The development of the basidiomycete Moniliophthora perniciosa in resistant and susceptible Theobroma cacao genotypes was analysed. The infection process leading to broom formation in shoot apexes was characterized by studying the kinetics of basidiospore germination, mode of penetration and colonization of the pathogen. Both resistant and susceptible cacao genotypes were inoculated with M. perniciosa and kept in the greenhouse for 90 days, explants were collected, treated for histological studies and meristematic tissues were observed by electron and light microscopy. Variation in the kinetics of germination between the cacao genotypes was detected 4 h after inoculation. The fungal penetration occurred through the star‐shaped trichome base, natural openings on the cuticular surface and stomata. Host responses between genotypes were found to be different. Compared with non‐infected plants, the swelling of all the stem tissues was evident at 60 days after inoculation. In the susceptible genotype, typical symptoms developed and fungal colonization was more intense than in resistant genotypes, which showed little or no fungal colonization. The investigations reported herein provide an important step in understanding the pattern of pre‐ and post‐penetration events of M. perniciosa in cacao genotypes with different levels of resistance to this disease.

Un projet international visant au sequencage d'une importante collection de 180 000 clones ADNc de cacao enrichis en pleine longueur, representant des genes exprimes dans T. cacao L. a ete realise. Ces clones ont ete isoles principalement a partir de deux genotypes, Scavina6 et ICS1, et a partir de differents organes avec et/ou sans traitement avec divers stress biotiques et abiotiques. Environ 45 bibliotheques d'ADNc ont ete elaborees a partir d'une large gamme d'organes : fleurs (auto-pollinisees et avec pollinisation croisee), coussinets floraux, graines a differents stades de developpement et durant la fermentation, cherelles, cortex de cabosse, pousses, bois, racines, graines germees et embryons de culture in vitro. Des bibliotheques ont egalement ete construites a partir d'organes soumis a des stress biotiques : feuilles et cabosses inoculees avec #Phytophthora palmivora# et #megakarya#, tiges et cabosses inoculees avec #Crinipellis perniciosa#, cabosse inoculee avec #Moniliophthora roreri#, tige inoculee avec #Ceratocystis fimbriata#, cabosses inoculees avec des endophytes utilises pour la lutte biologique, et tiges attaquees par des mirides. Des bibliotheques d'hybridations soustractives suppressives (SSH) ont aussi ete generalement construites a partir de ces interactions avec des pathogenes vegetaux pour faciliter l'identification ulterieure de genes de resistance ou de defense exprimes dans le cacaoyer au cours d'infections par les pathogenes. Par ailleurs, deux bibliotheques SSH ont ete realisees pour les fleurs en utilisant les deux conditions a la fois comme sondes et comme pilotes. Des bibliotheques ont egalement ete etablies a partir de boutures dans des conditions de secheresse. La construction et la gestion des bibliotheques d'ADNc, le picking et la replication ont ete realises avec l'aide de la plate-forme robotique, au sein du GENOPOLE Languedoc-Roussillon . Tous le travail de sequencage a ete realise par le GENOSCOPE (Ivry), le Centre national francais de sequencage. Pour stocker et exploiter efficacement les informations sur les genes, un pipeline bioinformatique (ESTtik) a ete utilise, qui traite automatiquement les sequences, les assemble, les annote, et compile les resultats dans une base de donnees sur Internet, ce qui permet aux chercheurs de consulter les resultats et d'effectuer des requetes. Apres l'annotation et la comparaison des donnees des sequences de cacao avec la base de donnees internationale des sequences (NCBI), environ 68 % des sequences de cacao faisaient apparaitre une similitude significative avec les sequences genetiques d'autres especes. Ces comparaisons ont permis l'annotation de la fonction genetique de beaucoup des sequences, ainsi qu'une classification generale des sequences d'ADNc de cacao en fonction du systeme d'ontologie genetique. Cette nouvelle ressource de sequences EST de cacao rend possible l'identification de centaines de nouveaux marqueurs microsatellites et de milliers de SNP (Polymorphismes d'un seul nucleotide), de nouveaux marqueurs tres efficaces, la recherche de genes candidats de cacao homologues a des genes specifiques d'interet qui ont ete caracterisees chez d'autres especes (impliques dans la resistance, la reaction de defense, la qualite...), et l'identification d'un vaste ensemble unigene convenant a l'analyse, par genomique fonctionnelle, des mecanismes moleculaires sous-tendant la resistance, la qualite ou d'autres caracteres interessants pour la selection genetique du cacao. (Resume d'auteur)

