β-glucronidase (GUS) staining

Arabidopsis roots with or without galls were fixed in 90% acetone for 24 h at −30°C and stained in GUS buffer (100 mM NaPO₄ buffer (pH 7), 10 mM EDTA (pH 8), 0.1% Triton X-100, 3 mM K₃Fe(CN)₆ and 3 mM K₄Fe(CN)₆) with 0.5 mg ml⁻¹, 5-bromo-4-chloro-3-indolyl-β-D-glucuronidase (Wako) for 4 h. The reaction was stopped with Carnoy’s solution (90% (v v⁻¹) methanol, 10% (v v⁻¹) acetic acid), and the roots were mounted in a chloral hydrate solution (8 g chloral hydrate, 2 ml water, and 1 ml glycerol). They were then observed using an Axio Imager M1 microscope (Carl Zeiss Microscopy) and photographed using a DP71 digital camera (Olympus). Here we have used Arabidopsis wild type with GUS reporter gene construct without any other mutation to examine the spatial expression after nematode inoculation.