1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

NEP1 (necrosis- and ethylene-inducing peptide 1)-like proteins (NLPs) have been identified in a variety of taxonomically unrelated plant pathogens and share a common characteristic of inducing responses of plant defense and cell death in dicotyledonous plants. Even though some aspects of NLP action have been well characterized, nothing is known about the global range of modifications in proteome and metabolome of NLP-treated plant cells. Here, using both proteomic and metabolomic approaches we were able to identify the global molecular and biochemical changes in cells of Nicotiana benthamiana elicited by short-term treatment with MpNEP2, a NLP of Moniliophthora perniciosa, the basidiomycete responsible for the witches' broom disease on cocoa (Theobroma cacao L.). Approximately 100 protein spots were collected from 2-DE gels in each proteome, with one-third showing more than twofold differences in the expression values. Fifty-three such proteins were identified by mass spectrometry (MS)/MS and mapped into specific metabolic pathways and cellular processes. Most MpNEP2 upregulated proteins are involved in nucleotide-binding function and oxidoreductase activity, whereas the downregulated proteins are mostly involved in glycolysis, response to stress and protein folding. Thirty metabolites were detected by gas spectrometry (GC)/MS and semi-quantified, of which eleven showed significant differences between the treatments, including proline, alanine, myo-inositol, ethylene, threonine and hydroxylamine. The global changes described affect the reduction-oxidation reactions, ATP biosynthesis and key signaling molecules as calcium and hydrogen peroxide. These findings will help creating a broader understanding of NLP-mediated cell death signaling in plants.


two-dimensional gel electrophoresis


1-aminocyclopropane-1-carboxylic acid


aconitate hydratase


alcohol dehydrogenase


alcohol dehydrogenase 1


Automated Mass Spectral Deconvolution and Identification System


l-ascorbate peroxidase 2 cytosolic




coenzyme Q


alpha chaperonin subunit alpha






elongation factor 2




endoplasmic reticulum


glyceraldehyde 3 phosphate dehydrogenase


gas chromatography


green fluorescent protein


Gene Ontology


glutathione peroxidase


reduced glutathione


oxidized glutathione


glutathione S transferase


high pressure liquid chromatography


hypersensitive response


isoelectric focusing


immobilized pH gradients




liquid chromatography tandem mass spectrometry


mitogen-activated protein


malate dehydrogenase




5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase


superoxide dismutase Mn mitochondrial


mass spectrometry.

MS medium

murashige and skoog medium




necrosis- and ethylene-inducing peptide 1


National Institute of Standards and Technology


NEP1 like proteins


nitric oxide


polyacrylamide gel electrophoresis


pathogen-associated molecular patterns


programmed cell death


pyruvate decarboxylase


2,3-bisphosphoglycerate-independent phosphoglycerate mutase


phenylmethylsulphonyl fluoride


PAMP-triggered immunity


reactive oxygen species


sodium dodecyl sulphate


superoxide dismutase Mn


trichloroacetic acid


N, N, N′, N′-tetramethylethylenediamine




UDP glucose 6 dehydrogenase


witches' broom disease


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Nep1 (necrosis- and ethylene-inducing peptide 1)-like proteins (NLPs) comprise a family of relatively small proteins that are microbial elicitors of plant necrosis. Firstly isolated from Fusarium oxysporum culture filtrates (Bailey 1995), they have since been identified in a broad range of taxonomically unrelated prokaryotic and eukaryotic micro-organisms that predominantly rely on heterotrophic (hemibiotrophic, necrotrophic or saprophytic) growth (Qutob et al. 2002, Pemberton and Salmond 2004). Members of the NLP family share a high degree of sequence similarity, such as a conserved seven-amino acid motif (GHRHDWE), a secretory signal sequence and the presence of two or four conserved cysteine residues that could divide them into two groups (Gijzen and Nürnberger 2006). NLPs are able to induce cell death response in a variety of dicotyledonous plants but not in any known monocotyledonous plant (Gijzen and Nürnberger 2006).

Moniliophthora perniciosa is a basidiomycete fungus that causes the witches' broom disease (WBD), one of the most important phytopathological diseases affecting cacao (Theobroma cacao L.) plantations in the Americas (Purdy and Schmidt 1996). A growing body of evidences has indicated that NLPs are key components in M. perniciosa pathogenicity (Garcia et al. 2007, Mondego et al. 2008, Silva et al. 2011, Zaparoli et al. 2011). Five members of NLP family (termed MpNEP1−5) have been identified in M. perniciosa based on genomic-scale studies (Mondego et al. 2008). Firstly characterized, MpNEP1 and MpNEP2 have been detected in tissues of cacao infected by the fungus and both elicited necrosis and ethylene production in leaves of tobacco and cacao (Garcia et al. 2007, Silva et al. 2011). MpNEP2 expression correlated with the necrotic symptoms of WBD (Zaparoli et al. 2011). Besides, MpNEP2 was the first described NLP to be able recovering necrosis activity after boiling (Garcia et al. 2007).

The plant cell responses to NLPs have been extensively described using biochemical, cytological and transcriptomic approaches. Such investigations have demonstrated that NLP treatment induces the accumulation of calcium and reactive oxygen species (ROS), increases the activity of mitogen-activated protein (MAP) kinase and the production of ethylene, phytoalexins and pathogenesis-related proteins, changes the K+ and H+ channel fluxes and cell respiration, and triggers nuclear DNA fragmentation and programmed cell death (PCD) (Jennings et al. 2001, Veit et al. 2001, Fellbrich et al. 2002, Keates et al. 2003, Bae et al. 2006, Qutob et al. 2006). Microarray analyses revealed that short-term treatment of Arabidopsis with NLPs affected the expression of a number of genes encoding proteins potentially localized in organelles, such as chloroplast and mitochondria, and induced the expression of several classes of genes, including those involved in signal transduction, ROS production, ethylene biosynthesis, membrane modification, apoptosis and stress (Bae et al. 2006, Qutob et al. 2006). These responses are similar to a great extent to those induced by PAMP-triggered immunity (PTI), although an elicitor-active minimal motif has not been identified in NLPs (Veit et al. 2001, Fellbrich et al. 2002, Qutob et al. 2006) and the NLP-induced cell death does not require the same molecular components (i.e. salicylic acid, jasmonic acid, ethylene, caspase activity or functional SGT1) which are required for PTI-associated hypersensitive cell death (hypersensitive response, HR) (Qutob et al. 2006). Structure-based analyses showed that NLPs are cytolytic toxins that mediate membrane disruption and subsequent activation of plant immunity-associated defenses (Ottmann et al. 2009), implying that plant cells recognize signal(s) generated by the toxin's action rather than the molecule itself.

