Plant-nematode Interactions Research Articles

Production and characterisation of monoclonal antibodies to antigens from Xiphinema index

AbstractWhen feeding, Xiphinema index induces remarkable cellular modifications at the feeding site in the root tips of its hosts, similar to those induced by root endoparasites such as Meloidogyne spp. The large size of X. index enables direct analysis of their proteins. Following immunisation of a mouse with macerated extracts of X. index bodies, a panel of monoclonal antibodies (MAbs) was obtained against this nematode. The MAbs were used in immunolocalisation studies and antibodies recognising epitopes present at the surface coat, inner cuticle, dorsal gland cell and dorsal duct, body wall muscle fibres, nervous system and reproductive system were identified. These antibodies provide useful tools for studying plant-nematode interactions.

Direct identification of stylet secreted proteins from root-knot nematodes by a proteomic approach

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 (p I) 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.

Cell cycle activation by plant parasitic nematodes.

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 specialized 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 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 into 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 of possible links between cell cycle activation and local, nematode-induced changes in auxin levels.

New approaches to control plant parasitic nematodes

Plant parasitic nematodes are a serious threat for crop production worldwide. This review summarizes our understanding of plant nematode interactions and presents new alternatives for nematode control in the field. Breeding for resistance has been a major goal for many important crop species like soybean, potato, tomato and sugar-beet. As a result numerous nematode-resistance genes have been identified, two of which have been cloned recently, Hs1 pro-1 from sugar-beet, giving resistance to the beet cyst nematode Heterodera schachtii, and Mi from tomato, giving resistance to the root-knot nematode Meloidogyne incognita. Also artificial resistance genes, coding for nematotoxic proteins or causing rapid death of feeding cells, have been elucidated. In the future, genetic engineering of nematode resistance will become more and more important for plant breeding. Transformation techniques will allow genes to be quickly introduced into susceptible breeding lines and then combined with each other to produce plant varieties with durable resistance.

In plantamonitoring of the activity of two constitutive promoters, CaMV 35S and TR2′, in developing feeding cells induced byGlobodera rostochiensisusing green fluorescent protein in combination with confocal laser scanning microscopy

Under the control of either the constitutive CaMV 35S or the mannopine synthase TR2′ promoter, the green fluorescent protein (GFP) from the jellyfishAequorea victoriawas expressed in transgenic potato (Solanum tuberosum) plants. Confocal laser scanning microscopy (CLSM) was applied to observe GFPin plantaand, subsequently, to investigate promoter activity in developing feeding cells developed during potato cyst nematode (Globodera rostochiensis) infection. Both the CaMV 35S and the TR2′ promoter were strongly upregulated in young feeding cells in less than 4 days upon infection byG. 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 ofG. rostochiensis. Provided CLSM is used, GFP was shown to be a powerful tool that allowsin vivomonitoring 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.

Changes in the lipid content and fatty acid composition of 2nd-stage juveniles of Globodera rostochiensis after rehydration, exposure to the hatching stimulus and hatch.

The total lipid content of the dry weight of whole cysts and 2nd-stage juveniles (J2) of Globodera rostochiensis was 17.1% in dry cysts, 20.9% in cysts soaked in distilled water (DW), 20.3% in cysts that had been in potato root diffusate (PRD) for 7 days, 7.3% in cysts that had been in PRD for 28 days and 29.2% for hatched J2. The fatty acid composition of the total lipid did not differ between dry cysts, cysts in DW and cysts in PRD for 7 days. However, major differences in the fatty acid composition of all lipid classes were found between rehydrated cysts in PRD and freshly hatched J2. After hatching, the degree of saturation and the percentage of monounsaturated fatty acids decreased and the percentage of polyunsaturated fatty acids increased considerably, especially in the free fatty acid fraction, where C20:1 showed an 8-fold decrease and C20:4 a 33-fold increase. There was a difference in the fatty acid composition of cysts in PRD for 7 days and cysts in PRD for 28 days (after most of the J2 had hatched); with increased time in PRD the percentage of polyunsaturated fatty acids increased and the percentage of monounsaturated fatty acids decreased in all lipid classes. Differences in the fatty acid profiles between cysts in PRD for 28 days and hatched J2 were found mainly in the free fatty acid and the non-acidic phospholipid fractions. The free fatty acid fraction of the cysts was dominated by monounsaturated fatty acids (52%), whereas the same fraction of hatched J2 contained large amounts of polyunsaturated fatty acids (60%). These polyunsaturated fatty acids, especially C20:4, might play an important part in nematode-plant interactions during infection.

