Dissecting host plant manipulation by cyst and root-knot nematodes
Cyst ( Globodera spp. and Heterodera spp.) and root-knot nematodes ( Meloidogyne spp.), one of the most damaging crop pests, are a perfect example of highly adapted, sophisticated root parasites. These nematodes induces specialized feeding structures (cyst nematodes-syncytia, root-knot nematode-giant cells) within the host root and benefits from guaranteed continues supply of food and water from their host plant and protection within the cyst or gall for their progeny. This dissertation aims at elucidation of the molecular mechanisms controlling the induction and maintenance of those feeding structures. This knowledge is indispensable to make novel advances in constructing durable host plant resistance.A number of different observations suggested a primary role of the phytohormone auxin in the feeding cell development. However, it has never been directly proven. Therefore, we have used the DR5 promoter element fused to gusA reporter gene to visualize spatial and temporal changes in auxin distribution, during nematode infection in Arabidopsis thaliana roots infected with cyst or root-knot nematodes. DR5 promoter element is considered to be a specific indicator for auxin and is activation points at an increase of the perceived auxin concentration. For both, cyst and root-knot nematode species, strong GUS expression was observed at the very onset of parasitism, already at 18 hours post inoculation. This high expression level was maintained until 3-5 days post inoculation and then gradually was reduced. Semi-thin sections of infected roots were investigated microscopically, revealing the presence of GUS inside the initial cells, and in case of cyst nematodes, in cells to-be incorporated into syncytium. This implies that expansion of syncytium is marked by the regions with high gusA expression. Activation of DR5 promoter element can be explained by: an accumulation or an increased sensitivity to auxin. However, based on formerly gathered data, a local accumulation seems to be the more probable explanation. Moreover, the feeding structures of cyst and root-knot nematodes arise by distance mechanisms, hence the highly similar DR5 activation patterns in both feeding structures, is remarkable. This promptthe conclusion that notwithstanding the highly dissimilar structure and ontogeny, both nematodes interfere with similar signal transduction pathways in root morphogenesis.Apart from phytohormones, small peptide signalling plays a very important role in diverse aspects of plant growth and development, and such a molecule(s) could be potentially involved in the establishment of nematode feeding cell. Early nodulin ( ENOD ) genes have been defined as genes expressed in legumes during nodule formation. Recent studies, however, revealed a number of homologues of ENOD40 s in non-legume plants. In both plants types, ENOD40 expression associates with the development of the vascular tissue. Feeding site development by root-knot and cyst nematodes is accompanied by the distortion of the vascular tissue, therefore a local increase in ENOD40 expression could be anticipated. This holds truth in interaction between legume Medicago truncatula and root-knot nematodes. However, in non-legume tomato infected with cyst and root-knot nematodes, no change in ENOD40 transcript levels could be detected using RT-PCR. In addition, activation of the Sl-ENOD40 promoter was only observed in infected regions showing endogenous ENOD40 expression. Similar results were obtained for a legume ENOD40 promoter (soybean) in a non-legumes background ( Arabidopsis ), and - contrary to M. truncatula - over-expression of Gm-ENOD40 in Arabidopsis had no effect on nematode development. Hence, ENOD40 expression in tomato is basically unaffected by the induction of feeding sites by root-knot and cyst nematodes. This unexpected difference in nematode-induced ENOD40 is apparently plant background-dependent, and suggests the existence of a legume-specific factor that boosts the ENOD40 expression in legume hosts-nematode interaction.One of the most striking characteristic of the cyst nematode induced feeding structure (syncytium) is the extensive cell wall dissolution, as the syncytium expands towards the vascular bundle and along the xylem vessels. Endo-1,4-ß-glucanases (cellulases) reside among plant enzymes that are involved in this progressive cell wall dissolution.RT-PCR experiments in tomato revealed that the potato cyst nematode induces the expression of two out of the eight described cellulases, precisely, Sl-cel7 and Sl-cel8 . In situ hybridization and immunodetection studies showed that both cellulases are expressed inside and adjacent to nematode-induced syncytia. Transgenic potato plants carryinghpRNA-silencing