Although the observations above brought important light to current understanding of the mode of action of NLPs, more clarifications are still needed. For example, there are no studies describing the NLP-mediated cell death signaling in plants using genomic tools such as proteomics and metabolomics. Such approaches could provide further insights about the biological processes involved in NLP susceptibility in plants and contribute to the control of plant diseases caused by their producing pathogens, such as M. perniciosa. Here, we have tested the hypothesis that protein and metabolic profiles could identify novel molecular and biochemical events underlying plant cell response to NLPs. Thus, the objective of this study was to characterize the early molecular and biochemical events induced by MpNEP2 in cells of Nicotiana benthamiana, by comparing quantitative and qualitative global changes within its proteome and metabolome.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Expression and purification of recombinant protein

The MpNEP2 gene was amplified from the genomic DNA of M. perniciosa using a specific pair of primers (5′-CGTCTCAGGATCCATTGCCGGC-3′ and 5′-CCAAGCTTTCACTACTACCACATCCAAGCC-3′) containing restriction sites for BamHI and HindIII. After purification and digestion, the resulting polymerase chain reaction product was inserted into pET28a + (Novagen, San Diego, CA). The recombinant plasmid was used to transform Escherichia coli strain BL21(DE3) pLysS. Protein expression was induced with 1 mM isopropyl-1-thio-b-d-galactopyranoside (IPTG) for 4 h at 28°C. Cells were then collected by centrifugation and resuspended in 0.01 volumes of buffer containing 10 mM Tris–HCl (pH 8.0), 500 mM NaCl, 5 mM imidazole, 2% Triton X-100 and a protease inhibitor mixture [1 mM phenylmethylsulphonyl fluoride (PMSF), 1 µg ml−1 pepstatin and 1 µg ml−1 leupeptin]. After two freeze–thaw cycles, 0.01 volumes of the buffer as described above, except for the absence of Triton X-100, was added and the cells were then subjected to sonication. Cell debris was excluded by centrifugation at 15 000 g for 1 h at 4°C. The supernatant was then loaded into a HisTrap™ FF crude (1 ml), charged with Ni2+ ions following the manufacturer's instruction (GE Healthcare, Piscataway, NJ), and equilibrated, at a flow rate of 1 ml min−1, with binding buffer containing 10 mM Tris–HCl (pH 8.0), 500 mM NaCl and 5 mM imidazole. After washing with the same buffer as describe above, a 30–200 mM imidazole gradient was used for elution of the protein. The eluted fractions containing purified protein were mixed and then loaded into a HiTrap desalting column (GE Healthcare), according to the manufacturer's protocol and using 10 mM phosphate buffer (pH 5.7). The purity of the protein was confirmed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) and the concentration was determined using the Bradford method (Bradford 1976).

Cell culture and microscopy analysis

The green fluorescent protein (GFP)-expressing N. benthamiana cell suspension was grown in liquid Murashige and Skoog medium (MS medium) (Murashige and Skoog 1962) under constant agitation (100 r.p.m.) at 25°C, and subcultured as described by Hemmerlin and Bach (1998). One microgram per ml MpNEP2 was added to the cell suspension at exponential growth phase. Cell death in the control and treated cells was analyzed by epifluorescent microscopy, 1 h after the treatments. Microscopic analysis was carried out using an epifluorescent microscope (Leica DMR, Heidelberg, Germany) and cells were observed in excavated blades using a Leica IM50 software. All experiments were performed in triplicate and in sterile conditions.

Protein extraction

Proteins of the cultured cells were extracted using a modified trichloroacetic acid (TCA)/acetone procedure (Wang et al. 2006, Smart et al. 2010). The cells were quenched using methanol water 50% (v/v) at −20°C and fast filtration under a vacuum (0.45 µm membrane). Cells were immediately ground to a fine powder in liquid nitrogen, resuspended in an ice-cold 10% (w/v) trichloroacetic acid solution in acetone and 0.07% (w/v) dithiothreitol (DTT) and centrifuged for 30 min at 35 000 g. The sediment was lyophilized and solubilized in lysis buffer, containing 32 mM Tris–HCl (pH 6.8), 7 M urea, 2 M thiourea, 0.2% (v/v) Triton X-100, 4% (v/v) CA-630, 1 mM PMSF and 14 M DTT, and then subjected to centrifugation at 12 000 g for 15 min. The supernatant was collected and the proteins were precipitated by adding four volumes of ice-cold acetone and centrifuged at 12 000 g for 15 min. The purified proteins were solubilized in rehydration buffer containing 8 M urea, 2% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 18 mM DTT, 0.5% (w/v) immobilized pH gradients (IPG) buffer (pH 4–7) and a trace of bromophenol blue. The 2-D Quant kit (GE Healthcare) was used to determine protein concentrations, with bovine serum albumin used as standard. Three samples per treatment were prepared from three individual biological experiments. All samples were maintained at −80°C prior their use in electrophoresis.

