involvement of calcium oxalate degradation during programmed … · 2019. 12. 20. · perniciosa...

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Plant Science 173 (2007) 106–117

Involvement of calcium oxalate degradation during programmed cell

death in Theobroma cacao tissues triggered by the hemibiotrophic

fungus Moniliophthora perniciosa

Geruza de Oliveira Ceita a, Joci Neuby Alves Macêdo a, Thais Bomfim Santos a,Laurence Alemanno b, Abelmon da Silva Gesteira a, Fabienne Micheli a,c,

Andrea Cristina Mariano d, Karina Peres Gramacho e, Delmira da Costa Silva f,Lyndel Meinhardt g, Paulo Mazzafera h, Gonçalo Amarante Guimarães Pereira i,

Júlio Cézar de Mattos Cascardo a,*a Laboratório de Genômica e Expressão Gênica, UESC, Rodovia Ilhéus-Itabuna, Km 16, Ilhéus 45662-000, BA, Brazil

b Cirad-CP, UMR BEPC, Avenue Agropolis TA80/03, 34398 Montpellier Cedex 5, Francec Cirad-CP, UMR PIA, Avenue Agropolis TA80/02, 34398 Montpellier Cedex 5, France

d Departamento de Educação, UNEB, Campus VII, Rodovia Lomanto Junior s/n, Senhor do Bonfim 48970-000, BA, Brazile Laboratório de Fitopatologia Molecular, CEPEC, Rodovia Ilhéus-Itabuna, Km 22, Ilhéus 45600-000, BA, Brazil

f Laboratório de Anatomia Vegetal, UESC, Rodovia Ilhéus-Itabuna, Km 16, Ilhéus 45662-000, BA, Brazilg Sustainable Perennial Crops Laboratory—USDA-ARS, 10300 Baltimore Av. BARC-W, Beltsville, MD 20705, USA

h Departamento de Fisiologia Vegetal—UNICAMP, 13083-970 Campinas, SP, Brazili Departamento de Genética e Evolução—UNICAMP, 13083-970 Campinas, SP, Brazil

Received 15 December 2006; received in revised form 5 April 2007; accepted 11 April 2007

Available online 19 April 2007

Abstract

Moniliophthora perniciosa, the causal agent of witches’ broom disease of Theobroma cacao, significantly affected cacao production in South

America and Caribbean countries. Host colonization by the pathogen exhibits a concerted succession of symptoms, starting with hypertrophic

growth and ‘‘broom’’ formation, followed by tissue degeneration and death. To understand mechanisms of host susceptibility, we investigated

fungal development during a compatible interaction with a susceptible genotype. Microscopic analysis revealed the initial fungal biotrophic

intercellular growth, followed by intracellular growth associated with the presence of an increasing number of host apoptotic nuclei and calcium

oxalate crystals, with subsequent accumulation of hydrogen peroxide and cell death. Active oxalate degradation and its possible source of origin

were detected in infected tissues. Together, these processes may increase the availability of nutrients for the fungal mycelia and may contribute to

the disease cycle in this plant–fungal hemibiotrophic interaction. Based on the histological and gene expression data, a novel role for calcium

oxalate in disease susceptibility is proposed.

# 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Apoptosis; Ascorbate; Cacao; Druses; H2O2; ROS

Abbreviations: COC, calcium oxalate crystals; DAB, 3,30-diaminobenzi-dine; DAI, days after inoculation; G-OXO, germin oxalate oxidase; HR,

hypersensitive response; APX, ascorbate peroxidase; PCD, programmed cell

death; ROS, reactive oxygen species; WBD, witches’ broom disease

* Corresponding author. Tel.: +55 73 3689 1082; fax: +55 73 3680 5226.

E-mail address: [email protected] (J.C. de Mattos Cascardo).

0168-9452/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.plantsci.2007.04.006

1. Introduction

Programmed cell death (PCD) is genetically controlled and

normally occurs during plant development and in response to

environmental stresses [1]. PCD can be seen as a counterpart of

cell division in determining shape and morphology of tissues

and organs during differentiation, and for this reason, has been

extensively studied in animal development. PCD has also been

associated with several plant processes, including senescence

mailto:[email protected]://dx.doi.org/10.1016/j.plantsci.2007.04.006

G. de Oliveira Ceita et al. / Plant Science 173 (2007) 106–117 107

[2], organ and cell development [3], response to environmental

changes [4] and hypersensitive response (HR) to pathogen [5].

HR is a PCD-related process characterized by a rapid host

response to pathogen infection that causes growth inhibition [6]

and induces the secretion of pathogen-induced components [7].

One of the most characteristic processes of PCD is

internucleosomal DNA degradation, a phenomenon described

in both animal and plant cells [4,8,9]. PCD-induced DNA

degradation can be detected [9,10], and PCD can be

characterized by nuclear morphology alterations, apoptotic

body formation [11,12], accumulation of toxic molecules,

callose deposition [13,14] and generation of reactive oxygen

species (ROS) [15], which are believed to prevent pathogen

development and colonization [16]. Conversely, in some plant–

hemibiotrophic fungal interactions, ROS production appears to

be an important factor for fungal colonization [17,18].

