511Mbp; 27 chromosomesGenome size: 511Mbp; 27 chromosomes
Genome sequence completed in 2007Genome sequence completed in 2007
Functional conservation with higher plants Functional conservation with higher plants ggand yeastand yeast
Undergoes high efficiency homologous Undergoes high efficiency homologous recombinationrecombination
Allows targeted gene replacement for gene Allows targeted gene replacement for gene knockout or siteknockout or site--specific mutation etc.specific mutation etc.
Model system for functional Model system for functional genomicsgenomicsOur Targets: Our Targets:
Disease, Cell Death, Toxin Action, Induced Disease, Cell Death, Toxin Action, Induced Immunity Cell WallImmunity Cell WallImmunity, Cell WallImmunity, Cell Wall
Gene knockout by homologous recombination
ATGATG TAATAA
AA BB CCGenomic DNA DD
NeoNeoBB CCBBBB wild type sequence
mutant sequence
NeoNeo Selectable marker
Double homologous recombination
lox loxLox site
NeoNeo Selectable marker
AA
ATGATG////////
TAATAA
DDNeoNeoBB CC Result: gene disruption by allele replacement
ATGATG TAATAA 3-10% reversion to Neos
AA DDBB CC
P ibl i f t t d
following transient expression of CRE recombinase
Possible reversion of targeted gene following site-specific recombination
Recombination rates in Physcomitrella
Efficiency (GT/GT+IR): Efficiency (GT/GT+IR): 4% (.5kb) up to 100% (24% (.5kb) up to 100% (2--4 kb) in 4 kb) in PhyscoPhysco
0.0050.005--0.1% in angiosperms0.1% in angiosperms
95% in95% in S cerevisaeS cerevisae95% in 95% in S. cerevisaeS. cerevisae
11--30% in 30% in N. crassaN. crassa
0.10.1--1% in mouse ES cells1% in mouse ES cells0.10.1 1% in mouse ES cells1% in mouse ES cells
•• Essentially any gene or genomic sequence in the Essentially any gene or genomic sequence in the Physcomitrella Physcomitrella genome genome b d l t db d l t d i kli kl dd i li lcan be deleted can be deleted quickly quickly and and preciselyprecisely..
•• Can complement knockout mutants with genes from other plants.Can complement knockout mutants with genes from other plants.
•• Foreign genes can be introduced into a specific locus and expressed in aForeign genes can be introduced into a specific locus and expressed in a•• Foreign genes can be introduced into a specific locus and expressed in a Foreign genes can be introduced into a specific locus and expressed in a predictable manner (no position effects).predictable manner (no position effects).
Can Fusarium infect Physcomitrella patens?
Is it aCan it be Infected?
Is it a relevant model for diseases of
www.wheatlifemagazine.com
diseases of crop plants?
Infection of Physcomitrella by Fusarium graminearum
24h
SymptomsFusarium::GFP
24h
48h
72h72h
96h
Physcomitrella is sensitive to multiple FHB mycotoxins
0h 24h 48h 72h
DONDON
DAS
toxi
n
T-2
Myc
o
T 2
ZON
(A)Tricothecenes contribute to F. graminearum virulence
(A) SymptomsFusarium-GFP Cell DeathFusarium-WT
WT
∆tri5
72h 48h48h 48h
(B)75
100
ATH WT
25
50
% C
ELL
DE
∆tri5The ∆tri5 strain of F. graminearum does not produce DON or DAS
00 24 48 72 96
hours post inoculation
produce DON or DAS
Programmed Cell Death (PCD)
Is Programmed Cell Death a target for Fusarium toxins?