2-D electrophoresis

Isoelectric focusing (IEF) was carried out with precast 13-cm IPG strips (pH 4–7, 130 × 3 × 0.5 mm; GE Healthcare) using an Ettan IPGphor IV (GE Healthcare). Total protein (250 µg) was mixed with rehydration buffer [8 M urea, 2% (w/v) CHAPS, 18 mM DTT, 0.5% (w/v) IPG buffer (pH 4–7)] totaling 250 µl. Rehydration occurred at room temperature for 12 h on a ceramic plate (Manifold/GE Healthcare) and the first dimensional IEF was performed under the following conditions: (1) 500 V for 1 h; (2) 1000 V for 1 h; (3) 8000 V for 2.5 h; (4) 8000 V for 30 min. After IEF separation, the gel strips were equilibrated for 2 × 15 min in an equilibration buffer containing 50 mM Tris–HCl buffer (pH 8.8), 6 M urea, 2% (w/v) SDS and 30% (w/v) glycerol. The first equilibration buffer contained DTT (1% w/v), while in the second equilibration buffer DTT was replaced by 2.5% (w/v) iodoacetamide. Equilibrated strips were placed on the top of a 12% SDS-PAGE [12 ml of 30%/0.8% (w/v) acrylamide/bis-acrylamide, 7.5 ml of 1.5 M Tris–HCl (pH 8.8), 0.15 ml of 10% (w/v) ammonium persulfate, 0.02 ml of N, N, N′, N′-tetramethylethylenediamine (TEMED) and 10.2 ml of water , 18 × 16 cm, 1 mm) and sealed with 1% agarose containing traces of bromophenol blue. The second dimensional 2-D SDS-PAGE was carried out at Hoefer SE 600 Ruby (GE Healthcare) in a running buffer (25 mM Tris, 192 mM glycine and 0.1% SDS) at 11°C for 30 min at 15 mA, and for about 3.5 h at 60 mA, until the bromophenol blue line was around 1-cm from the bottom. After two-dimensional gel electrophoresis (2-DE), gels were kept 1 h in a fixation solution [1.3% orthophosphoric acid (85%) and 20% methanol] and stained with a solution containing 1.5% orthophosphoric acid (85%), 7.7% ammonium sulfate and 0.01% Coomassie G-250. The stained gels were scanned with ImageSCanner™ II at 300 dpi (GE Healthcare) and analyzed with ImageMaster™ 2D Platinum v6.0 (GE Healthcare). Control group gels were compared with the MpNEP2-treatment group gels (three independent biological replicates, three technical replicates). Differentially acquired protein spots were compared to find common differential protein spots. The spots were quantified based on their relative volume; t-test was performed and data were expressed as mean ± SD. P values less than 0.05 were regarded as statistically significant. Protein spots achieving a ≥twofold increase in spot intensity and observed in three replicated gels from three independent experiments were scored and subjected to mass spectrometry.

Mass analysis and protein identification

The selected stained spots were excised from the gel followed by destaining with 50 mM NH4HCO3 and 50% acetonitrile. The supernatant was removed and gel pieces were completely dried at room temperature. The excised spots were then rehydrated in 15 µl digestion buffer containing 40 mM NH4HCO3, 10% acetonitrile and 15 ng µl−1 trypsin (sequencing grade modified trypsin; Promega, São Paulo, Brazil) in an ice-cold bath for 30 min. The digestion was performed at 37°C overnight. The resulting peptides from the digests were subjected to online nanoflow liquid chromatography tandem mass spectrometry (LC–MS/MS) on a nanoAcquity system (Waters, Milford, MA) coupled to an Q-ToF micro mass spectrometer (Waters). Peptide mixtures were loaded onto a 1.7-µm × 100-mm nanoAcquity UPLC BEH300 column packed with C18 resin (Waters) and were separated at a flow rate of 0.6 µl min−1 using a linear gradient of 1 to 50% solvent B (95% acetonitrile and 0.1% formic acid) in 23 min, followed by an increase to 85% solvent B in 4 min and held at 85% solvent B for additional 3 min. Solvent A was 0.1% formic acid in water. The eluant from the high pressure liquid chromatography (HPLC) column was directly electrosprayed into the mass spectrometer, which operated in a data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition. The MS and MS/MS raw data were processed in Masslynx v4.1 (Waters) and the resulting pkl-files were used for searching against a target protein sequence database (SwissProt/Viridiplantae) using the ProteinLynx Global server v2.3 (Waters) and the Mascot server v2.4 ( The search criteria used were trypsin digestion, variable modifications set as cysteine (Cys) and methionine (Met), max of one missed cleavages allowed and peptide mass tolerance of ±0.3 Da for the parent ion and 0.10 Da for the fragment ions.

Metabolomic analysis

The cultured cells metabolites were extracted using a procedure previously described (t'Kindt et al. 2009), with some modifications. The cells were quenched using 50% (v/v) methanol water at −20°C and fast filtration under a vacuum (0.45 µm membrane). Cells were immediately ground in liquid nitrogen to a fine powder and 70 mg was weighed in an Eppendorf tube. Plant material was extracted with 300 µl cold 80/20 (v/v) MeOH/H2O and ribitol (20 mg ml−1) as internal standard, in a thermomixer during 15 min at 4°C. The extracts were sonicated for 5 min (Bransonic Ultrasonic Cleaner 1210, Danbury, CT) and centrifuged 12 000 g for 15 min at 4°C; a 50 µl aliquot of the supernatant was concentrated to dryness in a vacuum concentrator. Dried extracts were maintained at −80°C for 1 day before derivatization and GC-TOF mass spectrometry. Samples were derivatized by methoxyamination, using 80 µl solution of methoxyamine hydrochloride in pyridine (20 mg ml−1), and incubated in a thermomixer during 90 min at 30°C in order to induce methoxymation reaction. Subsequently was performed a silylation reaction with 80 µl of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) and incubated again for 30 min at 37°C before injecting to the GC-TOF MS machine. Samples were analyzed in an Agilent 7683B Series Injector (Agilent, Santa Clara, CA) coupled to an Agilent HP6890 Series gas chromatograph system and a 5973 quadrupole type Mass Selective Detector (Agilent), using an Agilent J&W DB-5ms ultra inert column (30 m × 0.25 mm × 0.25 µm). Helium was applied as the carrier gas in a constant column flow rate of 1 ml min−1. The injector temperature was held at 230°C. Splitless injection (1:l) of samples was carried out during 1.5 min using a total flow of 39 ml min−1, followed by a reduction to 24 ml min−1 after 2 min. The column temperature program started at 70°C for 5 min, and then it was ramped to 325°C at 5°C min−1. After 1 min at 325°C, the oven was brought to the initial temperature of 70°C within 5 min. Before the next injection, a temperature equilibration phase of 5 min was used. The transferline and EI source temperature were 250 and 200°C, with the EI spectra acquired between 50 and 600 Da. The electron multiplier voltage was set on 1700 V. Raw chromatographic data acquired from GC-TOF-MS analysis were processed using AMDIS (Automated Mass Spectral Deconvolution and Identification System) software ( to identify the compounds searching the NIST (National Institute of Standards and Technology) 8.0 library. For control and MpNEP2-treated cells, the samples were analyzed in three independent experiments to determine the analytical error in the analysis of metabolites by GC–MS. Pre-processing of metabolic data included the composition of target lists of peaks detected in the samples based on retention time and mass spectra. The peaks were integrated for all samples and their areas were subsequently normalized to the internal standard (ribitol) and biomass. The resulting target lists were used for statistical analysis. Fold changes and P values (t-test, two-sided, unequal variance) were calculated for control and MpNEP-2-treated cells. Signals were considered as differential when fold change was ≥2.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Cytological effects of MpNEP2 treatment

