[2024]Digital Global Overview Report 2024 Meltwater.pdf
Exploiting pathogen biology for disease resistance breeding
1. Exploiting pathogen biology for
disease resistance breeding in
plants
Diane Saunders
BGRI 2012 technical workshop
2. Outline
• Phytophthora infestans -
a model system for studying
secreted proteins (effectors)
that perturb plant processes
• Durable resistance – the
use of effectoromics and
synthetic R genes in developing
durable resistance
• Rust fungi – identifying
effector proteins from newly
sequenced genomes
Sarah Gurr, University of Oxford Kupferchmidt, Science (337) 2012.
3. Phytophthora spp. – The "Plant Destroyers"
Cocoa Sudden oak
death
P. palmivora P. ramorum
Soybean Fruit rot
• Most important pathogens of dicot P. sojae P. capsici
plants
Late blight
• P. infestans most destructive on
crops – up to $6.7 billion in crop
losses annually
• Potato: third most important food
crop, critical to feeding the poor
P. infestans
4. The aggressive clonal lineage 13_A2 (blue-13)
blue-13 T30-4
• First recorded in the Netherlands in 2004, then the UK in 2005
• A2 genotype increase dramatically since 2005 to 2008 (from 12 to 79%)
• 2007: 324 outbreaks of blight in UK of which 82% contained blue-13
• Resistant to the fungicide metalaxyl
• Evades recognition by several key late blight resistance proteins
Cooke et al., PLoS Pathog, In press
5. Durable potato blight resistance?
• Classical approaches are ‘blind’ – R genes bred and
deployed without knowledge of the effectors they are
sensing
• Effectoromics – using core effector set of P. infestans
to identify natural R genes from wild potato germplasm
• Synthetic (non-natural) R genes – expanding
recognition spectrum of known resistance proteins
6. Effectors – secreted pathogen molecules that perturb plant
processes
• Effectors – described in parasitic bacteria, oomycete, fungi,
nematodes and insects
• Encoded by genes in pathogen genomes but function inside
plant cells – operate as plant proteins
• Target of natural selection in the context of coevolutionary
arms race between pathogen and plant
• Current paradigm – effector activities are key to
understanding parasitism
7. Microbes alter plant cell processes by secreting a
diversity of effector molecules
bacterium
targets
Alter plant cell
effectors
processes
haustorium Help microbe
colonize plant
oomycete
fungus
plant
cell
Modified from: Dodds and Rathjen 2010 NAT REV GENET
8. Some effectors “trip the wire” and activate immunity
in particular plant genotypes
bacterium
targets
Alter plant cell
effectors
processes
haustorium effector-
triggered
immunity
oomycete
fungus
intracellular
plant
cell
immune receptors
Modified from: Dodds and Rathjen 2010 NAT REV GENET
9. AVR effectors of P. infestans
• AVR1 and AVR4
are dispensable
• AVR2, AVR3a and
AVRblb2 are always
present and
expressed;
polymorphic families
Vleeshouwers et al. Annu Rev Phytopathol 2011
10. Effectoromics for durable blight resistance
Agro-infiltration
Identification of effectors for screening
• All P. infestans Avr genes identified belong to the RXLR effector class
• RXLR effectors are encoded in gene sparse regions of the genome
Functional screening
• Effectors cloned into expression vectors and expressed in planta by
agro-infiltration
Vleeshouwers et al. Annu Rev Phytopathol 2011
11. Effectoromics for durable blight resistance
(i) Cosegregation F1
(ii) Coinfiltration
• Focusing on cloning and breeding R genes that recognize "core" P. infestans
effectors, we maximize the potential for resistance durability in the field
Vleeshouwers et al. Annu Rev Phytopathol 2011
12. Effectoromics for durable blight resistance
