Exploiting pathogens' tricks of the trade for engineering of plant disease resistance: challenges and opportunities.

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by  Grant, Murray R

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Exploiting pathogens’ tricks of the trade for
engineering of plant disease resistance:
challenges and opportunities
MurrayR.Grant,1*KemalKazan2and still have substantial progress to make in understanding
JohnM.Manners2 how pathogen effectors would work. However, emerging
1CollegeofLifeandEnvironmentalSciences,University evidence suggests that a remarkably diverse range of
ofExeter,Exeter,StockerRoad,ExeterEX44QD,UK. plant processes can be potentially targeted by these
2CSIROPlantIndustry,QueenslandBioscience effectors. Identifying the structure of the molecular net-
Precinct,Brisbane,Qld4069,Australia. worksunderpinningthetwokeyplantdefenceprocesses;
effector triggered immunity (ETI) and pathogen-
associated molecular pattern (PAMP) triggered immunity
Summary
(PTI) would require substantial new efforts. Despite this,
With expansion of our understanding of pathogen recent insights into pathogen effector function provide
effector strategies and the multiplicity of their host new foundations for revisiting and reshaping biotechno-
targets,itisbecomingevidentthatnovelapproaches logical approaches to crop protection. This review will
toengineeringbroad-spectrumresistanceneedtobe address our current understanding of pathogen infection
deployed.Theincreasingavailabilityofhightemporal processesfromaglobalperspective,drawingonalimited
gene expression data of a range of plant–microbe selection of key examples of the defence networks tar-
interactionsenablesthejudiciouschoicesofpromot- geted by pathogens to illustrate both the complexity and
ers to fine-tune timing and magnitude of expression underlyingcommunalityinpathogenvirulencestrategies.
under specified stress conditions. We can therefore We briefly examine opportunities and challenges in
contemplate engineering a range of transgenic lines genetic-based disease intervention strategies and
designed to interfere with pathogen virulence strate- discuss the possibility of solutions that precisely target a
gies that target plant hormone signalling or deploy universal pathogen virulence strategy, i.e. modulation of
specific disease resistance genes. An advantage of plant hormone signalling networks. Specifically, we raise
such an approach is that hormonal signalling is the following questions. First, can we engineer plants to
genericsoifthisstrategyiseffective,itcanbeeasily overcome pathogen virulence strategies by targeted
implementedinarangeofcropspecies.Additionally, intervention of effector-mediated transcriptional repro-
multiple re-wired lines can be crossed to develop gramming? And second, based upon a systems level
moreeffectiveresponsestopathogens. understanding of plant hormone signalling during infec-
tion, can we intelligently design strategies to attenuate
pathogenvirulenceandthereforedevelopaframeworkfor
Introduction
generatingbroad-spectrumpathogen-resistantcrops?
Recenteffortsinsequencingofpathogengenomeshave
revealed numerous new insights into the processes Ageneralizedmoleculardescriptionofplant
employed by plant pathogens. One of such insights was diseaseresistance
the identification of surprisingly large numbers of candi-
Concomitant with entry into the host either through
dateeffectorproteinsencodedbypathogengenomes.We
stomata,woundsorviaaspecializedhaustorialstructure,
the pathogen betrays its presence through surface-
Received 28 October, 2012; accepted 17 November, 2012. *For exposed pathogen/microbe-associated molecular pat-
correspondence. E-mail [email protected]; Tel. (+44) 013
terns(P/MAMPs),suchasfungalchitin,bacterialflagellin,
92269166;Fax[phone].
MicrobialBiotechnology(2013)6(3),212–222 peptidoglycansorlipopolysaccharides(LPS).Thesemol-
doi:10.1111/1751-7915.12017 eculesactivatespecificplantpatternrecognitionreceptor-
FundingInformationSomeideasinthisreviewhaveevolvedfrom
like kinases (PRRs) (Jones and Dangl, 2006). PRRs,
British Biotechnology and Science Research Council-funded Grant
BB/F005903/1. oftenoftheleucine-richrepeat(LLR)orlysin-motif(LysM)
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd.Thisisanopenaccessarticleunder
thetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedthe
originalworkisproperlycited.