The hemibiotrophic basidiomycete Moniliophthora (=Cri-

nipellis) perniciosa (Stahel) Aime & Phillips-Mora is the causal

agent of witches’ broom disease (WBD) of cacao (Theobroma

cacao L.). This disease is a serious threat to cacao production in

South America and the Caribbean. In Brazil, the annual cacao

production decreased from 400,000 to 120,000 tonnes as a

consequence of the spread of WBD in the main cacao-growing

region of Brazil [19]. Basidiospores infect meristematic tissues

(shoots, flower cushions, single flowers, and developing pods),

and induce a range of symptoms depending on the organ

infected and the developmental stage: (i) infected apical

meristem presents hypertrophic growth (‘brooms’); (ii) infected

flower cushion usually revert to vegetative shoot production and

parthenocarpic pods; (iii) pod infection can directly result in

seed loss due to a pod rot. The pathogen life cycle is completed

by basidiocarp production on necrotic plant tissues (brooms and

pods). In all infected plant organs, tissue degradation is the

most important symptom of the cacao-M. perniciosa interac-

tion [20–22]. Some of the biochemical changes occurring in

infected tissues were recently described [23], with the

generated knowledge used to develop an artificial medium to

maintain the fungus on the biotrophic phase [24]. Currently, our

group is concentrating in the M. perniciosa genome initiative

(http://www.lge.ibi.unicamp.br/vassoura/) to understand this

complex disease. In the present work, we show in planta that

PCD is triggered by M. perniciosa infection, likely involving

calcium oxalate crystal (COC) accumulation and subsequent

degradation through activation of an oxalate oxidase gene (G-

OXO) expression and H2O2 production.

2. Materials and methods

2.1. Plant material

Seedlings of the susceptible cultivar Catongo and the resistant

clone TSH1188 were grown in sterile substrate in the greenhouse

of CEPEC/CEPLAC (Centro de Pesquisas do Cacau da

Comissão Executiva do Plano da Lavoura Cacaueira, Ilhéus,

Bahia, Brazil) from September 2002 to January 2003 under

natural light and 90% relative humidity. Apical meristems of one

hundred and fifty-four 4-week-old seedlings were inoculated by

spraying a 105 mL�1 basidiospores suspension from the M.

perniciosa Cp1441 CEPEC/CEPLAC strain, obtained as

described [25]. After inoculation, seedlings were kept for 24 h

at 25 � 2 8C in a water-saturated atmosphere [22,26]. Fifty-sixcontrol seedlings were inoculated with sterile water and

submitted to the same conditions as the inoculated plants.

Symptoms were evaluated of the Catongo susceptible plants 4

weeks after inoculation. Disease development was monitored for

90 days. Inoculated and non-inoculated apical meristems from

Catongo and TSH1188 plants were periodically sampled for the

various analyses. For DNA extraction and ascorbic acid

determination, additional plant material from infected and

non-infected Catongo seedlings were collected from a distinct

experiment, placed in liquid nitrogen and kept at �80 8C.

2.2. Histological analysis of cacao meristems during

infection

Meristems from inoculated and non-inoculated Catongo and

TSH1188 seedlings were harvested at 1, 2, 3, 8, 13, 19, 23, 28,

33, 36, 45, 50, 57, 60, 65 and 90 days after inoculation (DAI)

and immediately fixed for at least 24 h in 0.2 M phosphate

buffer pH 7.2, containing 2% paraformaldehyde, 1% glutar-

aldehyde and 1% caffeine (w/v). Plant tissues were then

dehydrated in an alcohol series. Samples were subsequently

transferred to Technovit 7100 resin (Kulzer, Wehrheim,

Germany) for 12 days, and finally embedded in the same

material. Three mm-thick sections were double stained withnaphthol blue-black and Periodic Acid-Schiff (PAS), which

were observed in a DMRXA microscope (Leica, Wetzlar,

Germany) under white and polarized light to localize calcium

oxalate crystals (COC), and photographed with a Coolpix 4500

camera (Nikon, Tokyo, Japan). COC were counted from stems

on sample surfaces of 0.4 mm2. For each day of sampling, one

to three sections were counted and for each section the crystals

were counted in three distinct areas.

2.3. Analyses of Catongo DNA degradation during

infection by M. perniciosa

DNAwas extracted from Catongo shoot tips using the CTAB

method, as described: approximately 300 mg of plant material

was frozen and ground in liquid nitrogen. After the addition of

800 ml of extraction buffer (2% CTAB; 1.4 M NaCl; 20 mMEDTA; 100 mM Tris–HCl pH 8.0; 2% PVP; 0.2% b-mercaptoethanol; 20 mg mL�1 proteinase K), samples wereincubated 40 min at 65 8C, and then centrifuged for 5 min at4000 rpm. The aqueous phase was extracted twice with 800 mlof chloroform–isoamyl alcohol (24:1), mixed for 5 min and

then centrifuged for 5 min at 14,000 rpm. DNA was pre-

cipitated with isopropanol and kept at 4 8C overnight. The DNApellet was washed twice with 300 ml of ethanol 70% and 96%,resuspended in 50 ml of TE (10 mM Tris–HCl; 1 mM EDTA,pH 8) containing 80 mg mL�1 of RNase-DNase free, and thenincubated for 30 min at 37 8C. DNA purity and concentrationwas determined spectrophotometrically (Novaspec II, Pharma-

cia Biotech). To determine DNA degradation during disease

http://www.lge.ibi.unicamp.br/vassoura/

G. de Oliveira Ceita et al. / Plant Science 173 (2007) 106–117108

development, calibrated samples of cacao DNA (10 mg) weresubjected to agarose gel electrophoresis (1% agarose), and

stained with ethidium bromide (0.5 mg mL�1).

