MLO and SBP genes from Theobroma cacao are differencially expressed between resistent and suscetible cacao plants infected with Moniliophthora perniciosa

Among sequences previously identified as potentially envolved in the resistance versus susceptibility of Theobroma cacao to the fungus Moniliophthora perniciosa, the MLO (Mildew resistance lócus O) and the SBP (selenium binding protein) genes were found. The MLO gene is characterized as a plant defense and programmed cell death modulator, and the SBP gene was successfully used to increase the rice resistance to Magnaporthe grisea by plant transformation, among other applications. The objective of this work was to evaluate the expression of the MLO and SBP genes from T. cacao in cacao plants infected by M. perniciosa. Varieties of cacao resistant (TSH1188) and suceptible (Catongo) to M. perniciosa were inoculated with a suspension of fungus basidiospores (2.105. ml-1). After inoculation, the plants were kept for 24h at 25±2ºC under 100% of humidity. Apical meristems were harvested in triplicates at 24, 48 and 72 hours after inoculation (hai) and 30, 60 and 90 days after inoculation (dai). Non-inoculated plants (controls) were kept and harvested in the same conditions. Total RNA was extracted using the RNAqueous Kit® (Ambion). First strand cDNA was obtained using the Revertaid Fisrt Strand cDNA Synthesis Kit (Thermo Scientific, Fermentas). Quantitative PCR (qPCR) of MLO and SBP was obtained using the standard settings of the ABI PRISM 7500 and System of Sequence Detection (SDS) software, v.1.6.3 (Applied Biosystems). The expression levels of MLO and SBP was analyzed on triplicates with the comparative Ct method (2-??Ct) using malate dehydrogenase and actin as endogenous reference genes, and non-inoculated plants (control) were used as a calibrator. At the early stages of infection and in the final stage of the disease, the MLO gene was more expressed in Catongo than in TSH1188. In TSH1188, the highest expression of MLO was observed at 30 dai. The SBP gene was highly expressed in TSH1188 at the late stages after infection while in Catongo, the expression was high at the early stages and then constant until the end of the disease. The involvement in the cacao-M. perniciosa interaction of both MLO and SBP genes is discussed. Financial Support: CNPq, BNB, FINEP/Renorbio, Cirad. (Résumé d'auteur

Expression analysis of Mildew Resistance Locus O of cacao in resistant and susceptible plants infected by Moniliophthora perniciosa : S01P03

A Mildew Resistance Locus O (MLO) cDNA was identified from a library of Theobroma cacao L. meristems (Catongo varieties) infected by Moniliophthora perniciosa, the fungus responsible for the witches' broom disease. In other plants, the MLO gene is characterized as a defense and programmed cell death (PCD) modulator, and for this reason may be a good candidate for functional studies aiming the increase of plant resistance. An in silico analysis of the cacao MLO (TcMLO) using the BLAST, Pfam, InterProScan and ORF-Finder programs, as well as a search on CocoaGenDB databank were performed. TcMLO belongs to a multigene family of proteins containing 19 sequences present in the cacao genome: 12, 5 and 2 of them showed one, two and three MLO domains, respectively. The complete TcMLO sequence (including UTRs and ORF) is 5712 bp in length with 13 exons and 12 introns, and is located on the chromosome 5. The TcMLO ORF is 1629 bp in length and encodes a protein with 542 amino acids containing 2 MLO domains. The expression of TcMLO was analyzed by quantitative PCR (qPCR) in resistant (TSH1188) and susceptible (Catongo) cacao varieties infected or not by Moniliophthora perniciosa. Plantlets of cacao were inoculated by the droplet method with a basidiospore suspension of M. perniciosa. After inoculation, the plantlets were kept for 24h at 25±2ºC and 100% humidity. Apical meristems were harvested in triplicates at 24, 48 and 72 hours after inoculation (hai), and 8, 15, 30, 45, 60 and 90 days after inoculation (dai). Non-inoculated plants (controls) were kept and harvested in the same conditions. The qPCR of Tc MLO was obtained using the standard settings of the ABI PRISM 7500 and using the System of Sequence Detection software. The TcMLO expression was analyzed with the comparative Ct method (2-??Ct) using malate dehydrogenase and actin as endogenous reference genes, and non-inoculated plants (control) as calibrator. The results showed that TcMLO was more expressed in Catongo than in TSH1188 at the early and final stages of disease. In TSH1188, the highest expression of MLO was observed at 15 dai. The expression of TcMLO at the final stage of the disease in the susceptible infected plants may be related to the PCD events occurring in this variety as a signal for the finalization of the fungus life cycle. Funding Agency: FAPESB, CNPq, CAPES, EMBRAPA, FINEP/Renorbio and CIRAD. (Texte intégral