Engineeringplantdiseaseresistance 213
domain class, function as part of an immune recognition Whole-genomesequencinghasprovidedaninvaluable
complex that perceives PAMPs and the signal is then experimental resource with a plethora of pathogen
transduced to downstream components through a phos- genomes are now available (e.g. http://cpgr.plantbiology.
phorylationcascade,leadingtoactivatedbasaldefences msu.edu/). However, rather than simplifying our under-
(Greeffetal.,2012).Successfulpathogenshaveeffector standing,comparativegenomicshasrevealedthatpatho-
complements evolved to suppress PTI. However, ETI, gen effector complements are diverse and, depending
typicallyorchestratedthroughtheactivitiesofcytoplasmi- upon the pathogen, either markedly reduced, such as in
cally localized R proteins of the NBS-LRR (nucleotide thepseudomonads,orexpanded,mostnotablyinoomyc-
binding site-LRR) class, recognizes the physical or bio- etes (Bozkurt etal., 2012). Indeed, ooymcete effector
chemical presence of one or more effectors, providing a complements extend to many hundreds per species and
robust, second layer of post-delivery protection. This exhibit little, if any, sequence homology although some
co-evolutionary battle selects for pathogens with effector structuralsimilarityexistsintheRxLReffectors(Boutemy
complements evolved to evade PTI and ETI recognition. etal., 2011; Win etal., 2012). While expansion is not so
In parallel, host R proteins evolve new recognition spe- evident in fungal effector repertoires (Koeck etal., 2011;
cificitiesleadingtohighlypolymorphicrepertoiresofboth Rafiqi etal., 2012), comparative genomics indicates
effectorsandRproteins. thatextensivetransposon-basedgenomeexpansionhas
occurred in powdery mildews despite little sequence
homology existing between pea (Erysiphe pisi), barley
Whatdefinesapathogen?
(Blumeria graminis) and Arabidopsis (Golovinomyces
Apathogenhastwokeygoalstoachievewheninvadinga orontii) powdery mildew isolates, indicative of strong
host; to initially disarm basal defence networks and sub- selective adaptation (Spanu etal., 2010). The consider-
sequently to liberate nutrients required for its own suste- able expansion of retro-transposon derived repetitive
nanceandmultiplication.Toachievethis,pathogenshave DNA in the Blumeria genome appears characteristic of
evolved a set of proteins and small-molecule virulence filamentous plant pathogens, particularly numerous Phy-
factors (e.g. toxins) to overcome plant defence.Asignifi- tophthora isolates, where this genome plasticity is pre-
cant proportion of current molecular plant pathology dicted to aid the emergence of new virulence traits
research focuses on the function of pathogen effectors (RaffaeleandKamoun,2012).
and over the past decade notable inroads have been Effector expansion is consistent with the complexity of
made into understanding their collective mechanisms of these pathogens’ lifestyles. The adoption of complex
action.Pathogeneffectorcomplementsarehighlyredun- infection strategies, including haustorial establishment
dant and dispensable, but are also highly evolved to and maintenance appears to have led to a remarkable
exploitadaptedhostdefences.Anelegantexampleisthe co-evolutionary adaptation of effector repertoires to spe-
phaseddeliveryofsmutfungus,Ustilagomaydis,effector cialized hosts. Despite the limited relatedness in primary
proteins into host cells during infection of maize. The sequence and effector complement, many taxonomically
expressionofsmuteffectorgenesduringinfectioniscon- distinct pathogens share similar infection strategies and
sistentwithinitialdeliveryofhighlyconservedsetofeffec- virulence mechanisms. Notably, bacterial pathogens,
tors to establish host compatibility, and subsequently despite a much reduced yet still redundant effector rep-
deployment of a more adapted effector set to modulate ertoire,successfullycausediseaseonmanyofthesame
host metabolic processes necessary for organ-specific hosts that also support oomycete and fungal infections.
tumorigenesis(Skibbeetal.,2010). Importantly,hostdefenceregulatoryhubs,suchasEDS1
(enhanceddiseasesusceptibility1),NPR1(nonexpresser
ofPRgenes1)andPAD4(phytoalexindeficient4),iden-
Doweyetunderstandwhatmakes
tifiedbygeneticscreens,arenecessaryforresistanceto
apathogenvirulent?
a range of diverse pathogens (Glazebrook, 2005). It is
There are many challenges to address before we fully possiblethatthesekeydefencecomponentsaredesired
understand pathogen virulence strategies, notwithstand- targets of effectors from multiple pathogens (Mukhtar
ing the interactions between effectors and the chemical etal.,2011).Forinstance,ArabidopsisEDS1,whichinter-
activities of the small molecules they induce to promote acts with the TIR-NB-LRR (Toll-interleukin-1 receptor-
disease. We still lack knowledge on the nature of the nucleotide binding-leucine-rich repeat) class bacterial
carbon and nitrogen compounds required for pathogen disease resistance proteins to initiate ETI, is targeted by
nutrition. One area that has received particularly little multiple effectors (e.g.AvrRps4 and HopA1) of the bac-
attention is role of host molecules that may specify the terial pathogen Pseudomonas syringae (Bhattacharjee
inductivesignalforregulatorypathwaysactivatingeffector etal., 2011; Heidrich etal., 2011; see also below). It is
cascades. therefore logical to conclude that plant defence signals
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,212–222
214 M.R.Grant,K.KazanandJ.M.Manners
converge on key host network components, which (Buelletal.,2003;Cunnacetal.,2009;2011;Kvitkoetal.,
are necessary and essential to elaborate an effective 2009;Lindebergetal.,2012)whichvariouslycontributeto
immune response. Thus, a detailed knowledge of viru- suppressionofplantdefenceandre-configurationofhost
lence mechanisms of a model pathogen will provide metabolismforpathogennutrition(Fig.1).
