Nicotiana benthamiana is widely used as a model plant to analyse cell biology and to obtain insight into the molecular host-pathogen interaction because it is susceptible to many pathogens. Since N. benthamiana can be transformed easily, it is also used to study pathogens for which it is not a known host. Meloidogyne graminicola has a fairly broad host range of mainly monocots and some dicots but no data were available on the ability of M. graminicola to infect N. benthamiana. In this study, we show that M. graminicola is able to infect and complete its life cycle in N. benthamiana, although our experiments demonstrate a lower susceptibility compared to rice. In addition, M. graminicola was also able to develop in N. tabacum but the reproduction was very low. Therefore, we conclude that N. benthamiana can be considered as a host, while this is not the case for N. tabacum.
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 126.96.36.199) 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.
In this research, Arabidopsis thaliana plants were transformed with hairpin constructs targeting cyst nematode (Heterodera schachtii) genes, driven by the cauliflower mosaic virus (CaMV) 35S promoter: two housekeeping genes (the splicing factor Hs-U2AF and the vacuolar Hs-H+ATPase) and one candidate effector gene (the ubiquitin extension protein Hs-ubi). Expression of the dsRNA appeared to be extremely variable between and within homozygous T3 lines and even between tissues. Infection experiments showed up to 50% reduction in nematode infection for some transgenic lines. The results varied not only between lines containing the same construct but also between independent repetitions of the experiment. Further focusing on the Hs-U2AF-RNAi lines revealed large variations and a general decline of construct expression levels over the generations. Bisulphite sequencing of a 197 bp part of the CaMV 35S promoter revealed substantial methylation in this region and a negative correlation between the methylation level and expression of the hairpin construct. Taken together, our results show that host-generated RNAi can suffer from high levels of transcriptional silencing of the construct, leading to varying expression levels within and between transgenic lines.
Plant parasitic nematodes are known to cause a severe reduction in crop yield. Recently much effort is being put to engineering new nematode-resistant crop cultivars. Plant parasitic nematodes occur in three widely separated orders: Triplonchida, Dorylaimida and Tylenchida. All triplonchid and dorylaimid plant parasitic nematodes are migratory ectoparasites of roots. Within the Tylenchida, several different types of plant parasitism can be recognised. The sedentary endoparasites have the most complex interaction with their host, and are responsible for the vast majority of the agricultural damage. This causes most research to be concentrated on two groups of the sedentary endoparasitic nematodes: cyst- and root-knot nematodes. Both induce specialised feeding structures in the vascular cylinder of the plant root. The mechanism of phytoparasitism of the cyst- and root-knot nematodes is reviewed, of which some aspects will be applicable to the study of the other plant parasitic nematodes. Les nématodes parasites de plantes sont connus pour provoquer de sévères réductions dans les rendements des cultures. Actuellement, un effort se développe pour créer de nouveaux cultivars résistants aux nématodes. Les nématodes parasite de plantes appartiennent à trois ordres très éloignés: Triplonchida, Dorylaimida et Tylenchida. Tous les nématodes parasites de plantes chez les Triplonchida et Dorylaimida sont des ectoparasites migrateurs. Chez les Tylenchida, plusieurs types différents de parasitisme peuvent être identifiés. Les endoparasites sédentaires ont l’interaction la plus complexe avec leur hôte et sont responsables de la plus grande part des dégâts agricoles. C’est la raison pour laquelle la plupart des recherches sont concentrées sur deux groupes de nématodes endoparasites sédentaires, les nématodes à kystes et les nématodes galligènes. Ces deux groupes induisent des structures d’alimentation spécialisées dans les tissus vasculaires de la racine végétale. Le mécanisme parasitaire des nématodes à kystes et galligènes est revu, certaines de leurs caractéristiques pouvant être applicables à l’étude des autres nématodes phytoparasites.
Upon pathogen attack, the plant defence response is mediated by a set of connected signal transduction pathways, guided by several classes of plant hormones. In this study, experiments were conducted to observe the role of the plant hormone gibberellic acid in the response of rice to infection by the migratory root-rot nematode Hirschmanniella oryzae. Foliar treatments with gibberellic acid showed a negative effect on H. oryzae infection in the roots. Analyses of mutant rice lines impaired in the production or signalling of gibberellic acid confirmed the effect of the plant hormone on H. oryzae infection. Taken together, the results clearly indicate that gibberellic acid has a positive effect on the capability of the rice plant to fend off an infection by the migratory nematode H. oryzae.
Hairy roots induced by Agrobacterium rhizogenes have been proposed as a versatile, easy and reproducible system for testing nematode resistance in crop plants. Here, A. rhizogenes was used to induce transgenic hairy roots on tomato (Lycopersicon esculentum) containing the LEMMI9 cDNA in sense and/or antisense orientation under control of a nematode responsive promoter. The purpose was to inhibit the expression of this gene that is strongly activated in the nematode-induced feeding sites in order to block their development and, thus, make the plants resistant. Several LEMMI9 transgenic lines produced fewer Meloidogyne incognita second-stage juveniles than the control lines, but a large variation in progeny was also seen among the control lines. Therefore, we recommend that several independent wild-type hairy root lines are generated and tested to obtain a solid control group for evaluating putative resistance constructs.
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.
Analysis of the genome sequence of the potato cyst nematode, Globodera pallida, has shown that a substantial gene family (approximately 300 sequences) of proteins containing a SPRY domain is present in this species. This is a huge expansion of the gene family as compared to other organisms, including other plant-parasitic nematodes. Some SPRY domain proteins from G. pallida and G. rostochiensis have signal peptides for secretion and are deployed as effectors. One of these SPRYSEC proteins has been shown to suppress host defence responses. We describe further analysis of this gene family in G. pallida. We show that only a minority (10%) of the SPRY domain proteins in this species have a predicted signal peptide for secretion and that the presence of a signal peptide is strongly correlated with the corresponding gene being expressed at the early stages of parasitism. The data suggest that while the gene family is greatly expanded, only a minority of SPRY domain proteins in G. pallida are SPRYSEC candidate effectors. We show that several new SPRYSECs from G. pallida are expressed in the dorsal gland cell and demonstrate that some, but not all, of the SPRYSECs can suppress the hypersensitive response induced by co-expression of the resistance gene Gpa2 and its cognate avirulence factor RBP-1 in Nicotiana benthamiana.
The root-lesion nematode, Pratylenchus zeae, is commonly found in upland rice fields. To measure the impact of the nematode on rice production, a screenhouse experiment was conducted using two farmer-adapted Oryza sativa cultivars, ‘Supa’ (‘SurinamV-880’) and ‘SARO-5’ (‘TXD 306’), under flooded, upland and drought water regimes imposed at 7 days post-inoculation of mixed-stage nematodes (200, 500, 1000, 3000 and 10 000 plant−1). Growth and yield parameters were recorded, and the experiment was terminated after 5 months. ‘Supa’ was shown to be resistant to P. zeae, while ‘SARO-5’ was susceptible. Pratylenchus zeae reduced the growth and yield of both cultivars, though more for ‘SARO-5’ than for ‘Supa’. Yield decreased with increasing final nematode densities. Pratylenchus zeae reproduction was highest at 200 and 500 inoculum levels and under upland water conditions. The yield of ‘SARO-5’ was greater than that of ‘Supa’ under flooded conditions and with no or 200 and 500 nematode inoculum levels, but with high P. zeae inoculum ‘Supa’ yield was better than ‘SARO-5’.