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This thesis describes a first attempt to investigate the biological activity of cyst nematode secretions on plant cell proliferation and the molecular mechanisms underlying feeding cell development in plant roots upon cyst nematode infection.To investigate the role of nematode secretions in feeding cell development, the in vitro induction and collection of putative nematode signalling molecules is needed. Chapter 2 describes the specific induction of nematode secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis upon exposure to potato root diffusate (PRD). In pre-parasitic juveniles (J2) of Globodera rostochiensis , six proteins with molecular weights of 30, 31a/b, 32, 39, and 49 kDa were recognized on western blots by a monoclonal antibody (MGR48) specific for the subventral esophageal glands. All these subventral gland proteins ( svp s) focused in the basic range (pI 6.8-8.6) of an immobilized pH gradient. Western blotting showed that the svp s were present in pre-parasitic and in parasitic J2, and not in following juvenile stages and adult females. Minor svp quantities were also observed in adult males. Immunogold labelling of pre-parasitic (J2) showed that the svp s were localized in the rough endoplasmic reticulum and secretory granules of the subventral esophageal glands. Potato root diffusate triggered the secretion of the svp s through the stylet and 5-methoxy-N,N-dimethyltryptamine-hydrogen-oxalate (DMT) was shown to have only a quantitative, additional effect. The forward flow of the svp s through the metacorporal pump chamber was confirmed by the presence of svp s in the circular lumen of the esophagus (procorpus), as established by immunoelectron microscopy. Our data provide conclusive evidence that secretory proteins of the subventral glands of G. rostochiensis can be secreted through the stylet and support the hypothesis that the subventral esophageal glands play an important role in the early events of this nematode-plant interaction.Using PRD as a host stimulus, relatively large quantities of naturally-induced secretions could be collected from infective juveniles. In Chapter 3, we describe the detection of an oligopeptide(s) in nematode secretions, which stimulate(s) the proliferation of plant cells using a bioassay based on tobacco leaf protoplasts. Naturally-induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones NAA and BAP. Using a protoplast-based bioassay, a low molecular weight peptide(s) (< 3 kDa) was shown to be responsible for the observed effect. This mitogenic oligopeptide(s) is functionally dissimilar to auxin and cytokinin and, in addition, it does not change the sensitivity of the protoplasts towards these phytohormones. In combination with the mitogen phytohaemagglutinin (PHA), cyst nematode secretions also co-stimulated mitogenesis in human peripheral blood mononuclear cells (PBMC). The stimulation of plant cells isolated from non-target tissue - these nematodes normally invade the roots of potato plants - suggests the activation of a general signal transduction mechanism(s) by an oligopeptide(s) secreted by the nematode. Whether a similar oligopeptide-induced mechanism underlies human PBMC activation remains to be investigated. Reactivation of the cell cycle is a crucial event in feeding cell formation by cyst nematodes. The secretion of a mitogenic low molecular weight peptide(s) by infective juveniles of the potato cyst nematode could contribute to the redifferentiation of plant cells into such a feeding cell.The analysis of early changes in gene expression in response to cyst nematode infection is strongly supported by the use of an in vivo reporter gene system. Chapter 4 describes the use of the green fluorescent protein (GFP) to monitor the transcriptional regulation of respectively the viral CaMV 35S and the bacterial TR2' promoter in early feeding cell development in transgenic potato roots infected with G. rostochiensis . Under the control of either the constitutive CaMV 35S or the mannopine synthase TR2' promoter, the Green Fluorescent Protein (GFP ) from the jellyfish Aequorea victoria , was expressed in transgenic potato ( Solanum tuberosum ) plants. Confocal laser scanning microscopy (CLSM) was applied to observe GFP in planta and, subsequently, to investigate promoter activity in developing feeding cells upon potato cyst nematode ( Globodera rostochiensis ) infection. Both the CaMV 35S and the TR2' promoter were strongly