In this study, N. benthamiana GFP-expressing cells were evaluated in an epifluorescence microscope 1-h after exposure to MpNEP2 in attempts to assess the early cytological effects of the protein. The control cells did not show any damage to cells membranes or lose their ability to express GFP (Fig. 1A, C, E). On the other hand, MpNEP2 treatment rapidly reduced the number of GFP-expressing cells and, when they were observed, the expression of GFP was restricted to areas within the cells, due to the detachment and condensation of the cytoplasm inside the cell (Fig. 1B, D, F).


Figure 1. MpNEP2 induces a detachment of cell membranes. GFP-expressing cells of Nicotiana benthamiana in exponential growth phase were exposed to 1 µg ml−1 MpNEP2 and observed in epifluorescent microscope. (A, C, E) Control cells, not exposed to MpNEP2, showing intact membranes. (B, D, F) Cells treated with MpNEP2 for 1-h showing a detachment of the cell membranes and GFP release. Magnification is 250×.

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Many NLPs have a characteristic ability to trigger cell death in dicotyledonous plants, although how this occurs, if by apoptosis, necrosis or intermediate forms of both, remains controversial (Fellbrich et al. 2002, Keates et al. 2003, Bae et al. 2006, Schouten et al. 2008). A classification of plant cell death based on morphological criteria has been recently suggested (van Doorn et al. 2011). In this classification the terms ‘apoptosis’ or ‘apoptotic-like’ are considered as incorrect and misleading, since the typical cytological characteristics of apoptosis are absent in plants. However, two major classes of PCD can be recognized in plants: vacuolar cell death and necrotic cell death. Vacuolar cell death involves a combination of autophagy-like process and release of hydrolases from collapsed lytic vacuoles, which rapidly destroys the protoplast or even the entire cell. On the other hand, necrotic cell death is characterized by swelling of various organelles, early rupture of the plasma membrane, shrinkage of the protoplast and lack of vacuolar cell death features. In this study, we observed a response similar to necrosis. MpNEP2-treated cells released GFP protein outside the cell (Fig. 1F), indicating direct damage to the plasma membrane. Another important observation was the total detachment of the plasma membrane, one of the first steps of protoplast shrinkage (van Doorn et al. 2011).

Proteomic analysis of MpNEP2-treated N. benthamiana cells and Gene Ontology annotation

To identify proteins that are important in the signaling process initiated in the early stages (1-h) of MpNEP2 treatment, we fractionated equal amounts of protein in control and MpNEP2-treated cells using 2-DE. Overall, about 100 differentially expressed protein spots were excised from 2-DE gels and 54 were identified by MS/MS (Table 1, Appendix S1). The setup of protein spots that increased or decreased in relative abundance (MpNEP2-treated/control) of at least twofold as compared with the control treatment in three replicate gels are shown in Fig. 2. Seventeen proteins were identified as upregulated [60 kDa chaperonin subunit alpha (CPN-60 alpha), pyruvate decarboxylase (PDC), UDP glucose 6 dehydrogenase (UDPG), enolase (ENO), alpha tubulin, adenosylhomocysteinase, malate dehydrogenase (MDH), heat shock protein 80, annexin, aconitate hydratase (ACO), elongation factor 2 (EF-2), NADH ubiquinone oxidoreductase isoform 1 and 2, superoxide dismutase Mn mitochondrial (MnSOD), 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (MetE), heat shock cognate 70 kDa, and alcohol dehydrogenase (ADH)]. Fifteen proteins were identified as downregulated [stromal 70 kDa heat shock, nucleosome assembly protein 1 like protein 2, glyceraldehyde 3 phosphate dehydrogenase (GAPDH), calreticulin, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (PGAM-i), 14-3-3-like protein GF14 epsilon, glutathione S transferase (GST), copper chaperone, glycine-rich RNA binding protein 3, heat shock cognate 70 kDa protein 2, heat shock protein 90 (HSP90), heat shock protein 81 (HSP81), fructose-bisphosphate aldolase cytoplasmic (aldolase), fructose bisphosphate aldolase like protein and l-ascorbate peroxidase 2 cytosolic (APX2)]. The changes in the stead-steady levels of these proteins were statistically significant (P ≤ 0.05). The most significant upregulated proteins [60 kDa CPN-60 alpha (fold change of 2.3), PDC (fold change of 2.1), enolase (fold change of 2.1), adenosylhomocysteinase (fold change of 2.8), aconitate hydratase (unique), EF-2 (unique), NADH ubiquinone oxidoreductase (unique), 5-methyltetrahydropteroyltriglutamate-homocystein (unique) and heat shock cognate 70 kDa protein (unique)] and downregulated proteins [heat shock cognate 70 kDa protein 2 (unique), HSP90 (unique), HSP81 2 (unique), nucleosome assembly protein (fold change of −3.8), calreticulin (fold change of −13.3), fructose-bisphosphate aldolase (unique) and L-ascorbate peroxidase 2 (unique)] are shown in Fig. 3.

