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MAJOR ADVANCES ON COFFEE RUST RESEARCH
MariadoCé[email protected]
Labs Greenhouses
The importance of coffee leaf rust as a threat to worldproduction led, in 1955, the Governments of the USA andPortugal (Agreement FO-PO-5, Project D. O. A. 72-11-004), toprovide for the foundation of CIFC in Portugal
CIFC– CentroInv.Ferrugens doCafeeiro (CoffeeRustsResearchCentre)
CIFC’smainobjectivesToprovideacentrefor international cooperationoncoffeeleafrust(CLR)researchandpre-breedingoutside thecoffeegrowingregionsandsoavoid
inadvertently spreadingofnewvirulentracestocoffeecountries
Morethan 3.000m2 of heated greenhouses
www.isa.ulisboa.pt/en/cifc
Researchers:AnaPaulaPereira,DoraBatista,Helena Azinheira,LeonorGuimarães,MariadoCéuSilva,PedroTalhinhas,Vitor Várzea
CIFCCollaborations – Coffee Leaf Rust Research
Anne-SophiePetitotDianaFernandezMichelNicolePhilippeLashermes
Sebastien Duplessis
FernandoCardoso
CândidoP.RicardoCarlaPinheiroRitaAbranches
Inês Chaves
Laércio ZambolimVagner T. Queiroz
Mário L. ResendeKátia Possa
Danielle R. Barros
Jenny RenautSébastien Planchon
LuxembourgInstituteofScienceandTechnology
Elijah GichuruRalf VoegeleTobiasLink
OctávioPaulo
James TeriD.Kilambo
Dr.Y.RaghuramuluNayani Prakash
Hongbo ZhangLiJinhong
Uthai NoppakoonwongChatnapa Khomarwut
Alvaro Gaitan
Molecularmarkers associatedwith Hv patotypesPopulation genomics
Hv PathogenicityMicroscopy,transcriptomics,
functional genomics
Coffee ResistanceMicroscopy,biochemistry/proteomics,
transcriptomics
Screening of disease resistanceSpectrumof coffee resistance to
CIFC’s rust races
H.vastatrix (Hv) and coffee uniqueworldcollections
CIFCResearchTopicsPathogenicity surveys
Spectrumof rust virulence onasetofcoffeedifferentials
Pathogenicity Surveys &Disease Resistance ScreeningMain Results of CIFCactivities
ØIdentification ofnineresistancegenes(SH1–SH9)oncoffeeandthematchingofvirulencegenes (v1– v9)inHvØA decisive step forward in the research programme of the CIFC was the discovery, inthe late 1950´s, of the Timor Hybrid (HDT), a spontaneous natural hybrid betweenArabica and Robusta
SomeHDTs ,with resistance toall known rust races,were used assources of resistance in the breedingprogrammes originating varieties such asCATIMOR,
SARCHIMOR
Both HDT and its derivatives as well as all the available coffeematerials of CIFC have been provided free of charge to allcoffee growing countries of the world
ØThegene-for-genetheoryisappliedtocoffee- H.vastatrix (Hv)interactions
CATIMOR,China
Morethan 90%of Arabica coffee varietiesresistant torust grown indifferentcoffee growing countrieswere created from studies carried outat CIFC
CATIMOR=Variety CaturraxHDT(CIFC832/1)
SARCHIMOR=Variety Villa Sarchi xHDT(CIFC832/2)
Rodrigueset al.(1975)AnnualReviewofPhytopathology 13:49-70.Bettencourt&Rodrigues Jr.(1988).InR.J.Clarke,&R.Macrae (Eds.),CoffeeAgronomyVol.4(pp.199-234).