Molecular aspects of plant-nematode interactions and their exploitation for resistance strategies

Cyst- and root-knot nematodes are economically the most important plant parasitic erop pests. They establish sophisticated feeding sites (syncytia or giant cells) in host roots and cause stunted growth, reduced yield or death of the host plants. This review focusses on molecular aspects of the host-parasite relationship and possibilities that arise from this knowledge to engineer nematode resistance in plants.

Potato Root Diffusate-Induced Secretion of Soluble, Basic Proteins Originating from the Subventral Esophageal Glands of Potato Cyst Nematodes

ABSTRACT In preparasitic second-stage juveniles (J(2)) of potato cyst nematode Globodera rostochiensis, six proteins with molecular masses 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 of these subventral gland proteins (svp's) focused in the basic range (pI 6.8 to 8.6) of an immobilized pH gradient. Western blotting showed that the svp's were present in preparasitic and parasitic J(2) and not in later juvenile stages and adult females. Minor svp quantities also were observed in adult males. Immunogold labeling of preparasitic J(2) 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 svp's through the stylet, and 5-methoxy-N,N-dimethyltryptamine-hydrogen-oxalate had only a quantitative, additional effect. The forward flow of 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.

Identification and in situ and in vitro characterization of secreted proteins produced by plant-parasitic nematodes.

Secretions of plant-parasitic nematodes which are released into plant tissue may play critical roles in plant-nematode interactions. The identification and characterization of these molecules are of fundamental importance and may help to facilitate the development of novel strategies to interfere with nematode infection of plants and thereby decrease nematode-induced damage to crops. An antibody-based approach was used to isolate molecules present on the nematode surface and in nematode secretions. Monoclonal antibodies (MAbs) were produced to secretions and to whole Heterodera avenae 2nd-stage juveniles; several of these MAbs recognized molecules present in nematode secretions produced in vitro. Three of these molecules have been partly characterized in H. avenae, Globodera rostochiensis, G. pallida and Meloidogyne incognita. A MAb reacting with the surfaces of these nematodes recognized antigens of different molecular weight in each of the species tested. This difference in antigenicity might be related to specific functions in these nematodes. Preliminary results show that this antibody also localized the antigen in root cells surrounding the feeding site induced by M. incognita in Arabidopsis thaliana.

PARASITIC STRATEGIES OF ROOT NEMATODES AND ASSOCIATED HOST CELL RESPONSES

Many species of plant-parasitic nematodes have evolved complex relationships with their hosts, inducing profound alterations in the morphology of the cells from which they feed. The availability of modem molecular techniques has recently enabled several breakthroughs in our understanding of plant-nematode interactions. This work underpins an emerging ability to deploy new resistance strategies against these pathogens. Our objective is to highlight recent advances in the areas of parasitic strategies, host cell responses and novel defense strategies.

Induction Patterns of an Extensin Gene in Tobacco upon Nematode Infection.

When sedentary endoparasitic nematodes infect plants, they induce complex feeding sites within the root tissues of their host. To characterize cell wall changes induced within these structures at a molecular level, we studied the expression of an extensin gene (coding for a major structural cell wall protein) in nematode-infected tobacco roots. Extensin gene expression was observed to be induced very early upon infection. This induction was weak, transient, and probably due to wounding during penetration and migration of the tobacco cyst nematode Globodera tabacum ssp solanacea-rum. In contrast, high extensin gene expression was observed during the whole second larval stage (an ~2-week-long phase of establishment of the feeding site) of the root knot nematode Meloidogyne javanica. During later stages of this interaction, expression gradually decreased. Extensin gene expression was found in at least three different tissues of the gall. We propose that distinct mechanisms lead to induced expression in these different cell types. The significance of these results for the understanding of plant-nematode interactions as well as the function of structural cell wall proteins, such as extensin, is discussed.