Hevein is involved in the coagulation of rubber particles and is known to be highly expressed in latex cells from Hevea brasiliensis. As all HEVEIN genes are highly conserved in their transcribed region, a promoter functional analysis was carried out in transgenic rubber tree in order to evaluate the expression of HEV2.1, one member of the HEVEIN multigenic family. Three out of the 14 established independent transgenic lines carrying the HEV2.1::GUS construct were selected and set to regenerate plantlets. In situ hybridization of transgenic and wild-type plant tissues was respectively carried out with gusA and HEVEIN antisense mRNA probes. This showed co-localized-expression of the HEV2.1 gene with the overall HEVEIN gene expression. Laticifer-specific expression was observed in roots and stems, but not in leaves where HEVEIN genes were expressed in all cell types. Since HEV2.1::GUS callus lines responded significantly to light stimulation, up-regulation of the expression of HEV2.1 genes by light was suggested in leaves.

To better understand the cocoa transcriptome and to provide a framework for future investigations on cocoa functional genomics, approximately 175 000 cocoa clones from 52 cDNA libraries were sequenced. These libraries were constructed with RNA isolated from 13 genotypes (mainly from the two genotypes Scavina6 and ISC1) and from various tissues and organs (flowers, seeds, cherels, pod, shoot, root or embryos) under various conditions (fermentation, abiotic and biotic stresses). Suppression subtractive hybridisation (SSH) libraries were also produced under various plant pathogen interactions and from flowers pollinated by self compatible or incompatible pollen. From these cDNA libraries, 149 650 good quality ESTs were generated and analyzed using our house bioinformatics pipeline. The assembly process produced 13837 contigs and 66746 singletons that represent 80583 different potential transcripts. These unigenes were assigned with BLASTX and BLASTN similarities, Gene Ontology annotation and protein domains. A comparison with public ESTs cocoa sequences already already available in public domain indicates that 80% of the unigenes may not have been described in public databases. (Texte integral)

Theobroma cacao L., an economically important crop for developing countries, can be experimentally propagated by somatic embryogenesis. Because of their potential roles in embryogenesis, a gene candidate strategy was initiated to find gene homologues of the members of the leafy cotyledon family of transcription factors. A homologue of the leafy cotyledon1-like gene, that encodes the HAP 3 subunit of the CCAAT box-binding factor, was found in the cocoa genome (TcL1L). The translated peptide shared a high amino acid sequence identity with the homologous genes of Arabidopsis thaliana, Phaseolus coccineus and Helianthus annuus. TcL1L transcripts mainly accumulated in young and immature zygotic embryos, and, to a lesser extent, in young and immature somatic embryos. In situ hybridization specified the localization of the transcripts as being mainly in embryonic cells of young embryos, the meristematic cells of the shoot and root apex of immature embryos, and in the protoderm and epidermis of young and immature embryos, either zygotic or somatic. Non-embryogenic explants did not show TcL1L expression. Ectopic expression of the TcL1L gene could partially rescue the Arabidopsis lec1 mutant phenotype, suggesting a similarity of function in zygotic embryogenesis.

BACKGROUND:Theobroma cacao L., is a tree originated from the tropical rainforest of South America. It is one of the major cash crops for many tropical countries. T. cacao is mainly produced on smallholdings, providing resources for 14 million farmers. Disease resistance and T. cacao quality improvement are two important challenges for all actors of cocoa and chocolate production. T. cacao is seriously affected by pests and fungal diseases, responsible for more than 40% yield losses and quality improvement, nutritional and organoleptic, is also important for consumers. An international collaboration was formed to develop an EST genomic resource database for cacao.RESULTS:Fifty-six cDNA libraries were constructed from different organs, different genotypes and different environmental conditions. A total of 149,650 valid EST sequences were generated corresponding to 48,594 unigenes, 12,692 contigs and 35,902 singletons. A total of 29,849 unigenes shared significant homology with public sequences from other species.Gene Ontology (GO) annotation was applied to distribute the ESTs among the main GO categories.A specific information system (ESTtik) was constructed to process, store and manage this EST collection allowing the user to query a database.To check the representativeness of our EST collection, we looked for the genes known to be involved in two different metabolic pathways extensively studied in other plant species and important for T. cacao qualities: the flavonoid and the terpene pathways. Most of the enzymes described in other crops for these two metabolic pathways were found in our EST collection.A large collection of new genetic markers was provided by this ESTs collection.CONCLUSION:This EST collection displays a good representation of the T. cacao transcriptome, suitable for analysis of biochemical pathways based on oligonucleotide microarrays derived from these ESTs. It will provide numerous genetic markers that will allow the construction of a high density gene map of T. cacao. This EST collection represents a unique and important molecular resource for T. cacao study and improvement, facilitating the discovery of candidate genes for important T. cacao trait variation.

