death be not proud: modulation of programmed cell death...
TRANSCRIPT
Death be not Proud: Modulation of Programmed Cell Death for
DiseaseDevelopment/Stress Tolerance in Plants
MARTY DICKMAN
Distinguished ProfessorInstitute for Plant Genomics and Biotechnology
Borlaug Center
Department of Plant Pathology and Microbiology
Texas A&M University
College Station
TOPICS-not discussed
1. Genome EDITING --CRISPR
2. RNAi
3. Computation –Pathway Analysis
4. Phytobiome
What are the key determinants of the “necrotrophic “ (and broad host range) lifestyle that enable Sclerotinia to achieve pathogenic success??
Sclerotinia -soybean
• Targeted gene silencing can be used to modulate biosynthetic pathways in a specific tissue to obtain a desired phenotype in a way that is not always possible with traditional breeding.
WT Transgenic2
Dpi
30 D
pi
DE Genes
Count TableReadsRNA
Assess pathway: KEGG
GO Term Pathway DatabaseCandidate
Illunimina Alignment
STARDE Seq
ORAFCSPathway
Gene set analysis
RNA Isolation
Arabidopsis mutants
Screen the mutant plant under drought and disease stress
Khatri,P.,Sirota,M.,andButte,A.J.(2012).Tenyearsofpathwayanalysis:currentapproachesandoutstandingchallenges.PLoS Comput Biol
Drought tolerance : 8 days after watering
Candidate mutant
Apoptosis:A Pre-programmed Cell Autodestruction
• An everyday routine…
10107107 cells turnover via cells turnover via apoptosis/mitosis dailydaily,
or 10or 109or 109-9-101010 cells per year
• Too little:…cancer --- cells that have forgotten how to dieToo much:...organ damage, e.g., stroke,
post heart attack, hepatitis, AIDS…
Jeff Rollins
C. elegans
EGL-1 CED-9
CED-4
CED-3
Apoptosis
Mammals
Bcl-2
Apaf-1
Caspase-9
Apoptosis
Bik
BaxIntracellularmembrane
Plants
?
?
?
Disease/HRSenescence
Abiotic Stress(Apoptosis)
1. Bcl-2 (Human)2. Bcl-xl (Chicken)3. Bcl-xl (G138A)4. CED-9 (C. elegans)5. Op-IAP (Baculovirus)6. Sf-IAP(S. frugiperda)
TRANSGENES
?28@C8
?28@C8%4F=EGH7
Bcl-2 Tobacco Sclerotinia inoculation
Bcl-2
WT
GAL- Bax
GAL- BaxX
Plant cDNA Library
GlucoseGlucoseYeast CellYeast Cell GalactoseGalactose
Bax/ Bak-Induced Lethality in Yeast:Bax/ Bak-Induced Lethality in Yeast:cDNA Library Screening AssaycDNA Library Screening Assay
Identification of the Arabidopsis BAG Family
A B
Kabbage and Dickman, 2008
Human BAGs Arabidopsis BAGs
Crystal structure of Human and ArabidopsisBAG Domains
BD/Hsp70 complex
hBAG4 BD
AtBAG4 BD JBC Doukhanina et al., 2006
Subcellular Localization of AtBAGs
AtBAG5-YFP ScCOX4-RFP Overlay
AtBAG5-YFP Mito-Tracker Overlay
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H$?HFQ%%VN.
H$?HFI%NJ
[?DW%K/;("#-,-#%'$%#8>W%AXXQ
IS THIS THE FUTURE OF
BANANA ?
Why Biotechnology?
DNA laddering - time and dose dependence
12 hours 24 hours 48 hours 72 hoursTreatments:
Lane 1: control (No AGL1)
Lane 2: AGL1 OD 0.1
Lane 3: OD 0.5
Lane 4: OD 1.0
Lane 5: OD 0.1
Lane 6: OD 0.5
Lane 7: OD 1.0
Lane 8: OD 0.1
Lane 9: OD 0.5
Lane 10: OD 1.0
Lane 11: OD 0.1
Lane 12: OD 0.5
Lane 13: OD 1.0
Banana embryogenic cell suspensions co-cultivated with Agrobacterium (AGL1)
Agrobacterium/Banana Interactions
External symptoms
LF-NI LF-I 1 LF-I 2 LF-I 3
Panama Wilt Resistance
ced-9 transgenicUntransformed
Fusarium oxysporum
GGPP
Phytoene
Lycopene
+!-carotene
psy1
crt I + transit peptide
psy2a
^#,b'%?ZE
H.;3,-#
Proposed genetic modification of !-carotene pathway in
banana
(Pro-vitamin A)
Exp1-Apsy2a(FT246) 23ug carotenoids/g dry weight
WT control (FT184) 3.8 ug carotenoids/g dry weight
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3456748$)+9,-./.,01,23!
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M 1 2 3 4 5 6Sugarcane callus before Agro-exposure (a) and after Agro exposure (b)
a b
Lane 1: Control callus (Q117)
Lane 2: Callus 72 hours after LBA4404 exposure
Lane 3: Callus 12 hours after AGL1 exposure
Lane 4: Callus 24 hours after AGL1 exposure
Lane 5: Callus 48 hours after AGL1 exposure
Lane 6: Callus 72 hours after AGL1 exposure
Jeff Rollins
Thanks Jeff
G E N O ME
E D I T I NG
Zinc-Finger NucleasesZFNs
Transcription Activator-Like Effector-based Nucleases
TALEN
Clustered Regularly-Interspaced Short Palindromic Repeats
CRISPR
Advantage of CRISPR/Cas system
• Target design simplicity.– gRNAs can be designed
readily and cheaply
• Efficiency. – eliminates the long processes
of transfecting and selecting.
• Multiplexed mutations.
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Heliocide H3
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