Table 1. Identification of proteins in GFP-expressing Nicotiana benthamiana cells treated with MpNEP2. aProtein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits. bMP, matching peptides. cPercentage cover of matching peptides of full-length predicted protein. dMascot Ions score is −10*Log(P), where P is the probability that the observed match is a random event. Individual ions scores >42 indicate identity or extensive homology (P < 0.05). eSpot ratio: N, present only in the MpNEP2 treatment; C, present only in the control treatment; N/C, ratio MpNEP2-treated/control. *Not statistically validated
Spot no.AccessionDescriptionMascot protein scoreaNo. MPb% CovercTwo most significant peptidesMascot ion scoredSpot ratioe N/C
1Q02028Stromal 70 kDa heat shock related protein538812.32K.SFAAEEISAQVLR.K122−3.2
2Q01899Heat shock 70 kDa protein mitochondrial6911320.15R.QAVTNPTNTVFGTK.R107*
3AAP96920.1Pyruvate decarboxylase17334.63R.VSAANSRPPNPQ.-692.1
4CAD27461.1Nucleosome assembly protein201413.79K.KGKNVTVK.I84−3.8
5P2123960 kDa chaperonin subunit alpha465816.3R.GYISPQFVTNPEK.L952.3
6Q9XI01Probable protein disulfide isomerase 115032K.QSGPASAEIK.S60 49*
8AAF26445.1Vacuolar H ATPase B subunit3971128.81K.AVVQVFEGTSGIDNK.Y90*
9P17614ATP synthase subunit beta chloroplastic25946.61R.FVQAGSEVSALLGR.M71*
      R.AVAMSATDGLTR.G + Oxidation (M)64 
12YP_173459.1ATP synthase F1 subunit 1195715.52R.VVDGLGVPIDGR.G70*
13Q96558UDP glucose 6 dehydrogenase16847.08K.LAANAFLAQR.I752.2
14CAA90011.1Alpha tubulin181311.26R.AVFVDLEPTVIDEVR.T922.3
15P17614ATP synthase subunit beta mitochondrial4131128.39R.FTQANSEVSALLGR.I97*
17P354942,3-Bisphosphoglycerate-independent phosphoglycerate mutase203614.31K.ALEYENFDKFDR.V R.GWDAQVLGEAPHK.F83 80−2.7
18Q43497Monodehydroascorbate reductase387310.16R.EIDDADQLVEALK.A125*
19P43282S-adenosylmethionine synthetase 3435723.08K. EQPMDEELKEAFQNAYLELGGLGER.A K.IDVLLHFQSAHIAQGVDNAADK.Q100 89*
20P27322Heat shock cognate 70 kDa protein 224859.16R.IINEPTAAAIAYGLDK.K117C
21AAP72158.1Heat shock protein 909322.17K.APFDLFESK.K56C
22P55737Heat shock protein 81 220846.72K.EVSNEWSLVNK.Q55C
24O04916Aconitate hydratase, cytoplasmic217611.2R.SNLVGMGIVPLCFK.A + Oxidation (M)89−2.2
25P50218Isocitrate dehydrogenase10537.95R.SLNLTLR.K60*
26AAU14833.1Adenosine kinase isoform 2S120310.29K.KPENWALVEK.A81*
27CAC12826.1Malate dehydrogenase336723.49K.LSSALSAASSACDHIR.D883.3
28P09094Glyceraldehyde-3-phosphate dehydrogenase cytosolic Fragment439829.75K.DAPMFVVGVNEK.E + Oxidation (M)K.KVVISAPSK.D102 86−2.3
29AF113545_1Vacuole associated annexin VCaB42363519.3K.LLVPLLTAFR.Y105*
30AAT42189.1Putative mitochondrial malate dehydrogenase118430.66K.DDLFNINAGIVK.- -83*
      VAILGAAGGIGQPLSLLMK.D + Oxidation (M)78 
31P8594214-3-3-like protein 489335.71R.NLLSVGYK.N51−2.7
32P42212Green fluorescent protein379829.83K.LEYNYNSHNVYIMADK.Q + Oxidation (M)95*
33Q03662Probable glutathione S transferase9634.04K.NLDFELVHVELK.D68*
34Q2LQC250S ribosomal protein L68336.15R.AYGGVLSGSAVR.E48*
35P32111Glutathione S transferase8334.15K.SPLLLQSNPIHK.K66−2.2
36P08440Fructose-bisphosphate aldolase, cytoplasmic isozyme10633.94K.EAAWGLAR.I R.TAAYYQQGAR.F77 58C
37ABC01905.1Fructose bisphosphate aldolase like protein9326.42K.AADGTPFVDIIR.A66C
38Q9FE01L-ascorbate peroxidase 2 cytosolic12249.16R.VDASGPEDCPEEGRLPDAGPPSPATHLR.I59C
39AAP04395.1Glutathione S transferase U1168310.08K.TPLLLELNPLHK.K90*
40AAT12488.1Copper chaperone364314.12K.GNVQPDAVLQTVSK.T112−2
41AF500588_1Heat shock protein 80293331.75R.ELISNSSDALDK.I107N
      K.GWGTNEK .L73 
43P84977Glycine-rich RNA binding protein 363350K.GFGFVTFGDEK.D68−3.2
44Q9S9P140S ribosomal protein S12 194218.75K.TPGPGAQSALR.A60*
45P49608Aconitate hydratase, cytoplasmic206811.58R.SNLVGMGIVPLCFK.A + Oxidation (M)103N
46O23755Elongation factor 2, EF 283810.79K.STLTDSLVAAAGIIAQEVAGDVR.M88N
47Q43644NADH ubiquinone oxidoreductase10269.76R.FFYDGLK.R107N
48P11796Superoxide dismutase Mn, mitochondrial precursor205411.4R.LVIETTANQDPLVSK.G K.YANEVYEK.E77 51N
49Q426625 methyltetrahydropteroyltriglutamate homocystein17142.62K.GMLTGPVTILNWSFVR.N K.YLFAGVVDGR.N87 46N
50P09189Heat shock cognate 70 kDa protein2671627.65R.IINEPTAAAIAYGLDK.K102N
51P09094Putative protein327718.71K.LVSWYDNEWGYSSR.V116N
52Q43644Putative protein6535.69K.GPDVVGSFGLLQPLADGLK.L59N
53P25141Alcohol dehydrogenase 182411.78K.FGVTEFVNPK.R68N
      R.GVMIHDGQTR.E + Oxidation (M)45 

