Accueil et encadrement du chercheur Abelmon Gesteira (bourse DESI) dans le cadre de l'étude de l'interaction cacao-Crinipellis : rapport de mission à Montpellier, France du 20 juin au 31 juillet 2004

Cette mission avait deux objectifs principaux: - l'accueil et l'encadrement au sein du département AMIS/Biotrop du chercheur Abelmon Gesteira (Universidade Estadual de Santa Cruz-UESC, Ilhéus-BA, Brésil) ayant obtenu une bourse DESI de 6 semaines; - développement d'expériences de biologie moléculaire (macroarrays et criblage de banque BAC) portant sur l'étude de l'interaction cacao-Crinipellis perniciosa. Les objectifs fixés pour cette mission ont été atteints. La mission a permis la bonne intégration de A. Gesteira au sein du laboratoire Biotrop ainsi que sa formation à des techniques de biologie moléculaire telles que l'obtention de macroarrays, l'utilisation de sondes radioactives et le criblage de banques BAC. Les expériences menées conjointement avec A. Gesteira ont permis d'obtenir des résultats significatifs pour l'étude de l'interaction cacao-Crinipellis telle qu'elle est actuellement entreprise dans la cadre de la collaboration entre le Cirad et l'UESC. (Résumé d'auteur

Use of Neurospora crassa as fungus model for systems biology of Moniliophthora perniciosa hyphae proteins: case study of polygalacturonases : S01P13

Moniliophthora perniciosa, the fungus responsible for the witches' broom disease, brought serious problems to cacao (Theobroma cacao L.) cultivation in the infected areas such as in Southern Bahia, Brazil. For this reason, several molecular studies have been recently developed from T. cacao and/or M. perniciosa, and subsequent computational analyses were developed. Among them, the systems biology provides a framework for assembling models of biological systems from systematic measurements obtained by experimental analysis. In the case of M. perniciosa, which is still little studied, the first step for protein-protein interaction (PPI) network analysis by systems biology consists in the identification of an adequate organism for ortholog search. Neurospora crassa is a well-known filamentous fungus, considered as a model organism that has been used for more than 90 years to study genetics, biochemistry and fungal biology. Moreover, lots of genomics and molecular data are available for N. crassa, including those related to architecture and hyphae development, as well as cell wall degradation apparatus. Here, the objective was to evaluate the possible use of N. crassa as model for determination of M. perniciosa PPI networks, and as an example to test our hypothesis, two polygacturonases from M. perniciosa (MpPG1 and MpPG2) were analyzed. First, a reciprocal BLASTp of MpPG1 and MpPG2 was performed on N. crassa database using stringent conditions (10-10). The respective N. crassa orthologs (NCU06961 and NCU02369) were used to build the PPI network in STRING 9.05. This network was analyzed in the Cytoscape 2.8.2 software with the Molecular Complex Detection, Biological Network Gene Ontology and CentiScaPe plugins. The PPI network contained 892 nodes (proteins), 43.035 connectors, and was organized in 11 modules corresponding to biological processes, such as ribosome biogenesis, regulation of gene expression, processes related to carbohydrate metabolism, among others. Twenty-two proteins interacted directly or indirectly with MpPG2- NCU02369 and were associated to post-translational protein modification or pectin degradation. From the 892 and 22 specific proteins, 544 (61%) and 14 (63.6%) were found by reciprocal BLASTp when we looked back on the M. perniciosa database. Considering that the M. perniciosa published sequence only represent 69% of the total genome, the results we obtained confirmed that N. crassa could be used as a good model for M. perniciosa in systems biology analysis of hyphae proteins. Work supported by BNB, CNPq, FAPESB, Cirad. (Texte intégral

Expression of genes involved in cell wall structural components in different genotypes of Theobroma cacao

