Arabidopsis formin AtFH6 is a plasma membrane-associated protein upregulated in giant cells induced by parasitic nematodes

Plant-parasitic nematodes Meloidogyne spp induce an elaborate permanent feeding site characterized by the redifferentiation of root cells into multinucleate and hypertrophied giant cells. We have isolated by a promoter trap strategy an Arabidopsis thaliana formin gene, AtFH6, which is upregulated during giant cell formation. Formins are actin-nucleating proteins that stimulate de novo polymerization of actin filaments. We show here that three type-I formins were upregulated in giant cells and that the AtFH6 protein was anchored to the plasma membrane and uniformly distributed. Suppression of the budding defect of the Saccharomyces cerevisiae bni1Delta bnr1Delta mutant showed that AtFH6 regulates polarized growth by controlling the assembly of actin cables. Our results suggest that AtFH6 might be involved in the isotropic growth of hypertrophied feeding cells via the reorganization of the actin cytoskeleton. The actin cables would serve as tracks for vesicle trafficking needed for extensive plasma membrane and cell wall biogenesis. Therefore, determining how plant parasitic nematodes modify root cells into giant cells represents an attractive system to identify genes that regulate cell growth and morphogenesis.     

Enhancement of Cylindrocladium crotalariae Root Rot by Meloidogyne arenaria (Race 2) on a Peanut Cultivar Resistant to Both Pathogens

Two populations of Meloidogyne arenaria (race 2, incompatible on peanut) enhanced development of Cylindrocladium black rot (CBR) on CBR-resistant peanut cv. NC 3033 in greenhouse factorial experiments. Nematode populations 256 and 486 (0, 10³, 10⁴ eggs per 15-cm pot) were tested in all combinations with Cylindrocladium crotalariae (0, 0.5, 5, 50 microsclerotia per cm³ of soil). Root-rot index increased in the presence of either population. Positions but not slope values of inoculum density-disease curves were changed by both populations, indicating increased efficiency of microsclerotia when peanuts were grown in the presence of these nematodes. Although little or no reproduction occurred with either nematode population on NC 3033, larvae of 256 and 486 penetrated roots. Meloidogyne arenaria 486 did not induce root galls and was not snccessful in establishing feeding sites. Meloidogyne arenaria 256 produced a few very small eliptical galls and had a range of success in establishing a feeding site, varying from no giant cell development to large giant cell with production of a few eggs.     

Cloning and characterization of an esophageal-gland-specific chorismate mutase from the phytoparasitic nematode Meloidogyne javanica

Root-knot nematodes are obligate plant parasites that alter plant cell growth and development by inducing the formation of giant feeder cells. It is thought that nematodes inject secretions from their esophageal glands into plant cells while feeding, and that these secretions cause giant cell formation. To elucidate the mechanisms underlying the formation of giant cells, a strategy was developed to clone esophageal gland genes from the root-knot nematode Meloidogyne javanica. One clone, shown to be expressed in the nematode's esophageal gland, codes for a potentially secreted chorismate mutase (CM). CM is a key branch-point regulatory enzyme in the shikimate pathway and converts chorismate to prephenate, a precursor of phenylalanine and tyrosine. The shikimate pathway is not found in animals, but in plants, where it produces aromatic amino acids and derivative compounds that play critical roles in growth and defense. Therefore, we hypothesize that this CM is involved in allowing nematodes to parasitize plants.     

Effects of Hydroxyurea on the Ultrastructure of Giant Cells in Galls Induced by Meloidogyne javanica

Hydroxyurea (HU) at concentrations of 10 or 20 mg/liter was included in a medium on which excised tomato roots infected with the root-knot nematode Meloidogyne javanica were grown. In the HU, treated roots, giant cells were small and contained large vacuoles. Giant cell nuclei were amoeboidal with relatively small nucleoli in treated roots, compared with giant cells of nontreated galls. In treated-root giant cells, the cytoplasm was diffuse and few organelles such as mitochondria, dictyosomes, and endoplasmic reticulum were detected; also, walls of giant cells were thin with less extensive ingrowths than in nontreated roots. We conclude that HU suppressed normal giant cell formation interfering with its function as a feeding cell.     

