Plant parasitic nematodes have been, so far, refractory to transformation or mutagenesis. The functional analysis of nematode genes relies on the development of reverse genetic tools adapted to these obligate parasites. Here, we describe the application of RNA interference (RNAi) to the root-knot nematode Meloidogyne incognita for the knock-down of two genes expressed in the subventral esophageal glands of the nematode and potentially involved in parasitism, the calreticulin (Mi-crt) and the polygalacturonase (Mi-pg-1) genes. Incubation in 1% resorcinol for 4 h induced double-stranded RNA uptake through the alimentary track of the nematodes and led to up to 92% depletion of Mi-crt transcripts. Timecourse analysis of the silencing showed different temporal patterns for Mi-crt and Mi-pg-1. The silencing of Mi-crt was optimal 20 h after soaking, whereas the silencing of Mi-pg-1 was optimal 44 h after soaking. For the two genes, the silencing effect was highly time-limited, since no transcript depletion was detectable 68 h after soaking.

Similarly to microbial pathogens, plant-parasitic nematodes secrete into their host plants proteins that are essential to establish a functional interaction. Identifying the destination of nematode secreted proteins within plant cell compartment(s) will provide compelling clues on their molecular functions. Here the fine localization of five nematode secreted proteins was analysed throughout parasitism in Arabidopsis thaliana. An immunocytochemical method was developed that preserves both the host and the pathogen tissues, allowing the localization of nematode secreted proteins within both organisms. One secreted protein from the amphids and three secreted proteins from the subventral oesophageal glands involved in protein degradation and cell wall modification were secreted in the apoplasm during intercellular migration and to a lower extent by early sedentary stages during giant cell formation. Conversely, another protein produced by both subventral and dorsal oesophageal glands in parasitic stages accumulated profusely at the cell wall of young and mature giant cells. In addition, secretion of cell wall-modifying proteins by the vulva of adult females suggested a role in egg laying. The study shows that the plant apoplasm acts as an important destination compartment for proteins secreted during migration and during sedentary stages of the nematode.

The ¢rst animal polygalacturonase (PG, EC 2.1.15) encoding cDNA, Mi-pg-1, was cloned from the plant parasitic nematode Meloidogyne incognita. The enzymatic activity of MI-PG-1 was con¢rmed after heterologous expression in Escherichia coli. The presence of a predicted signal peptide on the MI-PG-1 sequence together with the speci¢c localization of the transcripts of the Mi-pg-1 gene in the oesophageal glands of infective juveniles imply that MI-PG-1 could be secreted into plant tissues. The potential role of MI-PG-1 in parasitism is discussed. ß

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.

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