exploiting pathogen biology for disease resistance breeding
DESCRIPTION
Diane Saunders, The Sainsbury LabTRANSCRIPT
Exploiting pathogen biology for disease resistance breeding in
plants
Diane Saunders
BGRI 2012 technical workshop
Sarah Gurr, University of Oxford
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
Kupferchmidt, Science (337) 2012.
Phytophthora spp. – The "Plant Destroyers"
Fruit rot
P. capsici
Sudden oak death
P. ramorum
Soybean
P. sojae
Cocoa
P. palmivora
Late blight
P. infestans
• Most important pathogens of dicot plants • 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
The aggressive clonal lineage 13_A2 (blue-13)
• 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
T30-4 blue-13
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
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
effectors
bacterium
fung
us
oom
ycet
e
haustorium
plant cell
Alter plant cell processes
targets
Help microbe colonize plant
Microbes alter plant cell processes by secreting a diversity of effector molecules
Modified from: Dodds and Rathjen 2010 NAT REV GENET
effectors
bacterium
fung
us
oom
ycet
e
haustorium
Alter plant cell processes
targets
intracellular immune receptors
Some effectors “trip the wire” and activate immunity in particular plant genotypes
plant cell
Modified from: Dodds and Rathjen 2010 NAT REV GENET
effector- triggered immunity
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
Effectoromics for durable blight resistance
Vleeshouwers et al. Annu Rev Phytopathol 2011
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
Agro-infiltration
(i) Cosegregation F1
(ii) Coinfiltration
Vleeshouwers et al. Annu Rev Phytopathol 2011
Effectoromics for durable blight resistance
• Focusing on cloning and breeding R genes that recognize "core" P. infestans effectors, we maximize the potential for resistance durability in the field
Hendrik Rietman et al. Wageningen
HR +++ HR + No response Not tested
Effectoromics for durable blight resistance Effectors
Synthetic R genes with expanded effector sensing
- R3a
+ R3a
AVR3aKI AVR3aEM
Maria Eugenia Segretin
Mutagenesis
Agro-mediated gene expression
Moscou et al.; Boch et al. Science 2009 Marton et al. Plant Physiol 2010
Targeted genome mutagenesis and editing
Transcription activator-like (TAL) effectors
NLS AD N C
Repeat type: DNA base recognised:
NI HD NG NN A C T G
A
Vladimir Nekrasov
• Xanthomonas TAL effectors – directly modulate host gene expression
• Central repetitive region confers DNA-binding specificity
• Opportunity for designer DNA binding proteins
HD NI NG HD NN HD HD NI HD NG NI HD HD NN NG
A T C G C C C A C T A C C G T
DNA-‐binding domain
Target DNA
Targeted genome mutagenesis and editing
Vladimir Nekrasov
• 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
• 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?
Targeted genome mutagenesis to engineer resistant crops
More than 30 filamentous plant pathogen genomes sequenced
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
Screening for candidate rust effectors in Puccinia graminis and Melampsora larici-‐populina
Saunders et al. PLoS One 2012
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 ….
Cluster I RCP proteins
Cluster II SCRs
Cluster III Annotated
Cluster IV HESPs/AVRs
Cluster V Non-annotated
Cluster VI Effector motif
or NLS
Cluster VII Expressed in haustoria
Cluster VII Expressed during
infection
Identifying candidate Puccinia striiformis effectors
Tribe300
Tribe276
Tribe342
Tribe134
Tribe403
Tribe63
Tribe43
Tribe68Overall score
Expression during infection Expression in haustoria
HESP/AVR score FIR score
RCP score SCR score
Effector motif/NLS score Members showing polymorphisms
Absence of any members
low high
score
“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
Kamoun Group @ TSL Khaoula Belhaj Tolga Bozkurt Liliana Cano Angela Chaparro-Garcia Suomeng Dong Artemis Giannakopoulou Krissana Kowitwanich Vladimir Nekrasov Sylvain Raffaele Maria Eugenia Segretin Joe Win Kentaro Yoshida
Wageningen UR Vivianne Vleeshouwers
Hendrik Rietman
John Innes centre Cristobal Uauy Vanesa Segovia
Albor Dobon
UC Davis Jorge Dubcovsky
Dario Cantu
Sophien Kamoun
Acknowledgments