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 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

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

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