up-regulated in young feeding cells in less then 4 days upon infection by G. rostochiensis whereas the GFP level in the surrounding tissues remained low. Optical sectioning revealed intense green fluorescence in the dense cytoplasm of the entire syncytial cell, including the most distal cell. Furthermore, GFP was observed within the digestive system of the feeding nematode, showing that proteins with an apparent molecular weight of 32 kDa can be taken up by parasitic juveniles of G. rostochiensis . Provided CLSM is used, GFP was shown to be a powerful tool that allows in vivo monitoring of gene expression inside young developing feeding cells. Finally, the transcriptional regulation of the CaMV 35S and TR2' promoter in plant-nematode interactions is discussed. Unfortunately, the use of GFP in combination with the more subtle Arabidopsis cell cycle promoters cycB1;1 and cdc2a was complicated by inadequate expression levels necessary for proper GFP detection in infected potato roots. This obstacle was overcome by using the firefly luciferase gene luc as an in vivo reporter gene (Chapter 6).For decades, it is hypothesised that phytohormones could be involved in syncytium formation. In Chapter 5, a molecular genetic approach was used to study the role of auxin in cyst nematode-induced feeding cell development. The infection of the auxin-insensitive tomato mutant diageotropica and several A. thaliana auxin response mutants suggest that cyst nematodes manipulate the local auxin balance upon early feeding cell induction. Infection of the extreme auxin-insensitive tomato mutant diageotropica ( dgt ) and a number of Arabidopsis auxin-response mutants with the potato cyst nematode Globodera rostochiensis and the beet cyst nematode Heterodera schachtii , respectively, strongly point at a role for auxin in early feeding cell development. Nematode development was significantly reduced in the single gene, recessive mutant dgt and was accompanied by abnormal feeding cell development. For the Arabidopsis auxin-insensitive mutants, only a significant reduction of the infection rate was observed for the mutant axr2 . However, the majority of the mutants showed a reduction in feeding cell hypertrophy - like in dgt - and a reduction in nematode-induced lateral root formation. The induction of gus expression in expanding syncytia of the auxin-responsive Arabidopsis promoter trap line 5-1E1 upon infection with H. schachtii suggested that early feeding cell development is accompanied by a local auxin accumulation. In addition, the obstruction of polar auxin transport in the root either by the application of N -(1-naphthyl)phtalamic acid (NPA) or by the use of the Arabidopsis auxin efflux mutants pin1-1/ttg1 and eir1-1 resulted in a substantial reduction of cyst nematode infections. Moreover, abnormal feeding cell development was observed in the presence of NPA, which was accompanied by the disruption of radial feeding cell expansion, disorganized cell division and metaxylem formation.Chapter 6 gives an overview of cell cycle activation by plant-parasitic nematodes; especially cyst and root knot nematodes, and links the possible role of nematode secretions and auxin in feeding cell formation. Sedentary nematodes are important pests of crop plants. They are biotrophic parasites that can induce the (re)differentiation of either differentiated or undifferentiated plant cells into specialised feeding cells. This (re)differentiation includes the reactivation of the cell cycle in specific plant cells finally resulting in a transfer cell-like feeding site. For growth and development the nematodes fully depend on these cells. The mechanisms underlying the intriguing ability of these nematodes to manipulate a plant for its own benefit are unknown. Nematode secretions are thought to play a key role both in plant penetration and feeding cell induction. Research on plant-nematode interactions is hampered by the minute size of cyst and root knot nematodes, their obligatory biotrophic nature and their relatively long life cycle. Recently, insights in cell cycle control in Arabidopsis thaliana in combination with reporter gene technologies showed the differential activation of cell cycle gene promoters upon infection with cyst or root knot nematodes. In this review, we integrate the current views of plant cell fate manipulation by these sedentary nematodes and made an inventory about possible links between cell cycle activation and local, nematode-induced changes in auxin levels. |