2.4. In situ detection of DNA fragmentation using the

TUNEL method

For TUNEL (Terminal Deoxynucleotidyl Transferase-

mediated dUTP Nick End-Labeling) evaluation, control, 45

and 90 DAI inoculated and non-inoculated ‘Catongo’ mer-

istems were fixed in 4% paraformaldehyde in PBS buffer

(10 mM NaH2PO4; 150 mM NaCl, pH 7.2) and infiltrated for

10 min under vacuum. Samples were then fixed for 75 min and

dehydrated under vacuum in graded tert-butanol concentrations

(70%, 85%, 95% and 100% for 30 min at each step). The

samples were immersed in absolute tert-butanol for 8 h, and

then in 50% tert-butanol and paraffin for 2 h at room

temperature, and then for 2 h at 60 8C. Samples were incubatedtwice for 2 h in paraffin at 60 8C. Transverse sections of 12 mmwere obtained using a Leica RM 2145 microtome, and mounted

onto slides and kept for 2 days at 45 8C.Sections were deparaffinized (2� 10 min in xylene;

2� 5 min in 100%, 95%, 75% ethanol and 3 min in distillatedwater), washed five times with PBS buffer, incubated for

30 min at room temperature in a PBS solution containing 4%

pectinase and 2% cellulase Onozuka R-10 (Merck), washed for

5 min in PBS, incubated for 20 min in 1% acetic acid in

methanol, washed for 5 min in PBS, incubated with proteinase

K (20 mg mL�1 in 10 mM Tris–HCl, pH 7.5) for 15 min at37 8C, washed twice for 2 min in PBS, followed by immersionin TDT buffer (30 mM Tris base; 140 mM Na-cacodylate;

1 mM cobalt chloride, pH 7.2).

To detect DNA fragmentation, the TUNEL procedure was

applied, using the In situ Cell Death Detection Kit (Boehringer,

Mannheim) according to the manufacturer’s instructions. DNA

fragmentation was detected directly after the TUNEL reaction

by examining slides using an Axiovert microscope (Carl Zeiss,

Oberkochen, Germany) with a filter set for fluorescein

isothiocyanate (FITC), and photomicrographs were taken with

KODAK Ultra ASA 400 film.

Several slides were mounted; the pictures represent

accurately the average of the observed. As the differences

were striking, the apoptotic nuclei were not counted. A negative

control, incubated only with the labeling buffer, was included

in the experiment, whereas the positive control was permea-

bilized sections of non-inoculated meristems incubated with

DNase I (1 mg mL�1) for 10 min at room temperature before

processing.

2.5. Ascorbic acid determination

Dried healthy and infected plant samples were ground in a

knife mill, and extracted (300 mg per 3 mL) with cold 4 mM

H2SO4 containing 5 mM dithiothreitol and 0.01% polyvinyl-

polypyrrolidone (w/v) as described in [27]. Samples were

analyzed on the same day of extraction. The extracts were

centrifuged and filtered through 0.22 mm, and analyzed using a

Shimadzu HPLC equipped with a diode array detector,

operating at 190–370 nm. Ascorbic acid was separated on a

Supelcosil LC-18 reversed phase column (Supelco,

4 mm � 250 mm, 5 mm) using 1% aqueous acetic acid (solventA) and methanol (solvent B) in the following gradient: 0–25%

solvent B for 6 min; 25–100% solvent B for 1 min; 100%

solvent B for 12 min. The flow rate was set to 0.8 mL min�1 and

the detector was adjusted to monitor ascorbic acid elution at

210, 250 and 280 nm. Sample ascorbic acid was identified by

comparing elution time with pure standard, by co-injection of

the samples with the standard, and by the UV absorbance

spectrum obtained by the detector. Ascorbic acid concentration

in samples was determined by comparing the signals acquired

at 250 nm and integrated at the workstation (Class VP,

Shimadzu) with signals obtained from pure ascorbic acid

analysis at known concentrations.

2.6. Cacao RNA extraction and semi-quantitative RT-PCR

analysis

Total RNA from infected (3, 30 and 60 DAI) and non-

infected control ‘Catongo’ tissues was isolated [28]. RNA

samples were treated with DNase RNase-free (Invitrogen).