Three-dimensional ultrastructure of feeding tubes and interconnected endoplasmic reticulum in root-knot nematode-induced giant cells in rose balsam

We investigated the three-dimensional ultrastructure of feeding tubes and the surrounding region in giant cells induced in rose balsam (Impatiens balsamina L.) roots by the root-knot nematode Meloidogyne incognita, using osmium maceration coupled with field emission scanning electron microscopy (FE-SEM). In the roots of 35-day-old galled rose balsam plants, adult nematodes induced the formation of giant cells containing feeding tubes and numerous organelles, including tubular endoplasmic reticulum (ER), cisternal ER, and mitochondria. The feeding tubes were surrounded by fine tubular structures (20–50 nm in diameter), which were in turn surrounded by tubular ER (approximately 120 nm in diameter). The termini of the fine tubular structures appeared to be connected to the surface of the feeding tubes, suggesting that the fine tubular structures were continuous with narrow channels in the feeding tubes. The tubular ER arose from cisternal ER. Large bundles of tubular ER were present near the feeding tube, in the centers of the giant cells, and in the peripheral regions of the giant cells, such as cell wall ingrowths, while smaller bundles of tubular ER formed networks in the giant cells. These observations suggest that tubular ER functions as vascular bundles in giant cells, facilitating the transport of nutrients. We identified capsule-shaped structures (30 μm in diameter) in the giant cells that consisted of smooth, repeatedly branched ER tubules wrapped in several layers of cisternal ER. We propose that lipids and steroids are synthesized at the smooth branched ER and stored in these capsules until needed by the nematode.     

Expression of a plant expansin is involved in the establishment of root knot nematode parasitism in tomato

A group of plant proteins, expansins, have been identified as wall-loosening factors and as facilitators of cell expansion in vivo. The root knot nematode Meloidogyne javanica establishes a permanent feeding site composed of giant cells surrounded by gall tissue. We used quantitative PCR and in situ localization to demonstrate the induction of a tomato (Lycopersicon esculentum cv. VF36) expansin (LeEXPA5) expression in gall cells adjacent to the nematode feeding cells. To further characterize the biological role of LeEXPA5 we have generated LeEXPA5-antisense transgenic roots. The ability of the nematode to establish a feeding site and complete its life cycle, the average root cell size and the rate of root elongation were determined for the transgenic roots, as well as the level of LeEXPA5 expression in non-infected and nematode-infected roots. Our results demonstrated that a decrease of LeEXPA5 expression reduces the ability of the nematode to complete its life cycle in transgenic roots. We suggest that a plant-originated expansin is necessary for a successful parasitic nematode–plant interaction.     

Histological response of resistant tomato cultivars to infection of virulent Tunisian root-knot nematode (Meloidogyne incognita) populations

The response of a susceptible tomato cultivar (Solanum lycopersicum cv. Rio Grande) to infection by three populations of root-knot nematode (Meloidogyne incognita) was compared histologically with that of Lycopersicon esculentum cv. Monita, L. esculentum cv. VFN8 and Solanum lycopersicum cv. Nemador possessing the Mi-1 resistance gene and accession PI126443 of L. peruvianum possessing the Mi-3 gene. The resistant cultivars showed susceptibility to the Tunisian Meloidogyne populations. Feeding sites were characterised by the development of giant cells that contained granular cytoplasm and several hypertrophied nuclei. The cytoplasm of giant cells was aggregated along their thickened cell walls and consequently the vascular tissues within galls appeared disrupted and disorganised. Feeding site formed on resistant L. esculentum lines and susceptible cultivar Rio Grande are similar according to cell and nucleus number, and the nurse superficies. Resistant accession L. peruvianum PI126443, known to possess heat-stable nematode resistance, also showed susceptible reaction to Tunisian Meloidogyne incognita populations; however, nematode development was reduced in comparison with susceptible plants and less developed feeding cells were observed.     