Recentfindings
ØThe breakdown of resistance of several resistant cultivars in some coffeegrowing countries is associated with the increase of virulence in rust populations(new rust races)
Ø More than 50 rust races were already characterized
PerspectivesIn the near future new sources of resistance must be identified
Pathogenicity Surveys &Disease Resistance Screening
Varzea &Marques(2005) (InL.Zambolim,E.Zambolim,&V.M.P.Várzea (Eds.),Durableresistancetocoffeeleafrust(pp.53–74).Várzeaetal.(2009). Proc.22ndInt.ConfCoffeeSci (ASIC),1424-1429.Diniz etal.(2012)EuropeanJournalofPlantPathology 133:261-277
ØSome rust races (from Timor), recently characterized, revealed ability to attack all thesources of resistance used in the creation of the majority of resistant cultivars (Catimorand Sarchimor) including the original clone of HDT (CIFC’s collection)
IdentificationofmolecularmarkersassociatedwithH.vastatrix race-specificpathotypes
Differentapproachesarebeingapplied,mainly:
GenotypingofSSR(microsatellites) andAFLP
(Amplified FragmentLengthPolymorphism) loci
PopulationgenomicsusingNextGenerationSequencing(NGS)
tools
Analysisofpopulationgenotypicprofiles
DetectionofSNPmarkers(SingleNucleotidePolymorphism) andanalysisofpopulationgenotypic
profiles
Testforstatisticallysignificantcorrelationsbetweenspecificmarkergenotypesand
virulenceprofiles
Validation
Markersputativelyassociated
ResultsVerylowSSRpolymorphismNocorrelation foundfor
AFLPmarkers
Ongoinganalysisof110Hvisolatesfrom20 countries,differentvirulenceprofiles,collectionyearsandcoffee
hosts
Preliminaryresultswithinasub-sampleIdentificationofSNPsdifferentiatingHv
populationgroups
Silvaetal.(2015).Proc.25th Int.ConfCoffeeSci (ASIC), pb248
Anchor
A
Rijoet al.(1990)RevistadeCiênciasAgrárias XIII:169-178;Silvaet al.(1999)Int.JournalPlantSciences 160(1):79-91.Silvaet al.(2006)Braz JPlantPhysiol 18: 119-147
Germinateduredosporeandappressorium Appressorium
over stomata
Penetration hypha
Haustorial mothercell with haustoria
Intercellular hyphaewith haustoria
Uredosporic sorum
Rust fungi are biotrophic pathogens with a complex life cycle. They have evolved specialized
structures, haustoria, formed within the living host cells to efficiently acquire nutrients.
Large sporulatingpustules21days after inoculation
H.vastatrix Pathogenicity
Susceptibility:in themajority of the
infection sites(70-80%)the fungus growswithout apparent
inhibition
H.vastatrix Pathogenicity
Intensesignalling,transportandsecretoryactivitywereidentifiedingermtubes;thissuggeststheonsetofaplant-fungusdialogueasearlyasatthegermtubestage
Up-regulationofbiogenesis,lipidtransportandenergyproductionduringapressorium formation
DNAreplicationandcelldivisiontranscriptsincreaseattheonsetofsporulation
From9234genesreported,516werepredictedtoencodesecreted proteins(candidateeffectors),particularlyingermtubesandhaustoria
Transcriptomic analysis
From454Pyrosequencing andqPCR (quantitativepolymerase chainreaction)
Fernandezetal.(2012)MolecularPlantPathology 13:17-37.Vieiraetal.(2012)EuropeanJournalofPlantPathology133:261-277.Gonçalveset al.(2013)ActaPhytopathologica Sinica43(Suppl):335.Talhinhas etal.(2014)FrontiersinPlantScience5:88.Loureiro etal.(2015)Proc.25th Int.ConfCoffeeSci (ASIC),Colombia,pb250
Effectorsarepathogen-producedproteinsthatinduceorsuppressplantresponses
These candidateeffectorswillbefurthervalidatedandusedtoidentifytherespectivehosttargets formarkerassisted selection
Haustorial mothercell with haustoria
Anchor
A
Germinateduredosporeandappressorium Appressorium
over stomata
Penetration hypha
Intercellular hyphaewith haustoria
Uredosporic sorum
PrehaustorialResistance
PosthaustorialResistance
Large sporulatingpustules
CoffeeResistance
Previous cytological studies have shown that host plants with high levels of prehaustorialdefences to rust fungi may be sources of more durable resistance
Resistance – Restricted fungalgrowthMicroscopicanalysis
Reaction flt
Niks and Rubiales (2002) Euphytica 124: 201-216. Fernandez-Aparicio etal. (2011)Annals of Applied Biology159: 93-98.Diniz et al. (2012) European Journal of PlantPathology 133: 261-277
A
0
20
40
60
80
100
1 2 3 4 7%infectionsitesw
ithHR
Days after inoculation
CI1I2
C= Compatible interaction =Susceptibility