Figure 2. Two-dimensional PAGE reference gel profiles of the total cellular protein fraction isolated from GFP-expressing Nicotiana benthamiana cells. Samples were resolved in the first dimension using a linear IEF strip of pH 4–7. The approximate molecular weights are shown on the left of the gel. (A) Reference gel of proteins extracted from control cells. (B) Reference gel of proteins extracted from MpNEP2-treated cells. All proteins identified by mass are numbered and circled. The gels shown are representative of three such a replicates.

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Figure 3. Comparison of the most significantly up- and downregulated proteins from 2D-PAGE. Upregulated proteins (left panel): stromal 70 kDa heat shock related protein (1), PDC (3), 60 kDa CPN-60 alpha (5), enolase (11), adenosylhomocysteinase (16), aconitate hydratase (45), EF-2 (46), NADH ubiquinone oxidoreductase (47), 5 methyltetrahydropteroyltriglutamate homocystein (49), heat shock cognate 70 kDa protein (50), NADH ubiquinone oxidoreductase (52). Downregulated proteins (right panel): nucleosome assembly protein (4), calreticulin (7), heat shock cognate 70 kDa protein 2 (20), HSP90 (21), heat shock protein 81 2 (22).

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To aid in the interpretation of the experimental findings, the proteins accession numbers were uploaded to the Uniprot website ( and proteins were categorized by Gene Ontology (GO) cellular location, molecular function or biological process terms. Fifty-nine representative categories of interest are shown in Fig. 4. Most proteins that were upregulated by MpNEP2 treatment are involved in nucleotide binding function (47.1%), oxidoreductase activity (41.2%), metal ion binding function (35.3%) and GTP binding function (11.8%) and were located mostly in the cytosol (47.1%) and in the mitochondria (29.4%). Proteins that were downregulated are mostly involved in glycolysis (31.2%), response to stress process (31.2%) and protein folding (25%) and were located in the cytosol (56.2%), mitochondria (25%) and membrane (25%). Among the proteins that were upregulated due to treatment with MpNEP2, we highlight those that are involved in the oxidation-reduction process. NADH ubiquinone oxidoreductase (Complex I), UDP glucose 6-dehydrogenase (UDPGDH), malate dehydrogenase (MDH), superoxide dismutase Mn (SOD) and alcohol dehydrogenase 1 (ADH1) are some examples. The proteomic changes indicate that MpNEP2 treatment potentially increases the cell's capacity to reduce oxidized proteins. Among the proteins that were downregulated, most of them are involved in biological processes such as glycolysis, response to stress and protein folding. 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (PGAM-I), fructose-bisphosphate aldolase, heat shock cognate 70 kDa protein 2, HSP90, l-ascorbate peroxidase 2, calreticulin and stromal 70 kDa heat shock protein are some examples of these proteins.


Figure 4. Functional classification of proteins identified by 2D-PAGE and nano-LC-MS/MS analysis. GO classification (Cellular location, Molecular function and Biological process) were categorized using Uniprot website ( The percent of genes reflect the participation of the 53 gene products related to molecular function, cellular localization and biological process in which they are involved.

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In stress conditions, plants can change their metabolism from aerobic respiration to anaerobic fermentation (Peng et al. 2001, Fukao and Bailey-Serres 2004). As the respiration declines, the electron flow through the respiratory pathway is reduced, thus diminishing ATP production. Consequently, chemical oxidizing power (i.e. nicotinamide adenine dinucleotide, NAD+) is generated by alternative pathway. The fermentative pathway serve as a safe route and includes two steps: the first one is the carboxylation of pyruvate to acetaldehyde (catalyzed by PDC) and the second is the reduction of acetaldehyde to ethanol concomitantly with the oxidation of NAD(P)H to NAD(P)+, catalyzed by ADH. However, the fermentation metabolic route allows the synthesis of only 2 moles of ATP against 36 per mole of glucose produced during aerobic respiration. To compensate the deficit in energy, glycolysis is accelerated, leading to depletion of carbohydrate reserves (Parent et al. 2008). In this work, the upregulation of PDC and ADH give us an indication that this pathway is rapidly activated in MpNEP2-treated cells, demonstrating that MpNEP2 initially affects the ATP synthesis. The downregulation of the enzymes involved in the glycolytic pathway (PGAM-I, fructose-bisphosphate aldolase and GAPDH) further confirms this observation. Besides, the respiratory rates of Nep1-treated cells of tobacco were observed to be significantly reduced about 90 min after treatment, while the cell membranes were still intact (Jennings et al. 2001). A drop in ATP level as a result of mitochondrial dysfunction has been well documented in the downstream events of necrosis in both plant and animal cells (van Doorn et al. 2011). The upregulated proteins MDH and aconitate hydratase, involved in the citric acid cycle, could provide the precursors for biosynthesis of compounds such as amino acids and the reducing agent NADH, which is used in various biochemical reactions. The two upregulated proteins adenosylhomocysteinase and 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase demonstrate the importance of methionine biosynthesis in this process, since this amino acid regulates the one-carbon metabolism and also the methylation capacity (Ranocha et al. 2001, Kocsis et al. 2003).

