The pathogen Moniliophthora perniciosa promotes differential proteomic modulation of cacao genotypes with contrasting resistance to witches´ broom disease

Background: Witches' broom disease (WBD) of cacao (Theobroma cacao L.), caused by Moniliophthora perniciosa, is the most important limiting factor for the cacao production in Brazil. Hence, the development of cacao genotypes with durable resistance is the key challenge for control the disease. Proteomic methods are often used to study the interactions between hosts and pathogens, therefore helping classical plant breeding projects on the development of resistant genotypes. The present study compared the proteomic alterations between two cacao genotypes standard for WBD resistance and susceptibility, in response to M. perniciosa infection at 72 h and 45 days post-inoculation; respectively the very early stages of the biotrophic and necrotrophic stages of the cacao x M. perniciosa interaction. Results: A total of 554 proteins were identified, being 246 in the susceptible Catongo and 308 in the resistant TSH1188 genotypes. The identified proteins were involved mainly in metabolism, energy, defense and oxidative stress. The resistant genotype showed more expressed proteins with more variability associated with stress and defense, while the susceptible genotype exhibited more repressed proteins. Among these proteins, stand out pathogenesis related proteins (PRs), oxidative stress regulation related proteins, and trypsin inhibitors. Interaction networks were predicted, and a complex protein-protein interaction was observed. Some proteins showed a high number of interactions, suggesting that those proteins may function as cross-talkers between these biological functions. Conclusions: We present the first study reporting the proteomic alterations of resistant and susceptible genotypes in the T. cacao x M. perniciosa pathosystem. The important altered proteins identified in the present study are related to key biologic functions in resistance, such as oxidative stress, especially in the resistant genotype TSH1188, that showed a strong mechanism of detoxification. Also, the positive regulation of defense and stress proteins were more evident in this genotype. Proteins with significant roles against fungal plant pathogens, such as chitinases, trypsin inhibitors and PR 5 were also identified, and they may be good resistance markers. Finally, important biological functions, such as stress and defense, photosynthesis, oxidative stress and carbohydrate metabolism were differentially impacted with M. perniciosa infection in each genotype

Proteomic profile of necrotrophic mycelium of Moniliophthora perniciosa

The fungus Moniliophthora perniciosa, the etiologic agent of witches' broom disease of cacao (Theobroma cacao L.), has a hemibiotrophic life cycle, with a biotrophic phase and a necrotrophic phase. The biotrophic phase, initiating the disease, is characterized by a monokaryotic mycelium, while the necrotrophic phase is characterized by a dikaryotic mycelium leading to plant death. During the culture of Mp on cookies, six different developmental phases were observed according to the mycelium color or the organ produced: white (? 35 days), yellow (? 37 days), pink (? 40 days ), dark pink (? 45 days), primordium (? 60 days) and basidiocarp (? 75 days). In this study, we identify proteins involved in each stage of the fungus development focusing on basidiocarp formation. Proteins were extracted using the ADP method, followed by a simple cleaning using SDS-dense and phenol. The quantification was made using the 2-D quantification kit. The proteins were extracted in triplicate and separated using a 12% bi-dimensional SDS-PAGE gel. The 2-D maps showed approximately 300 spots per gel, and present differential protein expression patterns. Spots were cut from gels and analyzed by mass spectrometry. At the basidiocarp stage, we identified several proteins potentially involved in its formation, which may be good candidates for further analysis required to understand the mode of spread of the fungus. To our knowledge this is the first work analyzing Mp development by proteomics. (Texte intégral

Proteomic Analysis of Moniliophthora perniciosa necrotrophic phase : [Abstract R8990]

Moniliophthora perniciosa (Mp) is the causal agent of witches' broom disease of cacao (Theobroma cacao L.). Mp is a hemibiotrophic fungus; the biotrophic phase, initiates the disease and is characterized by a monokaryotic mycelium, while the necrotrophic phase is characterized by a dikaryotic mycelium and leads to the necrosis of the infected tissues. The study of the necrotrophic phase was conducted on artificial system (cookies), the only solid medium allowing basidiocarp and basidiospore production. During the culture of Mp in cookies, six different developmental phases were observed according to the mycelium color or the organ produced: white, yellow, pink, dark pink (or pre-water stress), primordium and basidiocarp. The objective of this work was to identify proteins specific of each phase, particularly the phases preceding the basidiocarp formation. Proteins were extracted using the ADP method, followed by a simple cleaning using SDS-dense and phenol. The quantification was made using the 2-D quantification kit. The proteins were extracted in triplicate and separated using a 12% Bi-dimensional SDS-PAGE gel. The 2D maps showed approximately 300 spots for gel, and present differential protein expression patterns. Spots were cut from gels and analyzed by mass spectrometry. At the basidiocarp stage, we identified several proteins potentially involved in its formation, which may be good candidates for further analysis required to understand the mode of spread of the fungus. To our knowledge, this is the first analysis of Mp development phases by proteomics. (Résumé d'auteur