broad insight into the nature and diversity of general Genomic sequencing of the three major pathovar
host signalling processes targeted during disease clades represented by DC3000, P.syringae pv. syringae
progression. B728a(brownspotofbean)andP.syringaepv.phaseoli-
cola 1448A (halo blight of bean) and numerous strains
within each clade identified core effector sets (Joardar
Pseudomonassyringae;studyofamodelpathogen
etal., 2005; Vencato etal., 2006; Vinatzer etal., 2006;
illuminatesgeneralvirulencemechanisms
Studholmeetal.,2009;Baltrusetal.,2011)andatotalof
One of the best-studied plant pathogens molecularly is 57 effector families within the pangenome (Baltrus etal.,
the hemi-biotrophic P.syringae pv. tomato DC3000 2011). Unexpectedly, these strains carry markedly differ-
(DC3000),thecausalagentofbacterialspeckdiseaseof ent effector repertoires and in silico analyses provide no
tomato. DC3000 entry into plant via stomata or wounds evidence for conservation of host-specific effectors.
triggersassemblyofafunctionaltypeIIIsecretionsystem These comparative analyses did however, provide an
(T3SS), encoded by hrc/hrp (hypersensitive response intriguing insight into the impact of the host–pathogen
conserved/hypersensitive response and pathogenicity) co-evolutionary arms race, revealing the birth, death,
genes which predominately reside in two main clusters; migrationandinactivationofvariouseffectors.
the conserved effector locus (CEL) containing universal Even within pathovar clades, diverse effector reper-
and highly conserved effector genes and the exchange- toires exist. Both DC3000 and P.syringae pv. syringae
able effector locus (EEL) containing more hypervariable strain T1 cause bacterial speck disease on tomato yet
determinants (Alfano and Collmer, 2004). DC3000 deliv- shareonly14effectors(Almeidaetal.,2009)highlighting
ers 28 diverse and internally redundant effector proteins thecapacityofplantpathogenicpseudomonadstoimple-
Fig.1. Pseudomonassyringaeisusedasanexampletohighlightpathogenvirulencestrategiesduringpathogeninfectionstrategiesandthe
timingofthoseevents.Thecartoondeciphersclassicalsignallingnetworksengagedpost-PTI,followingactivationofhostpatternrecognition
receptors.Pseudomonassyringaepv.tomatoDC3000delivers28variouseffectorproteinsthroughthetypeIIIsecretionsystemintotheplant
cell.Thesecollaboratetotargethostproteins,directlyorafterpost-deliverymodifications,whichmayincludephosphorylation,acetylationor
proteolyticcleavage.EffectortargetsmayincludecomponentsofbothETIandPTI,shownashubproteins.Aseffectorsmodulatehostsignal-
lingpathways,thereisatranscriptionalreprogrammingawayfromcomponentsunderpinningactivatedbasaldefencetowardsinductionof
pathwaysthatsuppressbasaldefence,andlater,reconfigurehostmetabolismforpathogennutrition.Thisreviewproposesthatjudicious
selectionofunique,earlyhostresponsivepromoterscanbeusedtopreciselycontrolexpressionofre-engineeredcomponentsofplant
hormoneresponsenetworkstonullifypathogenvirulencestrategies.
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,212–222
Engineeringplantdiseaseresistance 215
ment a range of virulence strategies. Interestingly, T1 networks challenging. The evolved complexity of patho-
carries a full-length HopAS1 effector and is non- geninterventioninP/ETIisillustratedinthefollowingtwo
pathogenicinnon-hostArabidopsisthaliana,whereasall examples.
P.syringae strains carrying a truncated hopAS1 variant
arepathogeniconArabidopsis,indicatingasingleeffector
HopAB–amultifunctionalbacterialeffector
can contribute to restricting host range (Sohn etal.,
2012). TheDC3000effectorHopAB(AvrPtoB)bestencapsulates
UsingNicotinabenthamianaasahost,elegantdecon- the evolved multifunctional complexity of effectors.