• Fusarium mycotoxins contribute to virulence on Physcomitrella and wheat• Both Fusarium and mycotoxins cause cell death on host plants• Programmed Cell Death (PCD) is a genetically controlled process• Programmed Cell Death (PCD) is a genetically controlled process• Mutating genes that control host cell death may suppress symptoms and
attenuate virulence
0h 24h 48h 72h
Fusarium graminearumMycotoxins
DON
DAS
T-2
ZON
PCD is required for host cell death and sensitivity to DON
Plant Cell Death
80
100
ATH
40
60
CEL
L D
EA
0
20
% C
0WT L3∆ ERG1KO AtBI-1OE PPBI-1KO VPEгKO
0 24 48 72
Deoxynivalenol (DON)
PCD mutant plants are resistant to multiple mycotoxins
Plant Cell Symptoms
Some PCD mutant plants are resistant to FHB
Fusarium-GFP
KO = Gene Knockout Plant
OE = Overexpressing Transgenic Plant
100
50
75
100
ath % 0 24 48 72 96
0
25
WT VPEγKO ERG1 KO MC2Pa KO SOX KO
Cell de
a
WT VPEγ-KO ERG1-KO MC2Pa-KO SOX-KO
Medium Throughput Assay for Gene Function
Assay Gene Knockout Plants for Reduction of Symptoms
100 100100 100WT HS-230HS-25HS-108%
0
25
50
75
0
25
50
75
0
25
50
75
0
25
50
75
Cel
l dea
th
50
75
100
50
75
100
50
75
100
50
75
100
HS-26 HS-55 HS-36HS-28
eath
%
0
25
50
0
25
50
0
25
50
0
25
Cel
l de
50
75
100
50
75
100
HS-51
50
75
100
HS-16 HS-6
50
75
100
HS-215
ll de
ath%
0
25
0
25
0
25
0
25
Ce
Plant Cell Death kinetics
Lots of ‘failures’ –you need a high capacity system in order to select those rare genes that do confer resistance Fusarium.
Fusarium graminearum infection in TP-KO plantsTP
-KO
24h 48h 72h 96h48h 72h 96h
0 24 48 72 96
0 24 48 72 96ath
%
75
100
Cel
l de
25
50
0WT TP-KO
Fusarium graminearum infection in PC-KO plantsO
PC-K
24h 48h 72h 96h
0 24 48 72 96
ath
%
75
100
Cel
l dea
25
50
0WT PC-KO
Fusarium graminearum infection in HP-OE plantsH
P-O
E
24h 48h 72h 96h
100
0 24 48 72 96
deat
h %
50
75
100
Cel
l d
0
25
50
0WT HP-OE
Fusarium graminearum infection of WT, tb1-KO and tb1-OE plants
tb1‐KO tb1‐OEWT
72h 72h 72h
Physcomitrella patens infected with Fusarium graminearum:GFP
Mutation of tb1 affects the ability of Fusarium to enter into cells.
Effect are manifested at or near the plant cell surface and vacuole.
Mechanism is not understood, but may be revealing something importantMechanism is not understood, but may be revealing something important about how Fusarium colonizes its host. Interactions at the cell surface may be important.
Pipeline for discovery and deployment of genes effective against FHB
Select Select potentialpotentialantifungalantifungal
Isolate Isolate selected selected genesgenes
PhyscomitrellaPhyscomitrella::Create gene Create gene knockouts andknockouts and
PhyscomitrellaPhyscomitrella::Assay plants for Assay plants for FHB resistanceFHB resistance
WheatWheat: assay : assay selected genes forselected genes forFHB resistanceFHB resistance
WheatWheat: Stable: StableTransgenicsTransgenicsgg
genesgenes genesgenes oc outs a doc outs a doverexpressorsoverexpressors
FHB resistanceFHB resistance FHB resistanceFHB resistance gg
d
Transient assay in
TransformedFHB ResistantBased on genetic,
molecular data from wheat and other species
Clone and sequence genes
Mutate or express genes in
PhyscomitrellaExpose mutant
plants to FHB, DON
assay in wheat using
VIGS(Scofield Lab)
FHB-ResistantWheat, Barley (Dahleen Lab)
Induced Immunityy
Chitosan induces immunity in Physcomitrella and wheat
al % 100
ell s
urvi
va
25
50
75
Ce
U F. g F. g + C0
100
n %
25
50
75
erm
inat
ion
0Ge
U F. g F. g +C
Chitosan pre-treatment induces immunity against Fusarium graminearum in both Physcomitrella and wheat. However, the response is more pronounced in Physcomitrella.
