Virus Inoculation of Nicotiana benthamiana

Seedlings of N. benthamiana were grown in a glasshouse and were infected with one of five viruses at 4 weeks of age. Virus inocula were first prepared in N. benthamiana as follows: BBSV and TNV-A^C were mechanically inoculated into N. benthamiana with previously described in vitro transcripts [87] (link), [88] ; BSMV and PVX were agro-inoculated into N. benthamiana according to published methods [65] (link); and BNYVV was propagated on N. benthamiana, and its total RNA was extracted from symptomatic leaves to be used as the inoculum [89] (link). At 7–14 dpi, systemic leaves that had typical symptoms of the corresponding viruses were sampled. These samples were ground in 20 vol. 0.1 M potassium phosphate (K₂HPO₄) buffer (pH 7.4) containing carborundum (as an abrasive). Mechanical inoculations were then undertaken using the sap from systemically infected N. benthamiana leaf tissue for the test. Simultaneously, mock-inoculated plants without infectious homogenate were created as controls.
All the N. benthamiana plants, except those inoculated with BBSV, were maintained in a controlled environmental climate chamber at 24±0.5°C with a photoperiod of 14-hours light (∼75 µmol/m²/s) and 10-hours dark. Plants infected by BBSV, together with four mock-inoculated controls, were grown at 18°C, since relatively low temperatures is required for BBSV to establish systemic infection in N. benthamiana[90] (link). At least 12 plants were inoculated for each virus. On days 7–14, typical symptoms appeared in the upper leaves and the infection was further confirmed by Western blotting using virus-specific antiserum (Figure S2). For each of the five viruses, three biological replicates were collected and subjected to RNA extraction. Each replicate consisted of upper leaf tissue pooled from four N. benthamiana plants.