struction and reassembly of the DC3000 effector reper- HopAB encodes multiple activities in PTI and ETI, these
toire by the Collmer lab demonstrated deletion of 15 activitiesbeingascribedtobothmodulardomainsandthe
DC3000 effectors had minimal effect on virulence but intact protein itself (He etal., 2006; Xiao etal., 2007;
identified two essential redundant effector groups, com- Shanetal.,2008;Xiangetal.,2008).Thefirst307of553
prisingAvrPto/AvrPtoB(HopAB) andAvrE/HopM1/HopR1 amino acids HopAB interact with the chitin binding LysM
(Kvitko etal., 2009; Cunnac etal., 2011), which target domainCERK1tointerferewithPTIsignalling(Gimenez-
PAMPimmunesignallingandvesicletraffickingpathways Ibanez etal., 2009; Zeng etal., 2012). This domain also
respectively. Significant progress has been made in elu- interactswiththetomatoRproteinkinasePtotoactivate
cidating the function of many of these constituent effec- ETI (Gimenez-Ibanez etal., 2009). The N-terminal 137,
tors and the emerging paradigm is that the collective butnot307aminoacidsinteractwith(i)thekinasedomain
activities of these effectors paralyse plant defences by of FLS2 and BAK1 suppressing signalling following flag-
either physical inhibition, elimination or post-translational ellinperception(Gohreetal.,2008;Shanetal.,2008)and
modificationofhostimmunityproteinsinvolvedinPTIand (ii)withthePtodiseaseresistancehomologueFen(Rose-
ETI. Detailed descriptions of these effector activities are brocketal.,2007).TheC-terminalRINGfingerandU-box
outside the scope of this review but are covered in the E3 ligase domains participate in the proteasomal degra-
following excellent reviews (Deslandes and Rivas, 2012; dationofFLS2andEFR,whereasfull-lengthHopABsup-
FengandZhou,2012;Lindebergetal.,2012). presses ETI by ubiquination of Fen, targeting it for
degradation(Gohreetal.,2008).
ChallengesinmodifyinghostPTIandETInetworks
MultipleeffectorscamouflageCladosporiumfulvumfrom
ThecomplexitiesintargetingPTInetworks
recognitionbytomato
Logic predicts targeting the apex of PAMP signalling
The fungal pathogen Cladosporium fulvum uses a
would potentially provide broad-spectrum immunity.
complexmulticomponentapproachasonetactictoevade
However,followingremarkableprogressinunderstanding
PTI. Chitin oligomers detected by PRRs are potent elici-
effector activation and effector intervention in signalling
tors of PTI (Kaku etal., 2006; Shimizu etal., 2010).
from innate immune receptor complexes, particularly
Cladosporium fulvum secretes Ecp6, a LysM chitin-
those involving the archetypal FLS2 (flagellin sensing 2)
bindingdomainprotein,whichselectivelybindschitinoli-
receptor,biotechnologicallyfeasiblesolutionsforenhanc-
gosaccharides preventing recognition by tomato PRRs
ing host resistance are less obvious. Experimental evi-
(Jonge etal., 2010). Moreover, C.fulvum secretes Avr4
dence shows that the kinase domains of FLS2/EFR
apoplastically.Avr4bindstothefungalwallchitin,prevent-
(elongation factorTu PAMPreceptor) and CERK1 (chitin
inghydrolysisbytomatochitinase(Burgetal.,2006;Esse
elicitor receptor kinase 1) exist in pre-formed immune-
etal., 2007). Cladosporium fulvum also secretes Avr2,
receptor complexes constitutively interacting with BIK1
which binds and inhibits plant extracellular cysteine pro-
(Brassinosteroid Interacting Kinase 1). Perception of the
teasesrequiredforbasaldefence(Esseetal.,2008).This
MAMPligands,elf18orflg22,recruitsthecytosolicBAK1
collectiveassaultonhostdefencesmeansitisdifficultfor
(Brassinosteroid Associated Kinase 1) and the resultant
a host to overcome multiple effector activities targeted
phosphorylation of BIK1’s activation loop initiates down-
towardscamouflagingthepathogen’spresence,thuscon-
stream signalling cascades. An emerging theme is that
tributingtothepathogendurability.
coreeffectorrepertoirestargetcomponentsoftheinnate
immune perception complex, including FLS2/CERK1/
EFR/BIK1, and downstream phosphorylation cascade, Strategiestoovercomeimmune
usingavarietyofdifferentstrategies(seeDeslandesand receptorintervention
Rivas, 2012; Feng and Zhou, 2012 for recent reviews).