In silico characterization and expression analysis of a Selenium-Binding Protein gene from cacao : S01P01

Witches' broom disease, caused by the fungus Moniliophthora perniciosa, is one of the main diseases of cacao (Theobroma cacao L.) and is responsible to severe economic losses in the production areas. Recently, expressed sequence tags (ESTs) from cacao-M. perniciosa interaction were obtained and differentially defense-related genes expressed during the cacao-M. perniciosa interaction were identified. Among them, a Selenium-Binding Protein (TcSBP) was found. In other organisms, SBP genes are related to the increase of plant defenses against abiotic and biotic stresses; in rice the SBP gene was successfully used to increase the plant resistance to Magnaporthe grisea by plant transformation. Here, in silico characterization and expression analysis of TcSBP were developed. Search on the Cacao Genome Database revealed the presence of only one SBP sequence of 4774 pb in length located on the chromosome 4. The TcSBP ORF is 1431 bp in length and encodes a protein of 476 amino acids which does not contain any signal peptide. Prediction of possible post-translational events allowed the identification of several glycosylation, phosphorylation and acetylation sites. The comparison of TcSBP sequence with SBP from other organisms using the BLASTP tool revealed identity from 62% to 91% and allowed the identification of specific conserved regions. The expression analysis of TcSBP in meristems of cacao plantlets varieties Catongo (susceptible) and TSH1188 (resistant to M. perniciosa), inoculated or not with M. perniciosa, was obtained by RT-qPCR using 3 biological and 3 experimental replicates. qPCR analysis of TcSBP gene was conducted using the standard settings of the ABI PRISM 7500 and the System of Sequence Detection software. The TcSBP relative expression was analyzed with the comparative Ct method (2-??Ct) using malate dehydrogenase and actin as endogenous reference genes, and noninoculated plants (control) as calibrator. The relative expression of TcSBP was significantly increased 8 and 15 days after inoculation in the resistant variety TSH1188 compared to susceptible Catongo. These data suggest the possible role of TcSBP in cacao resistance to M. perniciosa. This study is the first step to better understand the role of TcSBP in cacao resistance as well as for the development of control strategies of the witches' broom disease (e.g. using plant transformation). Work supported by FAPESB, CAPES, EMBRAPA, CNPq, FINEP/Renorbio and CIRAD. (Texte intégral

Bioinformatic analysis of glutathione peroxidase family from theobroma cacao and gene expression during Moniliophthora perniciosa infection.[Poster-B185]

Glutathione peroxidases (GPXs) are enzymes which are part of the antioxidant system of the cell. Mammalian GPXs are known as selenoproteins because containing the selenocysteine (Sec) amino acid. In plants, these proteins are less known. Here, were analyzed the protein structure and the gene expression of five GPXs from Theobroma cacao . The three-dimensional structure of the TcGPXs showed that the catalytic site of Tc PHGPX and TcGPX ( 2,4 and 5) contain a cysteine while the GPX8 contain a tryptophan. Interestingly, the T. cacao GPX did not show any selenocysteine in their structure. Docking analysis revealed that TcGPXs can bind to selenium. Phylogenetic analysis split plant and mammalian GPXs in two distinct branches. RT-qPCR analysis of TcGPXs during the T. cacao - Moniliophthora perniciosa interaction showed that TcGPX8 gene is overexpressed in the green broom phase of the susceptible cacao variety. In the resistant variety, the TcGPX5 was significantly more expressed in the final stages of the interaction. This study shows that TcGPXs are important targets for the understanding of the T. cacao - M. perniciosa interaction but also for the functionality of these proteins

Análise da expressão da proteina TcPR-10 na interação resistente e suscetível de cacau-Moniliophthora perniciosa