The pathogenesis-related protein PR-4 from Theobroma cacao has antifungal activity and induces ROS in Moniliophthora perniciosa : S03O02

The pathogenesis-related proteins class 4 (PR-4) are known to be involved in plant defense response and/or related stress situations. The objective of this study was to evaluate the antifungal activity and reactive oxygen species (ROS) production of the TcPR-4b protein in Moniliophthora perniciosa. The TcPR-4b gene was cloned into pET28a and the resulting in frame fusion plasmid was used to transform Escherichia coli Roseta (DE3) for protein expression. The expression of the TcPR-4b recombinant protein was induced by 0.4 mM isopropyl-?-D-thio-galactoside and purified by immobilized metal affinity chromatography with TALON® Metal Affinity Resin. The TcPR-4b protein was used for in vitro assays against dikaryotic M. perniciosa broken hyphae. Then, 1 ml of the broken hyphae suspension was incubated for 2h with: i) 10 ?g of TcPR-4b in phosphate buffer (PB); ii) 20 ?g of TcPR-4b in PB; iii) 40 ?g of TcPR-4b in PB; iv) PB (control). Then, 1 ml of each treatment was applied on CPD solid medium (2% glucose, 2% peptone, 2% of agar) and incubated for 7 days at 25°C. The inhibition of hyphal growth was examined by counting the number of pseudo-colonies on three experimental replicates. To detect the production of the ROS in living cells of M. perniciosa, 1 ml of hyphae suspension was treated with 10 ?g of TcPR-4b in PB (or not - control) overnight at 25ºC, and then incubated at 25°C for 30 min with dihydroethidium which selectively stains the mitochondrial superoxide (O2 -). The hyphae were mounted on slides and observed under fluorescence microscope DMRA2 (Leica). Images were captured under fluorescent filters using the IM50 software (Leica). The reduction of M. perniciosa survival was observed in all tested concentrations of TcPR-4b with a decrease of survival correlated to the increase of the protein concentration. The hyphae treated with TcPR-4b presented a bright red fluorescence with specific more intense fluorescence in some foci. The control did not present fluorescence emission comparing to the hyphae treated with TcPR-4b. This study showed the antifungal activity of TcPR-4b and the induction of ROS in M. perniciosa. Work supported by CNPq, FAPESB, FINEP/RENORBIO, CAPES, Cirad. (Texte intégral

Proteomic profile of the fungus Moniliophthora perniciosa in response to PR10 from Theobroma cacao : [Abstract R9140]

Witches' broom disease is caused by the hemibiotrophic basidiomycete Moniliophthora perniciosa. This pathogen is the main cause of the decline in cocoa production, and consequently of social, economic and environmental problems. The transcriptomic program of cacao allowed the identification of a pathogenesis-related 10 protein. The corresponding recombinant protein expressed in Escherichia coli BL21 showed a strong antifungal activity in vitro against M. perniciosa. Here, we developed a proteomic analysis of M. perniciosa proteins expressed in the presence of recombinant TcPR10. M. perniciosa was grown in CPD 2% agar medium; after 15 days, the fungal hyphae were broken and were brought together with 3 ?g/mL of TcPR10 for 1h. After this time, the total proteins of the hyphae were extracted using the ADP method, followed by a simple cleaning using the method of SDS-dense and phenol. The quantification was made using a 2-D quantification kit. The proteins were extracted in triplicate and separated using a 12% bi-dimensional SDS-PAGE gel. The 2D map analysis showed approximately 300 "spots" per gel (control and one hour treatment) with differential protein expression pattern. The analysis using a mass spectrometry (naniESI-Q-TOF) was made for the identification of the spots. We identified several proteins involved in fungal metabolism, carbohydrates/proteins metabolism, related proteins to growth and phytotoxics proteins. More spots have been identified to better understand the mechanism of fungi response to protein PR10. (Résumé d'auteur

The cysteine protease TcCYSPR04 T. cacao accumulates in senescent leaves and change the biotrophic phase for saprophytic tissues infected by M. perniciosa

