Joachim Jacob

An endosymbiotic bacterium in a plant-parasitic nematode: member of a new Wolbachia supergroup.

Wolbachia is an endosymbiotic bacterium widely present in arthropods and animal-parasitic nematodes. Despite previous efforts, it has never been identified in plant-parasitic nematodes. Random sequencing of genes expressed by the burrowing nematode Radopholus similis resulted in several sequences with similarity to Wolbachia genes. The presence of a Wolbachia-like endosymbiont in this plant-parasitic nematode was investigated using both morphological and molecular approaches. Transmission electronmicroscopy, fluorescent immunolocalisation and staining with DAPI confirmed the presence of the endosymbiont within the reproductive tract of female adults. 16S rDNA, ftsZ and groEL gene sequences showed that the endosymbiont of R. similis is distantly related to the known Wolbachia supergroups. Finally, based on our initial success in finding sequences of this endosymbiont by screening an expressed sequence tag (EST) dataset, all nematode ESTs were mined for Wolbachia-like sequences. Although the retained sequences belonged to six different nematode species, R. similis was the only plant-parasitic nematode with traces of Wolbachia. Based on our phylogenetic study and the current literature we designate the endosymbiont of R. similis to a new supergroup (supergroup I) rather than considering it as a new species. Although its role remains unknown, the endosymbiont was found in all individuals tested, pointing towards an essential function of the bacteria.

Expressed sequence tags of the peanut pod nematode Ditylenchus africanus: The first transcriptome analysis of an Anguinid nematode

In this study, 4847 expressed sequenced tags (ESTs) from mixed stages of the migratory plant-parasitic nematode Ditylenchus africanus (peanut pod nematode) were investigated. It is the first molecular survey of a nematode which belongs to the family of the Anguinidae (order Rhabditida, superfamily Sphaerularioidea). The sequences were clustered into 2596 unigenes, of which 43% did not show any homology to known protein, nucleotide, nematode EST or plant-parasitic nematode genome sequences. Gene ontology mapping revealed that most putative proteins are involved in developmental and reproductive processes. In addition unigenes involved in oxidative stress as well as in anhydrobiosis, such as LEA (late embryogenesis abundant protein) and trehalose-6-phosphate synthase were identified. Other tags showed homology to genes previously described as being involved in parasitism (expansin, SEC-2, calreticulin, 14-3-3b and various allergen proteins). In situ hybridization revealed that the expression of a putative expansin and a venom allergen protein was restricted to the gland cell area of the nematode, being in agreement with their presumed role in parasitism. Furthermore, seven putative novel candidate parasitism genes were identified based on the prediction of a signal peptide in the corresponding protein sequence and homologous ESTs exclusively in parasitic nematodes. These genes are interesting for further research and functional characterization. Finally, 34 unigenes were retained as good target candidates for future RNAi experiments, because of their nematode specific nature and observed lethal phenotypes of Caenorhabditis elegans homologs.

A unique genetic code change in the mitochondrial genome of the parasitic nematode Radopholus similis

BackgroundMitochondria (mt) contain their own autonomously replicating DNA, constituted as a small circular genome encoding essential subunits of the respiratory chain. Mt DNA is characterized by a genetic code which differs from the standard one. Interestingly, the mt genome of nematodes share some peculiar features, such as small transfer RNAs, truncated ribosomal RNAs and - in the class of Chromadorean nematodes - unidirectional transcription.FindingsWe present the complete mt genomic sequence (16,791 bp) of the plant-parasitic nematode Radopholus similis (class Chromadorea). Although it has a gene content similar to most other nematodes, many idiosyncrasies characterize the extremely AT-rich mt genome of R. similis (85.4% AT). The secondary structure of the large (16S) rRNA is further reduced, the gene order is unique, the large non-coding region contains two large repeats, and most interestingly, the UAA codon is reassigned from translation termination to tyrosine. In addition, 7 out of 12 protein-coding genes lack a canonical stop codon and analysis of transcriptional data showed the absence of polyadenylation. Northern blot analysis confirmed that only one strand is transcribed and processed. Furthermore, using nucleotide content bias methods, regions for the origin of replication are suggested.ConclusionThe extraordinary mt genome of R. similis with its unique genetic code appears to contain exceptional features correlated to DNA decoding. Therefore the genome may provide an incentive to further elucidate these barely understood processes in nematodes. This comprehension may eventually lead to parasitic nematode-specific control targets as healthy mitochondria are imperative for organism survival. In addition, the presented genome is an interesting exceptional event in genetic code evolution.

Arabinogalactan endo-1,4- β -galactosidase: a putative plant cell wall-degrading enzyme of plant-parasitic nematodes

Plant-parasitic nematodes secrete a plethora of enzymes to degrade polysaccharides of the recalcitrant plant cell wall. Here we report on the presence of a putative endo-1,4- β -galactosidase (EC 3.2.1.89) in cyst nematodes of the genus Heterodera . This enzyme hydrolyses β -1,4-galactan in the hairy regions of pectin and to our knowledge it is the first report of this class of enzymes in animals. The gene was cloned from H. schachtii and subjected to a detailed molecular characterisation. The deduced protein contains a putative signal peptide for secretion, being in agreement with the presumed extracellular function of the mature protein. It has a molecular mass of 33.78 kDa and folds into an ( α/β) 8 barrel structure typical for glycosyl hydrolases. The two glutamic acids that function as electron donor and acceptor in the active site are conserved. Whole mount in situ hybridisation revealed that the gene is expressed in the subventral pharyngeal glands and the expression was correlated with the onset of parasitism.

Structural and functional investigation of a secreted chorismate mutase from the plant-parasitic nematode Heterodera schachtii in the context of related enzymes from diverse origins.

In this article, we present the cloning of Hscm1, a gene for chorismate mutase (CM) from the beet cyst nematode Heterodera schachtii. CM is a key branch-point enzyme of the shikimate pathway, and secondary metabolites that arise from this pathway control developmental programmes and defence responses of the plant. By manipulating the plants endogenous shikimate pathway, the nematode can influence the plant physiology for its own benefit. Hscm1 is a member of the CM gene family and is expressed during the pre-parasitic and parasitic stages of the nematodes life cycle. In situ mRNA hybridization reveals an expression pattern specific to the subventral and dorsal pharyngeal glands. The predicted protein has a signal peptide for secretion in addition to two domains. The N-terminal domain of the mature protein, which is only found in cyst nematodes, contains six conserved cysteine residues, which may reflect the importance of disulphide bond formation for protein stabilization. The C-terminal domain holds a single catalytic site and has similarity to secreted CMs of pathogenic bacteria, classifying HsCM1 as an AroQ(gamma) enzyme. The presumed catalytic residues are discussed in detail, and genetic complementation experiments indicate that the C-terminal domain is essential for enzyme activity. Finally, we show how the modular design of the protein is mirrored in the genomic sequence by the intron/exon organization, suggesting exon shuffling as a mechanism for the evolutionary assembly of this protein.