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

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

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

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

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

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

Análise de uma cisteíno-protease de 39 kDa identificada em bancos de cDNA do cacau

As cisteíno-proteases são aquelas usam uma cisteína do sítio ativo como nucleófilo durante a catálise. Os genomas de plantas codificam cerca de 140 cisteíno-proteases. Estas enzimas podem estar envolvidas na ativação de outras enzimas, em defesa contra ataque de insetos, em morte celular programada, defesa contra patógeno e germinação de sementes. Além disso, elas apresentam importância biotecnológica em indústria de alimentos, têxtil, farmacêutica e de detergentes. Uma cisteíno-protease denominada TcCysPr04 foi identificada em bibliotecas de cDNA da interação entre Theobroma cacao e Moniliophthora perniciosa seqüenciadas na UESC. Esse trabalho objetivou novos homólogos da proteína CISPR04 identificadas em bibliotecas de cDNA coordenadas pelo CIRAD e publicadas recentemente. Os homólogos de TcCysPr04 apresentaram ORF´s com 1068 pb, conforme análise com o ORF Finder. A análise da sequencia de aminoácidos deduzida a partir da sequencia de nucleotídeos indica que: (i) a proteína possui massa molecular e ponto isoelétrico estimados de 39 kDa e 5.43, respectivamente, de acordo com programa pI/MW; (ii) possui um peptídeo sinal, com provável sítio de clivagem entre os aminácidos 19 e 20, revelados pelo programa SignalP 2.0; e (ii) possui um domínio inibitório entre os aminoácidos 56 e 112 e um domínio catalítico entre os aminoácidos 158 e 353, identificados mediante análise com os programas Pfam e ProDom. Primers foram engenheirados a partir de um mapa de restrição preparado com o WebCutter, visando a clonagem e expressão de três versões da proteína: (i) a proteína completa (eliminando o peptídeo sinal); (ii) somente o domínio inibitório; e (iii) somente o domínio catalítico. Na continuidade desse trabalho essas versões deverão ser expressas em bactéria e empregadas em estudos de caracterização bioquímica e funcional da proteína. (Résumé d'auteur

Etudes in silico et in vitro des polyganaturonases de Moniliophthora Perniciosa

Les pectinases consùtuent un groupe d'enzymes qui se dégradent en substances pectiques, hydrolysant les liaisons glycosidiques tout au long de la chaîne carbonée. Elles sont produites par les plantes, les champignons filamenteux, les bactéries et les levures. Ces enzymes ont un rôle important: i) dans la physiologie végétale, ii) dans les microorganismes pathogènes agissant comme un facteur de virulence pendant l'infection de la plante; iii) dans les industries agro-alimentaire, du textile et du papier. Des gènes homologues des polygalacturonases (PG, EC 3.2.1) ont été identifiés dans la base de données génomique du champignon Moniliophthora perniciosa, l'agent pathogène de la maladie des balais de sorcières du cacaoyer (Theobroma cacao L.). Nous avons réalisé une caractérisation in silico des gènes de polygalacturonase (PG) de M. pemiciosa et nous avons évalué leur expression dans le champignon cultivé dans un système artificiel (cookies) ainsi que dans un milieu de culture contenant différentes sources de carbone. Trois gènes PG ont été identifiés (PG1, PG2, PG3) et caractérisés in silico par analyse de séquence, alignement de séquence. construction de cladogramme et conception d'amorces pour l'étude de l'expression des gènes par PCR en temps réel (RT-qPCR), en utilisant différents logiciels. Pendant la croissance de l'agent pathogène sur les cookies, quatre étapes de développement ont été observées en fonction de la couleur du mycélium (blanc, jaune, rose clair et rose foncé). L'analyse par RT-qPCR a montré que l'expression était supérieure dans la phase jaune, suivie par la phase rose foncé et la phase rose clair. respectivement, pour les trois gènes PG. L'expression des gènes a aussi été analysée sur l'agent pathogène cultivé dans un milieu liquide contenant différentes sources de carbone (D-glucose, D-galactose, D-mannose, D-mannitol, D-sucrose, D-fructose et glycérol). Dans cette étude, nous avons observé que le gène PG2, suivis par le gène PGl, étaient ceux avec l'expression la plus élevée quand le champignon était cultivé dans un milieu contenant du D-mannose. Quand le champignon a été cultivé dans du D-galactose, les trois gènes étudiés ont présenté une expression significative. PG3 obtenant l'expression la plus élevée et PG2 la plus faible. Dans le D-mannitol, seul le gène PG2 a présenté une expression, les autres gènes ont été réprimés. Dans le D-sucrose, les gènes PG1 et PG2 ont montré une faible expression, et dans le D-fructose et le glycérol, nous avons observé une répression de tous les gènes étudiés. Nous pensons que cette étude peut servir à l'élaboration de différentes stratégies de lutte contre la maladie et également à une production à grande échelle de pectinase destinée à être utilisée dans le secteur des biotechnologies. (Résumé d'auteur