Immature seeds isolated from pods of carob (Ceratonia siliqua L.) were used as a source of explants for callogenesis and somatic embryogenesis. Suitable developmental Stages of immature seed explants, and in vitro culture conditions for the production of translucent callus were identified. The addition of 2,4-dichlorophenoxyacetic acid (2,4-D) to the culture medium was found to be necessary for callogenesis. Both the developmental Stage of the explant and the, 2,4-D concentration had significant effects on the percentage of callus production. A high proportion (67.0 +/- 1.0%) of immature seed at Stage 2 gave rise to callus when cultured on a medium containing 9.0 mu M 2,4-D, which was significantly higher than all the other treatments tested. Embryogenic calli consisted of a white-translucent mass proliferating all around the explants and gave rise to somatic embryos. On day-45 of culture, transverse sections of callus showed de-differentiated cells located on the surface, which gave rise to pro-embryos. On day-70, early somatic embryos were observed as globular white structures, and others were at the heart-shaped stage. On day-90, complete somatic embryos were initiated which had attained the cotyledonary stage, characterised by a defined meristematic area and separation of a protoderm and pro-vascular bundles.

Little is known of the mechanisms that induce the dedifferentiation of a single somatic cell into a totipotent embryogenic cell that can either be regenerated or develop into an embryo and subsequently an entire plant. In this Opinion article, we examine the cellular, physiological and molecular similarities and differences between different plant stem cell types. We propose to extend the plant stem cell concept to include single embryogenic cells as a totipotent stem cell based on their capacity to regenerate or develop into an embryo under certain conditions. Our survey suggests that differences in chromatin structure might ensure that meristem-localized stem cells have supervised freedom and are pluripotent, and that embryogenic stem cells are unsupervised, autonomous and, hence, freely totipotent.

An international project aiming to sequence a large collection of 180,000 cocoa cDNA enriched in full length cDNAs was carried out. CDNA were isolated from different organs, sometimes submitted to various biotic and abiotic stresses. About 45 cDNA libraries were constructed from a wide panel of organs: flowers, flower cushions, seeds at different stages of development and during fermentation, cherels, pod cortex, shoot, wood, root, germinated seeds, and embryos from vitro culture. Direct and SSH libraries were constructed from organs subjected to biotic or abiotic stresses: leaves and pods inoculated with Phytophthora palmivora and Phytophthora megakarya, shoots and/or pods inoculated with Crinipellis perniciosa, Moniliophthora roreri, Ceratocystis fimbriata, and shoots attacked by mirids. leaves and roots under drought conditions. The construction and management of cDNA libraries, was performed with the help of the robotic platform, part of the Montpellier Languedoc-Roussillon GENOPOLE. All the sequencing work was performed by GENOSCOPE (from the GENOPOLE Evry/France). A bioinformatic pipeline (ESTtik) was constructed; it automatically processes the sequences, assembles, annotates them, and integrates the results into a web-based database, allowing researchers browse and query of the results. About 62% of cocoa sequences showed significant similarity to gene sequences from other species and a part of them could be classified according to the Gene Ontology. This new molecular cocoa resource allowed the identification of hundreds of new microsatellite markers and thousands of SNPs (Single Nucleotide Polymorphisms). It will allow to identify an unigene set suitable for all functional genomic studies in cocoa. (Texte integral)