C OS
Induced Immunity in PhyscomitrellaCHITOSAN (12h )
APAF1MDAR
D
- CHITOSAN + CHITOSAN (12h pre)+ FHB + FHB
APAF1VPEγBI-1SHSP
PCD
s SHSPATG8Derlin1
ER s
tres
sSec61Calnexin
CA4HIPRbLAPef
ense
Sec61
RNS1LSDEIN1Hin2
De
unity
Hin2EDS5
0 12 24 0 12 24
Actin1Imm
u
WT plants inoculated
with F. graminearum
WT plants pre-treated with
chitosan for 12h and then
inoculated with F. graminearum
RT-PCR
Role of nucleases in infection of F. graminearum
56 kDa
RNase activity gel assay Gene Expression -Northern blot
RNS1
BEN1
35 kDa
32 kDa 0 6 12 24h
BEN1
rRNA
Gene Expression RT PCR
32KDa
DNase activity gel assay
0 6 12 24h
Actin1
RNS1BEN1
Gene Expression -RT-PCR
32KDa
24h0 6 12 -0 12 24 0 12 24
Role of nucleases in infection of F. graminearum
100
% 100TH
Sensitivity to DONSensitivity to FHB
25
50
75
Cel
l dea
th %
50
75
100
0 24 48 72
CEL
L D
EAT
WT0
100
th %
0
25
WT AtBI1-OE SCL3∆-OE
VPEγ-KO WD40-KO MC2Pa-KO
RNS1-OE
%
Plant Cell Death
0
25
50
75
Cel
l dea
t
Note that plants overexpressing RNS1 are i ifi tl i t t t FHB th WT b t
Plant Cell Death
RNS1-OE
0
100
h %
significantly more resistant to FHB than WT but are still sensitive to DON (as is the WT).
This is consistent with RNS1 having a direct tif l ff t F i
0
25
50
75
Cel
l dea
t antifungal effect on F. graminearum.
N.B. Exogenous RNase is lethal to F. graminearum
BEN1-OE0
0 24 48 72 96
Fusarium-GFP Plant Cell Death
Induced Immunity
Chitosan Induced Immunity
PAMP Signaling Genes
‐CHITOSAN + CHITOSAN (12h pre‐treatment)
+ FHB
RLK1
Chitosan
PAMPs0 h 12h 24h0 h 12h 24hRLK1
RLK2
C OS C OS ( p e t eat e t)
CEBP
RLK2RLK4/5
MAPK3
RLK4/5
CEBP
RLK4/5
MAPKinaseCascade
WRKY22
RT‐PCR*
0 h 12h 24h0 h 12h 24h
WRKY
RNA Transcription of
Chitosan pre-treatment induces many of the components invoved in chitosan and other PAMPRNA
Pol Innate ImmunityTarget Genes
in chitosan and other PAMP (elicitor) signaling.
PAMP = Pathogen Associated Molecular PatternMolecular Pattern
* “Non-quantitative” RT-PCR
Chitosan-Induced Immunity Against FHB
Chitosan-induction immunity in WT, CEBiP OE and CEBiP KO Physcomitrella plants
WT P. patens CEBiP-OE CEBiP-KO
F.g 75
100
ival
%
F g +0
25
50
F.g. F.g. + F.g. F.g.+ F.g. F.g. +
Surv
F.g. +Chitosan
g gChit
g gChit
g gChit
WT CEBiP-KOCEBiP-OE
Physcomitrella
Yeast Gene Knockout Library Screeny
Nilgun Tumer’s LabRutgers University
Screens for new cellular targets of trichothecene toxins
Screen for Screen for t it inew toxin new toxin
targets in targets in yeastyeast
T2 ToxinT2 Toxin
Yeast Gene Yeast Gene Knockout Knockout LibraryLibrary
••Arabidopsis TArabidopsis T--DNA KnockoutsDNA Knockouts••RNAi in wheat, barleyRNAi in wheat, barley
Selection of resistant strains on plates usingdifferent concentrations of T-cin
4 μM T‐cin 8 μM T‐cin
different concentrations of T-cin
4 μM T cin 8 μM T cin
Double printed omniplates identified mutants that were resistantDouble printed omniplates identified mutants that were resistant only at 4μM (circled white) or at 8 μM (circled black) T‐cin
McLaughlin, Tumer et al., 2009 PNAS in press.
A genome-wide high throughput screen of yeast gene deletion library to identify novel tricothecene targets
Controls (No toxin)
deletion library to identify novel tricothecene targets
Working GlycerolPlate
Rep1 Rep2 Rep3Rep1 Rep2 Rep3
Toxin TreatmentsPCR
Original GlycerolPlate
Rep1 Rep2 Rep3
PCRVerification“Barcode”
Backup GlycerolPlate
Phenotype Comparions(OD 600)
Inhibited Growth(Susceptibility)Increased Growth(Resistance)
4720 strains on 72 plates350 slow growing strains on 4 platesScreened twice at 4, 8, 12 and 24 µM
False Positive(Resistance)
McLaughlin, Tumer et al., 2009 PNAS in press.
Mitochondria play a critical role in Tricothecin toxicity
These NUCLEAR-encoded genes, targeted to the mitochondrion, represent novel tricothecene targets. Their contribution to the susceptibility of plants to Fusarium can now be tested in Physcomitrella, Arabidopsis and wheat.