First strand cDNA was synthesized from 2 to 5 mg total RNAusing SuperScript II Kit (Invitrogen) according to the

manufacturer’s instructions. PCR assays were performed with

TC-G-OXO (GenBank accession No. EF191355) specific

primers (forward: 50-CATTAGCATCGGCCTTTGACCC- 30

and reverse: 50-GCCCTCCACGACCACTAGG-30). TC-APX(GenBank accession No. EF533885) specific primers (forward:

50- ATGACGAAGTGTTACCCGACTG-30 and reverse: 50-GCTTGTCCTCTCTTCCGGGG- 30). As an internal controlfor the T. cacao transcript level, specific primers for the TC-S-

18 (GenBank accession no. EF140885) ribosomal protein were

used (forward: 50- CAAGCGATCTTTTCGTAGGC-30 andreverse: 50-CGAAGATAAAATCCGAGCTTGT-30). All geneswere identified in cDNA library with highly significative e-

values of 1e�44, 3e�83, and 3e�26, respectively. RT reactions

contained 2 ml of the reverse transcription reaction, 200 mM ofeach dNTP, 5 pmol of each gene-specific primer, 1� PCRbuffer (10 mM Tris–HCl pH 8.3; 50 mM KCl, 2.5 mM MgCl2)

and 1 U of Taq DNA polymerase (Invitrogen) in a total volume

of 25 ml. Presence of contaminating DNA was assessed incontrol reactions made without reverse transcriptase. Ampli-

fication reactions were performed for 35 cycles at the suitable

annealing temperatures for each set of primers. All of the

reaction products were resolved by electrophoresis on 1.2%

agarose-TBE gels, stained with ethidium bromide and

visualized under UV illumination.

2.7. H2O2 detection by DAB-uptake method

Necrotic brooms, as well as non-inoculated meristems of

Catongo plants were harvested and immersed in 1 mg mL�1

3,30- diaminobenzidene (DAB)-HCl, pH 3.8 (Sigma), infiltratedunder a vacuum for 4 h. Control samples were infiltrated and

incubated with distilled water. Samples were cleared in boiling


ethanol (96%) for 20 min, then hand-sectioned with a razor

blade, mounted in 50% glycerol and examined using a DMRA-

2 microscope (Leica, Wetzlar, Germany). Images were

obtained using a computer assisted image analysis. H2O2was visualized as a reddish-brown coloration.

3. Results

3.1. Disease symptom evaluation in compatible interaction

Disease symptoms on susceptible Catongo seedlings were

observed in the greenhouse (Fig. 1A). The first symptoms

appeared 30 days after inoculation (DAI), and corresponded to

wilting and partial necrosis of a few young leaves (Fig. 1C).

Symptoms of green-broom (stem swelling and axillary bud

elongation) were observed 45 DAI (Fig. 1D). Infected tissues

Fig. 1. Witches’ broom disease symptoms. (A) Culture conditions for the cacao plan

plantlets. (C) First symptoms 30 days after the inoculation with M. perniciosa spore

green brooms with petiole swelling (white arrows), 45 DAI. Black arrow: hypertrophy

60 DAI. (F) Completely dried brooms with dried leaves, 90 DAI. Bar = 2 cm.

(brooms) started to dry 60 DAI and reached their final stage at

90 DAI (Fig. 1E), with necrotic tissues with dried brown leaves

in heavily infected plants (Fig. 1E and F). Disease symptoms

were not found in any non-inoculated control seedlings

(Fig. 1B) or on inoculated resistant TSH1188 (not shown).

3.2. Histological analysis of M. perniciosa penetration and

development in susceptible Catongo tissues

In susceptible Catongo tissues, hyphae were first detected

2 DAI (Fig. 2A). Hyphae were located just under the epidermal

stem cells, far below the apical meristem, suggesting a

penetration through the stem epidermis. Colonization by

mycelium proceeded through the intercellular space of the

cortex (Fig. 2B and C), the sclerenchyma (Fig. 2D), vascular

tissues (Fig. 2E), and finally reaching the pith (Fig. 2F) around

tlets in the greenhouse at CEPLAC/CEPEC (Bahia, Brazil). (B) Non-inoculated

s: wilt and necrosis of the young leaves (white arrows). (D) Terminal and axial

of the apical meristem and formation of axial brooms. (E) dry broom formation,

Fig. 2. Penetration and progression of M. perniciosa mycelia into meristems and stems of susceptible Catongo plants. (A) The first hyphae observed were under the

epidermis of the stem 2 DAI (bar = 30 mm). (B) Penetration of the fungus into the stem cortex 13 DAI (bar = 60 mm). (C) Intercellular progression of the fungus

(bar = 30 mm). (D) Shows the fungus close to sclerenchyma cells, 36 DAI (bar = 30 mm). (E) Progression of the fungus through the vascular tissues as seen in

transversal section, 36 DAI (bar = 60 mm). (F) Fungal mycelium between medulla cells in a longitudinal section, 36 DAI (bar = 30 mm). (G) Colonization of the

fungus inside the medulla, 50 DAI (bar = 60 mm). (H) Shows an increase in the hyphal diameter, 50 DAI (bar = 30 mm). (I) Signs of differentiation within the stem,

50 DAI (bar = 120 mm). (J) Transversal section of a stem showing phenolics and necrosis, 50 DAI (bar = 30 mm). (K) Necrosis inside the medulla, 50 DAI

(bar = 60 mm). (L) Longitudinal section of a control meristem, 57 DAI (bar = 240 mm). (M) Longitudinal section of an inoculated meristem, 57 DAI (bar = 240 mm).

