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 genomesSarah Gurr, University of Oxford Kupferchmidt, Science (337) 2012.
Phytophthora spp. – The "Plant Destroyers" Cocoa Sudden oak death P. palmivora P. ramorum Soybean Fruit rot• Most important pathogens of dicot P. sojae P. capsiciplants Late blight• P. infestans most destructive oncrops – up to $6.7 billion in croplosses annually• Potato: third most important foodcrop, critical to feeding the poor P. infestans
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
Durable potato blight resistance?• Classical approaches are ‘blind’ – R genes bred anddeployed without knowledge of the effectors they aresensing• Effectoromics – using core effector set of P. infestansto identify natural R genes from wild potato germplasm• Synthetic (non-natural) R genes – expandingrecognition 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
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
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
AVR effectors of P. infestans• AVR1 and AVR4are dispensable• AVR2, AVR3a andAVRblb2 are alwayspresent andexpressed;polymorphic families Vleeshouwers et al. Annu Rev Phytopathol 2011
Effectoromics for durable blight resistance Agro-infiltrationIdentification 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 genomeFunctional screening• Effectors cloned into expression vectors and expressed in planta byagro-infiltration Vleeshouwers et al. Annu Rev Phytopathol 2011
Effectoromics for durable blight resistance (i) Cosegregation F1 (ii) Coinfiltration• Focusing on cloning and breeding R genes that recognize "core" P. infestanseffectors, we maximize the potential for resistance durability in the field Vleeshouwers et al. Annu Rev Phytopathol 2011
Effectoromics for durable blight resistanceHendrik Rietman Effectorset al. Wageningen HR +++ HR + No response Not tested
Synthetic R genes with expanded effector sensing Mutagenesis - R3a + R3a AVR3aKI AVR3aEM Agro-mediated gene expressionMaria Eugenia Segretin
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 specificityHD 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 AMoscou et al.; Boch et al. Science 2009Marton et al. Plant Physiol 2010 Vladimir Nekrasov
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
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?
More than 30 filamentous plant pathogen genomes sequenced
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
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 ….
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 VIEffector motif or NLS Cluster IV HESPs/AVRs score Cluster V low high Non-annotated
“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
AcknowledgmentsKamoun Group @ TSLKhaoula BelhajTolga BozkurtLiliana Cano John Innes centreAngela Chaparro-Garcia Cristobal UauySuomeng Dong Vanesa SegoviaArtemis Giannakopoulou Albor DobonKrissana KowitwanichVladimir NekrasovSylvain Raffaele UC DavisMaria Eugenia Segretin Jorge DubcovskyJoe Win Dario CantuKentaro Yoshida Wageningen UR Vivianne Vleeshouwers Hendrik Rietman Sophien Kamoun