A cysteine proteinase named TcCYSPR04 was identified in a cDNA library of the Theobroma cacao-Moniliophthora perniciosa interaction, in the ESTtik-CIRAD database and in the cacao genome of MARS. TcCYSPR04 presents an ORF of 1068 bp encoding protein with: (i) a molecular weight and isoelectric point of 39 kDa and 5;43, respectively; (ii) a signal peptide with a probable cleavage site between the amino acids 19 and 20; (iii) an inhibitory domain between amino acids 56 and 112; and (iv) a catalytic domain between amino acids 158 and 353. TcCYSPR04 may be secreted or cytoplasmic. According to the literature, the cysteine proteases may be involved in cell differentiation, senescence, and programmed cell death-PCD. The catalytic domain is highly conserved among cysteine proteases and was subcloned into pET28a expression vector . The corresponding protein was expressed in strain Escherichia coli BL21 (DE3) and purified by affinity column His-Trap. Polyclonal antibodies against the recombinant protein were produced in rabbits and purified by immunoadsorption. Total proteins were extracted from apoplastic fluid of healthy and infected leaves of resistant and susceptible varieties of cacau. Proteins were subjected to electrophoresis on SDS-PAGE 15%, and immunoblot using the serum anti-TcCYSPR04.: TcCYSPR04 was imunodetected in senescent leaves infected by M. perniciosa at different development stages and in apoplastic fluid. According to these results, TcCYSPR04 may participate in plant senescence, cell death and defense in response to pathogen attack. (Texte intégral

A new desiccation-related protein identified by proteomics in the phylloplane of Theobroma cacao

Currently, 20 millions of people from producing countries, such as Brazil, depend directly on cacao (Theobroma cacao L.) for their survival. The witches' broom disease caused by the fungus Moniliophthora perniciosa had drastic consequences on the socio-economic and environmental development of the affected regions, such as the Bahia State. The disease begins with the germination of the basidiospores on the leaf surface (or phylloplane), followed by the penetration of the germination tube into the intercellular space and the colonization of the plant tissues by the mycelium (biotrophic phase). It has been suggested that the phylloplane is one of the first battlefield of the host and pathogen, and the first interface between plant and environment. Here, we identify by SDS-PAGE/MS/MS, the cacao phylloplane proteins, using two different cacao varieties, one susceptible (Catongo) and one resistant (CCN51) to M. perniciosa. One of our objectives was to quantify the small glandular trichomes (SGTs) in relation with the plant resistance/susceptibility to M.perniciosa. Six hundred resistant cacao leaves were collected and washed in distilled water for 30 seconds. Proteins were extracted from filtered and dried washing, and analyzed on SDS-PAGE. The bands were excised from the gel, subjected to reduction/alkylation and tryptic digestion, and then the peptides were analyzed by mass spectrometry on Micromass ESI-Q-Tof Micro (Waters). The more abundant band (25-35 kDa) was sequenced by MS/MS and resulted in eight peptides, corresponding to a new basic protein of 310 amino acids, with a molecular mass of 33.7 kDa and a theoretical pI of 10.25. This protein contains a probable signal peptide cleavage site between the amino acids 24 and 25. The amino acid sequence revealed similarity to a protein related to desiccation tolerance characterized in pollen-grain of Medicago. Histology was performed on CCN51 and Catongo leaves, to obtain the rate of occurrence of SGTs. CCN51 and Catongo presented an average of 1500 and 700 SGTs/cm2, respectively. The role of the proteins involved in tolerance to desiccation or present in the phylloplane of T. cacao are discussed. (Texte intégral

Root protein interatomic network obtained from citrus seedlings subjected to water deficit : S08O03

Citrus Brazilian industry may be strongly affected by drought. Characterize rootstock behavior under different environmental conditions is crucial, since rootstocks may confer better adaption to environmental stresses. Plant responses to drought require the production of important functional and regulatory proteins. In the present study, we identified proteins related to water deficit response in tolerant ('Rangpur' lime, Citrus limonia) and susceptible ('Sunki Maravilha' mandarin, Citrus sunki) genotypes. From 105 differentially expressed proteins, twenty-nine proteins from roots of both genotypes were identified and sequenced by mass spectrometry. Protein interaction analysis of orthologous proteins from A. thaliana protein expressed in the two citrus varieties was obtained by ontological and cluster analysis.Among the 16 amino acid sequences found in 'Rangpur' lime and 'Sunki Maravilha' mandarin, 13 proteins were found in A. thaliana via BLAST, of which, 12 showed protein-protein interaction. It is possible to group these 12 proteins into a single network interatomic, comprising 3302 proteins. Among the different biological processes found in the protein networks, regulation of response to water deficit and osmotic stress, signaling pathway of abscisic acid were highlighted. A point to take into account is the importance role of the DREB2, a protein hub/bottleneck which participate in the stimulus and the response to water stress. (Texte intégral