Learning from the pathogen towards tailored-sustainable
resistance : the case study of Xanthomonas TAL effectors
Boris.szurek@ird.fr
Boris SZUREK
Montpellier
France
Group GTIPP, for Genomics & Transcriptomics of Plant-Procaryotes Interac>ons

Learning from the pathogen towards tailored-sustainable
resistance : the case study of Xanthomonas TAL effectors
Outline
Ø Some background about TAL effectors
Ø Story I : search for SWEET14 loss-of-S
Ø Story II : a blast NLR confers R against Xo

Tomato
Arabidopsis
Cabbage
Rice Pepper
Cassava
Citrus
Plant pathogenic Xanthomonas

TAL (Transcription Activator-Like) type III effectors

TAL (Transcription Activator-Like) type III effectors
Ø They localize to the plant nucleus
Ø They induce expression of host genes
Ø How do they bind to DNA ?
TALs are bonafide plant TFs :

• 12 + 13: “hypervariable”
• 4 repeat types in AvrBs3
HD, NI, NG, NS
modified from:
Schornack et al. (2006) J. Plant Physiol. 163:256.
TAL repeat types

picture: Adam Bogdanove
Boch et al. (2009) Breaking the code of DNA binding specificity of
TAL-type III effectors. Science 326:1509-1512; published online 29
October 2009 (10.1126/science.1178811).
Moscou and Bogdanove (2009) A simple cipher governs DNA
recogni>on by TAL effectors. Science 326:1501;
published online 29 October 2009 (10.1126/science.1178817).
The TAL code revealed

Correlation between repeat types and UPA box
UPA box consensus
AvrBs3
COOH NH2
17.5 x 34 aa
LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG
NI
NG
NS
1 12/13 34
T A T A T A A A C C T N N C C C T C TDNA
HD NG NS NG NI NI NI HD HD NG NS NS HD HD HD NG HD NGRepeats
1 AvrBs3-repeat corresponds to 1 bp

Different RVDs associate preferentially with different bases
Test the model !

Target specificity of Hax2, Hax3, and Hax4
Boch et al.,
Science, 2009

Artificial TAL effectors with novel specificities
Boch et al.,
Science, 2009
GUS gene
ArtX-box

GUS gene
ArtX-box
Boch et al.,
Science, 2009
Artificial TAL effectors with novel specificities

Crystal structure of a TAL bound to its DNA
Mak et al., Science, 2012

One can predict targets !
T
C
G
G
C
A
T
C
A
T
C
T
C
A
T
C
A
G

Xanthomonas oryzae
pv. oryzae (Xoo)
The Rice-Xoo interaction

The Rice-Xoo interaction

S gene
TAL EBE
DISEASE
Roles of TAL effectors in compatible interactions

5dpi
1cm
5 kb
4 kb
2 kb
MAI1 BAI3
3 kb
talC
Yu et al., MPMI, 2011
TalC is a major virulence TAL effector

5dpi
1cm
5 kb
4 kb
2 kb
MAI1 BAI3
3 kb
talC
Yu et al., MPMI, 2011
SWEET14 talC
TalCBAI3
TalC is a major virulence TAL effector

SWEET/nodulin-3 proteins
. 22 members in rice
. Membrane Transporters
. Glucose & sucrose export
Talbot, 2010, Nature

Clade III SWEET genes confer TAL-ETS to Xoo
Streubel, Pesce, et al., 2013, New Phytol.

SWEET14 talC
TalCBAI3
Burkina Faso
SWEET14 is targeted by multiple strains..

SWEET14 talC
TalCBAI3
tal5
Tal5MAI1
Burkina Faso
Mali
SWEET14 is targeted by multiple strains..

pthXo3
SWEET14 avrXa7talC
TalCBAI3
PthXo3PXO61
tal5
Tal5MAI1
AvrXa7PXO86
Philippines
Burkina Faso
…of different geographic origins and lineages
Mali
Susceptibility
SWEET14 is targeted by multiple strains..

SWEET14
TalCBAI3
PthXo3PXO61
Tal5MAI1
AvrXa7PXO86
Philippines
Burkina Faso
Mali
? ??
Loss of
Susceptibility

ü Unique collection of 132 wild and cultivated rice accessions
ü Sequencing of a 400-bp PCR product
3
65
pthXo3
SWEET14 avrXa7talC tal5
3034
Search for polymorphism in the SWEET14 promoter

PATTERN 1
PATTERN 2
PATTERN 3
AvrXa7 Tal5
Search for polymorphism in the SWEET14 promoter

SWEET14 avrXa7talC tal5
Δ
Does it work ?

No dele>on
Δ18bp
5 DPI
SWEET14 avrXa7talC tal5
Δ
Does it work ?

No dele>on
Δ18bp
5 DPI
SWEET14 avrXa7talC tal5
Δ
Does it work ?

No dele>on
Δ18bp
2 DPI
SWEET14 avrXa7talC tal5
Δ
Does it work ?