A doença vassoura de bruxa causada pelo fungo Moniliophthora perniciosa devastou plantações de cacau levando às mudanças econômicas, sociais e ambientais no sul da Bahia. Estudos moleculares da interação cacau-M. perniciosa revelou genes diferencialmente expressos envolvidos em eventos biológicos relacionados à patogênese. Entre os genes está o que codifica para a proteína relacionada à patogênese, classe 10 (TcPR-10). Esta proteína tem um potencial biotecnológico promissor, porque age in vitro como ribonuclease e apresenta atividade antifúngica. Análise da expressão de genes por RT-qPCR mostra que TcPr-10 tem sua expressão aumentada em plantas suscetíveis (Catongo) inoculadas com M. perniciosa nos estágios finais da infecção, enquanto que, no inicio não difere do controle. No entanto, a detecção de mRNA não fornece muitas evidências da presença da proteína. O objetivo deste trabalho foi analisar, por Western blot, a expressão da proteína TcPR-10 em meristemas e folhas de plantas de cacau resistente (TSH1188) e suscetível (Catongo) inoculadas com M. perniciosa. Meristemas apicais de plantas com 20-30 dias de idade, foram inoculados com uma suspensão de basidiósporos e mantidos em câmara úmida por 24 horas. As amostras foram coletadas em 24h, 48h, 72h, 15, 30, 60 e 90 dias após a inoculação (DAI). As amostras foram liofilizadas, trituradas em N2 líquido e homogeneizados em 100 mM Tris-HCl (pH 8,8) contendo 50 mM de ácido ascórbico, 1% (v/v) de ?-mercaptoetanol, 0,1% (m/v) de Triton-X e 2% (m/v) de polivinilpirrolidona. As proteínas foram precipitadas com solução TCA/acetona seguido de extração com fenol/SDS. A dosagem das proteínas foi feita utilizando o 2D-Quant-Kit (GEHealthCare®). As proteínas foram separadas por SDS-PAGE e transferidas para membrana de nitrocelulose, a qual foi incubada com anticorpos produzidos em coelho contra a TcPR-10. A imunodetecção foi realizada com o 5-bromo-4-cloro-3-indolil-fosfato e nitroazul de tetrazólio (BCIP/NBT, Promega®). A TcPR-10 foi expressa em meristemas inoculados de ambas variedades estudadas após o ponto 30 DAI, no entanto, apresenta diferença no nível de expressão. Aos 30 DAI a proteína é mais expressa na variedade resistente enquanto aos 90 DAI a expressão é maior na variedade suscetível. Verificou-se ainda a expressão em folhas de TSH1188 com 60 DIA o que nos leva a inferir uma resposta sistêmica na planta resistente, pois até 90 DAI a expressão na planta suscetível limitou-se apenas ao meristema. (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

Genetic transformation of tobacco plants with a cacao pathogenesis-related protein 4 for tolerance to water stress study : S03P06

Drought is an important environmental factor limiting the productivity of various crops worldwide. The development of crop cultivars with improved adaptation to drought is a major goal in many crop breeding programs. In addition to classical breeding approaches, genetic transformation to introduce candidate genes into plants for better tolerance to water deficit has been successfully developed. Pathogenesis-related proteins (PR proteins) are defined as plant proteins induced in response to pathogen attack. However, it is known that these proteins may also be involved in response to abiotic stresses. The objective of this study was to transform tobacco plants (as plant model for subsequent analysis of cultivated plant such as citrus) with a PR-4b protein from Theobroma cacao (TcPR-4b) and to select and test the tolerance of such transformed plants to water/osmotic stress. First, an in silico analysis of the TcPR-4b using the BLAST, Pfam,InterProScan, ORF-Finder programs, as well as a search on Cocoa GenDB databank were performed. The TcPR-4b belongs to a small family of PR-4 proteins whose members were mainly located on the chromosomes 5 (five genes) and 10 (one gene). The complete TcPR-4b sequence is 802 bp in length and contains two exons (171 and 258 bp), and one intron (82 bp); the corresponding protein is 142 amino acids in length. For plant transformation experiment, the TcPR-4b cDNA (from cacao-M. perniciosa interaction library) was cloned into the pGem- T Easy vector then subcloned on the pCambia binary vector 1390. Then, Agrobacterium tumefaciens strain EHA 105 was transformed with the35S::TcPR-4b::pCambia construction, and the transformed A. tumefaciens used for Nicotiana tabacum cv.Havana transformation by co-cultivation of leaf segments in selection medium. Transformed shoots from three transformation events are under selection for subsequent in vitro water/osmotic stress, using, among others, mannitol and NaCl. Funding Agency: FAPESB, CNPq, CAPES, EMBRAPA, FINEP/RENORBIO and 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

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