Major advances have been made in coffee rust (Hemileia vastatrix) research at the Coffee Rust Research Centre (CIFC) in Portugal since 1955. CIFC aims to study coffee rust outside of coffee growing regions to avoid spreading new virulent races. Through pathogenicity surveys and disease resistance screening, CIFC has identified nine resistance genes (SH1-SH9) in coffee and matching virulence genes (v1-v9) in the rust pathogen. A key discovery was the identification of the Timor Hybrid, which has resistance to all known rust races and was used to breed resistant varieties such as Catimor and Sarchimor. Current research at CIFC includes characterizing the genetic variability of rust

1. MAJOR ADVANCES ON
COFFEE RUST RESEARCH
Maria do Céu Silva
mariaceusilva@isa.ulisboa.pt

2. Labs
Greenhouses
The importance of coffee leaf rust as a threat to world
production led, in 1955, the Governments of the USA and
Portugal (Agreement FO-PO-5, Project D. O. A. 72-11-004), to
provide for the foundation of CIFC in Portugal
CIFC – Centro Inv. Ferrugens do Cafeeiro (Coffee Rusts Research Centre)
CIFC’s main objectives
To provide a centre for international cooperation on coffee leaf rust (CLR)
research and pre-breeding outside the coffee growing regions and so avoid
inadvertently spreading of new virulent races to coffee countries
More than 3.000m2 of heated greenhouses
www.isa.ulisboa.pt/en/cifc

3. Researchers: Ana Paula Pereira, Dora Batista, Helena Azinheira, Leonor
Guimarães, Maria do Céu Silva, Pedro Talhinhas, Vitor Várzea
CIFC Collaborations – Coffee Leaf Rust Research
Anne-Sophie Petitot
Diana Fernandez
Michel Nicole
Philippe Lashermes
Sebastien Duplessis
Fernando Cardoso
Cândido P. Ricardo
Carla Pinheiro
Rita Abranches
Inês Chaves
Laércio ZambolimVagner T. Queiroz
Mário L. Resende
Kátia Possa
Danielle R. Barros
Jenny Renaut
Sébastien Planchon
Luxembourg Institute of Science and Technology
Elijah Gichuru
Ralf Voegele
Tobias Link
Octávio Paulo
James Teri
D. Kilambo
Dr. Y. Raghuramulu
Nayani Prakash
Hongbo Zhang
Li Jinhong
Uthai Noppakoonwong
Chatnapa Khomarwut
Alvaro Gaitan

4. Molecular markers associated
with Hv patotypes
Population genomics
Hv Pathogenicity
Microscopy, transcriptomics,
functional genomics
Coffee Resistance
Microscopy, biochemistry/proteomics,
transcriptomics
Screening of disease resistance
Spectrum of coffee resistance to
CIFC’s rust races
H. vastatrix (Hv) and coffee unique world collections
CIFC Research Topics
Pathogenicity surveys
Spectrum of rust virulence on a set
of coffee differentials

5. Pathogenicity Surveys & Disease Resistance Screening
Main Results of CIFC activities
ØIdentification of nine resistance genes (SH1 –SH9) on coffee and the matching of virulence
genes (v1 – v9) in Hv
ØA decisive step forward in the research programme of the CIFC was the discovery, in
the late 1950´s, of the Timor Hybrid (HDT), a spontaneous natural hybrid between
Arabica and Robusta
Some HDTs , with resistanceto all known rust races,
were used as sources of resistance in the breeding
programmes originating varieties such as CATIMOR,
SARCHIMOR
Both HDT and its derivatives as well as all the available coffee
materials of CIFC have been provided free of charge to all
coffee growing countries of the world
ØThe gene-for-gene theory is applied to coffee - H. vastatrix (Hv) interactions
CATIMOR, China
More than 90% of Arabica coffee varieties resistant to rust grown in different
coffee growing countries were created from studies carried out at CIFC
CATIMOR = Variety Caturra x HDT (CIFC 832/1)
SARCHIMOR = Variety Villa Sarchi x HDT (CIFC 832/2)
Rodrigues et al. (1975) Annual Review of Phytopathology 13: 49-70.Bettencourt & RodriguesJr. (1988). In R. J.
Clarke, & R. Macrae (Eds.), Coffee Agronomy Vol. 4 (pp.199-234).

