MAJOR ADVANCES ON COFFEE RUST RESEARCH

MariadoCéuSilvamariaceusilva@isa.ulisboa.pt

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