constructs for cel7 and cel8 were infected with cyst nematode which resulted in a significant arrest of cyst nematodes development.Reduced development of juveniles into females (up to 60%) and increase in the fraction of females without eggs (up to 89%) was observed. This indicates that the recruitment of specific plant cellulases by potato cyst nematodes is essential for their development, and demonstrates how detailed knowledge of compatible plant-parasite relationships can lead to host plant resistance.The genomic sequence of potato, cel7 and cel8a was cloned and characterised, in order to enable the RNA interference experiments in the natural host of potato cyst nematodes. The analysis of sequences suggests the presence of multiple isoforms of cel8 in potato. That corresponding proteins consist solely of a catalytic core domain (St-CEL7) or of a catalytic domain linked to a carbohydrate binding module (CBM) (St-CEL8a). Only recently, the CBM domain of Sl-CEL8a was shown to belong to CBM family 2 and on the basis of shared features between CBM domains in Sl-CEL8 and St-CEL8a it can be concluded that St-CEL8a harbours a cellulose binding domain as well. Therefore, those findings imply that the potato cellulases recruited by the potato cyst nematode are able to hydrolyse both xyloglucan (CEL7) and crystalline cellulose (CEL8), and apart from cloning novel potato cellulases genes, provides an explanation why nematodes recruit exactly these two members of the cellulase family. Moreover, potato cellulase sequences were used to mine EST databases and this information was used for the generation of a phylogenetic tree of the Solanaceous endo-β-1,4-D-glucanase gene family. In this dissertation we take an effort to understand better the intimae relationship between the obligatory plant parasites, cyst and root-knot nematodes and host plants. We show that such a detailed knowledge can be beneficial in better understanding of plant physiology and can be used as a highly specific and bio-safe approach for pathogen management.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
| Published: |
Wageningen Universiteit
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| Subjects: | auxins, cellular biology, giant cells, globodera rostochiensis, heterodera schachtii, host parasite relationships, host plants, interactions, meloidogyne incognita, plant growth regulators, plant parasitic nematodes, auxinen, celbiologie, gastheer parasiet relaties, interacties, plantengroeiregulatoren, plantenparasitaire nematoden, reuzencellen, waardplanten, |
| Online Access: | https://research.wur.nl/en/publications/dissecting-host-plant-manipulation-by-cyst-and-root-knot-nematode |
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| Summary: | Cyst ( Globodera spp. and Heterodera spp.) and root-knot nematodes ( Meloidogyne spp.), one of the most damaging crop pests, are a perfect example of highly adapted, sophisticated root parasites. These nematodes induces specialized feeding structures (cyst nematodes-syncytia, root-knot nematode-giant cells) within the host root and benefits from guaranteed continues supply of food and water from their host plant and protection within the cyst or gall for their progeny. This dissertation aims at elucidation of the molecular mechanisms controlling the induction and maintenance of those feeding structures. This knowledge is indispensable to make novel advances in constructing durable host plant resistance.A number of different observations suggested a primary role of the phytohormone auxin in the feeding cell development. However, it has never been directly proven. Therefore, we have used the DR5 promoter element fused to gusA reporter gene to visualize spatial and temporal changes in auxin distribution, during nematode infection in Arabidopsis thaliana roots infected with cyst or root-knot nematodes. DR5 promoter element is considered to be a specific indicator for auxin and is activation points at an increase of the perceived auxin concentration. For both, cyst and root-knot nematode species, strong GUS expression was observed at the very onset of parasitism, already at 18 hours post inoculation. This high expression level was maintained until 3-5 days post inoculation and then gradually was reduced. Semi-thin sections of infected roots were investigated microscopically, revealing the presence of GUS inside the initial cells, and in case of cyst nematodes, in cells to-be incorporated into syncytium. This implies that expansion of syncytium is marked by the regions with high gusA expression. Activation of DR5 promoter element can be explained by: an accumulation or an increased sensitivity to auxin. However, based on formerly gathered data, a local accumulation seems to be the more probable explanation. Moreover, the