Nijat Imin - CRISPR-mediated gene editing in hairy roots of model … · 2017-12-04 · Nijat Imin - CRISPR-mediated gene editing in hairy roots of model legume Medicago truncatula:

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

Moss

Spike mossGymnosperm

Angiosperm

eudicots

Mono-cots

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

Saur et al. 2011. New Phytologist

Nodule Specific CLE peptides:Expression

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

***

sunn4

rdn1-1

MtCLE13TaP act on rhizobial infection processes

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

MtNAC82

Silencing multiple genes with limited success

Imin et al. 2013 JXB

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


Čermák et al. 2017 Plant CellCsy4: Csy-type (CRISPR system yersinia) ribonuclease 4

Cestrum: Yellow Leaf Curling Virus promoter

Curtin et al. 2017 Plant Phys.

0

2

4

6

8

10

12

14

A17 nin-CRISPR nac82-CRISPR

Nu

mb

er o

f n

odu

les

* *

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


• 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


CRISPR/Cas9 chromosome deletions

Medicago chromosomes

GW

AS

pv

al

Čermák et al. 2017 Plant Cell

CRISPR/Cas9 chromosome deletions

Čermák et al. 2017 Plant Cell

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

Kirchner et al. 2017 PLOS One

Screening of the transgenic roots

Kirchner et al. 2017 PLOS One

Acknowledgements

MtCLE12/MtCLE13

Richard Payne and Leo Cornelius(University of Sydney)

CRISPR-CAS system

Shaun Curtin and Nevin Young (University of Minnesota)

Michael Djordjevic Marie Oakes

Isabel Saur

Neha Patel

Collaborators

Kon Ping Leung

Documents

Nijat Imin - CRISPR-mediated gene editing in hairy roots of model … · 2017-12-04 · Nijat Imin - CRISPR-mediated gene editing in hairy roots of model legume Medicago truncatula: