Figure 4.

Polysome fractionation. (A) Flow chart illustrating the details of the separation of the 40S, the 60S and 80S ribosomes and of the polysomes. (B) RNA isolated from the fractionated non-translating ribosomes and from the polysomes were subjected to the RT-PCR assay using the Vid-FW/Vid-RE primer pair. Ladder (L); RNA extracted from mock inoculated tomato plants (TC), PSTVd inoculated tomato plants (TP), mock inoculated N. benthamiana plants (BC) and PSTVd inoculated N. benthamiana plants (BP). RNA extracted from non-translating ribosomes is indicated as NTR, and the RNA extracted from the polysome fraction is denoted by PS. ⁺ve, RT-PCR positive control; RT ⁻ve, RT negative control; and ⁻ve, PCR negative control. (C) Schematic representation of the differentiation of circular PSTVd RNA by RT-PCR assay. In the figure, the red right arrowhead indicates the Vid-FW primer, red left arrowhead indicates the Vid-RE primer, blue right arrowhead indicates the PSTVd-254F primer, and the blue left arrowhead indicates the PSTVd-253R primer. R indicates the reverse primer and F indicates the forward primer. The black dotted lines indicate the cRNA, the red dotted lines indicate the PCR product obtained with the Vid-FW/Vid-RE primer pair and the blue dotted lines indicates the PCR product obtained with the PSTVd-254F/253R primer pair. Vid-FW is complementary to nucleotide positions 355-16 of PSTVd^RG1, Vid-RE is complementary to positions 354-336 of PSTVd^RG1, PSTVd-254F is complementary to positions 254-273 of PSTVd^RG1 and, PSTVd-253R is complementary to positions 253-234 of PSTVd^RG1. The number 1 indicates the first nucleotide of PSTVd^RG1, and the number 359 indicates the last nucleotide of PSTVd^RG1. (D) PCR performed on the cDNA generated by the Vid-RE primer using the PSTVd-254F/PSTVd-253R primer set. The lanes are loaded as shown for (B).