6. Recent findings
ØThe breakdown of resistance of several resistant cultivars in some coffee
growing countries is associated with the increase of virulence in rust populations
(new rust races)
Ø More than 50 rust races were already characterized
Perspectives
In the near future new sources of resistance must be identified
Pathogenicity Surveys & Disease Resistance Screening
Varzea & Marques (2005) (In L. Zambolim, E. Zambolim, & V. M. P. Várzea (Eds.), Durable resistance to coffee leaf rust
(pp.53–74). Várzeaet al. (2009).Proc. 22nd Int. Conf Coffee Sci (ASIC), 1424-1429. Dinizet al. (2012) European Journal of
Plant Pathology 133: 261-277
ØSome rust races (from Timor), recently characterized, revealed ability to attack all the
sources of resistance used in the creation of the majority of resistant cultivars (Catimor
and Sarchimor) including the original clone of HDT (CIFC’s collection)

7. Identification of molecular markers associated with H.
vastatrix race-specific pathotypes
Different approaches are being applied, mainly:
Genotyping of SSR
(microsatellites) and AFLP
(Amplified Fragment
Length Polymorphism) loci
Population genomics
using Next Generation
Sequencing (NGS)
tools
Analysis of population genotypic
profiles
Detection of SNP markers (Single
Nucleotide Polymorphism) and
analysis of population genotypic
profiles
Test for statistically significant correlations
between specific marker genotypes and
virulence profiles
Validation
Markers putatively
associated
Results
Very low SSR polymorphism
No correlation found for
AFLP markers
Ongoing analysis of 110 Hv
isolates from 20 countries,
different virulence profiles,
collection years and coffee
hosts
Preliminary results within a sub-sample
Identification of SNPs differentiating Hv
population groups
Silva et al. (2015). Proc. 25th Int. Conf Coffee Sci (ASIC), pb248

8. Anchor
A
Rijo et al. (1990) Revista de Ciências Agrárias XIII: 169-178; Silva et al. (1999) Int. Journal Plant Sciences 160 (1): 79-91.
Silva et al. (2006) Braz J Plant Physiol 18: 119-147
Germinated uredospore
and appressorium
Appressorium
over stomata
Penetration hypha
Haustorial mother
cell with haustoria
Intercellular hyphae
with haustoria
Uredosporicsorum
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 sporulatingpustules
21 daysafter inoculation
H. vastatrix Pathogenicity
Susceptibility: in the
majority of the
infection sites (70-80%)
the fungus grows
without apparent
inhibition

9. H. vastatrix Pathogenicity
Intense signalling, transport and
secretory activity were identified
in germ tubes; this suggests the
onset of a plant-fungus dialogue
as early as at the germ tube stage
Up-regulation of biogenesis, lipid
transport and energy production
during apressorium formation
DNA replication and cell
division transcripts increase
at the onset of sporulation
From 9234 genes reported, 516 were predicted to encode secreted proteins
(candidate effectors), particularly in germ tubes and haustoria
Transcriptomic analysis
From 454 Pyrosequencing and qPCR (quantitative polymerase chain reaction)
Fernandez et al. (2012) Molecular Plant Pathology 13: 17-37. Vieira et al. (2012) European Journal of Plant Pathology
133: 261-277. Gonçalves et al. (2013) Acta PhytopathologicaSinica43 (Suppl): 335. Talhinhas et al. (2014) Frontiers in
Plant Science 5: 88 . Loureiro et al. (2015) Proc. 25th Int. Conf Coffee Sci (ASIC), Colombia, pb250
Effectorsare pathogen-
produced proteins that
induce or suppress
plant responses
These candidate effectors will be further validated and used to
identify the respective host targets for marker assistedselection

10. Haustorial mother
cell with haustoria
Anchor
A
Germinated uredospore
and appressorium Appressorium
over stomata
Penetration hypha
Intercellular hyphae
with haustoria
Uredosporicsorum
Prehaustorial
Resistance
Posthaustorial
Resistance
Large sporulating
pustules
Coffee Resistance
Previous cytological studies have shown that host plants with high levels of prehaustorial
defences to rust fungi may be sources of more durable resistance
Resistance – Restricted fungal growth
Microscopic analysis
Reaction flt
Niks and Rubiales (2002) Euphytica 124: 201-216.Fernandez-Aparicio etal.(2011) Annalsof Applied Biology159:93-98.
Dinizet al.(2012) European Journal of PlantPathology 133:261-277

11. A
0
20
40
60
80
100
1 2 3 4 7
%infectionsites withHR
Days afterinoculation
C
I1
I2
C= Compatible interaction =Susceptibility
I1= Incompatible interaction- PrehaustorialResistance
I2= Incompatible interaction- PosthaustorialResistance
Silva et al. (2002) Physiol Mol Plant Pathol 60 (4): 169-183; Guerra-GuimarãesL. (2004). PhD Thesis, FCUL
Pre- and Posthaustorial Resistance is associated with rapid plant cell death
(Hypersensitive Reaction - HR)
A - Appressorium
ØEarly accumulation of phenolic-like compounds
LM: Epifluorescencetest
41 hai 66 hai
A
A
Coffee Resistance
Microscopic analysis

