Fig. 3

Detection of PVX CP and genomic RNA in tomato plants inoculated with PVX using western (a) and northern blotting (b). a Total protein samples were prepared from non-inoculated upper leaves of RDR6-, AGO2-, and DCL2.4-knockdown tomato plants (hpRDR6, hpAGO2.3, and hpDCL4.2) at 15 dpi. Samples were also prepared from the empty vector-transformed plants inoculated with PVX (Empty) and buffer (Mock) as controls. The CBB-stained gel was used as a loading control (RUBISCO protein). b Total RNAs extracted from hpRDR6, hpAGO2.3, hpDCL2.4, Empty, and Mock plants at 15 dpi were fractionated using an agarose gel to detect PVX genomic (gRNA) and subgenomic RNAs (sgRNAs) in non-inoculated upper leaves through northern blotting. rRNA was used as loading control. c RT-qPCR to compare the DCL2s, DCL4, AGO2, AGO3, and RDR6 mRNA in the upper leaves of hpDCL2.4, hpAGO2.3, hpRDR6, and empty vector-transformed plants infected with PVX (Empty-PVX) with the levels measured in healthy empty vector-transformed plants (Empty-healthy) at 15 dpi. 18S ribosome RNA was used as a control. Error bars represent SE. Student’s t-test was applied to analyze the data. Each analysis was performed with three biological replicates; data were collected from three plants of each knockdown and empty vector-transformed transgenic lines. The values with the double asterisk (**p < 0.01) and single asterisk (*p < 0.05) were statistically significant at the 1% and 5% levels, respectively, compared with those of healthy empty vector-transformed plants