DeploymentofPTI
Moreover, multiple redundant effectors from a single
pathogen can act on PTI signalling networks, and many Despite these challenges, Lacombe etal. exploited the
also target ETI processes, making re-engineering of PTI findingthattheEFRwasevolutionarilyconstrainedtothe
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,212–222
216 M.R.Grant,K.KazanandJ.M.Manners
Brassicaceae (Lacombe etal., 2010), but that bacteria cally.Asalludedtoabove,mutationsinkeyimmunecom-
from diverse genera carried the highly conserved elf18 ponents such as EDS1, NPR1 and PAD4 lead to
epitopeoftheubiquitousEF-TuproteinrecognizedEFR. enhanced susceptibility to multiple pathogens with
They explored interfamily transfer of EFR. Expression in diverse infection strategies (Glazebrook, 2005). These
Solanaceae plants N.benthamiana and tomato provided proteins must represent components of a conserved
levels of enhanced resistance to a variety of bacterial branch of the plant defence network that integrates
pathogenswithdifferentlifestyles.Mostnotable,bacterial signals from activated immune complexes. Recently,
wilt conferred by the soil living Ralstonia solanacearum EDS1 has been shown be involved in complex nuclear
was dramatically attenuated in transgenic tomato cytoplasmic signalling that involved its intimate associa-
expressingA.thalianaEFR(Lacombeetal.,2010).While tion with multiple R proteins (RPS6, SNC1, RPS4 and
promising, this strategy is restricted to identifying those other components such as the tetratricopeptide repeat
PAMP receptors that, though host specialization, have protein SRFR1) (Bhattacharjee etal., 2011; Heidrich
evolveddivergedPTIcomponents. etal., 2011). Unfortunately, the potential to use these
components for improved disease resistance may be
limited, as they appear to integrate defence signals sub-
CombinatorialRgeneapproaches
sequenttoupstreameffectorintervention.
Stacking R genes involved in recognizing the same spe- Underpinningdiseaseprogressionpostsuppressionof
cific range of pathogen isolates remains a core plant basal defence is the requirement for the pathogen to
breedingstrategy.Therobustnessofpotentialbiotechno- extract nutritional resources from its host. Evidence that
logical approaches for R-Avr interactions was demon- common mechanisms may be engaged to alter primary
strated by delivery of the Hyaloperonospora parasitica metabolism comes from the identification of SWEET
effectorproteinATR13withoomycete,bacterialandviral genes(Chenetal.,2010;2012).Sugareffluxspecifiedby
pathogens into a host carrying the cognate resistance the SWEET class of hexose bidirectional transporters
protein RPP13, resulting in defence responses that are appears to be hijacked by effectors from both bacterial
effectiveagainstallthreepathogens(Renteletal.,2008). and fungal pathogens, despite their diverse lifestyles, to
The caveat, however, is that R genes are extremely deliver carbohydrate apoplastically or via specialized
vulnerabletoasingle-lossoffunctionmutationsincorre- haustorialfeedingstructures.
spondingAvrgenes.Thus,superimposedupontheirability
to target early events in PTI, pathogens also retain the
Targetinghormones–apre-emptivestrike
inherent dispensability of the effector repertoire enabling
the luxury of discarding ‘liability’effectors without signifi- As more and more functions are being transcribed to
cantfitnesscosts,leadingtotheemergenceofnewpatho- effectors, it is becoming increasingly clear that effectors
genraceslackingtheabilitytoelicitthesignal(s)monitored cantargetmultiplehostproteins,whichappeartofunction
by the cognate R protein(s). Indeed the majority of the in unrelated pathways. Therefore, while there are many
currentlydefinedAvrgenesconstitutepartoftheP.syrin- potentialwaystointervene,pathogenshaveevolvedmul-
gae ‘variable effector repertoire’ (Cunnac etal., 2009; tiple mechanisms to promote disease. Thus, a judicious
2011;Lindebergetal.,2012)whichundergostrongdiver- approachisnecessarytoensurebroad-spectrumchoices
sifying selection to avoid detection by the host. This tooutsmartpathogens.Onepotentialstrategyistonullify
mechanismisnotrestrictedtobacterialpathogens.Multi- effector modulation of host signalling networks down-
pleallelesofflaxAvr456Lcarryingalterationsofsurface- streamofoverriddenPTIandETIdefences.Anemerging
exposed residues have been identified that evade direct theme is that diverse pathogens hijack host hormone
recognition by the L resistance genes (Ellis etal., 2007; biosynthetic or signalling pathways to overcome innate
Koecketal.,2011).InH.parasiticatheATR13allelesshow immunity and reconfigure metabolic pathways for their
extremelevelsofaminoacidpolymorphism,enablingthem nutrition. How effectors perturb hormonal signalling
to evade recognition by the highly polymorphic RPP13 remain to be clarified but all evidence suggest hormonal
resistanceproteininArabidopsis(Allenetal.,2004). perturbationunderpinsmost,ifnotall,virulencestrategies
(Robert-Seilaniantzetal.,2011).