Resistant in this main way to control witches´ broom resistance, therefore the understanding of pathogen infection and its disease resistance mechanism is very important in order to obtain durable resistance. Through the technique of in situ hybridization, the current study aimed to determine the expression of genes involved in possible mechanisms of resistance of cacao and in which stages of the infection they are acting. Apical meristems of susceptible and resistant genotypes of Theobroma cacao to witches' broom disease were artificially inoculated by placing a drop with a 5x105 basidiospore/mL. Meristems were collected at intervals of 3, 6, 12, 24, 48 and 72 hours, 5 and 15 days after the inoculation day, under free RNAse. The samples were fixed and sent for analysis of gene expression by in Situ Hybridization. HRGP genes (Hidroxyproline-rich glycoprotein) and RGC2 related to cell wall metabolism or plant defense mechanism, were chosen from cDNA libraries available at UESC/ CEPLAC/CEPEC. The analysis showed the localization in the in cells of the vascular system and in parenchymatous cells of the apical meristem in both resistant as well as susceptible genotype. However, no significant variation in the accumulation of HRGP and RGC2 genes were observed between genotypes. Thus, we suggest further investigation with earlier timein order to ascertain whether there is variation in the accumulation of such genes between the genotypes under study. (Texte intégral

Systems biology of proteins expressed during the Moniliophthora perniciosa necrotrophic phase : S01P12

The fungus Moniliophthora perniciosa, the etiologic agent of witches' broom disease of cacao (Theobroma cacao L.) has a hemibiotrophic life cycle, with a biotrophic 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 necrosis. During the culture of M. perniciosa on bran-based solid medium, six different developmental phases were observed according to the dikaryotic mycelium color or the organ produced: white, yellow, pink, dark pink, primordium and basidiomata. A proteomic analysis of the different M. perniciosa development stages associated to mass spectrometry allowed the identification of about 250 differentially expressed proteins. In this study, using such differentially expressed proteins, we developed a systems biology analysis to identify physical protein-protein interaction (PPPI) networks related to the fungus development focusing on basidiocarp formation. First, orthologous protein sequences of M. perniciosa were obtained in N. crassa using the BLASTX tool. The data mining screening and PPPI network design associated with fungal development was performed using the Cytoscape software, version 2.5.0. These data were downloaded from the STRING 8.3 database. The interactome networks obtained from this first screening were analyzed with the Molecular Complex Detection software, a Cytoscape plugin, in order to evaluate potential subgraphs that were used further for network expansion. Gene ontology clustering analysis was performed using the Biological Network Gene Ontology software. Moreover, an analysis of centrality was performed using the software Centiscape 1.2.1.; several hub-bottlenecks, hub and bottlenecks proteins, as well as proteins involved in biological processes important for the M. perniciosa development were identified. The main biological processes encountered were anatomy and morphology, reproduction, oxidative stress, cell wall biosynthesis, pigmentation, development and cell differentiation. The identification of proteins involved in the formation of basidiomata, as well as the knowledge about their interactions, may contribute to the future development of witches' broom control strategies. To our knowledge, this is the first system biology analysis of proteins involved in the M. perniciosa life cycle. Supported by: CAPES, CIRAD, BNB, PRONEM/FAPESB. (Texte intégral

Identification, classification and phylogenetic analysis of bZIP proteins from Theobroma cacao for subsequent studies of resistance to witches' broom disease : S01P02

Biotic and abiotic stresses are a major factor in decreased production of various cultures around the world. The culture of cacao (Theobroma cacao) has been suffering for many years with one of the diseases that most affect their crops, the witches' broom caused by the fungus Moniliophthora perniciosa. Studies have identified several transcription factors as promising candidates for developed roles in the regulation and signaling via various stresses in plants. bZIP family proteins are transcription factors (TF) that regulate various physiological and development processes, such as seed maturation, vascular development, and responses to biotic and abiotic stresses. Here, we performed an in silico analysis of the bZIP family from Theobroma cacao to subsequently develop a comprehensive phylogenetic analysis in four angiosperms species. For this, bZIP protein sequences of Arabidopsis thaliana were downloaded from the Phytozome database. BLASTP search was performed to identify Solanum lycopersicum, Oryza sativa, Sorghum bicolor and Theobroma cacao bZIP homologs. Analysis of protein domains, search for conserved motifs and alignment of the bZIP proteins were conducted using the PFAM, MEME, CLUSTALW, respectively. The distribution of the bZIP sequences on cacao chromosomes was obtained on the CacaoGenDB site (http://cocoagendb.cirad.fr) using the Interpro number. Protein sequences of the species under study were subjected to a multiple alignment using the software MEGA v5.0, and a Neighbor-joining tree was constructed based on the genetic distance matrix JTT. A classification of the A. thaliana bZIP TFs according to biological function was obtained using the TAIR site (http://www.arabidopsis.org/). We identified 65 bZIPs in cacao, 75 in tomato, 90 in rice and 89 in sorghum. The bZIPs found in cacao were distributed across all 10 chromosomes (Chr), except on chromosome 6, with higher abundance in Chr 01, 02 and 09. According to the study, some region of QTLs related to cacao resistance to witches' broom was located on Chr 2 and 9, may seek markers within genes in this region and use in plant breeding. The bZIP family proteins were grouped into 13 possible orthologous groups based on the classification performed in Arabidopsis thaliana. Five of the cacao bZIP were clustered with proteins highly related to defense to pathogens, being promising candidates for functional study in cacao plants, especially against the witches' broom disease. Funding Agency: FAPESB, CNPq, CAPES and CIRAD. (Texte intégral