In planta secretion of a calreticulin by migratory and sedentary stages of root-knot nematode

Esophageal secretions from endoparasitic sedentary nematodes are thought to play key roles throughout plant parasitism, in particular during the invasion of the root tissue and the initiation and maintenance of the nematode feeding site (NFS) essential for nematode development. The secretion in planta of esophageal cell-wall-degrading enzymes by migratory juveniles has been shown, suggesting a role for these enzymes in the invasion phase. Nevertheless, the secretion of an esophageal gland protein into the NFS by nematode sedentary stages has never been demonstrated. The calreticulin Mi-CRT is a protein synthesized in the esophageal glands of the root-knot nematode Meloidogyne incognita. After three-dimensional modeling of the Mi-CRT protein, a surface peptide was selected to raise specific antibodies. In planta immunolocalization showed that Mi-CRT is secreted by migratory and sedentary stage nematodes, suggesting a role for Mi-CRT throughout parasitism. During the maintenance of the NFS, the secreted Mi-CRT was localized outside the nematode at the tip of the stylet. In addition, Mi-CRT accumulation was observed along the cell wall of the giant cells that compose the feeding site, providing evidence for a nematode esophageal protein secretion into the NFS.     

Description of a Unique,Complex Feeding Socket Caused by the Putative Primitive Root-Knot Nematode,Meloidogyne kikuyensis

Meloidogyne kikuyensis produces unique galls that form on one side of the root resembling nitrogen-fixing nodules that are produced on legumes in response to infection by Rhizobium and related bacteria. The gall caused by this root-knot nematode is made up of a complex feeding socket composed of several giant cells that are ramified with xylem vessels extending perpendicular from the vascular cylinder. The anterior portion of the second-stage juvenile, which develops into an adult, plugs into this unique feeding socket. The socket and the surrounding parenchyma together form a gall that is very different in morphology from those typically caused by other species of root-knot nematodes. Even though M. kikuyensis was considered to be a primitive species because of its low chromosome count, the complexity of its feeding site and minor plant damage suggests a more derived systematic position.     

Plant parasitic nematode proteins and the host parasite interaction.

This review focuses on the proteins and secretions of sedentary plant parasitic nematodes potentially important for plant-nematode interactions. These nematodes are well equipped for parasitism of plants. Having acquired the ability to manipulate fundamental aspects of plant biology, they are able to hijack host-cell development to make their feeding site. They feed exclusively from feeding sites as they complete their life cycle, satisfying their nutritional demands for development and reproduction. Biochemical and genomic approaches have been used successfully to identify a number of nematode parasitism genes. So far, 65 204 expressed sequence tags (ESTs) have been generated for six Meloidogyne species and sequencing projects, currently in progress, will underpin genomic comparisons of Meloidogyne spp. with sequences of other pathogens and generate genechip microarrays to undertake profiling studies of up- and down-regulated genes during the infection process. RNA interference provides a molecular genetic tool to study gene function in parasitism. These methods should provide new data to help our understanding of how parasitic nematodes infect their hosts, leading to the identification of novel pathogenicity genes.     

Enhanced levels of plant cell cycle inhibitors hamper root-knot nematode-induced feeding site development

Root-knot nematodes (RKN) are highly specialized, obligatory plant parasites. These animals reprogram root cells to form large, multinucleate, and metabolically active feeding cells (giant cells) that provide a continuous nutrient supply during 3–6 weeks of the nematode’s life. The establishment and maintenance of physiologically fully functional giant cells are necessary for the survival of these nematodes. As such, giant cells may be useful targets for applying strategies to reduce damage caused by these nematodes, aiming the reduction of their reproduction. We have recently reported the involvement of cell cycle inhibitors of Arabidopsis, named Kip-Related Proteins (KRPs), on nematode feeding site ontogeny. Our results have demonstrated that this family of cell cycle inhibitors can be envisaged to efficiently disrupt giant cell development, based on previous reports which showed that alterations in KRP concentration levels can induce cell cycle transitions. Herein, we demonstrated that by overexpressing KRP genes, giant cells development is severely compromised as well as nematode reproduction. Thus, control of root-knot nematodes by modulating cell cycle-directed pathways through the enhancement of KRP protein levels may serve as an attractive strategy to limit damage caused by these plant parasites.