Recently it has been shown that pathogens hijack
Convergenceofvirulencestrategies
abscisic acid (ABA) signalling pathways to promote viru-
Despite the plethora of effectors deployed by a diverse lence,andthismechanismissharedbybothnecrotrophic
range of pathogens, it is clear virulence strategies con- and biotrophic pathogens (Asselbergh, De Vleesschau-
vergeoncommonsignallingpathways.Anumberofsuch wer0026;Hofte2008;Tonetal.,2009).ABAbiosynthetic
loci, other than classical R proteins, representing core mutants show reduced susceptibility to virulent P.syrin-
defencenetworkcomponentshavebeenidentifiedgeneti- gae,whereasABAaccumulationcompromisedresistance
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,212–222
Engineeringplantdiseaseresistance 217
to biotrophs and necrotrophs such as Botrytis cinerea. gistically mimic Fusarium graminearum induced produc-
Thisstrategyappearstoberobust.Forexample,ectopic tion zealexins, acidic sesquiterpenoid phytoalexin, in
expression of HopAB induces de novoABAbiosynthesis maize. Both these phytoalexins confer antifungal activity
(Torres-Zabala etal., 2007) but pathogen induced against numerous phytopathogenic fungi at physiologi-
ABA is not attenuated following challenge with cally relevant concentrations (Huffaker etal., 2011).
DC3000DAvrPtoBDAvrPto (M. Grant, unpublished), Experiments specifically investigating the impact of
indicating multiple redundant pathways can result in hormone modulation on phytoalexin production are
enhancedABA. limited.Transgeneinducedcytokininincreasesintobacco
Othereffectorshavebeenshowntospecificallymodu- ledtoSAandJAindependentinducedresistancetoP.sy-
latephytohormonestopromotevirulence.TheP.syringae ringae pv. tobacco. Resistance was associated with the
effectorAvrBtargetsbothPTIandETIthroughphospho- induction of the phytoalexins scopoletin and capsidiol,
rylation of RIN4 (RPM1 interacting protein 4) and MPK4 which could substitute in planta for the cytokinin signal
(MAP kinase 4), which leads to the induction of JA (Grosskinskyetal.,2011;2012).
response genes, classically associated with antagonism In contrast, ABA negatively regulates the synthesis of
ofSAsignalling,andenhancedbacterialgrowth(Heetal., elicitor-inducedcapsidiolintobacco(Mialoundamaetal.,
2004;Cuietal.,2010). 2009)providinganelegantexampleofhowABAinduced
Hereweproposethatprecise,temporallyandspatially susceptibility can antagonize the key bioactives in plant
controlled modulation of pathogen induced hormonal defence.
changescouldbeaneffectivestrategytonullifypathogen
virulence.Oneoftenoverlookedroleofphytohormonesin Alternativeapproachestoachieving
defenceistheirpotentialregulationofphytoalexinproduc- robustimmunity
tion (see Grosskinsky etal., 2011; Ahuja etal., 2012 for
Re-wiringhormonalnetworks
recent reviews). Therefore, despite the species-specific
nature of many phytoalexins, a major consequence of Based upon both structural and predictive modelling it is
pathogenmodulationofhormonepathwaysislikelytobe now possible to contemplate targeting and neutralizing
restrictionofphytoalexinproduction. pathogen virulence strategies that antagonize hormone-
regulated immune pathways. This will necessitate preci-
sionre-engineeringofcorepathogen-modulatedhormone
Hormonalcontrolofphytoalexins
signallingpathwaycomponentsandre-wiringthesemodi-
The genetic and molecular revolution in plant molecular fiedsignallingcomponenttoaspecificpathogeninduced
pathology in the late 1980s effectively overlooked a promoterthatconfersearlierandstrongertemporalregu-
fundamental and critical aspect of plant defences, the lationthanitscognatewild-typepromoter.Thisapproach
exquisitelycontrolledinductionofaplethoraofplantanti- reliesuponthenaturalpathogeninfectionprocesstoacti-
microbial defence compounds derived from secondary vate the interference strategy designed to attenuate
metabolism or of proteogenic origin. The latter include pathogen virulence. To circumvent this strategy, the
antimicrobial peptides, proteinase inhibitors, chitinases, pathogen would need to reconfigure its own virulence
glucanases and the archetypal Pathogenesis-Related 1 programmewiththecollateralfitnesscosts.
protein (PR1), whose function, despite being universally Engineering a proactive response to locally increased
used as a marker of salicylic-based defence, remains hormone concentrations could be achieved by re-wiring
unknown. The secondary metabolites are derived from hormonenetworksbydeploymentofpromotersspecifying
pathways transcriptionally induced by PAMP receptor highly localized, temporally and spatially controlled,
activation,includingtheshikimate(phenylpropanoids,stil- precise responses to pathogens linked to re-engineered
benes,terpenes),isopropenoids(diterpeneandsequiter- components of hormone signalling pathways. We will
penederivatives)andvariousalkaloidpathways. first consider promoter selection, then the possible
Hormones appear to generically induce host-specific re-engineeringstrategies.