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

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

Data mining and systems biology for identifying key genes involved in citrus quality

Quality in citrus is mainly characterized by fruit and juice colour, fruit and skin size, juice percent, total soluble solids, titrable acidity, and carotenoid/flavonoid contents. Moreover, studies of biosynthetic pathway of the metabolites/proteins involved in quality at transcriptional and translational levels may give relevant information for subsequent functional studies and quality improvement. Data mining of ESTs from HarvEST database allowed the selection of 17 cDNA libraries from albedo, flavedo, peel, pulp and juice sac of different orange, mandarin, clementine and grapefruit varieties. In order to select key genes involved in quality we used systems biology that offers mathematical tools that include the analysis of the structure, clustering and centralities of the network. In order to have information regarding physical protein-protein interactions (PPPI) from citrus sequences, orthologous sequences of Arabidopsis thaliana were used (BLASTX; reciprocal BLASTP). Literature data mining was performed, and PPPI network design was obtained using the Cytoscape software. The interactome networks thus obtained were analyzed with MCODE. Gene ontology clustering analysis was performed using BiNGO. Specific algorithms were applied to identify modules and central nodes within the citrus libraries associated network. The obtained results will be used as a guideline to select specific genes/proteins from citrus for further functional studies as gene expression or plant transformation. (Résumé

In silico characterization of a pathogenesis-related protein PR-1 from Theobroma grandiflorum : S01P11

The cupuassu (Theobroma grandiflorum [Wild. Ex Sperg.] K. Schum) is a native species of Brazil with a large industrial potential related to the use of the fruit pulp and seeds. In particular, the cupuassu presents a great economic value for the Pará State, which invested in cupuassu sweet (e.g. ice-cream) and cupulate (chocolate obtained from cupuassu seeds) production and commercialization. Theobroma grandiflorum belongs to the same genus than cacao (Theobroma cacao), and, unfortunately, both suffer from the attack of the fungus Moniliophtora perniciosa responsible for the witches' broom disease. Several molecular studies of the interaction between cacao and M. perniciosa were previously developed, while little is still known in regards to cupuassu resistance to witches' broom disease. Among the well known genes involved in plant-pathogen interactions, the pathogenesis-related proteins (PR proteins) could be highlighted. In particular, the PR-1 family proteins presented several functions that vary according to the organism and that may be involved in different ways in defense to pathogen infection. Recently Next Generation Sequencing of cupuassu expressed sequences tags allowed the identification of several PR proteins, and here we developed an in silico analysis of a TgPR-1 sequence. The TgPR-1 ORF encoded a 161 amino acid protein that showed homology with the PR-1 proteins belonging to the serine-carboxyl proteinase superfamily. A multiple alignment using the ClustalW program allowed the identification of domains conserved between the TgPR-1 and its homologs from other organisms. The TgPR-1 protein presented a peptide signal (24 amino acids identified by the SignalP 4.1 software), and had an isoelectric point and a molecular weight of about 8.75 and 17.3 kDa, respectively. The protein presented possible post-translational modification sites such as 10 phosphorylation sites (encountered using the NetPhos 2.0 software) - but no glycolsylation sites were found. The systems biology analysis of TgPR-1 was performed using the BLASTO and OMA browser to find a corresponding ortholog. The best score protein was found for the PRB1 from Arabidopsis thaliana. The network was set up using the software Cytoscape 2.8.2. The protein showed direct interaction with other proteins involved in responses to biotic stimuli, fungus, bacteria and stress as well as involved in mechanisms of resistance and plant defense. The in silico characterization of TgPR-1 constitute the first steps towards understanding the mechanisms of T. grandiflorum defense. Funding Agency: FAPESB, CNPq, CAPES, EMBRAPA and CIRAD. (Texte intégral