Nucleic acid extraction and virus/viroid enrichment

Five grams of leaf tissue from each infected plant were ground in liquid nitrogen and stored at -80°C until further extraction. For extraction, 100 mg leaf materials were mixed with 20 mg leaf discs from P. vulgaris infected with PvEV1 and PvEV2. The mix was used for three different RNA extraction methods (Fig 1):

The steps are indicated in orange boxes. The sequencing Illumina platforms are in green.

Double-stranded RNA extraction (dsRNA):

dsRNA was extracted using Double-RNA Viral dsRNA Extraction Mini Kit (for plant tissue) (iNtRON Biotechnology, USA) according to the manufacturer’s protocol.

Total RNA extraction followed by ribo-depletion (ribo-depleted totRNA):

Total RNA extraction was performed using innuPREP Plant RNA Kit as described by the manufacturers’ instructions. The ribosomal RNA (rRNA) was depleted using the RiboMinus™ Plant Kit for RNA-Seq (Invitrogen) according to the manufacturers’ protocol.

Total RNA extraction followed by small RNA extraction (sRNA):

Total RNA was extracted as described above, then DNase treated using innuPREP DNase I Digest Kit (Analytik Jena AG) according to the manufacturers’ protocol. sRNA was extracted using polyacrylamide gel selection at Fasteris Life Sciences SA (Plan-les-Ouates, Switzerland).

Additionally, DNA extraction followed by rolling circle amplification (RCA) was carried out for the nanovirus infected plants. Genomic DNA was extracted as described before followed by RCA using TempliPhiTM 100 Amplification Kit (GE Healthcare Limited, UK).