Making plants resistant to plant parasitic nematodes: no access - no feeding | NONEMA | Project | Results | FP5 | CORDIS

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.

In a search for plant proteins recruited by plant parasitic nematodes we concentrated on cell wall degrading and modifying proteins. Two expansins genes are activated by the presence of the cyst nematode. One isoform was strongly up-regulated in the immediate vicinity of the developing syncytium (= vascular cylinder), but not in the feeding cell itself. Another isoform was shown to be activated early during syncytium development. Both in situ hybridisation experiments and immunolocalisation studies were done to further confirm the involvement of these plants proteins nematode-induced feeding site formation. For the immunolocalisation approach specific polyclonal antibodies were produced against individual isoforms of plant expansins.

A large scale procedure was set up in order to purify stylet secreted proteins from M. incognita infective juveniles after secretion induction in vitro. The secreted proteins whose pI ranged from 5,5 to 7,0 were analysed by 2D-electrophoresis. Proteins were identified by trypsin digestion and mass spectrometry. A 14-3-3 protein was among the most abundant proteins. The 14-3-3 protein identified in Meloidogyne incognita stylet secretions was further analysed. 14-3-3 proteins are involved in signal transduction (e.g. activation or inhibition of protein kinases), in the control of the cell-cycle (e.g. by sequestering cdc25 in the cytoplasm), stress responses and cytoskeleton organisation (e.g regulation of Rho GTPases). From the 14-3-3 peptide sequences, two coding sequences were obtained, which correspond to two 14-3-3 isoforms: Mi-14-3-3-a and Mi-14-3-3-b. The tissue localisation of Mi-14-3-3-a and b transcription was analysed by in situ hybridisation on freshly hatched L2s with specific DNA probes. Mi-14-3-3-a transcription was observed exclusively in primordium germinal cells, whereas Mi-14-3-3-b transcripts were detected specifically in the dorsal oesophageal gland cell. The peptide sequence AFDDAIAE identified by mass spectrometry in the secreted 14-3-3 isoform was present in the deduced amino acid sequence of MI-14-3-3-B and absent from that of MI-14-3-3-A. This is consistent with the expression pattern of Mi-14-3-3-b in the dorsal oesophageal gland of infective juveniles.In order to investigate whether the secreted 14-3-3 could be targeted to a particular subcellular compartment of the plant cell or could play a role in the sequestering of plant cell factors, we analysed the localisation of the nematode 14-3-3s in tobacco BY2 cells. MI-14-3-3-A and MI-14-3-3-B fused at their N-terminal end to EGFP (enhanced green fluorescent protein) were expressed in BY2 cells and localised by confocal microscopy. Both EGFP-MI-14-3-3 fusion proteins exhibited a strong EGFP signal in the cytoplasm of the BY2 cells and a weak signal in the nucleus. The relative weakness of the signal observed in the nucleus as compared to the signal obtained with the diffusing free EGFP could result from the exclusion of the fusion protein from the nucleus.

Southern analysis showed that Gr-EXPB1, a functional expansin from the potato cyst nematode Globodera rostochiensis, is member of a multigene family, and EST data suggest expansins to be present in other plant parasitic nematodes as well. Homology modelling predicted that Gr-EXPB1 domain 1 (D1) has a flat b-barrel structure with surface-exposed aromatic rings, whereas the 3D structure of Gr-EXPB1-D2 was remarkably similar to plant expansins. Gr-EXPB1 shows highest sequence similarity to two extracellular proteins from saprophytic soil-inhabiting Actinobacteria, and includes a bacterial type II carbohydrate-binding module. These results support the hypothesis that a number of pathogenicity factors of cyst nematodes is of procaryotic origin and were acquired by horizontal gene transfer.

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.

Plant parasitic nematodes have been, so far, refractory to transformation or mutagenesis. The functional analysis of nematode genes relies on the development of reverse genetic tools adapted to these obligate parasites. Here, we describe the application of RNA interference (RNAi) to the rootknot nematode Meloidogyne incognita for the knock-down of two genes expressed in the subventral esophageal glands of the nematode and potentially involved in parasitism, the calreticulin (Mi-crt) and the polygalacturonase (Mi-pg-1) genes. Incubation in 1% resorcinol for 4h induced doublestranded RNA uptake through the alimentary track of the nematodes and led to up to 92% depletion of Mi-crt transcripts. Timecourse analysis of the silencing showed different temporal patterns for Mi-crt and Mi-pg-1. The silencing of Mi-crt was optimal 20h after soaking, whereas the silencing of Mi-pg-1 was optimal 44 h after soaking. For the two genes, the silencing effect was highly time-limited, since no transcript depletion was detectable 68 h after soaking.

