The following previously described Arabidopsis mutant lines were used: dcl1-8 [in Col-gl1, originally: sin1-2; (43 (link))], dcl1-9 [in La-er; originally: caf-1; (43 (link))], dcl2-5 [in Col-0; (19 (link))], dcl3-1 [in Col-0; (29 (link))], dcl4-2 [in Col-0; (12 (link))], rdr2-1 [in Col-0; (29 (link))], rdr6-15 [in Col-0; (10 (link))], nrpd1a-3 [in Col-0; (14 (link))], ago4-1 [in La-er; (16 (link))], hyl1-2 and hen1-5 [both in Col-0; (44 (link))]. To obtain the double mutant dcl2 dcl3 (d2d3), dcl2-5 was crossed with dcl3-1. Homozygous d2d3 was then crossed with dcl4-2 to obtain homozygous double mutants dcl2 dcl4 (d2d4) and dcl3 dcl4 (d3d4) and the triple mutant dcl2 dcl3 dcl4 (d2d3d4) in the F2 segregating population. Homozygous lines were selected using PCR with allele-specific primers (see Supplementary Data).
Arabidopsis wt and mutant plants were grown from seeds in soil in a phytochamber (Sanyo) at 20°C with 12 h day and 12 h night. Four to five weeks post-germination, unless otherwise stated, seedlings were inoculated with CaLCuV or CaMV by biolistic delivery of a plasmid mixture (0.5 μg each) of pMTCbLCVA.008 and pCPCbLCVB.002 (42 (link)) or 1 μg plasmid pCa122 [the CaMV strain CM1841; (45 (link))], respectively, as described earlier (19 (link)).
One month post-inoculation, unless otherwise stated, virus-infected plants were harvested in pools and ground in liquid nitrogen for total RNA (19 (link)) and DNA (46 (link)) preparations. Titers of the viruses were determined by semi-quantitative duplex PCR (see Supplementary Data).
Mechanical inoculation of Arabidopsis plants with ORMV (41 (link)) was performed at around 5 weeks post-germination using celite 545 (Merck) and sap of ORMV-infected Nicotiana benthamiana.
Arabidopsis wt and mutant plants were grown from seeds in soil in a phytochamber (Sanyo) at 20°C with 12 h day and 12 h night. Four to five weeks post-germination, unless otherwise stated, seedlings were inoculated with CaLCuV or CaMV by biolistic delivery of a plasmid mixture (0.5 μg each) of pMTCbLCVA.008 and pCPCbLCVB.002 (42 (link)) or 1 μg plasmid pCa122 [the CaMV strain CM1841; (45 (link))], respectively, as described earlier (19 (link)).
One month post-inoculation, unless otherwise stated, virus-infected plants were harvested in pools and ground in liquid nitrogen for total RNA (19 (link)) and DNA (46 (link)) preparations. Titers of the viruses were determined by semi-quantitative duplex PCR (see Supplementary Data).
Mechanical inoculation of Arabidopsis plants with ORMV (41 (link)) was performed at around 5 weeks post-germination using celite 545 (Merck) and sap of ORMV-infected Nicotiana benthamiana.
