In situ hybridization (ISH) in preparasitic and parasitic stages of the plant-parasitic nematode Meloidogyne spp.

[Abstract] The spatio-temporal expression pattern of a gene provides important indications to better understand its biological function. In situ hybridization (ISH) uses a labeled complementary single-stranded RNA or DNA probe to localize gene transcripts in a whole organism, a whole organ or a section of tissue. We adapted the ISH technique to the plant parasite Meloidogyne spp. (root-knot nematode) to visualize RNAs both in free-living preparasitic juveniles and in parasitic stages settled in the plant tissues. We describe each step of the probe synthesis, digoxigenin (DIG) labeling, nematode extraction from plant tissue, and ISH procedure.

4. After digestion, pour the liquid through a 2.5 mm sieve and collect the filtrate.
5. Pour the liquid through a 250 µm sieve and collect the filtrate.
7. Collect the parasitic RKNs on the 40 µm sieve with a minimum volume of tap water (~8 ml).
8. Purify the nematodes by sucrose gradient centrifugation: add slowly 4 ml of the collected nematode on the top of cold 50% sucrose/M9 mix (2 tubes) (Figure 1).9. Centrifuge at 720 x g, at 4 °C for 5 min, without a break.10.Collect the layer of nematodes with a P1000 and wash them carefully adding 40 ml M9 buffer in a 50 ml glass beaker (Figure 1). 2. Clean a glass plate with soap, rinse with nuclease-free water, spray RNaseZap ® and rinse with 70% ethanol.Dry well with a paper towel.
3. Centrifuge the 50 ml tube containing nematodes soaked in fixative buffer at 1,620 x g, slow deceleration.
4. Withdraw fixative buffer but keep 500 µl and transfer to 1.5 ml Eppendorf tubes.
5. Under the fume hood, wearing gloves, spread the nematodes on the glass plate, and cut the nematodes into two to three pieces using the cutting system (vibrating aquarium pump + autoclaved razor blades) described in Figure 2 and Video 1. Hold it with both hands.Check under the microscope the efficiency of the cutting.Do not cut too much, in particular the parasitic stages.

Figure 1 .
Figure 1.Extraction of parasitic stages by sucrose gradient

Figure 2 .
Figure 2. Nematode cutting before ISH.Diagram (A) and picture of the experimental device (B).