Hendrik Rietman Effectors
et al. Wageningen
HR +++
HR +
No response
Not tested
13. Synthetic R genes with expanded effector sensing
Mutagenesis
-
R3a
+
R3a
AVR3aKI
AVR3aEM
Agro-mediated
gene expression
Maria Eugenia Segretin
14. Targeted genome mutagenesis and editing
Transcription activator-like (TAL)
effectors • Xanthomonas TAL effectors –
directly modulate host gene
DNA-‐binding
domain
NLS
AD
expression
N C
• Central repetitive region confers
DNA-binding specificity
HD NI NG HD NN HD HD NI HD NG NI HD HD NN NG
• Opportunity for designer DNA
C A T C G C C A C T A C C G T
Target DNA binding proteins
Repeat type: NI HD NG NN
DNA base recognised: A C T G
A
Moscou et al.; Boch et al. Science 2009
Marton et al. Plant Physiol 2010 Vladimir Nekrasov
15. Targeted genome mutagenesis and editing
• TAL effectors can be fused to
FokI nuclease to target DNA
breaks
• NHEJ often induces deletions/
insertions
• Expression of TALENs (TAL
nucleases) can be used to induce
R gene mutagenesis in planta
Vladimir Nekrasov
16. Targeted genome mutagenesis to engineer resistant
crops
• TALEN (TAL-nuclease) technology - greatly facilitates
genome engineering
• Mutant plants are recombinant DNA-free – no
transgenic sequences, indistinguishable from naturally
occuring mutations
• Opportunity to further integrate biotechnology with plant
breeding
• Can we generate and deploy new resistance traits
faster than the pathogen can evolve?
17. More than 30 filamentous plant pathogen genomes
sequenced
18. Screening
for
candidate
rust
effectors
in
Puccinia
graminis
and
Melampsora
larici-‐populina
Identified based on known features:
• Secreted
• Similar to haustorial proteins
• Small cysteine rich proteins
• May contain effector motif/NLS
• Encoded by genes in gene sparse regions
• Repeat-containing proteins (microbial adhesins)
• Contain PFAM domains enriched in secretomes
The in silico approach
• Reduces complexity of whole genome datasets
• Is highly flexible and can easily accommodate
new criteria
Saunders et al. PLoS One 2012
19. Identifying candidate Puccinia striiformis effectors
Integration of Puccinia striiformis (PST)
• PST secretome data derived from 5 PST races
with different virulence profiles
Additional criteria
• mRNAseq analysis of haustoria and plant
material infected with PST
• Sequence polymorphisms, presence/absence,
copy number variations, positive selection ….
20. Identifying candidate Puccinia striiformis effectors
Tribe276
Tribe300
Tribe342
Tribe63
34
Tribe1
3
Overall score
4
Tribe
68
Expression during infection
e
Cluster VII
03
Trib
e4
Expression in haustoria
Trib
Expressed during Cluster I HESP/AVR score
infection RCP proteins FIR score
RCP score
SCR score
Effector motif/NLS score
Cluster VII Members
showing
polymorphisms
Expressed Absence
of
any
members
in haustoria
Cluster II
SCRs
Cluster III
Annotated
Cluster VI
Effector motif
or NLS
Cluster IV
HESPs/AVRs
score
Cluster V low
high
Non-annotated
21. “To secure ourselves against defeat lies in our own
hands, but the opportunity of defeating the enemy
is provided by the enemy himself.”
Sun Tzu – The Art of War
Our vision
Utilize knowledge of the pathogen to develop a framework to
rapidly generate new resistance specificities and introduce these
traits into crop genomes
22. Acknowledgments
Kamoun Group @ TSL
Khaoula Belhaj
Tolga Bozkurt
Liliana Cano John Innes centre
Angela Chaparro-Garcia Cristobal Uauy
Suomeng Dong Vanesa Segovia
Artemis Giannakopoulou Albor Dobon
Krissana Kowitwanich
Vladimir Nekrasov
Sylvain Raffaele UC Davis
Maria Eugenia Segretin Jorge Dubcovsky
Joe Win Dario Cantu
Kentaro Yoshida
Wageningen UR
Vivianne Vleeshouwers
Hendrik Rietman
Sophien Kamoun