12. Silva et al. (2002) Physiol Mol Plant Pathol 60 (4): 169-183
0
1
2
3
4
5
6
7
8
9
0 20 40 60 80 100 120 140 160 180
C
T
0
1
2
3
4
5
6
7
0 20 40 60 80 100 120 140 160 180
I1
T
0
1
2
3
4
5
6
7
8
9
0 20 40 60 80 100 120 140 160 180
Hours after inoculation
I2
T
ActivityofPAL (mg trans-cinnamicacidh-1
g-1
dryweight)
ØEarly accumulation of phenolic-like compounds
Coffee Resistance
LM: Epifluorescencetest
A - Appressorium
Phenylalanine ammonia-lyase (PAL) activity
H
H
H
First enzyme of the phenylpropanoid pathway
Biochemical analysis
41 hai 66 hai
A
A

13. Other enzymes involved in the resistance response:
Maxemiuc-Naccache et al. (1992). Rev Bras Bot; 15:145-150. Rojas et al. (1993). PhysiolMol Plant Pathol43: 209-219. Silva et al. (2008) Physiol Mol
Plant Pathol 72: 29-38; Guerra-Guimarães et al. (2009), Proceedings ASIC,1036-1039 pp ; Guerra-Guimarães et al. (2009) Biol. Plantarum 53:702-6
ØSuperoxide dismutases
ØLipoxigenases
ØPeroxidases
ØChitinases
ØGlucanases
Coffee Resistance
36 hai
96 hai
Biochemical analysis

14. Coffee Resistance
Control
Resistant
Susceptible
(24, 48, 72 and 96hai)
2-DE electrophoresis
Circled spots changed in abundance
between samples (Anova p-value < 0,05
and fold change >1,5)
Proteins of the extracellular space
vacuum infiltration
Guerra-Guimarães et al. (2015). Front. PlantSci. 6:478.
performed for
the spots
whose volume
significantly
changed in
abundance
Principal Component Analysis (PCA)
C
S
R
Proteins up-regulated in the resistant genotype and
identified as: Chitinases, Pectin methylesterase, Serine
carboxypeptidase, Reticuline oxidase, Subtilisin protease
were selected as antigens for antibody production
against these putative protein biomarkers
for resistance.
116 proteins were identify by MS
(Mass Spectrometry)
Proteomic analysis

15. The reliability of these putative resistant biomarkers are being tested in different
coffee genotypes from CIFC collection. These antibodies will be used to develop
an ELISA kit assay, as a potential diagnostic tool to assist in the selection of
varieties with resistance to H. vastatrix.
Higher level of detection in the resistant samples
Guerra-Guimarães et al. (2015) Front. PlantSci. 6:478
Coffee Resistance
Proteomic analysis

16. Different approaches (e.g. suppression subtractive
hybridisation method, 454 pyrosequencing and
qPCR) allowed the identification of several genes
putatively involved in coffee resistance
(e.g. receptor-like kinases, WRKY transcription
factors, chitinases, PRs, lipoxygenase, germin-like)
Coffee Resistance
Transcriptomic analysis
Expression profileof Ca_22853 a Germin-
like protein previously described
(proteomic studies) as up-regulated in
incompatible interaction
Relativeexpression
§ Incompatible
§ Compatible
Time after inoculation
Fernandez et al. (2004). Molecular Plant Pathology 5: 527-536. Ganesh et al. 2006. Plant Science 170:1045-1051. Fernandez et al.
(2004). Molecular Plant Pathology 5: 527-536. Ganesh et al. 2006. Plant Science 170:1045-1051. Azinheira et al 2013; XIII Congresso
Luso-Espanhol de Fisiologia Vegetal, Lisboa.

17. In the near future new sources of resistance
must be identified and characterized
combining the screening tests with the study
of coffee resistance mechanisms along with
the characterization of pathogen effectors
and genetic variability
Molecular Markers Associated
with Hv patotypes
Hv Pathogenicity
Coffee Resistance
Disease Resistance Screening
H. vastatrix (Hv) and coffee
unique world collections
Conclusions and Perspectives
Pathogenicity Surveys
More than 50 rust races were characterized
(time consuming work)
The breakdown of resistance of HDT derivatives is
associated with the appearance of new rust races
Prehaustorial resistance =
= more durable resistance
Early plant responses associated:
HR, accumulation of phenolic-like
compounds and up-regulation of
genes and proteins putatively
involved in plant defenses
Identification of SNPs differentiating Hv virulence profiles
Discovery of fungal effectors as
tools to uncover R-genes
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