Root-knot nematodes feed from specialized giant cells induced in the plants that they parasitize. We found that the promoter of the Hahsp17.7G4 gene, which encodes a small heat-shock protein involved in embryogenesis and stress responses, directed GUS expression in tobacco galls induced by the root-knot nematode Meloidogyne incognita. In roots containing a GUS reporter fusion to the Hahsp17.7G4 promoter, 10% of the galls stained for GUS expression 1 to 3 days after infection and the fraction stained increased to 60 to 80% 17 to 20 days after infection. A DNA fragment from 83 to +163, which contains heat-shock element (HSE) core sequences, is sufficient to support a promoter activity largely restricted to giant cells within the galls. Two-point mutations in HSE cores, previously reported to abolish the heat-shock response and to strongly reduce the embryogenesis response of the same promoter, did not reduce expression in giant cells. This suggests a distinct regulation of the promoter by nematodes. However, additional point mutations located at positions crucial for binding of heat-shock transcription factors (HSFs) caused a severe decrease in the nematode response. These results demonstrate that HSEs are involved in the promoter activation in giant cells and suggest that HSFs may mediate this response.

The feeding sites induced by sedentary root endoparasitic nematodes have long fascinated researchers. Nematode feeding sites are constructed from plant cells, modified by the nematode to feed itself. Powerful new techniques are allowing us to begin to elucidate the molecular mechanisms that produce the ultrastructural features in nematode feeding cells. Many plant genes that are expressed in feeding sites produced by different nematodes have been identified in several plant species. Nematode-responsive plant genes can now be grouped in categories related to plant developmental pathways and their roles in the making of a feeding site can be illuminated. The black box of how nematodes bring about such elaborate cell differentiation in the plant is also starting to open. Although the information is far from complete, the groundwork is set so that the functions of the plant and nematode genes in feeding site development can begin to be assessed.

In a search for plant proteins recruited by plant parasitic nematodes we concentrated on cell wall degrading and modifying proteins. In two target crops, potato and tomato, two cellulose isoforms were shown to be recruited by cyst nematodes. One was induced early in the infection process (till 5 days post inoculation), whereas the other was expressed over a more extended period of time. Moreover, the latter appeared to be expressed in cells that are about to be incorporated in the proliferating syncytium. In addition, two expansins genes are activated by the presence of the cyst nematode. One isoform was strongly up-regulated in the immediate vicinity of the developing syncytium (= vascular cylinder), but not in the feeding cell itself. Another isoform was shown to be activated early during syncytium development. Both in situ hybridisation experiments and immunolocalisation studies were done to further confirm the involvement of these plants proteins nematode-induced feeding site formation. For the immunolocalisation approach specific polyclonal chicken antibodies (IgYs) were produced against individual isoforms of plant cellulases.

RNA interference (RNAi) has been used widely as a tool for examining gene function and a method that allows its use with plant-parasitic nematodes recently has been described. Here, we use a modified method to analyze the function of secreted â-1,4, endoglucanases of the potato cyst nematode Globodera rostochiensis, the first in vivo functional analysis of a pathogenicity protein of a plantparasitic nematode. Knockout of the â-1,4, endoglucanases reduced the ability of the nematodes to invade roots. We also use RNAi to show that gr-ams-1, a secreted protein of the main sense organs (the amphids), is essential for host location.

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.

To study the role of the phytohormone auxin in nematode feeding cell induction and early development, the transcriptional regulation of the auxin-responsive promoter element DR5 was monitored in Arabidopsis thaliana roots infected with cyst or root-knot nematodes. For both nematode species, a specific and strong activation of DR5:gusA was observed inside the initial cells at 18 hours post inoculation, pointing at an increase of the perceived auxin concentration. This high expression was maintained until 3-5 days post inoculation and subsequently the GUS staining was reduced. Cyst and root-knot nematodes are distantly related (belonging to different families) and the feeding sites they induce are highly dissimilar. In this respect, the similarities between the two nematode-induced DR5 activation patterns in A. thaliana roots are remarkable. A transient and local increase in auxin perception could be due to an accumulation or to an increased sensitivity. Based on previously published data, a local auxin accumulation seems to be the more probable explanation. The observed specific and transient increase of the perceived IAA concentration in the initial feeding structure could be an important clue in the elucidation of the molecular mechanisms underlying feeding cell induction by plant parasitic nematodes, a fascinating but still poorly understood phenomenon.