Gene Knockouts Help Define Cellular Mechanisms
PCDPCD
Reactive Oxygen Species
Induced Immunity
yg p
y
Cell Wall
Can we simulate the effects of these mutants by chemical treatment?
Chemical suppression of PCD controls Fusarium
100 Fusarium only
(A) Prevention of plant cell death in Physcomitella
25
50
75
Cel
l Dea
thFusarium + CC1
00 24 48 72 96/h
Fusarium + CC2
CCCCCCCC
(C) Reduced Fusarium growth in planta(B) Suppression of Fusarium growth in planta
WTWT WT + CC2WT + CC2WT+ CC1WT+ CC1F.g. F.g. + CC2 F.g. + CC1
P.p. Actin1
F.g. Actin1
0h 12h 24h 0h 12h 24h 0h 12h 24h
RT-PCR
F.g. Fusarium graminearumP.p. Physcomitrella patens
0h 12h 24h 0h 12h 24h 0h 12h 24h
Physcomitrella plants infected with GFP-labeled Fusarium graminearum
CC1 and CC2 PCD suppressors are NOT toxic to Fusarium
Chemical suppression of PCD and Fusarium
Physcomitrella Wheat
Uninoc Uninoc
Fg Fg
Fg CC1
Fg CC2
Fg CC2
Fg CC1
PCD suppressors protect plants from Fusarium infectionPCD suppressors protect plants from Fusarium infection
Chemically Inducted Defense Against FHB (non-PCD)
GFP Autofluorescence Merge GFP Autofluorescence MergeWheatPhyscomitrella patens
FHB FHB
FHB +G17-1
FHB +G17-1
FHB + FHB + B49 B49
50
6040
% C
ell d
eath
10
20
30
40
50
10
20
30
% C
ell d
eath
C F.g. C F.g.0
10
Control Fg Fg+G17-1Fg+B490
Control Fg Fg+G17-1 Fg+B49
An end-run around the gene discovery and deployment pipeline?
SelectSelect PhyscomitrellaPhyscomitrella:: Wh tWh t VIGSVIGSSelect Select potentialpotentialantifungalantifungalgenesgenes
Isolate Isolate selected selected genesgenes
PhyscomitrellaPhyscomitrella::Create gene Create gene knockouts andknockouts andoverexpressorsoverexpressors
PhyscomitrellaPhyscomitrella::Assay plants for Assay plants for FHB resistanceFHB resistance
WheatWheat: VIGS : VIGS assay of selected assay of selected genesgenes
Wheat, BarleyWheat, BarleyTransgenicsTransgenics
Design chemical interventions Design chemical interventions based on mechanisms revealed by based on mechanisms revealed by mutant studiesmutant studiesmutant studiesmutant studies
Test efficacy in Physcomitrella, Test efficacy in Physcomitrella, h t th t twheat, etc.wheat, etc.
Chemical studies can help us Chemical studies can help us understand mechanisms of FHB understand mechanisms of FHB susceptibility and resistancesusceptibility and resistance
May provide novel approaches for May provide novel approaches for chemical control of FHBchemical control of FHB
Conclusions
• Physcomitrella is a rapid and sensitive assay for genes that control sensitivity to DON and susceptibility to Fusarium.
• We have identified a number of genes whose mutation alters sensitivity to Fusarium.
• There are multiple ways to enhance resistance to Fusarium.– Suppress PCD pathway– Enhance innate immunity/defense responsesy p– Alter the plant cell wall/cell surface
• It is important to test the efficacy of these genes in crop plantsIt is important to test the efficacy of these genes in crop plants – Test by VIGS assay in wheat (Scofield, Purdue University)– Test by in transgenic crop plants (Dahleen, BioEn-USP)
• Chemical approaches to controlling FHB– Physcomitrella gene mutants can suggest novel approaches for the
chemical control of FHB (non-toxic, non-fungicidal chemicals)– This may have some practical applications
Support
US Wh t d B lUS Wheat and BarleyScab Initiative (USDA)
Acknowledgements
Hemalatha SaidasanMark Diamond
Eric LamNilgun Tumer
Steve ScofieldLynn Dahleen
Overexpression of the Ribosomal Protein L3 confers resistance to FHB
Fusarium-GFP Plant Cell DeathFusarium-GFP Plant Cell Death
deat
h 75
100WTTaL3∆-OEScL3∆-OE
WT
TaL3∆
% c
ell d
0
25
50TaL3∆ScL3∆
00 24 48 72 96
96h TaL3∆: Wheat L3∆ geneScL3∆: Yeast L3∆ gene