Two other responses need to be considered in this process, the calcium (Ca2+) influx from endoplasmic reticulum (ER) and the generation of ROS. They are well described responses of dicot cells to NLPs (Fellbrich et al. 2002, Jennings et al. 2001, Schouten et al. 2008, Silva et al. 2011). The mitochondrial NADH ubiquinone oxidoreductase and Mn SOD were some of the proteins upregulated in this work and that could be involved in the production of ROS (Gechev et al. 2006). SODs catalyze the conversion of superoxide (O2) into O2 and H2O2, which are less harmful reactants. NADH ubiquinone oxidoreductase, also known as NADH dehydrogenase or complex I, is an enzyme that catalyzes the transfer of electrons from NADH to coenzyme Q (CoQ) in the inner mitochondrial membrane. It has been described as the main source of ROS in the mitochondria (Moller 2001). Here, the induction of these two enzymes as a result of MpNEP2 treatment may contribute to generation of a highly oxidative environment for the mitochondria, with direct impact on the ATP synthesis. The overproduction of ROS, which are common byproducts of the respiration in mitochondria, can easily lead these organelles to death (Seo et al. 2006, van Doorn et al. 2011).

One of the most important signal transduction pathways described in response to NLPs is the calcium signaling (Pemberton and Salmond 2004). A signaling cascade involving calcium and MAP kinases appears to be triggered following NLP recognition (Fellbrich et al. 2002). Among the Ca2+ binding proteins potentially involved in this pathway are the upregulated annexin and the downregulated calreticulin. Annexin, a Ca2+-binding protein, can behave as cytosolic, peripheral or integral membrane protein. The characteristic feature of this protein is that it can bind membrane phospholipids in a Ca2+ sensitive and insensitive manner (Talukdar et al. 2009). In this way, annexin can be upregulated in response to a calcium rich environment and could be involved in the maintenance of cellular homeostasis in MpNEP2-treated cells. Calreticulins are Ca2+-binding chaperones localized in the ER of eukaryotes acting in glycoprotein folding quality control and Ca2+ homeostasis (Del Bem 2011). The downregulation of calreticulin by MpNEP2 could favor the calcium availability outside ER, contributing to the oxidative stress.

It has been previously reported in the literature that the NLP-induced cell death differs from both PCD and HR, since mutants lacking components of the PCD pathway are still sensitive to NLPs (Arenas et al. 2010, Qutob et al. 2006) and the genes induced by NLPs or during HR are not the same (Bae et al. 2006, Qutob et al. 2006). Comparing our data with those reporting proteomic changes during PCD (Chaves et al. 2011, Swidzinski et al. 2004) or HR (Jones et al. 2004, Jones et al. 2006, Liao et al. 2009, Mahmood et al. 2006, Mukherjee et al. 2010) in plant cells or leaves, only a few proteins changing in abundance were similar. These included aconitate hydratase, (Mn)-SOD, GST, calreticulin, MDH, GAPDH, HSP70 and HSP60. The remaining differentially expressed proteins identified in our study have not been observed to change during PCD or HR. Therefore, our data reinforce that NLP-induced changes are distinct from other known types of cell death or defense that are triggered in plants.

Metabolomic profile

To examine the effects of MpNEP2 treatment on different metabolic pathways of N. benthamiana cells, global biochemical profiles were also compared between control and MpNEP2-treated cells in the first hour post-inoculation. A total of 30 metabolites were detected and semi-quantified based on the NIST8.0 library (chromatogram TOF, Fig. 5), of which eleven showed significant differences between the treatments (P ≤ 0.05). Among the compounds that were upregulated in MpNEP2-treated cells are myo-inositol, ethylene, the amino acids proline and alanine, (+)-delta-cadinene, propionic acid and the saturated fatty acid lauric acid. Among the compounds that were downregulated are hydroxylamine, the amino acid threonine, phosphoric acid and glucopyranose. A list of all the compounds identified by GC/MS is shown in Table 2 and Appendix S2. Changes in abundance of these metabolites may constitute the signal(s) required to trigger the observed plant immunity-associated defenses (Ottmann et al. 2009) that succeed NLP-driven membrane disruption.


Figure 5. Metabolite identification of 30 chemical constituents (Table 2). Reference chromatogram (GC-TOF) of control and MpNEP2-treated cells.

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Table 2. Identified metabolites and spectrometric data of 30 chemical compounds analyzed by GC-TOF-MS. Bold numbers represent the highest intensity mass ion of referred compound and are organized in descending order
Identified metabolitestR (min)Mass fragments
1Lactic acid9.5673, 117, 147, 191, 219
2Hydroxylamine10.6973, 133, 146, 249, 119
3Phosphoric acid12.84241, 73, 133, 242, 45, 256
4Malonic acid13.56147, 73, 75, 148, 45, 233, 248
5Glycine16.34174, 73, 147, 175, 248, 276
6Succinic acid16.52147, 73, 75, 148, 247, 149, 262
7Glyceric acid17.2073, 147, 189, 103, 45, 292, 133, 307
8Fumaric acid17.40245, 73, 147, 75, 45, 246, 143, 115, 133
9l-threonine18.7173, 117, 218, 219, 101, 45, 291, 320
10Alanine19.55174, 73, 248, 147, 290
11Malic acid21.3673, 147, 233, 75, 245, 350
12Cadinene21.73161, 119, 105, 41, 91, 134, 204
13l-Proline21.89156, 73, 147, 157, 258, 230, 45, 273
14Propionic acid22.11174, 73, 175, 147, 248, 86, 176, 304
15Threonic acid23.2873, 147, 292, 205, 220, 117, 103, 319, 379, 409
16Lauric acid24.1343, 60, 57, 41, 102, 61, 55, 183, 200, 201, 242
17Glutamic acid24.37246, 73, 128, 147, 247, 75, 156, 230, 348, 363
18l-Asparagine25.5373,116, 231, 132, 75, 147, 188, 316, 348
19l-Glutamine27.7573, 156, 155, 245, 147, 347, 362
20Ribonic acid28.2973, 147, 103, 292, 217, 75, 333, 429
21Citric acid28.9373, 147, 273, 75, 45, 347, 363, 375, 465
22d-Fructose30.2573, 103, 217, 307, 147, 133, 277, 390
23d-Fructose30.4673, 103, 217, 307, 147, 133, 277, 393, 464, 554
24Glucose30.7673, 147, 205, 319, 160, 45, 364
25Glucose31.0873, 147, 205, 319, 160, 45
26α-d-Glucopyranose32.36204, 73, 191, 147, 205, 217, 206, 393, 435
27l-Mannose33.2973, 204, 205, 147, 191, 206, 75, 305, 319
28Ethylene33.5573, 147, 102, 292, 45, 74, 75, 130
29Myo-inositol34.2973, 147, 217, 305, 191, 133, 318, 432, 507
30d-Glucopyranoside43.68361, 73, 362, 217, 147, 363, 103, 437, 451