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.

Expressed sequence tags (EST) have been widely used to assist in gene discovery in various organisms (e.g., Arabidopsis thaliana, Caenorhabditis elegans, Mus musculus, and Homo sapiens). In this paper we describe an EST project, which aims to investigate gene expression in Meloidogyne incognita at the onset of parasitism. Approximately 1000 50-end sequence tags were produced from a cDNA library made of freshly hatched preparasitic second stage juveniles (J2). The EST were identifed in the primary transformants of the cDNA library, and assigned to nine different functional groups, including (candidate) parasitism genes. A large fraction of the EST (45%) did not have a putative homologue in public databases. Sixty five percent of the EST that could be clustered into a functional group had putative homologues in other nematode species. EST was found for virtually all parasitism related genes that have been cloned from M. incognita to date. In addition, several novel genes were tagged, including a xylanase and a chitinase gene. The efficiency of EST projects, which produce sequence data for thousands of genes in a month¿s time without any difficult pre-selections of mRNA pools, makes random sequencing cDNA libraries a superior method to identify candidates for parasitism related genes in plant-parasitic nematodes. The sequences in this paper are retrievable from Genbank with the accession numbers BE191640 to BE191741, BE217592 to BE217720, BE225324 to BE225598, BE238852 to BE239221, and BE240829 to BE240865.

The stylet secretions produced by plant parasitic root-knot nematodes are thought to be pathogenicity factors involved in the invasion of the root tissue and in the induction and maintenance of feeding cells. A new procedure was established that allowed the direct qualitative analysis of proteins secreted by Meloidogyne incognita infective juveniles. Purified proteins whose isoelectric point (pI ) ranged from 5.0 to 7.5 were separated by two-dimensional (2D) electrophoresis and the seven most abundant proteins were identified by micro-sequencing. A calreticulin (CRT) was isolated and transcription of its gene in infective juveniles and adults was demonstrated. Moreover, evidence for expression of the CRT in the subventral oesophageal glands of infective juveniles was obtained. The potential roles of this secreted protein in pathogenesis and the advantages of developing this strategy to obtain new insights into plant-nematode interactions are discussed.

The ¢rst animal polygalacturonase (PG, EC 2.1.15) encoding cDNA, Mi-pg-1, was clonedfrom the plant parasitic nematode Meloidogyne incognita. The enzymatic activity of MI-PG-1 was con¢rmedafter heterologous expression in Escherichia coli. The presence of a predicted signal peptide on the MI-PG-1 sequence together with the speci¢c localization of the transcripts of the Mi-pg-1 gene in the oesophageal glands of infective juveniles imply that MI-PG-1 couldbe secretedinto plant tissues. The potential role of MI-PG-1 in parasitism is discussed.

The á- and â-expansins represent a class of plant proteins with characteristic pH-dependent in vitro cell wall-loosening and stress relaxation activity on cell walls of higher plants. Expansins, which have, until now, only been identified in land plants, induce cell wall extension within seconds and are thought to promote polymer slippage by weakening non-covalent interactions. Sequences with some degree of similarity to expansins have been found in a variety of taxa outside the plant kingdom. However, the corresponding proteins have not yet been tested for expansin activity and therefore cannot be considered as representing functional expansins. Here we report the characterisation of a secretory protein (Gr exp-1) expressed in the esophageal glands of the plant parasitic nematode Globodera rostochiensis with both the structural and functional characteristics of a â-expansin. The structure of the Gr-exp-1 protein resembles that of plant expansins with a bacterial type cellulose-binding domain linked to a catalytic expansin domain. The latter shows an overall sequence identity of 38% with a pistil-specific â-expansin from tobacco. Recombinant expression of Gr-exp-1 in tobacco allowed demonstration of a typical and rapid pH dependent cell wall loosening activity on heat inactivated wheat coleoptiles (a type II cell wall). The presence of â-expansin in their stylet secretions shows that these obligate plant parasites combine symbiont-independent enzymatic and non-enzymatic cell wall modifying activities to facilitate intracellular migration through plant roots.

Making plants resistant to plant parasitic nematodes: no access - no feeding

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