(N) No starch grains in cortex cells of an inoculated plant, 65 DAI (bar = 60 mm). (O) Presence of starch grains in the cortex of a control plant, 65 DAI (bar = 60 mm).

(P) Necrosis and proliferation of the fungus 90 DAI (bar = 30 mm). (Q) Presence of both biotrophic and saprophytic fungal hyphae, 90 DAI (bar = 30 mm). (R) Clamp

connection 90 DAI (bar = 30 mm). AM: apical meristem; CC: clamp connection; CW: cell wall thickening; Ep: epidermis; Hp: biotrophic hyphae; HpN: necrotrophic

hyphae Ne: necrosis; Sc: sclerenchyma; SG: starch grain; SP: saprophytic phase; Ph: phenolics; PP: parasitic phase; VT: vascular tissues.



36 DAI. Mycelium also spread progressively toward the apical

meristem. From 45 to 65 DAI, the fungus continued to grow

and colonize all the tissue (Fig. 2G), while hyphal diameter

increased (Fig. 2H). Concurrently, the infected tissues showed

evidences of differentiation, such as cell wall thickening, cortex

necrosis (Fig. 2I and J) and pith necrosis (Fig. 2K). At the

green-broom stage (up to 50 DAI), meristems of inoculated

seedlings were significantly larger than the controls (Fig. 2L

and M). Infected stem cortical tissues lacked starch reserves

(Fig. 2N) when compared to control tissues (Fig. 2O).

Differentiation and necrosis increased up to 90 DAI, when

brooms dried, and cell necrosis was commonly observed in the

tissues (Fig. 2P). The majority of the mycelia changed from

monokaryotic hyphae to the dikaryotic phase (Fig. 2P),

concurrent with a tissue disarrangement, typical of the

necrotrophic phase. Tissue areas with both mycelia phases

of the fungus were also observed (Fig. 2Q), as well as clamp

connections (Fig. 2R), which are markers of dikaryotization in

this basidiomycete [21,29,30].

Neither mycelium nor morphological or histological

modifications, typical of the disease symptoms, were observed

in control or inoculated resistant seedling (TSH 1188; not

shown). Other morphological and anatomical modifications

were observed during tissue colonization by the fungus, such as

cell wall thickening and cell necrosis. Phenolic compounds,

which have been associated with other fungal infections [23],

were observed in both inoculated and non-inoculated plant cells

(Fig. 2I and J).

3.3. Analysis of DNA degradation and in situ detection of

DNA fragmentation in meristems infected by M. perniciosa

DNA integrity during disease development was evaluated by

agarose gel electrophoresis. Meristems of non-infected control

plants showed minor DNA degradation (Fig. 3A, lane 1), in

Fig. 3. (Left panel) Analysis on electrophoresis gel of the DNA degradation in Caton

branch. Lane 2: initial stage of the green broom formation (45 DAI). Lane 3: late st

panel) Analysis of meristematic tissue sections from Catongo using the TUNEL meth

45 DAI, 100� (C); infected meristematic tissues 90 DAI, 100� (D); non-inoculatecellular contour remains (black arrows), 200� (F).

contrast to DNA from infected meristems, which showed a

remarkable higher level of degradation, proportional to

symptom severity (Fig. 3A; lane 2 = 45 DAI; lane 3 =

90 DAI). DNA degradation culminated at the dry broom stage,

reaching a typical DNA ladder pattern (Fig. 3, lane 3 and

arrows).

TUNEL analysis was applied to thin meristem sections to

verify the occurrence of cell death events during the witches’

broom disease development. Nuclei undergoing DNA

fragmentation appeared yellow-green under UV fluorescence

[10]. Sections of non-inoculated tissues did not show any

positive TUNEL reactions (Fig. 3B). In non-inoculated

meristem cells treated with DNase I (positive control), a

large number of fluorescent nuclei were observed, indicating

extensive nuclear DNA degradation (Fig. 3C). Sections of

susceptible meristems analyzed at 45 DAI (Fig. 3D) and

90 DAI (Fig. 3E) displayed intense yellow-green fluores-

cence, corresponding to TUNEL positive nuclei. The amount

of these positive nuclei at the final stage of the infection

(90 DAI) was significantly higher than at 45 DAI, indicating

an increase in DNA degradation.

3.4. Calcium oxalate crystal accumulation and degradation

during infection of susceptible cacao by M. perniciosa

Cacao can accumulate high amounts of calcium oxalate, as

previously described [31]. In agreement with this observation,

we detected large quantities of COC in stem tissues of the non-

inoculated susceptible Catongo seedlings. COC were located

mostly in the cortex (Fig. 4A and B), but also in the pith (Fig. 4C)

of mature stem tissue, distant from the apical meristem. In the

region closest to the apical meristem, only a few COC were

present and these were restricted to cortex cells (Fig. 4D). The

amount and localization pattern of the crystals remained more or

less constant over time in control plants (Fig. 4F and N).

go meristematic tissues during witches’ broom disease (A). Lane 1: non-infected

age of the dry broom formation (90 DAI). (M) 1 kb ladder (Invitrogen). (Right

od: non-inoculated meristematic tissue, 100� (B); infected meristematic tissuesd meristematic tissue, DNase treated (positive control), 100� (E); evidence of

