Tobacco rattle virus mediates gene silencing in a plant parasitic root-knot nematode.

Root-knot nematodes (RKNs) are sedentary biotrophic parasites that induce the differentiation of root cells into feeding cells that provide the nematodes with the nutrients necessary for their development. The development of new control methods against RKNs relies greatly on the functional analysis of genes that are crucial for the development of the pathogen or the success of parasitism. In the absence of genetic transformation, RNA interference (RNAi) allows for phenotype analysis of nematode development and nematode establishment in its host after sequence-specific knock-down of the targeted genes. Strategies used to induce RNAi in RKNs are so far restricted to small-scale analyses. In the search for a new RNAi strategy amenable to large-scale screenings the possibility of using RNA viruses to produce the RNAi triggers in plants was tested. Tobacco rattle virus (TRV) was tested as a means to introduce double-stranded RNA (dsRNA) triggers into the feeding cells and to mediate RKN gene silencing. It was demonstrated that virus-inoculated plants can produce dsRNA and siRNA silencing triggers for delivery to the feeding nematodes. Interestingly, the knock-down of the targeted genes was observed in the progeny of the feeding nematodes, suggesting that continuous ingestion of dsRNA triggers could be used for the functional analysis of genes involved in early development. However, the heterogeneity in RNAi efficiency between TRV-inoculated plants appears as a limitation to the use of TRV-mediated silencing for the high-throughput functional analysis of the targeted nematode genes.

Application of RNA interference to root-knot nematode genes encoding esophageal gland proteins.

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.