Moniliophthora perniciosa, the causal agent of witches’ broom disease of Theobroma cacao, significantly affected cacao production in South America and Caribbean countries. Host colonization by the pathogen exhibits a concerted succession of symptoms, starting with hypertrophic growth and “broom” formation, followed by tissue degeneration and death. To understand mechanisms of host susceptibility, we investigated fungal development during a compatible interaction with a susceptible genotype. Microscopic analysis revealed the initial fungal biotrophic intercellular growth, followed by intracellular growth associated with the presence of an increasing number of host apoptotic nuclei and calcium oxalate crystals, with subsequent accumulation of hydrogen peroxide and cell death. Active oxalate degradation and its possible source of origin were detected in infected tissues. Together, these processes may increase the availability of nutrients for the fungal mycelia and may contribute to the disease cycle in this plant–fungal hemibiotrophic interaction. Based on the histological and gene expression data, a novel role for calcium oxalate in disease susceptibility is proposed.

Ethylene is a major stimulating factor for natural rubber production in Hevea brasiliensis and is often applied in the form of Ethephon, an ethylene releaser. In a positive feedback type mechanism, the application of ethylene leads to the enhanced expression of genes involved in ethylene biosynthesis such as ACC oxidases, which are also responsive to wounding. We studied the regulation of ethylene biosynthesis in rubber tree through the characterization of the ACC oxidase multigene family and the responses of individual genes to ethylene and wounding. Three full-length cDNAs, HbACO1 (EMBL accession AM743170), HbACO2 (EMBL accession AM743171), and HbACO3 (EMBL accession AM743172) were isolated. All of the HbACO genes were expressed at all stages of development studied, from in vitro callus to the exploited plant, but with different expression profiles (Kuswanhadi et al. 2005). HbACO1 was expressed at a higher level than the other genes, and was down-regulated by ethylene and wounding in both leaf and bark tissues. In contrast, HbACO2 and HbACO3 were transiently induced in response to ethylene and wounding. Treatment with 1-MCP, an ethylene action inhibitor, abolished the ethylene induction of HbACO2 expression, showing that this gene was under positive feedback regulation. The expression of HbACO3 was also induced by ethylene treatment and wounding, again with an antagonistic effect of 1-MCP. In conclusion, HbACO1 appears to be responsible for basal levels of ethylene production while HbACO2 and HbACO3 are up-regulated in response to external factors. (Resume d'auteur)

Since the dawn of the genomics era, much research has focused on functional studies of genes of interest. In situ hybridization is a method that can be used to precisely localize the expression of a gene in tissues and cells. This article describes how the method has been adapted to the analysis of Hevea tissues. Initially, the conventional method of digoxigenin detection with NBT/BCIP revealed the expression of strongly expressed genes in tissues of different differentiation intensity. A new digoxigenin detection method using Alexa488 fluorochrome-labelled antibodies has been used to detect the expression of more weakly expressed genes. This method, combined with observation under a confocal microscope, has enabled very precise localization of expression. Some examples of in situ hybridization use are described for Hevea gene expression in somatic plantlets and shoot bark: the uidA gene in callus and transgenic somatic plantlets, the HEV2.1 gene encoding hevein and the ACO-H5 gene involved in ethylene metabolism. Cell imaging methods therefore open up fundamental prospects for studying the different molecular mechanisms involved in some agronomic traits of Hevea. (Resume d'auteur)

Applying Ethephon, an ethylene releaser, to increase rubber production in H. brasiliensis has been practised for a long time. The effect of ethylene on latex production has been amply described, notably for lengthening latex flow and its regeneration. Nevertheless, little is known about the expression of genes involved in ethylene biosynthesis and response to ethylene. In this paper, we isolated and characterized genes encoding ACC oxidase, a key enzyme in ethylene biosynthesis in the plant. We then studied the effect of ethylene stimulation on the expression of ACO genes in various tissues during plant development. Three members of the ACO multigenic family were isolated from a bark cDNA library with RACE technology: HbACO-H4, HbACO-H5, and HbACO-O48. Full length cDNA sequences encoded for peptides of 318, 315, and 318 amino acids respectively for these 3 members, which had 79 to 92% protein identity and 75 to 86% nucleotide homology between them. Two genomic sequences were isolated: HbACO-H4, which was 1504 bp long and consisted of 2 introns and 3 exons, while Hb-ACO-H5 was 1456 bp long and consisted of 3 introns and 4 exons. These three genes were differentially expressed in different plant organs in response to ethylene stimulation. (Resume d'auteur)