Fig. 4. Localization of calcium oxalate crystals in the meristems and stems of susceptible Catongo and resistant TSH1188 using polarized light. T. cacao inoculated

with M. perniciosa. (A) Stem cortex of a control Catongo plant, 2 DAI. (B) COC in the cortex of a control Catongo plant, 3 DAI. (C) Stem medulla of a control

Catongo plant, 2 DAI. (D) Transversal section of a control Catongo stem showing the localization of COC outside the vascular bundles, 2 DAI. (E) Apical region of a

Catongo control plant, 19 DAI without COC. (F) Decrease of COC in the cortex of a control Catongo plant, 57 DAI. (G) Presence of many COC in the cortex of an

inoculated Catongo plant, 2 DAI; (H) in the medulla of an inoculated Catongo plant, 2 DAI. (I) Higher magnification of COC in the cortex of an inoculated Catongo

plant, 2 DAI. Absence of COC (J) in the cortex of an inoculated Catongo plant, 50 DAI; (K) in the medulla of an inoculated Catongo plant, 42 DAI; (L) in the cortex of


Fig. 5. Ascorbic acid content was determined by HPLC (250 nm traces) in health shoots (A), green broom (B) and dried broom (C) from plants growing in the field.

Ascorbic acid was identified in the samples by its UVabsorbance spectrum, elution time (D) and by co-injection with standards. Ten microlitres of each sample were

injected into the HPLC. Arrows indicate ascorbic acid retention time in the samples. Semi-quantitative RT-PCR of oxalate oxidase (G-OXO) and ascorbate peroxidase

(APX) genes throughout disease development (0, 3, 30 and 60 DAI). The constitutive expressed TCS-18 gene was used as internal control for semi-quantitative RT-

PCR calibration (E). SP (catongo) and RT (TSH 1188).


In the inoculated susceptible plants, the amount of COC

increased rapidly in both cortex (Fig. 4G–N) and pith cells

(Fig. 4H, I and N) until around 36 DAI. After day 36,

corresponding to overwhelming plant colonization by the

mycelium, the amounts of crystal progressively decreased.

Forty-five DAI, only a few crystals were observed in the

infected tissues (Fig. 4J, K and N). The most juvenile stem

tissue near the apical meristem was always devoid of crystals in

healthy and infected plants whatever the disease stage, a pattern

that was also observed in the susceptible control. No COC were

observed in the resistant control genotype (Fig. 4L and M)

under the experimental conditions for all of the evaluated

periods. The pattern of the T. cacao oxalate oxidase (G-OXO)

gene expression in the susceptible genotype followed the

accumulation and degradation of COC (Fig. 5E, SP).

Conversely, in the resistant TSH 1188 (Fig. 5E, RT), G-

OXO expression remained high for up to 60 DAI.

Only 0.126 mg mg�1 of ascorbic acid was detected in non-inoculated shoots (Fig. 5A), while an increase in accumulation

an inoculated TSH1188 plant, 2 DAI; (M) in the medulla of an inoculated TSH1188

control (dashed line) and inoculated (thick line) Catongo plants. The transition pha

fungus phase, which is not synchronized in the infected tissues. Error bars represent s

counted and for each section the crystals were counted in three distinct areas. AM: a

starch grain; VT: vascular tissues. Bar = 60 mm in B and I, and 120 mm in all oth

was detected in infected tissues, when compared to control

plants (Fig. 5B). The ascorbate amount in green brooms

(Fig. 5B) was significantly higher (averaging 2.135 mg mg�1)than in dried brooms (ascorbate almost non-detectable;

0.015 mg mg�1; Fig. 5C). A contrasting expression patternof cacao ascorbate peroxidase gene (APX) between Catongo

and TSH1188 plants could also be observed, with an increased

expression in the resistant plants (Fig. 5E, SP and RT,

respectively).

3.5. In situ detection of H2O2

Tissues from non-inoculated and inoculated susceptible

control plants at 42 DAI, when infiltrated with distilled water

did not present any positive reaction to H2O2 (Fig. 6A and B).

Samples from non-inoculated susceptible controls, infiltrated

with DAB were also free of brown precipitates (Fig. 6C), while

those from inoculated susceptible plants infiltrated with DAB,

displayed an intense overall reddish-brown precipitate,

plant, 2 DAI. (N) Number of COC per surface unit (0.4 mm2) in the cortex of

se corresponds to the major transition from the biotrophic to the necrotrophic

tandard error of the mean for 0.4 mm2. For each point, one to three sections were

pical meristem; COC: calcium oxalate crystal; Ep: epidermis; Hp: hyphae; SG:

er pictures.