Myo-inositol has been proposed to act as an important stress protector (Bohnert et al. 1995, Casati et al. 2011), and may also regulate PCD in Arabidopsis. Mutants in a myo-inositol-1-phosphate synthase (AtIPS1) gene exhibited spontaneous cell death (Meng et al. 2009). The encoded protein has a putative nuclear localization signal, suggesting that nuclear pools of myo-inositol may have a critical role in the regulation of PCD (Meng et al. 2009). Myo-inositol is phosphorylated by lipid-dependent and -independent pathways, leading to the generation of a variety of derivatives with multifunctional roles. One of these derivatives, phosphatidylinositol, may act as both membrane structural lipid and signal transducing molecule, which plays a key role in numerous stress-related responses in plants (Valluru and Van den Ende 2011). In Arabidopsis, a NLP from Fusarium oxysporum was able to induce an increase in the levels of myo-inositol, which raised the possibility that inositol is synthesized in response to membrane damage and has an important role in membrane biogenesis (Bae et al. 2006).

Ethylene is another signaling molecule that has long been recognized in plant stress responses. Ethylene synthesis and action is regulated by both endogenous signals during development and environmental cues from pathogen attack and abiotic stresses (Kevin et al. 2002). The ethylene biosynthetic pathway has been focus of intensive investigation in plants (Kende 1993, Wang et al. 2002). These studies have established S-adenosylmethionine (SAdoMet) and 1-aminocyclopropane-1-carboxylic acid (ACC) as the precursors of ethylene (Wang et al. 2002). Here we observed that the enzyme that participates in the biosynthesis of l-methionine (5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase) is upregulated, indicating a direction for the synthesis of l-methionine and ethylene. Indeed, an increase of ethylene emission has been observed in tobacco leaves treated with NLPs (Bailey et al. 1997, Jennings et al. 2000).

Other compounds induced by treatment with MpNEP2 have been described to be involved in response to general stress, such as the amino acids proline and alanine (Goolish and Burton 1998) and (+)-delta-cadinene. The latter compound is involved in a cyclization reaction in terpenoid biosynthesis (Rosenkranz and Wink 2008). Some natural products, such as alkaloids, polyphenols, terpenoids and saponins, have been implicated in the induction of apoptosis (Rosenkranz and Wink 2008). These compounds have the ability to interact with important molecular targets in the cells, including membranes, receptors, microtubules and DNA (Rosenkranz and Wink 2008). It may be possible that terpenoid biosynthesis has an active role in cell death triggered by MpNEP2.

Hydroxylamine levels were reduced in MpNEP2-treated cells. This compound appears to be involved in formation of nitric oxide (NO), another important signaling molecule. NO plays critical functions in a variety of physiological processes in animals. Many advances have been made in the past years about the role of NO in plants. Nitrite has been established as the main precursor for NO synthesis in plants, which is mediated by enzymatic (nitrate reductase) and non-enzymatic reactions (Besson-Bard et al. 2008). Thus, reducing levels of hydroxylamine as shown here could be a direct implication of increased synthesis of NO, as previously observed in plant cells treated with NLPs (Qutob et al. 2006, Schouten et al. 2008). In Arabidopsis, the production of NO was mentioned as a very early response to NLP (within 30 min after stimulation), which occurs at time points preceding the onset of NLP-induced necrosis (Qutob et al. 2006).


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Here we used high throughput techniques such as proteomics and metabolomics to decipher the early responses of plant cells to MpNEP2. We show that most proteins that were upregulated are involved in nucleotide binding function and oxidoreductase activity, which potentially increases the cell's capacity to reduce oxidized proteins. The downregulated proteins are mostly involved in glycolysis, which is supposed to affect the ATP biosynthesis and to contribute to cell death. Collectively, these data indicate that MpNEP2 induces a rapid change in the plant cell metabolism, from aerobic respiration to anaerobic fermentation, thus diminishing ATP production, leading then to the ATP depletion, total detachment of the plasma membrane and consequently to necrosis (Fig. 6). This interactive approach will help to create a general view of the molecular and biochemical events affected during the early stages of NLP treatment.


Figure 6. Diagram representing the main events that occur in plant cells following MpNEP2 treatment. Downregulated proteins and metabolites are in red text, while those upregulated are in blue text. Solid-line arrows represent the main pathways involved in the metabolic response to MpNEP2, while dotted-line arrows represent the major signaling molecules involved in signal transduction, as evidenced in our experiment. See text for abbreviations and further details. GPX, glutathione peroxidase; GSSG, oxidized glutathione; GSH, reduced glutathione.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

This research was supported by the Moniliophthora perniciosa Proteomic Network granted by the Financiadora de Estudos e Projetos (FINEP; project # 01.07.0074-00) and the Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB; project # 7912/2007).


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information
ppl12061-sup-0001-AppendixS1.docWord document145KAppendix S1. Original proteome data set.
ppl12061-sup-0002-AppendixS2.docWord document78KAppendix S2. Original metabolome data set.
ppl12061-sup-0003-AppendixS3.jpgJPEG image559KAppendix S3. Symptoms of witches' broom disease in Theobroma cacao.

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