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Unveiling the involvement of oxidases in the resistance of
Coffea spp. to Colletotrichum kahawae
DINIZ, Inês1,2, FINO, Joana1,3, LOUREIRO, Andreia1,2, FIGUEIREDO, Andreia4,
AZINHEIRA Helena1,2, PEREIRA, A. Paula1, TALHINHAS, Pedro1,2, VÁRZEA,
Vítor1,2, GICHURU, Elijah K5, MONCADA Pilar6, OLIVEIRA, Helena2,
BATISTA, Dora1,3, GUERRA-GUIMARÃES, Leonor1,2, SILVA, Maria C.1,2
1-CIFC/Biotrop/IICT - Centro de Investigação das Ferrugens do Cafeeiro/Instituto de
Investigação Científica Tropical, Quinta do Marquês, Oeiras, Portugal; 2-CEER, Instituto
Superior de Agronomia, University of Lisbon, Tapada da Ajuda, Lisbon, Portugal, 3-
Computational Biology and Population Genomics group, Centro de Biologia Ambiental,
Faculty of Sciences, University of Lisbon, Lisbon, Portugal, 4-Plant Systems Biology Lab,
Centre for Biodiversity, Functional and Integrative Genomics, Faculty of Sciences, University
of Lisbon, Lisbon, Portugal, 6-Coffee Research Foundation (CRF), Ruiru, Kenya, 6-Cenicafe,
Manizales, Colombia.
e-mail: inesdiniz@gmail.com
SUMMARY
Cytological, biochemical and molecular studies were undertaken to elucidate the role of
oxidases in coffee resistance to Colletotrichum kahawae (Ck). Hypocotyls of the coffee
variety Catimor 88, resistant to Ck isolate Que2 (from Kenya), were used and compared
with the susceptible variety Caturra. Coffee resistance was characterized by a restricted
fungal growth associated with hypersensitive-like cell death (HR), monitored by cell
autofluorescence and/or browning. The activity of the oxidative enzymes peroxidase
(POD) and polyphenol oxidase (PPO) was evaluated. For both genotypes the activity of
POD and PPO measured in the infected tissues was, on average, higher than in control
samples. Moreover, in the resistant genotype, POD activity started to increase at 24hai
which was coincident with the beginning of the observation of HR.
At the molecular level, 33 unigenes with oxidative-related function were identified in an
Illumina RNA-seq coffee - Ck database as differentially expressed in Catimor 88 and
Caturra infected by C.kahawae comparatively with their controls, and grouped into six
main classes: multicopper, peroxidase, polyphenoloxidase, germin-like, redoxin domain
and isoflavone reductase-like protein. For 20 unigenes, a predominant expression
profile showing an increase of activation at 48 and/or 72hai in Catimor 88 when
compared to Caturra was detected. For the other 13 unigenes, the main expression
profile revealed repression at all time points, for both genotypes. Gene validation and
expression profiling is being performed through qPCR during key stages of the
infection process.
INTRODUCTION
Colletotrichum kahawae (Ck), the causal agent of Coffee Berry Disease (CBD), is
responsible for the most devastating Arabica coffee disease in Africa at high altitude,
and represents an imminent threat for coffee cultivation in America and Asia [1,2,3]. It
has long been recognized that increased knowledge on the key mechanisms of plant
resistance is necessary to breed efficiently for durable resistance. Coffee resistance to
Ck is characterized by restricted fungal growth associated with rapid hypersensitive-like
cell death (HR) [2, 4]. In different pathosystems, the rapid loss of cell integrity during
the HR, has been associated with the production of reactive oxygen species (ROS) and
an increase in oxidizing enzymes [5,6]. This work aims to elucidate the role of oxidases
in coffee resistance to Ck. Based on field resistance to Ck in Kenya, Catimor 88 (Timor
Hybrid derivative) was selected as resistant genotype to the isolate Que2 from Kenya,
comparatively with the susceptible variety Caturra.
MATERIALS AND METHODS
Hypocotyl inoculation: Coffee hypocotyls from the resistant genotype Catimor 88
(from Kenya) and the susceptible genotype Caturra (CIFC 19/1) were inoculated with
the C. kahawae isolate Que2, from Kenya according to the technique previously
described [4]. For the different studies, samples were collected at 12, 24, 48 and 72
hours after inoculation (hai).
Light microscopy: For time-course studies of fungal growth and plant cell responses, cross
sections of infected hypocotyl fragments, made with a freezing microtome, were submitted
to cotton blue lactophenol staining and epifluorescence test [4,7]. Observations were made
with a microscope Leica DM-2500 equipped with a mercury bulb HB 100W, blue light.
POD extraction and activity evaluation: Proteins were extracted from hypocotyls of both
genotypes [6] and protein content was measured using a modified Bradford assay [8].
The activity of guaiacol peroxidase (POD) and catechol polyphenol oxidase (PPO) was
determined by the increase in absorbance at 480 and 410nm, respectively [9].