• Reduced disease symptoms
• Impaired Xoo multiplication
• No induction of SWEET14
Ø The xa41(t) allele confers resistance by loss of susceptibility
to Xoo strains relying on Tal5 and AvrXa7
Summary

Asia
Africa
50% of Xoo strains are defeated by xa40(t)

PATTERN 1
PATTERN 2
PATTERN 3
AvrXa7 Tal5
What about pattern 3 ?

PATTERN 1
PATTERN 2
PATTERN 3
AvrXa7 Tal5
What about pattern 3 ?

A portion of SWEET14 is duplicated in O. glaberrima..

A duplication of pSWEET14 occurred in O. barthii ancestor

A duplication of pSWEET14 occurred in O. barthii ancestor
Hu>n et al., 2015, Plant J

Conclusions – Part I
Ø Based on the TAL code, an allele with an 18bp deletion was
predicted to resist to Xoo strains relying on AvrXa7 and Tal5
Ø New HTP methodology for the isolation of loss-of-S
resistance alleles
Ø Avoid laborious germplasm phenotyping

Hu>n et al., Fron>ers in Plant Science, 2015

Ralf Koebnik
Lionel Gagnevin
Valérie Verdier
Sébastien Cunnac
Servane Baufume
Jonathan Jacobs
Florence Auguy
Mathilde Hutin
Céline Pesce
Alvaro Perez
Tu Tran Tuan
Hinda Doucoure
Carolina Flores
Cheick Tekete
Sandrine Fabre
Alain Ghesquière
François Sabot

New Fron>er Project « Menergep »
(IRD, Cirad, JIRCAS & AfricaRice)
Funding

A Rice Blast Resistance Gene Confers
Resistance Against BLB and BLS
Thomas Kroj
Stella Cesari
Véronique Chalvon
Mathilde Hu>n
Tuan Tu Tran
Ralf Koebnik

Xoo - X. oryzae pv. oryzae
Xoc - X. oryzae pv. oryzicola
BLB
BLS
Hydathode
Wounds
Stomate
Wounds
Vascular bundle
Mesophyll intercellular space
Xanthomonas oryzae pathovars profiles

Xoo - X. oryzae pv. oryzae
Xoc - X. oryzae pv. oryzicola
BLB
BLS
Hydathode
Wounds
Stomate
Wounds
Vascular bundle
Mesophyll intercellular space
Xanthomonas oryzae pathovars profiles
Cloned R genes :
Xa1
Xa21
Xa27
Xa10
Xa23

Xoo - X. oryzae pv. oryzae
Xoc - X. oryzae pv. oryzicola
BLB
BLS
Hydathode
Wounds
Stomate
Wounds
Vascular bundle
Mesophyll intercellular space
Xanthomonas oryzae pathovars profiles
Cloned R genes :
Xa1
Xa21
Xa27
Xa10
Xa23
No R gene available (in rice)

Bogdanove et al., COPB, 2010
TAL effectors during resistance and disease

Boch et al., New Phytol, 2014
Engineered resistance against TALEs
Bs3, Xa27, Xa10

Boch et al., New Phytol, 2014
Engineered resistance against TALEs
Bs3, Xa27, Xa10

Boch et al., New Phytol, 2014
Engineered resistance against TALEs
Bs3, Xa27, Xa10

Ø Causal agent : Magnaporthe
oryzae
Ø Host : Oryza spp.
Ø Hemibiotrophic pathogen
Rice Blast

Rice
M. oryzae
Pi-CO39 pi-CO39
Avr1-CO39
avr1-CO39
The Avr1-C039 / Pi-CO39 interaction

Rice
M. oryzae
Pi-CO39 pi-CO39
Avr1-CO39
avr1-CO39
The Avr1-C039 / Pi-CO39 interaction

Cesari et al., Plant Cell, 2013
Cesari et al., EMBO, 2014
Pi-CO39 resistance is mediated by a pair of NB-LRR proteins

Kitaake
Pi-CO39
Kanto51
pi-CO39
AVR1-CO39
UAS
AVR1-CO39
UAS
uidA
UAS
uidA
UAS
DEX-inducible AVR1-CO39 transgenic lines

Kitaake (Pi-CO39)
Kanto51 (pi-co39)
pUASgal4::uidA pUASgal4 ::AVR1-CO39
DEX
+
-
+
-
Line 1 Line 2
DEX-inducible AVR1-CO39 transgenic lines

DEX-inducible AVR1-CO39 transgenic lines

pINDEX::AVR1-CO39:HA pINDEX::uidA Kanto51 (pi-co39)
DEX + - + - + -
Line 1 Line 2
Kitaake (Pi-CO39)
DEX-induction of AVR1-CO39 triggers cell death and ROS

Kitaake (Pi-CO39) Kanto51 (pi-co39)
+ - + -
pINDEX::AVR1-CO39 pINDEX::uidA
DEX
DEX-induction of AVR1-CO39 triggers cell death and ROS

