Precellys 24 homogeniser

L-Lactate was measured in conditioned medium and tissue samples using the L-Lactate Assay kit (abcam, #ab65331) according to the manufacturer's instructions. All samples were deproteinized with the Deproteinizing Sample Preparation Kit–trichloroacetic acid (TCIA; abcam, #ab204708) before assaying.
Tissue samples were weighed into Precellys tubes prefilled with ceramic beads (Bertin Instruments, #P000911-LYSKO-A). The exact 6x volume (1 mg tissue/6 µL buffer) of Lactate Assay Buffer was added on ice and samples were lysed using a Precellys 24 homogeniser (Bertin Instruments).

Using a Precellys 24 Homogeniser (Bertin Technologies, Montigny-le-Bretonneux, France), tissues were homogenized in RIPA buffer (50 mM tris, 150 mM sodium chloride, 1 mM Ethylenediaminetetraacetic acid, 0.5% Triton X-100, 0.5% Sodium deoxycholate, pH 7.4) plus cocktail protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA), thereafter homogenates were centrifuged at 13,000× g RPM for 30 min. The supernatants of the homogenates were collected, and the protein concentration was estimated using a bicinchoninic acid protein assay kit (Thermo-scientific, Rockford, IL, USA) according to the manufacturer’s instructions. The remaining supernatant was used for subsequent western blot analyses.

Plant material grown hydroponically for 60 days both under conditions of molybdate availability and deprivation, respectively, was separated into root, as well as into young and old leaves. A total of 100 mg of material was mixed with 500 µL GUS extraction buffer (0.1 M monosodium phosphate, 1 mM DTT) and homogenized using a Precellys 24 Homogeniser (Bertin Instruments, Montigny-le-Bretonneux, France) for two steps at 5500 rpm, each lasting 15 s. Subsequently, the samples were centrifuged for 20 min at 21,000× g and 4 °C, and 20 µL of the supernatant was loaded onto a 96-well plate in triplicates. After the addition of 100 µL prewarmed (37 °C) reaction buffer [50 mM Monosodium phosphate; 1 mM EDTA; 0.1% TritonX-100; 7 µL/mL ß-Mercaptoethanol; 0.33 mg/mL Methylumbelliferylglucuron (MUG); pH 7.0] fluorescence of the cleaved product MU (Methylumbelliferyl; excitation: 365 nm, emission: 455 nm) was measured every 2 min over a total time span of 40 min using a Tristar LB941 multimode reader (Berthold Technologies, Bad Wildbad, Germany). GUS activity was calculated from the gain of fluorescence over a time normalised to the amount of total protein and determined by Bradford assay using Roti^®Quant reagent (Carl Roth, Karlsruhe, Germany). Root samples were measured with a dilution factor of 10.

DNA for microbiome analysis was extracted from the dorsal skin and anterior gut using a DNeasy Blood & Tissue Kit (Qiagen). Modifications in the manufacturer’s protocol included pre-digestion with lysozyme, RNAse treatment and mechanical disruption with 0.1 mm zirconia/silica beads (Biospec) using a Bertin Precellys 24 homogeniser (20 s at 6,800 rpm). Total RNA for RNA-seq was extracted using an E.Z.N.A. Total RNA Kit I (Omega-Biotek) following mechanical disruption of approx. 30 mg of skin and 10–30 mg of gut using 5 mm iron beads (Qiagen) for 2 × 20 sec at 6,800 rpm in a Precellys 24. RNA samples were treated with DNAse to remove possible DNA contamination. DNA/RNA quality and integrity were analysed using a Nanodrop spectrophotometer and 1% agarose gel electrophoresis.

RNA was isolated from tissues using TRIzol reagent (Thermo Fisher Scientific) as per provided instructions, with an initial step of homogenizing the tissues in TRIzol reagent within a bead-containing Lysing Matrix D tube (MP Biomedicals) using the Precellys 24 homogeniser (Bertin Technologies). Complementary DNA (cDNA) was prepared from 2 μg of RNA with the Maxima First Strand cDNA Synthesis Kit (K1641, Thermo Fisher Scientific). qPCR was ran using the Maxima SYBR Green qPCR Master Mix (K0221, Thermo Fisher Scientific) with 10 ng of cDNA per reaction. Analysis was done with the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)) using Eef2 as loading control. Primers used are provided in Supplementary file 9.

Frozen tissue samples were thawed in 1 ml Trizol (Invitrogen) per 30–50 mg tissue and immediately homogenised using a Precellys 24 homogeniser (Bertin). 200 µl of chloroform was added to each sample and mixed by vortex. The aqueous phase was separated by centrifugation at 13,000 × g at 4 °C and RNA was purified using the RNeasy Mini Kit (Qiagen) with on-column DNAse digestion, as per manufacturer’s protocol. A Qubit Fluorimeter (Thermofisher) and/or a Nanodrop instrument was used to assess RNA quality and quantity.

Cells were suspended in protein extraction buffer (50 mM Tris-HCl, 2% Triton-X, 0,4% SDS, 12.5 mM EDTA) containing a protease inhibitor cocktail (ProteaseArrest, G-Biosciences, St. Louis, MO, USA). 0.2 mL acid-washed 425–600 μm diameter glass beads (Sigma-Aldrich) were mixed with the cells. Cells were disrupted using the Precellys-24 homogeniser (Bertin Instruments, Montigny-le-Bretonneux, France) during 4 × 30 s. Centrifugation was done twice at 18 000 × g, for 10 min at 4°C. Protein concentration was determined by using the DC protein assay (Bio-Rad, Hercules, CA, USA). Ten micrograms of proteins, per well, were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), using Any kD gels (Bio-Rad), and transferred to 0.2 μm Polyvinylidene fluoride (PVDF) membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). Strep-tags were detected by Anti-Strep-tag ΙΙ (Abcam, Cambridge, UK) by Clarity Western ECL substrate (Bio-Rad) using standard techniques.