I1=Incompatible interaction - PrehaustorialResistance
I2=Incompatible interaction - PosthaustorialResistance
Silvaet al.(2002)PhysiolMolPlantPathol 60(4):169-183;Guerra-Guimarães L.(2004).PhDThesis,FCUL
Pre- and Posthaustorial Resistance is associated with rapid plant cell death(Hypersensitive Reaction - HR)
A- Appressorium
ØEarly accumulation of phenolic-like compounds
LM:Epifluorescence test
41hai 66hai
A
A
CoffeeResistanceMicroscopicanalysis
Silvaet al.(2002)PhysiolMolPlantPathol 60(4):169-183
0
1
2
3
4
5
6
7
8
9
0 20 40 60 80 100 120 140 160 180
CT
0
1
2
3
4
5
6
7
0 20 40 60 80 100 120 140 160 180
I1T
0123456789
0 20 40 60 80 100 120 140 160 180
Hoursafterinoculation
I2
T
Activity
ofPA
L(mgtrans-cinnam
icacidh-1g-1dryweight)
ØEarly accumulation of phenolic-like compounds
CoffeeResistance
LM:Epifluorescence testA- Appressorium
Phenylalanine ammonia-lyase (PAL) activity
H
H
H
First enzyme of the phenylpropanoid pathway
Biochemicalanalysis
41hai 66hai
A
A
Other enzymes involved inthe resistance response:
Maxemiuc-Naccache etal.(1992).RevBrasBot;15:145-150.Rojasetal.(1993).PhysiolMol Plant Pathol43:209-219.Silvaetal.(2008)Physiol MolPlantPathol 72: 29-38;Guerra-Guimarães et al.(2009),ProceedingsASIC,1036-1039pp ;Guerra-Guimarães et al.(2009)Biol.Plantarum 53:702-6
ØSuperoxide dismutases
ØLipoxigenases
ØPeroxidases
ØChitinases
ØGlucanases
CoffeeResistance
36 hai
96 hai
Biochemicalanalysis
CoffeeResistance
ControlResistantSusceptible
(24,48,72and 96hai)
2-DEelectrophoresis
Circled spots changed in abundancebetween samples (Anova p-value < 0,05and fold change >1,5)
Proteins of the extracellular spacevacuum infiltration
Guerra-Guimarães et al.(2015).Front.PlantSci.6:478.
performedforthespotswhosevolumesignificantlychangedinabundance
Principal ComponentAnalysis(PCA)
C
S
R
Proteinsup-regulated intheresistant genotypeandidentified as:Chitinases, Pectinmethylesterase, Serinecarboxypeptidase, Reticuline oxidase,Subtilisin proteasewereselected asantigensfor antibodyproductionagainsttheseputativeproteinbiomarkersforresistance.
116proteins were identify by MS(Mass Spectrometry)
Proteomicanalysis
The reliability of these putative resistant biomarkers are being tested in differentcoffee genotypes from CIFC collection. These antibodies will be used to developan ELISA kit assay, as a potential diagnostic tool to assist in the selection ofvarieties with resistance to H. vastatrix.
Higherlevelofdetectionintheresistantsamples
Guerra-Guimarães et al.(2015)Front.PlantSci.6:478
CoffeeResistanceProteomicanalysis
Different approaches (e.g. suppression subtractivehybridisation method,454pyrosequencing andqPCR)allowedtheidentification ofseveralgenesputatively involvedincoffeeresistance(e.g. receptor-like kinases,WRKYtranscriptionfactors, chitinases, PRs,lipoxygenase,germin-like)
CoffeeResistanceTranscriptomic analysis
Expression profileof Ca_22853aGermin-like protein previously described
(proteomic studies)asup-regulated inincompatible interaction
Relativeexpression
§ Incompatible
§ Compatible
Time after inoculation
Fernandezetal.(2004).MolecularPlantPathology 5:527-536.Ganesh etal.2006.PlantScience 170:1045-1051.Fernandezetal.(2004).MolecularPlantPathology 5:527-536.Ganesh etal.2006.PlantScience 170:1045-1051.Azinheiraet al 2013;XIIICongressoLuso-EspanholdeFisiologiaVegetal,Lisboa.
In the near future new sources of resistancemust be identified and characterized
combiningthescreening testswiththestudyofcoffeeresistancemechanisms alongwiththecharacterizationofpathogeneffectors
andgenetic variability
MolecularMarkers Associatedwith Hv patotypes
Hv Pathogenicity
Coffee Resistance
Disease Resistance Screening
H.vastatrix (Hv) and coffeeuniqueworldcollections
ConclusionsandPerspectives
Pathogenicity SurveysMorethan 50rust raceswere characterized
(time consuming work)
The breakdown of resistance of HDTderivatives isassociated with the appearance of new rust races
Prehaustorial resistance ==moredurable resistance
Early plant responsesassociated:HR,accumulation of phenolic-likecompounds and up-regulation ofgenes and proteins putativelyinvolved in plant defenses
IdentificationofSNPsdifferentiatingHv virulenceprofiles
Discovery of fungal effectors astools touncoverR-genes
THANKYOU