Fig. 6. In vivo DAB reaction of susceptible cacao 45 DAI. (A) Non-infected susceptible cacao infiltrated with distilled water (control), 100�. (B) Infected susceptiblecacao infiltrated with distilled water (control), 100�. (C) Non-infected susceptible cacao infiltrated with DAB, 100�. (D) Infected susceptible cacao infiltrated withDAB, 100�, cortex (CX). (E) Non-infected susceptible cacao infiltrated with DAB (cortex region), 400�. White arrows: COC. (F) Infected susceptible cacaoinfiltrated with DAB, 400�.


corresponding with the presence of H2O2 (Fig. 6D). The brown

staining was more intense in the epidermis, cortex and pith

(Fig. 6D), and it was generally co-localized near the regions

containing COC, particularly the cortex (Fig. 6F). Noteworthy,

H2O2 diffusion was not homogeneous, but it appeared to be

dispersed from defined sites, possibly from the idioblasts

(Fig. 6F, arrows). In samples from non-inoculated susceptible

control plants infiltrated with DAB, some COC were observed

(as in Fig. 4), but no H2O2 could be detected (Fig. 6E).

4. Discussion

The morphological characteristics of the infected suscep-

tible plants were identical to those previously described

[22,23]. We observed hyphal development in susceptible plant

tissues, both from the stem base to the apical meristems, and

from the external (epidermis and cortex) to the inner tissues

(vascular bundles), confirming the observation of [30]. In our

experimental inoculation conditions, M. perniciosa penetrated

stem and leaves, probably by stomata or wounding, and

invaded the tissues around the apical meristems, where the

main disease reactions occurred. During the early phase of

tissue colonization, the mycelia grew intercellulary, and

because they did not have appressoria, the fungus must subsist

on nutrients present in the apoplast. Between 50 and 90 DAI,

most of the hyphae became intracellular, coinciding with the

onset of the visible necrotic symptoms (Fig. 2K, P–R). This

condition correlated with the major morphological transition

of the fungus, from biotrophic to the necrotrophic phase. The

simultaneous presence of mono- and dikaryotic M. perniciosa

hyphae within the infected cacao leaf tissue was observed

during the transition from biotrophic to necrotrophic phase

(Fig. 2P), an observation consistent with the findings of [30].

This detailed morphological description of the disease is

highly relevant to the understanding of this pathosystem, since

it involves a fungal growth phase switch from biotrophic to

necrotrophic.

In a previous work [23], it was shown that during the

transition of M. perniciosa from the biotrophic to the

necrotrophic phases in infected tissues, amino acids were

converted into amides. This conversion process had been shown

to be a physiological signal for the induction of PCD [32], and it

could be interpreted as a physiological effort by the plant to

mobilize nutrients from the diseased tissues. In this work, we

provided molecular evidences of PCD in the witches’ broom

disease through the detection of DNA fragmentation, a clear

indicator of this process [9,33]. To strengthen our results, we

have identified a protein from M. perniciosa capable to induce

cell death in cacao [34].

The first evidence of fragmentation was the presence of a

DNA ladder (200 bp band multiples) in DNA extracted from

dry broom cells (Fig. 3A, lane 3). When the DNA was extracted

from other infected sample (Fig. 3A, lane 2), smears were

detected, but it was not clear whether they were the


consequence of DNA degradation by nucleases that did not cut

at regular restriction sites [14], or due to the presence of high

gum levels in cacao [28,35], which affects the DNA migration.

Alternatively, the lack of a clear ladder in DNA extracted from

green-broom tissues could be due to a limited number of cells

undergoing PCD at this phase (Fig. 3D), which could prevent

the detection [12]. In addition, Schwartz et al. [36] suggested

that there is more than one PCD pathway and that the

activation of each pathway is directly connected to its

stimulation. For example, PCD induced by KCN leads to the

formation of a DNA ladder in plant cells, which is not

detectable when cells are treated with H2O2 [9,12]. Similarly,

Pasqualini et al. [37] observed PCD in tobacco cells treated

with ozone, but without the generation of the degraded DNA

ladder pattern.

To further confirm DNA fragmentation, TUNEL analysis

was applied [38]. This method clearly showed an increase in the

presence of apoptotic nuclei during the progression of the

disease, reinforcing the biochemical interpretation that there

was an orchestrated cell death in the infected tissues [23].

During a comprehensive microscopic analysis of cacao

tissues, susceptible cacao plants revealed to contain COC

(Fig. 4), which were not present in the resistant cultivar

TSH1188, as well as in five other WBD resistant cacao clones

analyzed (data not shown). During the infection process, the

number of these crystals increased in susceptible seedlings,

peaking at 33 DAI, followed by a rapid decrease in the final

disease stages (50 DAI, Fig. 4N). Therefore, it appeared that

COC were associated with the progression of the WBD

symptoms. Because one of the main sources of oxalic acid in

plants is ascorbic acid [27], the formation of the COC might

be related with the accumulation of ascorbic acid in infected

tissues, as confirmed by the HPLC analyses (Fig. 5B). In

relation to the decrease of COC, recent studies in animal

systems showed that high concentrations of oxalate were toxic

to cells, not only because of crystal formation, but also

because COC degradation can produce H2O2 [39,40]. In

plants, ROS production (such as H2O2) has been observed in

response to pathogens aggression, and it has been linked to the

inhibition of plant infection [41]. Using the DAB method, we

detected an accumulation of hydrogen peroxide in infected

tissues from susceptible plants (Fig. 6D and F), which could

be a functional part of the plant defense mechanism.

