Solanum torvum responses to the root-knot nematode Meloidogyne incognita.

BACKGROUND: Solanum torvum Sw is worldwide employed as rootstock for eggplant cultivation because of its vigour and resistance/tolerance to the most serious soil-borne diseases as bacterial, fungal wilts and root-knot nematodes. The little information on Solanum torvum (hereafter Torvum) resistance mechanisms, is mostly attributable to the lack of genomic tools (e.g. dedicated microarray) as well as to the paucity of database information limiting high-throughput expression studies in Torvum. RESULTS: As a first step towards transcriptome profiling of Torvum inoculated with the nematode M. incognita, we built a Torvum 3' transcript catalogue. One-quarter of a 454 full run resulted in 205,591 quality-filtered reads. De novo assembly yielded 24,922 contigs and 11,875 singletons. Similarity searches of the S. torvum transcript tags catalogue produced 12,344 annotations. A 30,0000 features custom combimatrix chip was then designed and microarray hybridizations were conducted for both control and 14 dpi (day post inoculation) with Meloidogyne incognita-infected roots samples resulting in 390 differentially expressed genes (DEG). We also tested the chip with samples from the phylogenetically-related nematode-susceptible eggplant species Solanum melongena. An in-silico validation strategy was developed based on assessment of sequence similarity among Torvum probes and eggplant expressed sequences available in public repositories. GO term enrichment analyses with the 390 Torvum DEG revealed enhancement of several processes as chitin catabolism and sesquiterpenoids biosynthesis, while no GO term enrichment was found with eggplant DEG.The genes identified from S. torvum catalogue, bearing high similarity to known nematode resistance genes, were further investigated in view of their potential role in the nematode resistance mechanism. CONCLUSIONS: By combining 454 pyrosequencing and microarray technology we were able to conduct a cost-effective global transcriptome profiling in a non-model species. In addition, the development of an in silico validation strategy allowed to further extend the use of the custom chip to a related species and to assess by comparison the expression of selected genes without major concerns of artifacts. The expression profiling of S. torvum responses to nematode infection points to sesquiterpenoids and chitinases as major effectors of nematode resistance. The availability of the long sequence tags in S. torvum catalogue will allow precise identification of active nematocide/nematostatic compounds and associated enzymes posing the basis for exploitation of these resistance mechanisms in other species.

Low cryoprotectant concentrations and fast cooling for nematode cryostorage.

Cryopreservation protocols based on slow freezing or vitrification often result in cell injury due to ice formation, cell dehydration and/or toxic concentrations of cryoprotectant (CPA). In this study, we present a cryopreservation technique based on low, non-toxic concentrations of cryoprotectants (≈ 2-4M) combined with a rapid cooling rate in the liquid nitrogen phase (-196°C). Protocols for successfully cryopreserving the plant parasitic nematodes Globodera tabacum tabacum, Heterodera schachtii and Meloidogyne incognita were developed, as demonstrated by the high survival rates and reproducibility of cyst and root-knot nematode species post-cryostorage. This approach for effective cryopreservation of viable plant-parasitic nematodes was developed by inducing an "apparent vitrification" by rapid cooling of the microscopic samples in less than 2M of cryoprotectant. The extremely thin structure (15-20 µm width, 350-400 µm length) of these nematodes, in combination with a direct and rapid exposure to LN(2), likely prevents the formation of damaging ice crystals. Moreover, this procedure results in viability of both short- and long-cryostorage samples. These techniques could potentially be used for the near-indefinite preservation of thousands of different nematode species. A cryo-nematode collection produced in our lab is available and presented here.