Murashige and skoog basal medium

To carry out GUS reporter gene staining assays, iqd1-2 (GT6935 line) seeds were sterilized in (70% ethanol, 0.05% tween 20) for 5 min, washed with 100% ethanol and left to air dry. Seeds were germinated in 12-well microtiter dishes sealed with parafilm, each well containing 3 seeds and 2 ml seedling growth medium [SGM; 0.5× Murashige and Skoog basal medium with vitamins (Duchefa, Haarlem, The Netherlands) containing 0.5 g/L MES hydrate and 1% sucrose at pH 5.7]. Seedlings were grown for 14 days in a growth chamber with continuous shaking at 100 rpm before treatment with elicitors. Elicitors were used at the following concentrations: 100 μM SA, 100 μM JA, 100 nM Flg22, and 500 μg/ml chitin. 18 h after treatment with elicitors, seedlings were either taken for RNA isolation (see below) or 2 ml of GUS substrate solution [125 mM sodium phosphate pH 7, 1.25 mM EDTA, 1.25 mM K₄[Fe(CN)₆], 1.25 mM K₃[Fe(CN)₆], 0.5 mM X-Gluc and 1.25% Triton X-100] was poured in each well. The plants were vacuum-infiltrated for 10 min and then incubated at 37°C overnight covered in aluminum foil. Tissues were de-stained with 100% ethanol overnight and placed in 70% ethanol before digital pictures were taken.

J2s of G. pallida were first sterilised with 0.1% chlorhexidine digluconate, 0.5 mg/ml CTAB for 25 mins and washed three times with sterile 0.01% Tween-20. The J2s were then incubated with gentle agitation for 24 h in water, 100 μM reserpine phosphate, 100 μM methiothepin or 100 μM CPA prior to inoculation of potato hairy root cultures with 25 J2s per infection point and 3 infection points per root system. Hairy root cultures were generated using Agrobacterium rhizogenes R1000 and multiplied as previously described [50 (link)]. Individual root systems of equivalent size growing on 9 cm plates containing Murashige and Skoog basal medium (Duchefa, Suffolk, UK) with 2% sucrose were selected for inoculation. Nine to 11 replicate plates were used for each treatment or control. Roots were stained with acid fuchsin 13 days after infection as described previously [51 (link)] and the number of nematodes in each root system counted. Each complete invasion assay was carried out on two separate occasions.