Moreover, the majority of H2O2 in the cortex was found

where COC had previously been detected (Fig. 6F, arrows),

suggesting that the hydrogen peroxide was produced at

specific defined points in the tissue through the degradation of

the oxalate crystals. As described by [39,40], degradation of

COC is recognized to be due to germin oxalate oxidase

activity (G-OXO), and a corresponding T. cacao gene (G-

OXO) was identified in a cDNA library produced from mRNA

from infected tissue [42]. The expression of this gene was

analyzed by semi-quantitative RT-PCR using mRNA col-

lected from meristems of infected stems, with a significant

expression in the diseased tissues. Expression of germin

oxalate oxidase had already been observed in: (i) Gramineae

involved in an oxalate related senescence process [39]; (ii)

Arabidopsis thaliana PCD induced by an Alternaria alternata

toxin [7]; and (iii) sunflower, where the over-expression of

germin oxalate oxidase allowed a high rate of oxalate

degradation that triggered high levels of H2O2 and defense

gene activation [40]. These findings corroborated that the G-

OXO gene might be expressed in response to C. perniciosa

infection, and the corresponding enzyme might play an

important role in the production of the hydrogen peroxide

found in the infected tissues.

Despite of the observed increase of ascorbate levels on

infected tissues (Fig. 5B), we detected a decrease in the

expression of the T. cacao H2O2-detoxifying APX gene

(Fig. 5E), which might indicate a depletion of ascorbate

availability in these tissues, suggesting its conversion to oxalic

acid and subsequently to H2O2. This result corroborated recent

findings [43,44] that proposed a new role for ascorbic acid as a

pro-oxidant molecule, capable of triggering an oxidative burst.

Noteworthy, the expression pattern of G-OXO and APX genes

were opposite in the resistant genotype (Fig. 5, RT), where G-

OXO and APX gene expression increased during infection. The

higher level of G-OXO expression in the resistant genotype

must explain the absence of visible COC, due to a continuous

degradation of oxalic acid. It must be highlighted that the

pattern of APX expression of the susceptible genotype was

different, when compared to other compatible interactions [45],

but similar to the results of [46]. Our results suggested that M.

perniciosa triggers significant changes in the antioxidant

system of cacao, leading to a collapse of the protective

mechanism during the infection in susceptible genotypes.

These changes appeared to be partly due to the effect of

pathogen-promoted tissue cell death. Conversely, it has been

suggested that the degradation of oxalate to hydrogen peroxide

may have beneficial effects for the plant by counteracting the

strategy of some pathogens [47], with several roles, depending

on the tissue concentration [7,48]. Therefore, we can

hypothesize that sensitive cacao plants produced hydrogen

peroxide in a concentration that triggers PCD of some cells,

which in turn increases the availability of nutrients for the

fungal cells, which causes the biotrophic to the necrotrophic

phase conversion.

In fungal diseases, transgenic tobacco plants expressing

negative regulators of apoptosis were found to block cell death

associated with HR. Plants were resistant to necrotrophic fungi,

including Sclerotinia sclerotiorum and Botrytis cinerea,

suggesting that these fungi require HR-induced cell death to

successfully infect the plants [49]. Recently, transgenic peanut

plants expressing an oxalic acid-degrading oxalate oxidase

have shown an increased resistance to the necrotrophic fungus

Sclerotinia minor [50]. Similarly, in lettuce, tobacco and

tomato, it was demonstrated that plant susceptibility to fungal

pathogens was abolished by COC elimination in transgenic

plants over-expressing a fungal oxalate decarboxylase [51,52].

Considering our data showing differences on COC amounts

between susceptible and resistant genotypes, and the recent

findings using transgenic plants, we can strongly suggest the

involvement of oxalate in the dynamics of the witches’ broom

disease.


Acknowledgements

The authors wish to thank Dr. Lise Labejof and Christine

Sanier for technical assistance, and Michel Vincentz, Nicolas

Carels, Martin Brendel, Antonio Figueira and Carter Miller

for critically reading the manuscript. G.O. Ceita and J.N.

Macêdo were supported by CAPES (Coordenação de

Aperfeiçoamento de Pessoal de Nı́vel Superior, Brazil); Dr.

A.S. Gesteira and T.B. Santos were supported by FAPESB

(Fundação de Amparo à Pesquisa do Estado da Bahia, Brazil);

and Dr. A.C. Mariano was supported by a CNPq Post-doctoral

Fellowship. This work was supported by FAPESB and Banco

do Nordeste.

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Involvement of calcium oxalate degradation during programmed cell �death in Theobroma cacao tissues triggered by the hemibiotrophic �fungus Moniliophthora perniciosaIntroductionMaterials and methodsPlant materialHistological analysis of cacao meristems during infectionAnalyses of Catongo DNA degradation during infection by M. perniciosaIn situ detection of DNA fragmentation using the TUNEL methodAscorbic acid determinationCacao RNA extraction and semi-quantitative RT-PCR analysisH2O2 detection by DAB-uptake method

ResultsDisease symptom evaluation in compatible interactionHistological analysis of M. perniciosa penetration and development in susceptible Catongo tissuesAnalysis of DNA degradation and in situ detection of DNA fragmentation in meristems infected by M. perniciosaCalcium oxalate crystal accumulation and degradation during infection of susceptible cacao by M. perniciosaIn situ detection of H2O2

DiscussionAcknowledgementsReferences

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