Pellet pestle

Drosophila: Three biological replicates of L3 wandering larvae (n = 30) and 10‐day‐old flies (n = 10) treated for 12 h with rapamycin or ethanol were snap‐frozen and stored at −80°C. Total RNA from frozen samples was isolated with TRIzol Reagent (Invitrogen), using pellet pestles (Sigma‐Aldrich, cat. #Z359971‐1EA). RNA was reverse‐transcribed using iScript cDNA synthesis kit (Bio‐Rad, cat. #1708891) according to the manufacturer's instructions, and quantitative PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, cat. #4367659). The Ct values were normalized to the housekeeping gene tubulin. Primer sequences used for qRT–PCR are listed in Table EV1.

Leaf tissues from Pfo-infiltrated tomato or Arabidopsis plants were harvested at 6 hpi and snap frozen. Samples were ground in collection tubes using pellet pestles (Sigma) and total RNA extracted using Trizol (Invitrogen) as per the manufacturer’s instructions. The 260/280 ratios of all samples were checked using a Nanodrop spectrophotometer and all were between 1.8 and 2.1 μg of total RNA was treated with DNaseI (NEB) and samples were subsequently tested for gDNA contamination by PCR amplification using either UBI3 (tomato) or PEX4 (Arabidopsis) primer pairs. First strand cDNA synthesis was performed using the SuperScript III kit (Invitrogen). cDNA was diluted 1:10 with dH₂O prior to qRT-PCR and 1 μl of this dilution was used per reaction.
Duplicate reactions for each sample/primer-pair combination were conducted using clear 96-well PCR plates (Bio-Rad) and iQ SYBR Green Supermix (Bio-Rad). Reactions were carried out using an iCycler real-time PCR system (Bio-Rad) using the following thermocycle (5 min at 95°C followed by 30 s at 95°C, 30 s at 58°C, 30 s at 72°C × 40 cycles). Relative expression values for defense-related genes were calculated using the formula 2^-ΔCt (Pfaffl, 2001 (link)) relative to the TIP41 reference gene (tomato) or PEX4 (Arabidopsis). Expression values were rescaled for presentation such that the buffer treatment is equal to 1.

Intracellular metabolites were extracted as previously described with some modifications (Dettmer et al., 2011 (link)). 800 μl of chilled MeOH/H₂O (− 20 °C, 80:20(v/v)) was added to the frozen cell pellet, homogenized for 1 min using Pellet pestles (Sigma-Aldrich, UK). The sample was centrifuged at 4 °C and 7000×g for 5 min and the supernatant was collected into a new Eppendorf tube. The pellet was re-extracted three times with 400 μl cold methanol/water (− 20 °C, 80:20 (v/v)) to produce 2 ml of extract. The supernatants were combined, dried under a gentle flow of N₂ gas and stored at -80° C until dissolution for NMR analysis.