phytoalexins. For example, JA signalling largely influ-
ences camalexin production in Arabidopsis (Rowe etal., Promoter selection. Specific temporal/spatial control is
2010), exogenous application of MeJA induced stilbene the key to re-wiring host signalling pathways to nullify
accumulation (Faurie etal., 2009) and in grapevine cell pathogen virulence strategies. Transcriptional activation
culture, the combined application of sucrose and MeJA underpinspathogenvirulencestrategies,butoccurssub-
stimulatedtheaccumulationoftrans-resveratrolandres- sequent to PTI initiated phosphorylation cascades and
veratrolglucosides(Belhadjetal.,2008).EthyleneandJA post-deliveryeffectormodifications.Asyet,nosystematic
application collectively induced accumulation of maize analyses of early effector responsive genes have been
kauralexins (Schmelz etal., 2011) and could also syner- reported. Ideal candidate genes are those that are not
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,212–222
218 M.R.Grant,K.KazanandJ.M.Manners
PAMP responsive but are rapidly induced by virulent structure predict it is possible to engineer high-affinity
pathogens. In an on-going project (http://www2.warwick. ABAreceptorsthatcan‘mopup’largequantitiesofpatho-
ac.uk/fac/sci/lifesci/research/presta/) analysis of high- gen induced ABA but not bind to and inactivate PP2Cs
resolution time-course microarrays reporting P.syringae (Santiagoetal.,2012).
infectionofArabidopsisusingthevirulentDC3000strain,
acognatehrpmutantandmockchallengehasrevealeda InterferingwithABAbiosynthesis. Inadualstrategythat
large range of promoter dynamics. Thus it is possible to complementsthePYLengineering,itispossibletoabro-
identify examples of early (within 3 hpi) effector respon- gate pathogen induced ABA accumulation by re-wiring
sivegeneswhoselow/undetectablebasaltranscriptlevels ABA8′-hydroxylase(Umezawaetal.,2006)toapathogen
are induced well in excess of an order of magnitude, responsive promoter. Coupled to an effector regulated
through to promoters specifying moderate expression promoter this strategy will specifically catabolize patho-
levels which subsequently saturate the microarray. Can- geninducedABA.
didategenescanbeidentifiedshowingeithersustainedor
transientinductionthatarenotPAMP,drought,high-light,
circadian or senescence responsive, providing a tightly
Re-wiringJAresponsenetworks
controlledeffectorresponsivetranscriptionalunittodrive
re-wiringofplantdefenceresponsenetworks. JAsignalling antagonizes SAsignalling and a number of
biotrophic pathogens exploit this property to attenuate
Re-wiring ABA signalling. Hijacking ABA signalling is a host defences (Glazebrook, 2005).Astriking example is
key pathogen virulence strategy (Anderson etal., 2004; theproductionofthephytotoxicpolyketidecoronatineby
Torres-Zabala etal., 2007; Grant and Jones, 2009; many P.syringae pathovars. This virulence factor is a
Robert-Seilaniantzetal.,2011).ABAbiosyntheticmutants structural mimic of the plant bioactive JA, 3R,7S-
show reduced susceptibility to both biotrophs (e.g. P.sy- jasmonoyl-isoleucine (JA-Ile) (Fonseca etal., 2009a).
ringae)andnecrotrophs(e.g.B.cinerea).Thusprecision Pathogen coronatine production interferes with function-
re-wiringABAsignallingattheapexofthenetwork(ABA ally antagonistic SA and JA signalling networks to
perception)offerspotentialforbroad-spectrumresistance disrupt plant immune responses and confer a fitness
tobothbiotrophsandnecrotrophs. advantage (Brooks etal., 2005; Laurie-Berry etal.,
2006). The recently identified JAZ (jasmonate-ZIM
The ABA perception network. Upon ABA binding, domain-containing) proteins repress jasmonate (JA)
cytosolic pyrabactin resistance 1 (PYR1)/PYR1-like responsivetranscriptionfactors,mostnotablyAtMYC2,a
(PYL)/regulatory components of ABA receptors (RCAR) key regulator of JA responses (Anderson etal., 2004;
ABA receptors interact with the active site of the nega- Lorenzoetal.,2004;Fonsecaetal.,2009b).Inthepres-
tive regulators of ABA signalling, Clade A protein phos- ence of coronatine, JAZs are ubiquitinated by the F-box
phatase 2Cs (PP2C) and inactivates them. In the component COI1 of the E3 ubiquitin ligase complex
absence of ABA, Clade A PP2Cs interact with (SCFCOI1) and subsequently degraded by the 26S pro-
and inhibit the sucrose non-fermenting related kinase teasome, freeing AtMYC2 to activate JA signalling net-
(SNRK) kinases (Cutler etal., 2010; Weiner etal., works (Lorenzo and Solano, 2005; Chini etal., 2007;
2010). The loss of function triple mutant (snrk2.2/ Thines etal., 2007; Melotto etal., 2008; Fonseca etal.,
snrk2.3/snrk2.6)isdefectiveinallknownABAresponses 2009b).