Differential expression analysis from RNA-Seq data: Differential expressed unigenes
previously identified and annotated as bearing an oxidative-related function were
retrieved from an Illumina RNA-Seq database [10]. Identification of those genes
resulted from a previous analysis of RNA-seq data generated from Catimor 88 and
Caturra hypocotyls, inoculated with Ck Que2, at 24, 48 and 72hai. Only unigenes with a
posterior probability of being differentially expressed (PPDE) > 0.95 and a -1.0 ≥ log2
fold change ≥ 1.0 were considered as such.
RESULTS AND DISCUSSION
In both genotypes, the fungus began to penetrate the hypocotyl tissues by 48hai and the
hyphal length was significantly higher in the susceptible genotype than in the resistant
one, at 72hai (Fig.1 and Fig.2). As shown in Fig. 2 in the resistant genotype, the first
cytological changes were displayed at 24hai in 4% of infection sites and corresponded
to the hypersensitive-like cell death – HR (associated with the presence of
autofluorescent and/or browning cells). At 48hai and 72hai, HR spread to adjacent cells
of the epidermis and of the first layer of cortex, being observed in 16% and 36% of
infection sites, respectively. In the susceptible genotype this response was also
observed, but in a significantly lower percentage of infection sites (1%- 12%, at 24hai
and 72hai, respectively) (Fig.2, Fig.3A-C). The analysis of post-penetration fungal
growth stages and host responses were similar to those previously described for the
same coffee- Ck interactions [4].
Fig. 1. Hyphal length in resistant (Catimor 88) and susceptible (Caturra) hypocotyls, at different time points. The
mean values of hyphal length did not differ significantly at 48hai (t= 1.32) but were significantly higher in the
susceptible that in the resistant hypocotyls at 72hai (t= 5.05; P0.001). Fig. 2. Percentage of infection sites with HR.
The mean values did not differ significantly at 24hai (t= 1.33) but were significantly higher in the resistant than in
the susceptible hypocotyls at 48hai (t= 2.22; P0.05) and 72hai (t= 5.76; P0.01). Each value (Figs 1 and 2) is the
mean±standard deviation of 2 different experiences (100-150 infection sites were observed per experiment per time).
Fig. 3. Fungal post-penetration
growth stages and plant responses.
Light microscope observations,
cotton blue lactophenol staining
(A and B), epifluorescence test
under blue light (C). (A) Infection
site showing a melanised
appressorium (a) and hyphae
(arrow) inside an epidermal cell of
the susceptible hypocotyl, 48hai.
(B) Infection site showing a
melanised appressorium (a) and a
vesicle (arrow) inside the epidermal cell of the resistant hypocotyl, 72hai. (C) Infection site showing an apressorium
(a) associated with HR-like in one epidermal cell (arrow) of the resistant hypocotyls, 72hai (bars = 10µm)
For both genotypes the activity of PPO measured in the infected tissues was, on
average, higher than in control samples (data not shown). In the resistant genotype
Catimor 88 (Fig.4A) POD activity started to increase by 24hai reaching the highest value at
72hai in the infected tissues, when compared to the control (non-inoculated hypocotyls). In
the susceptible (Fig.4B) genotype Caturra, no differences in POD activity were detected
between samples (inoculated vs control). These results suggest the involvement of POD in
the resistance mechanism of Coffea spp. to Ck.
Fig.4. POD activity in healthy (control) and infected hypocotyls of the resistant genotype Catimor 88 (A) and the susceptible
genotype Caturra (B). POD activity was expressed as O.D.480nm min-1 g-1 dry weight and hypocotyls were harvested at
different times after inoculation.
Based on an Illumina RNA-seq coffee - Ck database previously generated [10], 33
unigenes with annotation as oxidative-related function were identified as differentially
expressed in Catimor 88 and Caturra infected by Ck comparatively with their controls.
These unigenes can be grouped into six main classes of oxidases: multicopper,
a)
1
2
A
B
peroxidase, polyphenoloxidase, germin-like, redoxin domain and isoflavone reductase-
like protein. An integrative in silico analysis of their expression profiles revealed that, in
general for 20 unigenes, Catimor 88 presents a higher expression in genes that are
activated at 48 and/or 72hai, being normally repressed at 24hai (Fig.5A). Also, for the
other 13 unigenes for both genotypes, the predominant expression profile found is
repression in all time points (Fig.5B). To the best of our knowledge, this study
represents the first integrative attempt to understand the involvement of oxidative
enzymes in coffee resistance to Ck.
ACKNOLEDGMENTS
This work was funded by Portuguese national funds through Fundação para a Ciência e a Tecnologia
(project PTDC/AGR-GPL/112217/2009, grants SFRH/BPD/47008/2008, SFRH/BPD/63641/2009,
SFRH/BD/84188/2012, SFRH/BPD/88994/2012, attributed to AL, AF, ID and PT, respectively).
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Fig.5. Predominant expression profiles found in the oxidase classes studied, peroxidase (A) and isoflavone
reductase-like protein (B), based on a differential expression analysis previously performed with Illumina
RNA-seq data from coffee genotypes susceptible (Caturra) and resistant (Catimor 88) to Ck.
A
B
