Jan De Meutter

A New Class of Ubiquitin Extension Proteins Secreted by the Dorsal Pharyngeal Gland in Plant Parasitic Cyst Nematodes

By performing cDNA AFLP on pre- and early parasitic juveniles, we identified genes encoding a novel type of ubiquitin extension proteins secreted by the dorsal pharyngeal gland in the cyst nematode Heterodera schachtii. The proteins consist of three domains, a signal peptide for secretion, a mono-ubiquitin domain, and a short C-terminal positively charged domain. A gfp-fusion of this protein is targeted to the nucleolus in tobacco BY-2 cells. We hypothesize that the C-terminal peptide might have a regulatory function during syncytium formation in plant roots.

Identification of cytokinins produced by the plant parasitic nematodes Heterodera schachtii and Meloidogyne incognita

SUMMARY The presence of different types of cytokinins was analysed in exudates and lysates of stage-2 juveniles of Heterodera schachtii and Meloidogyne incognita and in mixed stages of Caenorhabditis elegans. For all species, cytokinins were detected in lysates and exudates in which benzyladenine and zeatin-type cytokinins were the most prominent forms. The production of cytokinins by Meloidogyne was much higher than by Heterodera, and the detected levels were in a range which interfered with the physiological activities of the host plant. The presence of 5-methoxy-N,N-dimethyltryptamine hydrogen oxalate did not affect hormone production by H. schachtii, whereas resorcinol slightly stimulated hormone production by M. incognita. The exuded cytokinins may play a role in feeding site induction, more particularly in cell cycle activation and in establishing the feeding site as a nutrient sink.

Molecular characterization and functional importance of pectate lyase secreted by the cyst nematode Heterodera schachtii.

SUMMARY To analyse the parasitic behaviour of the plant-parasitic nematode Heterodera schachtii, proteins secreted by this nematode were purified and separated by two-dimensional gel electrophoresis. Mass spectrometric analysis identified one of the spots as a pectate lyase (EC 4.2.2.2). The corresponding gene was cloned from a cDNA library using primers derived from the peptide tag. A second pectate lyase was cloned based on similarity to known pectate lyases of related cyst nematodes. The predicted proteins are only 29% identical. Despite the low homology, the proteins have a similar secondary structure and it is likely that they fold into a similar right-handed beta-helix. Both proteins have a putative signal peptide for secretion, and in situ hybridization revealed that expression of the genes was limited to the subventral secretory glands. RT-PCR showed that both genes were expressed in the migratory preparasitic stage although the level of expression between the two genes was different. Post-transcriptional gene silencing by soaking the nematodes in double-stranded RNA against the gene with the highest expression level affected the infection process of the nematode, which is in agreement with the general idea that pectate lyases are essential during migration of the nematode in the plant root.

Isolation of the LEMMI9 gene and promoter analysis during a compatible plant-nematode interaction

Plant-endoparasitic root-knot nematodes feed on specialized giant cells that they induce in the vascular cylinder of susceptible plants. Although it has been established that a number of plant genes change their expression pattern during giant cell differentiation, virtually no data are available about the mechanisms involved in that change. One possibility is differential promoter recognition by the transcription factor(s) responsible for the expression of specific genes. We have isolated and characterized a genomic clone from tomato containing the promoter region of LEMMI9, one of the few plant genes that have been reported to be highly expressed in galls (predominantly in giant cells). The analysis of transgenic potato plants carrying a LEMMI9 promoter-beta glucuronidase (GUS) fusion has demonstrated that the tomato promoter was activated in Meloidogyne incognita-induced galls in a heterologous system. We have located putative regulatory sequences in the promoter and have found that nuclear proteins from the galls formed specific DNA-protein complexes with the proximal region of the LEMMI9 promoter. The nuclear protein-binding sequence mapped to a region of 111 bp immediately upstream from the TATA box. This region contains a 12-bp repeat possibly involved in the formation of DNA-protein complexes, which might be related to the LEMMI9 transcriptional activation in the giant cells.

An SXP/RAL-2 protein produced by the subventral pharyngeal glands in the plant parasitic root-knot nematode Meloidogyne incognita

Meloidogyne incognita is a major parasite of numerous plant families, including many crop species. Upon infection of the plant root, it induces several multinucleate giant cells by the injection of pharyngeal gland secretions into the root cells. In order to obtain a better understanding of the nematode-plant interaction, characterization of the pharyngeal gland secretions is a necessity. By differential display, a nematode gene was identified that encodes a new member of the SXP/RAL-2 protein family. The gene is specifically expressed in the subventral pharyngeal glands and the protein is most likely secreted.

Differential activation of ABI3 and LEA genes upon plant parasitic nematode infection

SUMMARY Promoter activity of ABI3 and of three LEA genes was monitored in Arabidopsis transgenics infected with Heterodera schachtii and Meloidogyne incognita. ABI3::GUS expression was induced (in four different promoter deletion constructs) during early infection stages with H. schachtii. Similar GUS expression patterns, though slightly later in time compared with ABI3, were observed for one of the LEA promoter constructs, whereas the other two were not induced by H. schachtii. Expression was mainly observed in the syncytia. In contrast, little or no reporter gene expression was observed upon infection with M. incognita. The data suggest a role for ABI3 during the formation and active growth of the syncytium and demonstrate a marked difference between syncytium and giant cell ontogenesis.

An improved method for whole-mount in situ hybridization of Heterodera schachtii juveniles

Abstract. An optimized protocol is presented to visualize gene expression in the sedentary beet cyst nematode, Heterodera schachtii, by whole-mount in situ hybridization. Two different probes were used for genes with known expression pattern in other nematodes. Vacuum infiltration of the fixative significantly increased its efficiency and resulted in a nicely preserved morphology. Additional modifications were introduced to simplify and standardize the process.

Development and pharyngeal gland activities of Heterodera schachtii infecting Arabidopsis thaliana roots

As plant-nematode research continues to use the model host plant Arabidopsis thaliana more and more, we made a detailed life cycle study of Heterodera schachtii on this host, and investigated pharyngeal gland activities at the most crucial steps during the establishment of the parasitic interaction. In our experimental set up, in most cases induction of syncytia occurred during day 2, and the second moult during day 6. In pre-parasitic juveniles, both subventral glands contained numerous secretory granules, which gradually disappeared during intracellular migration of the juvenile through the roots. At the same time, both glands decreased in size and, after induction of the feeding cell, new kinds of secretory granules could be observed in the cytoplasm. During migration and induction of the feeding cell, the dorsal pharyngeal gland continuously increased in size. Secretory granules were already present in the dorsal gland of pre-parasitic juveniles, and a continuous synthesis of these granules was observed thereafter. Once the syncytium was induced, however, different types of secretory granules were formed.