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