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 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

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

Biochemical characterization of recombinant serine-protease inhibithor from cacao (Theobroma cacao L.) : [Abstract R8979]

Proteinase inhibitors (PIs) are widely expressed in plants, especially in seeds of Cruciferae, Leguminosae, Solanaceae, Graminaceae and Sterculiaceae. Their expression is regulated at the transcriptional level in response to different conditions, including germination and insect attack. Plant PIs are also involved in response to various biotic and abiotic stresses, e.g., pathogen invasion, wounding and environmental stress. PIs are common in plants and could be used to develop transgenic crop variety resistant to pathogens. Recently, molecular and genomic studies of cacao have been developed by different research team around the world and about 180,000 ESTs are now available on public databases, in which we identified a trypsin-like serine-protease inhibitor. The ORF contains 660 bp and encodes a protein of 219 amino acids. The predict protein has an estimated molecular weight of the 22 kDa and a theoretical pI of 5.9, and contain a peptide signal with a predict cleavage site at position 26. The His-Tag fused protein was expressed in Escherichia coli and the recombinant protein (named TcTI) accumulated in soluble fraction from bacterial extract. Purified His-tagged recombinant proteins showed inhibitory activity against porcine trypsin with a Ki of 406 nM. TcTI also showed thermostability up to 70°C. Polyclonal antibody raised against the recombinant TcTI detected that the endogenous protein was more abundant in cacao tissues infected by M. perniciosa than in healthy ones. Our data shows that recombinant TcTI is functional and presents potential biotechnology applications. (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

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

Dual enzymatic activity of the pathogenesisrelated protein TcPR-4 from Theobroma cacao: ribonuclease and Ca+2 and Mg+2 dependent deoxyribonuclease activities

The class 4 pathogenesis-related proteins (PR4) are classified as chitinases and contain a conserved Barwin domain. The TcPR-4b cDNA identified from a library of Theobroma cacao L. pod (genotype TSH1188) infected by Moniliophthora perniciosa also presents the Barwin domain with six conserved cysteine residues, but lacks the chitin-binding site and for this reason was classified as class II PR4. 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. To determine the DNase activity of the purified recombinant TcPR-4b protein, 1 ?g of purified pGEM-T® Easy Vector DNA was incubated with different protein amounts (2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 ?g) in the presence or absence of 10 mM of MgCl2 or 1 mM of CaCl2 overnight at room temperature. RNase activity of recombinant TcPR-4b was performed using different protein amounts (5, 10, 15, 20 and 25 ?g) incubated for 30 min with 5 ?g of RNA extracted from Solanum lycopersicum leaves. The reaction products were analyzed in 1.5% agarose electrophoresis gel. The TcPR-4b protein recombinant showed both DNase and RNase activity. DNase activity was observed only in the presence of Mg+2 and Ca+2 ions. The results of this study suggest that TcPR-4b may act as nuclease during the infection of cacao plants with M. perniciosa. Financial Support: CNPq, BNB, FINEP/RENORBIO, CAPES. (Résumé d'auteur

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

Action of the pathogenesis-related protein PR10 from Theobroma cacao in triggering response mechanisms of Moniliophtora perniciosa

Cacao (Theobroma cacao L.) is an important commodity worldwide, but its production is limited by destructive diseases such witches' broom, due to the fungus Moniliophthora perniciosa. The high recombination rate and genetic variability of this fungus promoted resistance breakdown of cacao and annihilated the efforts made by the Brazilian government to reduce the disease impact on plantations. According to the literature, pathogenesis related-proteins (PRs) plays an important role in defense/resistance mechanisms of the plant submitted to various biotic and abiotic stresses. A cDNA clone encoding a PR10 (named TcPR10) was obtained from a cacao-M. perniciosa EST library. The corresponding recombinant protein (expressed in Escherichia coli BL21(DE3)) present a strong antifungal activity against M. perniciosa, as previously demonstrated. In this work, we developed a proteomic analysis of the fungus cultivated in the presence of TcPR10, using 2DE-MS/MS. M. perniciosa was grown in CPD 2% agar medium; after 15 days, the fungal hyphae were broken and were grown in presence of 3 ?g/mL of TcPR10 for 1h.Then, the total proteins 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. (Texte intégral