nijat imin - crispr-mediated gene editing in hairy roots of model … · 2017-12-04 · nijat imin...
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CRISPR-mediated gene editing in hairy roots of model legume Medicago
truncatula:The role of peptide hormones
regulating nitrogen demand signalling
Nijat Imin
Peptide hormones: a new frontier for coordinating development, growth and yield
Czyzewicz et al. 2013. JXB
Phytohormones:Auxin, cytokinin, abscisic acid, gibberellin, Ethylene, strigolactone etc
Peptide hormones: Control of stem cell niche size and differentiation in the SAM
Controls balance between stem cell renewal and differentiation
Galli and Gallavotti 2016. Trends in Genetics
thick tassel dwarf1: regulator of inflorescence size
Bommert et al. Development 2005
PTM modification of CLV3 controls meristem and fruit size
Xu et al. 2015. Nature Genetics
The importance of nitrogen• Plant sequestered N is the source, directly and
indirectly, of all human nutritional N.
• Lack of N fertiliser availability limits crop production.
• Overuse of N fertilisers poses environmental threats.
Rockström et al. 2009 Nature
1900 1950 2000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0
Worl
d p
op
ula
tio
n (
mill
ion
s)
50
40
30
20
10
0
% W
orld
po
pu
latio
n/
Ave
rag
e fe
rtilize
r inpu
t: kg
N h
a–
1yr
World population
World population(no Haber Bosch nitrogen)
% World population
fed by Haber Bosch nitrogen
Average fertilizer input
–1
Trends in human population and nitrogen use throughout the twentieth century
Erisman et al. 2008 Nature Geoscience
The plastic plant: root responses to heterogeneous supplies of nitrogen sources
Drew 1975 New Phytologist
Plant provides fixed carbon
Rhizobia provide fixed nitrogen in
nodules
The symbiosis between legumes and rhizobia
Autoregulation of root nodulation
Tanabata and Ohyama 2014In: Advance in Biology and Ecology of Nitrogen Fixation
Over nodulation deters growth
Nod++ receptor mutants:• sunn in M. truncatula
• Gmnark in soybean• har1 in L. japonicus
• Pssym29 in pea
Ectopic expression of MtCLE12/13 suppress nodulation in SUNN dependent manner
Saur et al. 2011. New Phytologist
Schnabel et al. 2011 Plant Physiol.
ROOT DETERMINED NODULATION1 regulates nodule number in roots of Medicago truncatula
RDN1 is a hydroxyproline O-arabinosyltransferase
Nodule Specific CLE peptides:Structures
MtCLE13 R L S P A G P D P Q H N
MtCLE13-Hydroxy R L S P A G P D P Q H N
MtCLE13-MaP R L S P A G P D P Q H N
MtCLE13-TaP R L S P A G P D P Q H N
MtCLE12
MtCLE42
GmNIC1
GmNIC2
MtCLE13
LjCLE-RS1/2
GmRIC1
LjCLE-RS3
LjCLE40
R L S P G G P N H IH N
R L S P G G P D A H H H
R L S P G G P D Q K H H
R L S P G G P D H K H H
R L S P A G P D P Q H N
R L S P G G P D P Q H N
R L A P E G P D P H H N
W I S P G G P D P K H N
R L S P Q G P D P R H H
Up by ‘+CLE13TaP’ Down in sunn-4
Up in sunn-4 Down by ‘+CLE13TaP’
0
0
0
0
0
0
0
316 2,898
1,263267
13 188 253
29
In triplicates; 2-fold change with P < 0.05
0
500
1000
1500
2000
2500
3000
Rel
etiv
e ex
pre
ssio
n le
vel
Gene Expression Difference in Distinct Groups (Up_WT)
WT WT_CLE sunn sunn_CLE
0
200
400
600
800
100
0
120
0
140
0
160
0
180
0
Medtr0034s0170
Medtr1g080780
Medtr2g091125
Medtr2g435720
Medtr3g087740
Medtr4g040330
Medtr4g083130
Medtr4g087850
Medtr4g095700
Medtr0189s0040
Medtr4g129580
Medtr4g133720
Medtr4g134390
Medtr5g005770
Medtr2g084195
Medtr5g011950
Medtr7g405710
Medtr5g082750
Medtr8g023840
Medtr6g036620
Medtr6g086870
Medtr7g055730
Medtr7g090035
Medtr2g084190
Medtr4g118855
Medtr2g084180
Medtr8g075510
Medtr1g109650
Medtr8g446520
Relative Expression level
Gen
e Expressio
n D
ifference in
Distin
ct Gro
up
s (Dn
_WT)
WT
WT_C
LEsu
nn
sun
n_C
LE
0
0.5
1
1.5
2
2.5
3
3.5
A17 sunn4
Down-regulated by CLE13-TaP
(-) (+)
0
0.5
1
1.5
2
2.5
3
3.5
A17 sunn4
Up-regulated by CLE13-TaP
(-) (+)
A
B
C C
A
B
C C
SUNN receptor-dependent induction/suppression of target genes by CLE13TaP
TAL Effector Nucleases
(TALENs)Zinc Finger Nucleases
(ZFNs)
Homing Endonucleases
(HEs)
CRISPR/Cas9
Site-specific nuclease platforms
Wang et al. 2017 Progress in Molecular Biology and Translational Science. 149
ZFNs, TALENs and CRISPR-Mediated Genome Editing in Legumes
Whole-plant root transformation
CIM ~ 3 weeks CIM ~ 6 weeks CIM ~ 9 weeks SIM ~ 12 weeks
SIM ~ 15 weeks SIM ~ 15 weeks RIM ~ 20 weeks
Medicago whole plant transformation
Curtin et al 2017 Plant Physiology
Adapted from Wang et al. 2017 Progress in Molecular Biology and Translational
Science. 149
Workflow for CRISPR–Cas9-
mediated genome editing in
legumes
and/or At7SL
RNA polymerase III
CRISPR/Cas9 Platforms
Golden-gate vector
Gateway™ vector
• single gene knock-out
• multi-gene knock-out and chromosome deletions
• Twice higher mutation frequencies
Gmubi Pro 7sLCas9 U6
Cas9
35S Cas9 CestrumCsy4
P2A
Csy4Cas9
Curtin et al 2017 Plant Physiology
Čermák et al. 2017 Plant CellCsy4: Csy-type (CRISPR system yersinia) ribonuclease 4
Cestrum: Yellow Leaf Curling Virus promoter
Validation of GWAS candidates with CRISPR/Cas9
• Identify candidate genes that contribute to naturally occurring variation in nodulation
• GWA analysis using 6 million SNPs on a panel of 226 Medicago accessions
Yoder JB et al 2014 Genetics
Curtin et al 2017 Plant Physiology
• 16x T0 recovered from TC
• 5x T0 bi-allelic homozygous mutants
• 1x T0 double mutants
• Sequence confirmed
• Rapid phenotype screening
Validation of Pho2 candidate
Pho2a
Nla
IVN
laIV
Pho2b
Curtin et al 2017 Plant Physiology
0
2
4
6
8
10
12
14
A17 Chr2~58 Kb deletion Chr2~58 Kb deletion (no
transgene)
Nu
mb
er o
f n
od
ule
s
*
*
Summary
• Key AON downstream genes are identified
• NAC82 may regulate CLE-SUNN mediated AON
• Chr2 ~58Kb region control nodulation, yet independent from CLE-
SUNN mediated AON
• There are challenges with CRISPR-Cas9 gene editing in hairy root
transformation
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