Qubit fluorometry assay

Total RNA was extracted from the plant tissues (stem segment) and fungal material using the standard Trizol protocol (Invitrogen Corp., Carlsbad, CA, USA). Total RNA samples were examined quantitatively using the Qubit fluorometry assay (Invitrogen Corp., Carlsbad, CA, USA) and qualitatively using agarose gel electrophoresis.
Two different RNA mixtures were prepared as the source of the tester and driver samples for the suppressive subtractive hybridization (SSH) library construction. For the tester samples, equal amounts of the total RNA isolated from the stem tissues inoculated with S. sclerotiorum at 6, 12, 24, 48, and 72 hpi were mixed. In the case of the driver samples, RNA from non-inoculated stem tissues harvested at the same times (6–72 h) plus RNA extracted from the mycelium of the fungus [grown separately in minimal medium (2 g/L NH₄NO₃, 1 g/L KH₂PO₄, 0.1 g/L MgSO₄.7H₂O, 0.5 g/L yeast extract, 3 g/L DL-malic acid, 1 g/L NAOH) supplemented with 1% glucose] were mixed at a 2:1 ratio (estimated based on qPCR experiments – data not shown).

Protein extraction was performed using urea buffer (8 M urea, 150 mM NaCl, 50 mM Tris-HCl, pH 8) and mechanical disruption with 1.0-mm silica beads in a Bullet Blender (Next Advance). Samples were centrifuged at 10,000 x g for 10 min and the supernatant removed. The protein concentration was determined using a Qubit fluorometry assay (Invitrogen). Protein concentrations were normalized with lysis buffer. Five hundred micrograms of protein from each sample was reduced with 10 mM dithiothretiol for 30 min at room temperature and alkylated with 10 mM iodoacetamide for 45 min at room temperature. Trypsin was added at a 1:30 enzyme-to-substrate ratio, and urea was diluted to 1.6 M with 25 mM ammonium bicarbonate. The reaction was terminated with 0.1% trifluoroacetic acid (TFA) and the suspension clarified by centrifugation at 10,000 x g for 10 min.

Fungal mats and the infected plant tissues beneath it were flash-frozen in liquid nitrogen and stored at -80 °C. The samples were ground to a fine powder with an RNAse-free mortar and pestle precooled with liquid nitrogen. Total RNA was extracted using an Illustra RNAspin mini RNA isolation kit (Illumina, San Diego, USA). RNA quantity and quality was assessed using a Qubit fluorometry assay (Invitrogen Corp., Carlsbad, CA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), respectively. Libraries were prepared using a Truseq stranded mRNA kit (Illumina, San Diego, USA) following the manufacturer’s instructions. Sequencing was conducted on an Illumina MiSeq sequencing system using the Illumina MiSeq reagent kit V3 (Illumina, San Diego, USA) following the manufacturer’s instructions.