Kitaake
Pi-CO39
Kanto51
pi-CO39
AVR1-CO39
dTALEUAS
UAS
AVR1-CO39
dTALEUAS
UAS
uidA
dTALEUAS
UAS
uidA
dTALEUAS
UAS
Using dTALEs to induce AVR1-CO39

0,00
0,05
0,10
0,15
0,20
mock
EV
dTALE
RelaNve AVR1CO39 expression ∅ ∅ ∅
Kitaake
(Pi-CO39)
Kanto51
(pi-co39)
uidA AVR1-CO39
Line 1
AVR1-CO39
Line 2
AVR1-CO39
Using dTALEs to induce AVR1-CO39

0,00
0,05
0,10
0,15
0,20
mock
EV
dTALE
RelaNve AVR1CO39 expression ∅ ∅ ∅
Kitaake
(Pi-CO39)
Kanto51
(pi-co39)
uidA AVR1-CO39
Line 1
AVR1-CO39
Line 2
AVR1-CO39
The AVR1-CO39 and uidA genes are induced
by Xanthomonas oryzae carrying the dTALEUAS
Using dTALEs to induce AVR1-CO39

The induction of AVR1-CO39 confers resistance to Xoo

.. and Xoc

The induction of AVR1-CO39 confers resistance to Xoo
0
5
10
15
20
25
30
35
Line 1 Line 2 Line 1 Line 2 Line 1 Line 1
pUASgal4::AVR1-CO39 pUASgal4::uidA pUASgal4::AVR1-
CO39
pUASgal4::uidA
Kitaake Kanto51
lesion length (cm)
PXO99 EV
PXO99 ArTAL
PXO
99
PXO99 (EV)
PXO99 (dTALE)
PXO99 PXO99 Kitaake (Pi-CO39) Kanto51 (pi-CO39)

The induction of AVR1-CO39 confers resistance to Xoo
0
5
10
15
20
25
30
35
Line 1 Line 2 Line 1 Line 2 Line 1 Line 1
pUASgal4::AVR1-CO39 pUASgal4::uidA pUASgal4::AVR1-
CO39
pUASgal4::uidA
Kitaake Kanto51
lesion length (cm)
PXO99 EV
PXO99 ArTAL
PXO
99
PXO99 (EV)
PXO99 (dTALE)
PXO99 PXO99 Kitaake (Pi-CO39) Kanto51 (pi-CO39)

The induction of AVR1-CO39 confers resistance to Xoo
0
1
2
3
4
5
6
7
8
9
Line 1 Line 2 Line 1 Line 2 Line 1 Line 1
pUASgal4::AVR1-CO39 pUASgal4::uidA pUASgal4::AVR1-
CO39
pUASgal4::uidA
Kitaake Kanto51
Bacterial popualNons log10 (CFU)
PXO99 EV
PXO99 ArTAL
PXO99
PXO99 (EV)
PXO99 (dTALE)
PXO99 PXO99
Kitaake (Pi-CO39) Kanto51 (pi-CO39)
2
1

The induction of AVR1-CO39 confers resistance to Xoo
0
1
2
3
4
5
6
7
8
9
Line 1 Line 2 Line 1 Line 2 Line 1 Line 1
pUASgal4::AVR1-CO39 pUASgal4::uidA pUASgal4::AVR1-
CO39
pUASgal4::uidA
Kitaake Kanto51
Bacterial popualNons log10 (CFU)
PXO99 EV
PXO99 ArTAL
PXO99
PXO99 (EV)
PXO99 (dTALE)
PXO99 PXO99
Kitaake (Pi-CO39) Kanto51 (pi-CO39)
2
1

Cesari et al., Plant Cell, 2013
Cesari et al., EMBO, 2014
Pi-CO39 resistance is mediated by a pair of NB-LRR proteins

Induction of RGA4 in Pi-CO39 lines confers resistance to Xoo
RGA4
dTALERGA4
0
2
4
6
8
10
12
lesion length (cm)
Co39
0
2
4
6
8
10
12
14
16
18
20
Kitaake
PXO
99
Lesion length (cm.)
Lesion length (cm.)
PXO99
PXO99
PXO99
PXO99
PXO99 PXO99
PXO99
PXO99 Co39 Kitaake
0
5
10
15
20
25
Kanto51 PXO99 Kanto51
PXO99
PXO99
PXO99
PXO99
Lesion length (cm.)
Hu>n et al., Plant J, 2016

Ø The RGA4/RGA5-mediated resistance is able to control BLB
and BLS
Ø Autoac>ve forms of NB-LRRs and AVRs can be used for
synthe>c promoter-traps resistance strategies
tal2 RGA4 tal1
Tal1
Tal2
tal3
Tal3
Conclusions

Thomas Kroj
Stella Cesari
Véronique Chalvon
Mathilde Hu>n
Tuan Tu Tran
Ralf Koebnik
Thanks !