feeding structures of cyst and root-knot nematodes arise by distance mechanisms, hence the highly similar DR5 activation patterns in both feeding structures, is remarkable. This promptthe conclusion that notwithstanding the highly dissimilar structure and ontogeny, both nematodes interfere with similar signal transduction pathways in root morphogenesis.Apart from phytohormones, small peptide signalling plays a very important role in diverse aspects of plant growth and development, and such a molecule(s) could be potentially involved in the establishment of nematode feeding cell. Early nodulin ( ENOD ) genes have been defined as genes expressed in legumes during nodule formation. Recent studies, however, revealed a number of homologues of ENOD40 s in non-legume plants. In both plants types, ENOD40 expression associates with the development of the vascular tissue. Feeding site development by root-knot and cyst nematodes is accompanied by the distortion of the vascular tissue, therefore a local increase in ENOD40 expression could be anticipated. This holds truth in interaction between legume Medicago truncatula and root-knot nematodes. However, in non-legume tomato infected with cyst and root-knot nematodes, no change in ENOD40 transcript levels could be detected using RT-PCR. In addition, activation of the Sl-ENOD40 promoter was only observed in infected regions showing endogenous ENOD40 expression. Similar results were obtained for a legume ENOD40 promoter (soybean) in a non-legumes background ( Arabidopsis ), and - contrary to M. truncatula - over-expression of Gm-ENOD40 in Arabidopsis had no effect on nematode development. Hence, ENOD40 expression in tomato is basically unaffected by the induction of feeding sites by root-knot and cyst nematodes. This unexpected difference in nematode-induced ENOD40 is apparently plant background-dependent, and suggests the existence of a legume-specific factor that boosts the ENOD40 expression in legume hosts-nematode interaction.One of the most striking characteristic of the cyst nematode induced feeding structure (syncytium) is the extensive cell wall dissolution, as the syncytium expands towards the vascular bundle and along the xylem vessels. Endo-1,4-ß-glucanases (cellulases) reside among plant enzymes that are involved in this progressive cell wall dissolution.RT-PCR experiments in tomato revealed that the potato cyst nematode induces the expression of two out of the eight described cellulases, precisely, Sl-cel7 and Sl-cel8 . In situ hybridization and immunodetection studies showed that both cellulases are expressed inside and adjacent to nematode-induced syncytia. Transgenic potato plants carryinghpRNA-silencing constructs for cel7 and cel8 were infected with cyst nematode which resulted in a significant arrest of cyst nematodes development.Reduced development of juveniles into females (up to 60%) and increase in the fraction of females without eggs (up to 89%) was observed. This indicates that the recruitment of specific plant cellulases by potato cyst nematodes is essential for their development, and demonstrates how detailed knowledge of compatible plant-parasite relationships can lead to host plant resistance.The genomic sequence of potato, cel7 and cel8a was cloned and characterised, in order to enable the RNA interference experiments in the natural host of potato cyst nematodes. The analysis of sequences suggests the presence of multiple isoforms of cel8 in potato. That corresponding proteins consist solely of a catalytic core domain (St-CEL7) or of a catalytic domain linked to a carbohydrate binding module (CBM) (St-CEL8a). Only recently, the CBM domain of Sl-CEL8a was shown to belong to CBM family 2 and on the basis of shared features between CBM domains in Sl-CEL8 and St-CEL8a it can be concluded that St-CEL8a harbours a cellulose binding domain as well. Therefore, those findings imply that the potato cellulases recruited by the potato cyst nematode are able to hydrolyse both xyloglucan (CEL7) and crystalline cellulose (CEL8), and apart from cloning novel potato cellulases genes, provides an explanation why nematodes recruit exactly these two members of the cellulase family. Moreover, potato cellulase sequences were used to mine EST databases and this information was used for the generation of a phylogenetic tree of the Solanaceous endo-β-1,4-D-glucanase gene family. In this dissertation we take an effort to understand better the intimae relationship between the obligatory plant parasites, cyst and root-knot nematodes and host plants. We show that such a detailed knowledge can be beneficial in better understanding of plant physiology and can be used as a highly specific and bio-safe approach for pathogen management. |
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