Plant-nematode interactions.

Recent breakthroughs in research on plantnematode interactions underpinned by the availability of modern molecular techniques is attracting attention to this particular area of phytopathology. In contrast with most bacterial and fungal pathogens, sedentary nematodes are true parasites and establish a long-lasting interaction with their host. They induce profound alterations in host cell morphology (Fig. 1) that are scientifically intriguing and to a large extent a black box. It is the purpose of this paper to review the latest results that allow us an opportunity to peek inside that box. Eventually, more detailed knowledge of the molecular basis of the interaction will enable us to design elegant strategies to engineer nematode resistance in a range of crop plants.

The genetics of plant-nematode parasitic systems

The genetic basis of plant-nematode interactions is discussed with special emphasis on potato-Globodera rostochiensis/pallida, barley/oat-Heterodera avenae, soybean-H, glycines and tomato-Meloidogyne incognita parasitic systems. The basis of physiological specialization and its role in breaking down cultivar resistance is explored. There are numerous reports on the inheritance of resistance as compared to a few on the inheritance of virulence. These reports indicate certain general characteristics of the host-parasite genes. The genes for resistance and virulence are conditional in the sense that the expression of one is dependent on the presence of the other in the association. Therefore, host-parasite genes, often exhibit a gene-for-gene interaction, the basis of which is discussed and illustrated. Also, the genetics of the complex parasitic systems is briefly mentioned. Fungal and nematode parasitic systems are compared throughout the review.

Predisposition of Tomato To the Wilt Fungus (Fusarium Oxysporum Lycopersici) By the Root-Knot Nematode (Meloidogyne Incognita)

Root-knot nematodes (Meloidogyne incognita) often occur together with the wilt fungus (Fusarium oxysporum f. lycopersici) on tomato in a plant disease complex. An interaction between the plant and the nematode may transform a genetically resistant host plant into one that is susceptible to the wilt-fungus and which subsequently develops wilt symptoms. Bridging and grafting experiments indicate that a factor emanating from this nematode-plant interaction is translocated considerable distances to the upper foliage across a resistant scion. Disease indices based on chlorosis of the foliage and on propagule counts from stem exudates showed primarily distal rather than proximal translocation of the factor.

Aspects of the Host-Parasite Relationship of Plant-Parasitic Nematodes

Publisher Summary This chapter reviews the three aspects of the resistant/susceptible host plant response to the nematode parasite—namely, interpretation of the ultrastructure of the response, further explorations into the physiology and biochemistry of the response, and the genetics of the response. The response of plants to members of the Heteroderidae is morphologically the most complex of all nematode-plant interactions, and the morphological changes suggest a very close physiological relationship between the nematode parasite and plant host. In response to a stimulus from the developing sedentary nematode larva, the plant produces the typical giant cell response. Examining the ultrastructure helps in developing an understanding of the mechanisms of this host response. The response of the resistant plant to larvae that have already penetrated it varies greatly, but the plant endeavors either to remove the larvae by producing a chemical that functions as a repellent or to kill them by isolating, starving, or poisoning them. A hypersensitive reaction (HR) in the tissues of resistant plants is a common cell response resulting from an incompatible host–parasite interaction. The nature of the response, whether the initial penetration is in the leaf, stem, or root tissue, is determined by the plant species, the behavior of the nematode, and the introduction of chemicals into the cell by the nematode. Given that the host–parasite relationship is a compatible one, the biochemical interaction between the two components ensures mutual development resulting in successful parasite development and various degrees of host tolerance. Resistance of plant to plant-parasitic nematodes is not manifested until the nematode penetrates the plant tissue, and in this sense, it is equivalent to induced resistance that occurs in animals.