(Fujii and Zhu, 2009). Thus, ABA/PYL-induced PP2C
inhibition depresses SnRK2s, activating downstream InterferingwiththejasmonateCOI1receptor. Differential
ABA signalling networks. Below we discuss possibilities splicing of JAZs can lead to the splice variants lacking
of re-engineering the PLY receptors and PP2C-negative the Jas domain PY motif resulting in enhanced resistant
regulators to attenuate ABA induced virulence. to ubiquitin mediated proteasomal degradation (Chung
etal., 2010).To attenuate JAsignalling and its attendant
Re-engineeringPP2CstoattenuateABAsignalling. Mu- suppression of SA defences, one could envisage
tationoftheactivesiteofselectedCladeAPP2Cs(glycine re-wiring engineered JAZ splice variants lacking the
toasparticacidsubstitution)willdisruptPYL–PP2Cinter- C-terminal Jas domain, thus generating a JAZ variant
actions (Melcher etal., 2009; Miyazono etal., 2009) but that is resistant to ubiquitin mediated proteasomal deg-
retaintheabilitytodephosphorylatetargetSNRKsthere- radation. These modified JAZs would bind to and effec-
foreinactivatingABAsignallingattwokeynodes. tively ‘poison’ COI1, preventing other JA repressors
binding, thus creating a dominant JA-insensitive pheno-
EngineeringPYLreceptorstofunctionasABA‘sponges’. typeandpreventpathogensexploitingJAsignallingpath-
Based upon the PYL/ABA/PP2C complex crystal ways through COI1.
©2013TheAuthors.PublishedbySocietyforAppliedMicrobiologyandBlackwellPublishingLtd,MicrobialBiotechnology,6,212–222
Engineeringplantdiseaseresistance 219
RevisitingthedeploymentofRgenesin egies.Pathogensfirstreconfigurethehosttranscriptome
plantdefence in a precise, temporally controlled manner. With major
progressbeingachievedinunderstandingphytohormone
The AvrBs3/PthA family of Transcription Activator Like
perception and signalling pathways, it is an opportune
(TAL) effectors are found in plant pathogenic Xan-
time to consider wiring highly specific pathogen respon-
thomonas spp. and R.solanacearum. TALs contain an
sivepromoterstore-engineeredcomponentsofhormone
acidicactivationdomain,aC-terminalnuclearlocalization
signalling to target and nullify pathogen virulence strate-
signalandacentraldomaincontainingavariablenumber
gies that antagonize hormone-regulated immune path-
of34aminoacidrepeatmodules(BochandBonas,2010;
ways. This approach relies upon the natural pathogen
Bogdanove etal., 2010). These modules contain repeat
infection process to activate the interference strategy
variable diresidues (RVD) at positions 12 and 13. Crys-
designedtoattenuatepathogenvirulence.Tocircumvent
tallizationofTALrevealedthattheserepeatmodulesfold
this strategy, the pathogen would need to reconfigure its
intotwonearlyidenticalalphahelicesconnectedbyaloop
ownvirulenceprogrammewiththecollateralfitnesscosts.
formed by the RDV.The amino acid at position 12 stabi-
Ifsuccessful,thegenericnatureofthisapproachmeansit
lizestheloopbetweenrepeatswhereastheaminoacidat
canbeimplementedacrossarangeofcropspecies,and
position 13 makes base-specific contact with the DNA
lendsitselftostackingmultiplere-wiredlinesbycrossing
sensestrand(Maketal.,2012).
toenhancepathogenresistance.
Thus the ability design TALs that target specific
Using judicious selection of promoters, a similar strat-
genomicregions,andlinkthistofunctionaldomainssuch
egydeployingprecise,temporallyandspatiallycontrolled
as nucleases holds huge biotechnological promise for
modulationofengineeredTALeffectorstargetingselected
precision/customizableengineeringandsyntheticbiology
R genes could be an alternative strategy to generate
(BogdanoveandVoytas,2011).Ofrelevancetoengineer-
broad-spectrumresistance.
ing broad-spectrum pathogen resistance, artificialTALEs
could be constructed that recognize selected disease
resistance gene promoters to activate ETI. These Acknowledgements
designertranscriptionalactivatorscouldbedrivenbythe
This review was drafted during a McMaster Fellowship
same repertoire of promoters discussed above. Like any
awarded to M.R.G. by the Office of the Chief Executive
newtechnologycaveatsapply.Notably,spatialconsidera-
(OCE),CSIRO,Australia.
tions appear to lead to RVDs having different strengths.
These issues have been recently explored (Garg etal.,
2012) and recommendation to construct reliably func-
Conflictofinterest
tionalTALEshavebeensuggested(Streubeletal.,2012)
and tools such as TAL Effector-Nucleotide Targeter Nonedeclared.
(TALE-NT) are being developed for TAL effector design
and target prediction (Doyle etal., 2012). Alternatively,
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